CN117098841A

CN117098841A - Method for purifying recombinant viral particles

Info

Publication number: CN117098841A
Application number: CN202280023507.1A
Authority: CN
Inventors: 塔玛拉·泽科维奇; 康纳·史密斯; 保罗·格雷巴克-克拉克; 埃里克·沃斯特; 伊娃·格雷厄姆; 雅各布·史密斯; 伊伦拉·巴杰洛维奇; 约旦·霍布斯; 罗伯特·提克卡恩; 乔什·格里格
Original assignee: Asklepios Biopharmaceutical Inc
Current assignee: Asklepios Biopharmaceutical Inc
Priority date: 2021-01-21
Filing date: 2022-01-21
Publication date: 2023-11-21

Abstract

Provided herein are methods of purification, production, and manufacture of recombinant viral vector particles, e.g., recombinant adeno-associated viral (rAAV) vector particles that are substantially free of empty viral particles; a population of recombinant adeno-associated virus (rAAV) particles purified using the methods described herein, and pharmaceutical compositions comprising the purified rAAV.

Description

Method for purifying recombinant viral particles

Cross Reference to Related Applications

According to 35 U.S. c. ≡119 (e), the application claims the benefits of U.S. provisional application No. 63/139,997 filed on day 21 in year 2021, U.S. provisional application No. 63/222,087 filed on day 15 in year 2021, and U.S. provisional application No. 63/282,001 filed on day 22 in year 2021, each of which is incorporated herein by reference in its entirety.

Technical Field

The present disclosure relates generally to methods of purification, production, and manufacture of recombinant viral vector particles, such as recombinant adeno-associated virus (rAAV) vector particles.

Background

One challenge faced in the isolation and purification of recombinant viral vectors is to ensure efficient separation of functional viral particles from contaminating, closely related molecular species (e.g., inactive vector forms, including empty and incompletely packed viral capsids, helper viruses and cell membrane vesicles). Accordingly, there is a need in the art for methods, systems, and compositions for separating and/or purifying recombinant viral particles from contaminated impurities. The present disclosure addresses some of these needs in part.

Disclosure of Invention

In one aspect, provided herein are methods of purifying or isolating recombinantly expressed viral particles (e.g., recombinant adeno-associated viruses (rAAV)) from a formulation comprising recombinant vector particles, empty capsids, and host cell impurities, thereby providing a product substantially free of empty viral particles, optionally comprising a transgene. Typically, the method comprises contacting the formulation (e.g., a harvest medium comprising recombinant viral particles) with an affinity chromatography medium under conditions that allow binding of the viral particles to the affinity chromatography medium. The bound virus particles are eluted from the affinity chromatography medium using an elution buffer, and an eluate comprising the eluted virus particles is recovered. In some embodiments, the affinity elution buffer comprises a weak acid or salt thereof. In some embodiments, the affinity elution buffer is substantially free of a weak acid or salt thereof. The eluate from the affinity chromatography is also referred to herein as an affinity eluate.

In some embodiments of any of the aspects, the affinity eluate comprises glycine. In some embodiments, the affinity elution buffer comprises glycine, optionally with acetic acid or a salt thereof; or succinic acid or a salt thereof; or citric acid or a salt thereof; or propionic acid or a combination of salts thereof. In one aspect of an embodiment, the affinity elution buffer comprises glycine and citric acid or a salt thereof (e.g., citrate).

In some embodiments of any of the aspects, the affinity eluate comprises histidine. In some embodiments, the affinity elution buffer comprises histidine.

In some embodiments of any of the aspects, the affinity eluate comprises glycine and histidine. For example, the affinity eluate comprises glycine and histidine.

In some embodiments of any of the aspects, the eluate from the affinity chromatography comprises a predetermined amount of an anionic compound. For example, the method includes adjusting the affinity eluate such that the adjusted eluate contains a predetermined amount of the anionic compound. "anionic compound" refers to a compound that includes a negatively charged moiety. The predetermined amount of anionic compound may be at least about 0.5mM or greater. It should be noted that the anionic compound may be any suitable anionic compound. For example, the anionic compound may be an acid or a salt thereof. In some embodiments of any of the aspects, the anionic compound is a weak acid. For example, the anionic compound is citric acid or a salt thereof, acetic acid or a salt thereof, or succinic acid or a salt thereof.

In some embodiments of any of the aspects described herein, the method comprises adding an anionic compound to the affinity eluate. For example, the method comprises adding an acid or salt thereof to the affinity eluate. In some embodiments of any aspect, the method comprises adding a predetermined amount of a weak acid or salt thereof, e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof, to the affinity eluate. For example, the method comprises adding a predetermined amount of citric acid or a salt thereof, acetic acid or a salt thereof, or succinic acid or a salt thereof to the affinity eluate. In some embodiments, the method comprises adding a predetermined amount of citric acid or a salt thereof, e.g., citrate, to the affinity eluate.

The affinity eluate may be diluted prior to contact with the anion exchange chromatography medium. For example, the affinity eluate may be diluted by a factor of 2X or more, e.g., 3-6X or 12-15X. In some embodiments of any of the aspects described herein, the affinity eluate may be diluted by adding a dilution buffer to the affinity eluate comprising a predetermined amount of the anionic compound. For example, the dilution buffer comprises a predetermined amount of an acid or salt thereof. In some embodiments of any of the aspects, the dilution buffer comprises a predetermined amount of a weak acid or salt thereof. For example, the dilution buffer comprises a predetermined amount of citric acid or a salt thereof, acetic acid or a salt thereof, or succinic acid or a salt thereof. In some embodiments, the dilution buffer comprises a predetermined amount of citric acid or a salt thereof, e.g., citrate. The predetermined amount of anionic compound in the dilution buffer may be at least about 0.5mM.

The affinity eluate is contacted with an anion exchange chromatography medium under conditions that allow binding of the virus particles to the anion exchange chromatography medium. The inventors have found in particular that the presence of an ionic compound (e.g., an anionic compound, such as an acid or a salt thereof, e.g., a weak acid or a salt thereof) in a buffer for equilibration and/or adjustment of chromatography prior to contact with an affinity eluate surprisingly and unexpectedly results in preferential binding of genome-containing viral particles (e.g., adeno-associated viral (AAV) particles having a packaged genomic sequence) (i.e., whole viral particles) over AAV particles lacking the genome (i.e., empty). Preferential binding of such whole viral particles (e.g., whole AAV particles) and the consequent preferential elimination of empty viral particles (e.g., empty AAV particles) is referred to herein as a partitioning effect (partitioning effect/partition effect). Thus, in some embodiments of any aspect, the anion exchange chromatography medium is equilibrated with a buffer, e.g., an equilibration buffer or AEX equilibration buffer for anion exchange chromatography, comprising a predetermined amount of ionic compound (e.g., anionic compound and/or cationic compound). For example, the AEX equilibration buffer comprises a predetermined amount of an acid or salt thereof, e.g., a weak acid or salt thereof. In some embodiments of any of the aspects, the AEX equilibration buffer comprises a predetermined amount of citric acid or a salt thereof, acetic acid or a salt thereof, or succinic acid or a salt thereof. In some embodiments, the AEX equilibration buffer comprises a predetermined amount of citric acid or a salt thereof, e.g., citrate. In some aspects provided herein, adding an ionic compound (e.g., an anionic compound, such as an acid or salt thereof, e.g., a weak acid or salt thereof) to a dilution buffer used to dilute an affinity eluate results in preferential elimination of empty viral particles (e.g., empty AAV particles) that bind to anion exchange chromatography. In some embodiments, adding an ionic compound (e.g., an anionic compound, such as an acid or salt thereof, e.g., a weak acid or salt thereof) to a buffer used to equilibrate and/or adjust chromatography and/or adding an ionic compound (e.g., an anionic compound, such as an acid or salt thereof, e.g., a weak acid or salt thereof, to a dilution buffer used to dilute an affinity eluate prior to contact with the affinity eluate results in preferential elimination of empty viral particles (e.g., empty AAV particles) bound to anion exchange chromatography, i.e., results in a partitioning effect.

The bound virus particles may be eluted from the anion exchange chromatography medium using a suitable elution buffer. Typically, at least about 70%, or at least about 75%, or at least about 80% or more of the viral particles in the anion exchange eluate are all rAAV viral particles. In some preferred embodiments, at least about 85%, or at least about 86%, or at least about 88%, or at least about 90%, or at least about 92%, or at least about 95% or more of the viral particles in the anion exchange eluate are all rAAV viral particles. For example, less than 30%, less than 20%, less than 19.5%, less than 19%, less than 18.5%, less than 18%, less than 17.5%, less than 17%, less than 16.5%, less than 16%, less than 15.5%, less than 15%, less than 14.5%, less than 14%, less than 13.5%, less than 12%, less than 11.5%, less than 11%, less than 10.5%, less than 9.5%, less than 9%, less than 8.5%, less than 8%, less than 7.5%, less than 7%, less than 6.5%, less than 6%, less than 5.5%, less than 5%, less than 4.5%, less than 4%, less than 3.5%, less than 3%, less than 2.5%, less than 1.5%, less than 1%, less than 0.75%, less than 0.5%, less than 0.25%, less than 0.2%, less than 0.15%, less than 0.1% or less than 0.05% of the AAV viral particles in the eluate from the anion exchange are empty viral particles. In some embodiments, the anion exchange eluate comprises less than 0.04%, or preferably less than 0.02%, empty AAV viral particles. In some embodiments, the eluate from the anion exchange is substantially free of empty AAV viral particles. In some embodiments, about 10% to about 15% of AAV viral particles in the eluate from the anion exchange are empty viral particles. In some embodiments, the methods described herein produce AAV particles with a reduction in hollow particles by more than 86-fold, or more than 90-fold, or more than 95-fold, or preferably more than 99-fold. In some embodiments, 30% or less, or 25% or less, or 20% or less, or 15% or less, or 10% or less, or 5% or less, or even less of AAV particles purified with the methods disclosed herein are incompletely packed. In one aspect of an embodiment, the eluate of the anion exchange column comprises less than 11%, less than 10%, less than 9%, less than 8%, less than 6%, or less than 5% of the incompletely packed AAV particles.

In some embodiments of any of the aspects described herein, the ratio of total rAAV viral particles (e.g., intact, incomplete, and empty AAV viral particles) to empty rAAV viral particles in the anion exchange eluate is at least about 1.25× higher than the ratio of total rAAV viral particles to empty particles in the affinity eluate. For example, the number of the cells to be processed, the ratio of total rAAV viral particles to empty rAAV viral particles in the anion exchange eluate is at least about 1.5×, at least about 1.6×, at least about 1.7×, at least about 1.8×, at least about 1.9×, at least about 2×, at least about 2.1×, at least about 2.2×, at least about 2.3×, at least about 2.4×, at least about 2.5×, at least about 2.6×, at least about 2.7×, at least about 2.8× greater than the ratio of total rAAV viral particles to empty rAAV viral particles in the affinity eluate at least about 2.9×, at least about 3×, at least about 3.1×, at least about 3.2×, at least about 3.3×, at least about 3.4×, at least about 3.5×, at least about 3.6×, at least about 3.7×, at least about 3.8×, at least about 3.9×, at least about 4×, at least about 4.1×, at least about 4.2×, at least about 4.3×, at least about 4.4×, at least about 4.5×, at least about 4.6×, at least about 4.7×, at least about 4.8×, at least about 4.9× at least about 2.9×, at least about 3×, at least about 3.1×, at least about 3.2×, at least about 3.3×, at least about 3.4×, at least about 3.5×, at least about 3.6×, at least about 3.7×, at least about 3.8×, at least about 3.9×, at least about at least about 4×, at least about 4.1×, at least about 4.2×, at least about 4.3×, at least about 4.4×, at least about 4.5×, at least about 4.6×, at least about 4.7×, at least about 4.8×, at least about 4.9×, at least about, at least about 9.3×, at least about 9.4×, at least about 9.5×, at least about 9.6×, at least about 9.7×, at least about 9.8×, at least about 9.9×, or at least about 10×.

In some embodiments, the ratio of total rAAV viral particles to empty rAAV viral particles in the anion exchange eluate is at least about 2×, at least about 2.5×, at least about 3×, at least about 3.5×, at least about 4×, at least about 4.5×, at least about 5×, at least about 5.5×, at least about 6×, at least about 6.5×, at least about 7×, at least about 7.5×, at least about 8×, at least about 8.5×, at least about 9×, at least about 9.5×, or at least about 10× greater than the ratio of total rAAV viral particles to empty rAAV particles in the affinity eluate. For example, the ratio of total rAAV viral particles to empty rAAV particles in the purified population is at least about 2.5× higher than the ratio of total rAAV viral particles to empty rAAV particles in the affinity eluate. In another non-limiting example, the ratio of total rAAV viral particles to empty rAAV viral particles in the purified population is at least about 3× higher than the ratio of total rAAV viral particles to empty rAAV viral particles in the affinity eluate. In yet another non-limiting example, the ratio of total rAAV viral particles to empty rAAV viral particles in the purified population is at least about 3.5× higher than the ratio of total rAAV viral particles to empty rAAV viral particles in the affinity eluate. In yet another non-limiting example, the ratio of total rAAV viral particles to empty rAAV viral particles in the purified population is at least about 4× higher than the ratio of total rAAV viral particles to empty rAAV viral particles in the affinity eluate. In yet another non-limiting example, the ratio of total rAAV viral particles to empty rAAV viral particles in the purified population is at least about 5× higher than the ratio of total rAAV viral particles to empty rAAV viral particles in the affinity eluate.

In some embodiments of any of the aspects described herein, the ratio of intact and incompletely intact rAAV particles to empty rAAV viral particles in the anion exchange eluate is at least about 1.25× higher than the ratio of intact and incompletely intact rAAV particles to empty particles in the affinity eluate. For example, the number of the cells to be processed, the ratio of intact and incompletely filled rAAV particles to empty rAAV virus particles in the anion exchange eluate is at least about 1.5×, at least about 1.6×, at least about 1.7×, at least about 1.8×, at least about 1.9×, at least about 2×, at least about 2.1×, at least about 2.2×, at least about 2.3×, at least about 2.4×, at least about 2.5×, at least about 2.6×, at least about 2.7×, at least about 2.8×, at least about 2.9×, at least about 3×, at least about 3.1×, at least about 3.2×, at least about 3.3×, at least about 3.5×, at least about 3.6×, at least about 3.7×, at least about 3.8×, at least about 3.4×, at least about 4.4×. At least about 4.8×, at least about 4.9×, at least about 5×, at least about 5.1×, at least about 5.2×, at least about 5.3×, at least about 5.5×, at least about 5.6×, at least about 5.7×, at least about 5.8×, at least about 6.9×, at least about 6.1×, at least about 6.2×, at least about 6.3×, at least about 6.4×, at least about 6.5×, at least about 6.6×, at least about 6.7×, at least about 6.8×, at least about 6.9×, at least about 7.1×, at least about 7.2×, at least about 7.3×, at least about 7.4×, at least about 7.6×, at least about 7.7×, at least about 7.8×, at least about 8×, at least about 8.8× At least about 9.1×, at least about 9.2×, at least about 9.3×, at least about 9.4×, at least about 9.5×, at least about 9.6×, at least about 9.7×, at least about 9.8×, at least about 9.9×, or at least about 10×.

In some embodiments, the ratio of intact and incompletely intact rAAV particles to empty rAAV viral particles in the anion exchange eluate is at least about 2×, at least about 2.5×, at least about 3×, at least about 3.5×, at least about 4×, at least about 4.5×, at least about 5×, at least about 5.5×, at least about 6×, at least about 6.5×, at least about 7×, at least about 7.5×, at least about 8×, at least about 8.5×, at least about 9×, at least about 9.5×, or at least about 10× greater than the ratio of intact and incompletely intact rAAV particles to empty rAAV particles in the affinity eluate. For example, the ratio of intact and incompletely intact rAAV particles to empty rAAV particles in the purified population is at least about 2.5× higher than the ratio of intact and incompletely intact rAAV particles to empty rAAV particles in the affinity eluate. In another non-limiting example, the ratio of intact and incompletely intact rAAV particles to empty rAAV viral particles in the purified population is at least about 3× higher than the ratio of intact and incompletely intact rAAV particles to empty rAAV viral particles in the affinity eluate. In yet another non-limiting example, the ratio of intact and incompletely intact rAAV particles to empty rAAV viral particles in the purified population is at least about 3.5× higher than the ratio of intact and incompletely intact rAAV particles to empty rAAV viral particles in the affinity eluate. In yet another non-limiting example, the ratio of intact and incompletely intact rAAV particles to empty rAAV viral particles in the purified population is at least about 4× higher than the ratio of intact and incompletely intact rAAV particles to empty rAAV viral particles in the affinity eluate. In yet another non-limiting example, the ratio of intact and incompletely intact rAAV particles to empty rAAV viral particles in the purified population is at least about 5× higher than the ratio of intact and incompletely intact rAAV particles to empty rAAV viral particles in the affinity eluate.

In some embodiments, the anion exchange eluate is UV ₂₆₀ With UV ₂₈₀ Is at least 1.25 or higher. For example, UV of anion exchange eluate ₂₆₀ With UV ₂₈₀ At least 1.25, at least 1.26, at least 1.27, at least 1.28, at least 1.29, at least 1.3, at least 1.31, at least 1.32, at least 1.33, at least 1.34, or at least 1.35 or more.

In some embodiments, the affinity eluate is UV ₂₆₀ With UV ₂₈₀ The ratio of (2) is lower than 1.15. For example, UV of affinity eluate ₂₆₀ With UV ₂₈₀ A ratio of 1.10 or less, 1.05 or less, 1 or less, 0.95 or less, 0.9 or less, 0.85 or less, or 0.8 or less.

In some embodiments, the affinity eluate is UV ₂₆₀ With UV ₂₈₀ Is 1.15 or less, and the anion exchange eluate obtained from the affinity eluate has a UV ₂₆₀ With UV ₂₈₀ Is at least 1.25 or higher. For example, UV of affinity eluate ₂₆₀ With UV ₂₈₀ Is 1.10 or less, and the anion exchange eluate obtained from the affinity eluate is UV ₂₆₀ With UV ₂₈₀ Is at least 1.25 or higher. In some embodiments, the affinity eluate is UV ₂₆₀ With UV ₂₈₀ Is 1.05 or less, and the anion exchange eluate obtained from the affinity eluate is UV ₂₆₀ With UV ₂₈₀ Is at least 1.30 or higher.

In some embodiments, the UV in the anion exchange eluate ₂₆₀ With UV ₂₈₀ Ratio of the ratio of UV in the adjusted affinity eluate ₂₆₀ With UV ₂₈₀ Is at least about 1.15 x higher. For example, compared to UV in a conditioned affinity eluate ₂₆₀ With UV ₂₈₀ Is the ratio of UV in the anion exchange eluate ₂₆₀ With UV ₂₈₀ Is at least about 1.2×, at least about 1.25×, at least about 1.3× 0, at least about 1.35× 1, at least about 1.4× 2, at least about 1.45× 3, at least about 1.5× 4, at least about 1.55× 5, at least about 1.6× 6, at least about 1.65×, at least about 1.7×, or at least about 1.75×, at least about 1.8×, at least about 1.859×, at least about 1.9×, at least about 1.95×, or at least about 2× or higher. In some embodiments, the UV is compared to that in a conditioned affinity eluate ₂₆₀ With UV ₂₈₀ Is the ratio of UV in the anion exchange eluate ₂₆₀ With UV ₂₈₀ Is at least about 1.15 x to at least about 2 x or more, or at least about 1.15 x 0 to at least about 1.95 x 1 or more, or at least about 1.15 x 2 to at least about 1.9 x 3 or more, or at least about 1.15 x 4 to at least about 1.85 x 5 or more, or at least about 1.15 x 6 to at least about 1.8 x 7 or more, or at least about 1.15 x to at least about 1.7 x 0 or more, or at least about 1.15 x 1 to at least about 1.65 x 2 or at least about 1.15 x 3 to at least about 1.6 x 4 or more, or at least about 1.15 x 5 to at least about 1.55 x 6 or more, or at least about 1.15 x to at least about 1.5 x or more, or at least about 1.15 x 1.45 x or more, or at least about 1.15 x 0 or at least about 1.15 x 1 to at least about 1.15 x 2 or more, or at least about 1.15 x 4 or more About 1.35 x or greater, or at least about 1.15 x to at least about 1.3 x or greater, or at least about 1.15 x to at least about 1.25 x or greater, or at least about 1.15 x to at least about 1.2 x or greater, or at least about 1.15 x to at least about 1.15 x or greater.

The methods of purifying or separating AAV particles as described herein allow fewer empty particles to bind to the anion exchange column, and the stream passing through the anion exchange column contains more empty particles. In some embodiments, less than 10%, less than 8%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, less than 0.1%, less than 0.05%, or less than 0.02% or even less of the empty AAV particles bind to the anion exchange column. In preferred embodiments, the empty AAV particles do not bind to an anion exchange column. In some aspects of embodiments, the anion exchange column comprises at least 5%, at least 6%, at least 8%, at least 10%, at least 15%, or at least 20%, or more empty AAV particles.

In some embodiments, the presence of an amino acid or the presence of a combination of an amino acid and a weak acid or salt thereof in an affinity elution buffer is used to purify the recombinant AAV particles. In some aspects of embodiments, the affinity elution buffer comprises glycine, optionally in combination with acetic acid or a salt thereof, or succinic acid or a salt thereof, or citric acid or a salt thereof, or propionic acid or a salt thereof. In one aspect of an embodiment, the affinity elution buffer comprises glycine and citric acid or salts thereof, e.g., citrate. In another aspect of an embodiment, the affinity elution buffer includes glycine and other amino acids (e.g., histidine). In some embodiments, the affinity eluate disclosed herein comprises from about 10% to about 50% empty AAV particles in the affinity eluate. For example, the affinity eluate disclosed herein comprises less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 8%, or less than 5% empty AAV particles in the affinity eluate.

The methods disclosed herein can include producing a harvesting medium for contact with an affinity chromatography medium by a method that includes upstream processing (such as, for example, harvesting a cell culture and/or clarifying a harvested cell culture). Thus, in either aspect, the method comprises the step of clarifying the cell culture medium. For example, harvested cell cultures are clarified by depth filtration.

In some embodiments of any of the aspects, the method comprises the step of lysing the host cells in the harvested cell culture prior to clarification. Methods and compositions for lysing host cells are well known in the art. For example, a surfactant (e.g., a nonionic surfactant) can be added to the harvested cell culture to lyse host cells present in the harvested cell culture. In some embodiments of all aspects, the method does not include the step of lysing the host cells in the harvested cell culture prior to clarification. In one aspect of an embodiment, recombinant viral particles (rAAV) are isolated or purified from the supernatant of the host cell culture.

The harvested cell culture may contain impurities, for example host cell DNA (hcna). Thus, the method may include a step of removing or reducing the amount of impurities from the harvested cell culture, e.g., prior to the clarification step. Methods and compositions for reducing the amount of host cell DNA in a harvested medium are well known in the art. For example, cationic amines or nucleases can be added to the harvested medium.

In some embodiments, the methods disclosed herein further comprise treating virus particles eluted from the anion exchange chromatography by a downstream treatment step, e.g., tangential flow filtration and/or sterile filtration, or any combination thereof. It should be noted that the upstream and/or downstream processes may be used alone or in various combinations.

The methods described herein are readily adaptable to different viruses and serotypes. Thus, in some embodiments of any aspect, the recombinant viral particle is a recombinant adeno-associated virus (rAAV) particle. For example, the rAAV particle can be AAV-1, AAV-2i8, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAVrh10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, AAV-16, or a chimeric, derivative, modification, or pseudotyped thereof. In some embodiments, the rAAV is a reasonably polyploid (also referred to as haploid) AAV. In some embodiments, the methods described herein are used to purify/isolate a rAAV, wherein the rAAV comprises at least one capsid protein (e.g., VP1, VP2, or VP 3) from an AAV serotype listed in table 1.

In some embodiments of any of the aspects, the viral particle comprises a heterologous polynucleotide, e.g., a transgene or portion thereof.

In some aspects, provided herein are purified populations of recombinant adeno-associated viruses (rAAV), optionally lacking prokaryotic sequences, wherein the purified viruses have less than 2 x 10 ⁴ Particle to infectivity ratio of vg/TCID50, wherein the purified rAAV population comprises less than about 10% empty viral capsids. In some embodiments, the purified virus is obtained by a method comprising transfecting a suspension mammalian cell line, wherein the cells are transfected in suspension. In some embodiments, the purified rAAV population comprises less than about 9.5%, less than about 9%, less than about 8.5%, less than about 8%, less than about 7.5%, less than about 7%, less than about 6.5%, less than about 6%, less than about 5.5%, less than about 5%, less than about 4.5%, less than about 4%, less than about 3.5%, less than about 3%, less than about 2.5%, less than about 2%, less than about 1.5%, less than about 1%, less than about 0.75%, less than about 0.5%, less than about 0.25%, less than about 0Less than about 2%, less than about 0.15%, less than about 0.1%, less than about 0.05%, less than about 0.03%, less than about 0.02%, or less than about 0.01% of empty viral capsids. In several embodiments, the purified rAAV population is substantially free of empty viral capsids. In some embodiments, the purified virus has less than 1.5X10 ⁴ vg/TCID50, less than 1X 10 ⁴ vg/TCID50, less than 9X 10 ³ vg/TCID50, less than 8X 10 ³ vg/TCID50, less than 6X 10 ³ vg/TCID50, less than 5X 10 ³ vg/TCID50, less than 4X 10 ³ vg/TCID50, less than 3X 10 ³ vg/TCID50, less than 2X 10 ³ vg/TCID50, less than 9X 10 ² vg/TCID50, less than 8X 10 ² vg/TCID50, less than 7X 10 ² vg/TCID50, less than 6X 10 ² vg/TCID50, less than 5X 10 ² vg/TCID50, less than 4X 10 ² vg/TCID50, less than 3X 10 ² vg/TCID50, less than 2X 10 ² vg/TCID50, or less than 1X 10 ² vg/TCID50, or less than 0.5X10 ² Even lower particle to infectivity ratios are seen with vg/TCID 50. In some embodiments, provided herein are purified populations of recombinant adeno-associated viruses (rAAV) that do not lack prokaryotic sequences.

In some aspects, provided herein are purified populations of recombinant adeno-associated viruses (rAAV), optionally lacking prokaryotic sequences, wherein the purified viruses have less than 2 x 10 ⁴ Particle to infectivity ratio of vg/TCID 50. In some embodiments, the purified virus is obtained by a method comprising transfecting a suspension mammalian cell line, wherein the cells are transfected in suspension. In some embodiments, the purified virus has less than 1.5X10 ⁴ vg/TCID50, less than 1X 10 ⁴ vg/TCID50, less than 9X 10 ³ vg/TCID50, less than 8X 10 ³ vg/TCID50, less than 6X 10 ³ vg/TCID50, less than 5X 10 ³ vg/TCID50, less than 4X 10 ³ vg/TCID50, less than 3X 10 ³ vg/TCID50, less than 2X 10 ³ vg/TCID50, less than 9X 10 ² vg/TCID50, less than 8X 10 ² vg/TCID50, less than 7X 10 ² vg/TCID50, less than 6X 10 ² vg/TCID50, less than 5X 10 ² vg/TCID50, less than 4X 10 ² vg/TCID50, less than 3X 10 ² vg/TCID50, less than 2X 10 ² vg/TCID50, or less than 1X 10 ² vg/TCID50, or less than 0.5X10 ² vg/TCID50 or even lower particle to infectivity ratio.

Several aspects described herein provide a purified population of recombinant adeno-associated viruses (rAAV), wherein the purified population of rAAV comprises less than about 50% empty viral capsids, wherein the population of rAAV is optionally purified by the process described herein. For example, the purified rAAV population comprises about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, or 10% or less empty viral capsids. In some embodiments, the purified rAAV population comprises less than about 10%, less than about 9.5%, less than about 9%, less than about 8.5%, less than about 8%, less than about 7.5%, less than about 7%, less than about 6.5%, less than about 5.5%, less than about 5%, less than about 4.5%, less than about 4%, less than about 3.5%, less than about 3%, less than about 2.5%, less than about 2%, less than about 1.5%, less than about 1%, less than about 0.75%, less than about 0.5%, less than about 0.25%, less than about 0.2%, less than about 0.15%, less than about 0.1%, less than about 0.05%, less than about 0.03%, less than about 0.02%, or less than about 0.01% empty viral capsids. In several embodiments, the purified rAAV population is substantially free of empty viral capsids.

In some embodiments, the purified rAAV population comprises: (i) about 35% or less empty viral capsids; and/or (ii) the purified rAAV has less than 2X 10 ⁴ Particle to infectivity ratio of vg/TCID 50; and/or (iii) UV in anion exchange eluate ₂₆₀ With UV ₂₈₀ Ratio of the ratio of UV in the adjusted affinity eluate ₂₆₀ With UV ₂₈₀ Is at least about 1.15 x to at least about 1.5 x higher.

A further aspect described herein provides a pharmaceutical composition, wherein the pharmaceutical composition comprises a purified population of recombinant adeno-associated viruses (rAAV) as described herein. Pharmaceutical compositions comprising the rAAV comprise a buffer having a pH of about 6.5 to about 8.0. In some embodiments, the pH is about 6.5 to about 7.5. For example, the pH is about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5. In some preferred embodiments, the pH is below about 7.5. For example, the pH is below about 7.4, below about 7.3, below about 7.2, below about 7.1, below about 7.0, below about 6.9, below about 6.8, below about 6.7, or below about 6.6. In some embodiments, the pharmaceutical composition comprises one or more excipients comprising one or more multivalent ions and/or salts thereof. In some embodiments, the multivalent ion may be selected from or optionally selected from the group consisting of citrate, sulfate, magnesium, and phosphate. In some embodiments, the pharmaceutical composition comprises one or more excipients comprising one or more ions selected from or optionally selected from the group consisting of sodium, potassium, chloride, ammonium, carbonate, nitrate, chlorate, chlorite, and calcium. In some embodiments, the pharmaceutical composition comprising the rAAV further comprises a nonionic surfactant. In some embodiments, the nonionic surfactant is selected from the group consisting of: polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (alkyl phenol ethoxylates), preferably polysorbates, polyoxyethylene alkylphenyl ethers, and any combination thereof. In some embodiments, the nonionic surfactant is selected from the group consisting of: TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, brij 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, PF-68, ECOSURF SA-15, TERGOL 15-S-9, TERGITOL 15-S-12, TERGOL L-64, TERGOL P-7, TERGITOL NP-8, GINP-9, TERGOL-5, TERGOL-10, TERGNP-10, TERGToL-12, TERGNP-12, TERGIN-4, TERGOL-4, TERGIN-12, and any combination thereof. In some embodiments, the pharmaceutical composition further comprises a polyol, or a sugar or the like.

Drawings

The patent or application document includes at least one drawing in color. The patent application publication with the color drawing or a copy of this patent will be provided by the patent office upon request and payment of the necessary fee.

FIGS. 1A-1D show analyses of empty and complete capsids using CryoTEM (FIGS. 1A and 1B), analytical ultracentrifugation (AUC, FIG. 1C) and SEC-HPLC UV/260/280 ratios (FIG. 1D).

Figure 2 shows the partitioning of empty (E) and intact (F) capsids by anion exchange chromatography with different anion exchange patterns.

Figures 3A and 3B show separation of empty and intact capsids from different batches by anion exchange chromatography. In FIGS. 3A and 3B, the dotted line (. Cndot. Cndot.) is UV 260nm, DNA is shown, and solid (- -) is UV 280nm, protein is shown.

Figures 4A-4D show that increasing the amount of citric acid in the dilution buffer and anion exchange equilibration buffer to purify from affinity eluate comprising glycine and citrate transferred empty particles (E) to unbound fraction. FIG. 4A, control without added citric acid in equilibration buffer and dilution buffer; FIG. 4B, 0mM citric acid in equilibration buffer and 1.5mM citric acid in dilution buffer; FIG. 4C, 1.5mM citric acid in equilibration buffer and 1.5mM citric acid in dilution buffer; and FIG. 4D, 3mM citric acid in equilibration buffer and 1.5mM citric acid in dilution buffer. Fig. 4D shows that only intact particles (F) are in the bound fraction. In FIGS. 4A-4D, the dotted line (. Cndot. Cndot.) is UV 260nm, which represents DNA, and the solid line (. Cndot. -) is UV 280nm, which represents protein.

Figures 5A-5D show that increasing the amount of citric acid in the dilution buffer to purify from affinity eluate comprising glycine and histidine but lacking citrate, transferred empty particles (E) to unbound fraction. Fig. 5A, control; FIG. 5B, 3mM citric acid was added to the dilution buffer; FIG. 5C, 5mM citric acid was added to the dilution buffer; and FIG. 5D, 7mM citric acid was added to the dilution buffer. In FIGS. 5A-5D, the dotted line (. Cndot. Cndot.) is UV 260nm, which represents DNA, and the solid line (. Cndot. -) is UV 280nm, which represents protein.

Figure 6 shows SEC HPLC analysis of separation of empty and intact capsids from affinity eluate comprising glycine and histidine but lacking citrate by anion exchange chromatography, which is transferred with increasing amount of citric acid in dilution buffer.

Fig. 7 is an exemplary anion exchange separation chromatography for purification from an affinity eluate comprising glycine and histidine but lacking citrate.

FIG. 8 is a contour plot showing the effect of histidine and glycylglycine concentrations in affinity eluate on vector genome titres for affinity purification.

Figure 9 shows the separation of empty particles and intact particles using different anion exchange buffers.

Fig. 10A and 10B are different anion exchange chromatography modes showing reproducibility of anion exchange purification amplification. In FIGS. 10A and 10B, the dotted line (. Cndot. Cndot.) is UV 260nm, which represents DNA, and the solid line (. Cndot. -) is UV 280nm, which represents protein.

FIG. 11 shows the effect of gradient length (CV) and elution Q (CV/min) on yield (viral particles (VP) and viral genome (Vg)).

Fig. 12 is an exemplary anion exchange separation chromatography for purification from an affinity eluate comprising glycine and histidine but lacking citrate.

Fig. 13 is a graph showing SEC-HPLC elution profiles using different modes of capsids.

FIG. 14 shows UV _260/280 The ratio is related to MALS% complete.

Figures 15A-15C show that in the absence of weak acid in the dilution buffer, complete binding and complete elution of intact (F) and empty (E) capsids are present regardless of the% of intact capsids in the starting material. FIG. 15A, starting Material (S/M) UV _260/280 Is 0.96; FIG. 15B, starting Material UV _260/280 The ratio of (2) is 1.05; and FIG. 15C, starting material UV _260/280 The ratio of (2) was 1.19. In FIGS. 15A-15C, the dotted line(. Cndot. Co. Cndot. Is the UV light of 260nm and the ultraviolet light of the ultraviolet light is the ultraviolet light of the ultraviolet the expression "DNA" means a DNA, the solid line (- -) is UV 280nm, representing protein.

Fig. 16A and 16B show that the inclusion of a weak acid (e.g., citric acid) in the dilution buffer used to dilute the affinity eluate of the AEX chromatograph results in the fragmentation of the empty capsids in two different test starting materials.

Figures 17A-17C show that adjustment of citric acid in anion exchange dilution buffer results in a reduction of empty shoulder elution. FIG. 17A, 8mM citric acid in anion exchange dilution buffer and 3mM citric acid in equilibration buffer, showing UV _260/280 The ratio is 1.31 and the empty shoulder inflection point at-42 mAU; FIG. 17B, 9M citric acid in anion exchange dilution buffer and 3mM citric acid in equilibration buffer, showing no empty shoulder and UV _260/280 The ratio was 1.30; and FIG. 17C, 10mM citric acid in anion exchange dilution buffer and 3mM citric acid in equilibration buffer, showing no empty shoulder and UV _260/280 The ratio was 1.30. In FIGS. 17A-17C, the dotted line (. Cndot. Cndot.) is UV 260nm, DNA is shown, and solid (- -) is UV 280nm, protein is shown.

FIGS. 18A-18C show scale comparability of empty/complete separations on 1mL, 4mL, and 8mL monolith scales according to an exemplary embodiment of the invention. FIG. 18A,1mL of monoliths containing 8mM citric acid in anion exchange dilution buffer and 3mM citric acid in equilibration buffer; FIG. 18B,4mL of monoliths containing 9mM citric acid in anion exchange dilution buffer and 3mM citric acid in equilibration buffer; and FIG. 18C,8mL of monoliths containing 10mM citric acid in anion exchange dilution buffer and 3mM citric acid in equilibration buffer. In FIGS. 18A-18C, the dotted line (. Cndot. Cndot.) is UV 260nm, which represents DNA, and the solid line (. Cndot. -) is UV 280nm, which represents protein.

Fig. 19A-19C show the effect of viral particle packaging of starting material on empty/complete separation by AEX purification according to an exemplary embodiment of the invention. FIG. 19A, starting material UV _260/280 Is 0.87; FIG. 19B, starting Material UV _260/280 Is 0.96; fig. 19C, starting material UV _260/280 The ratio of (2) was 1.04. In FIGS. 19A-19C, the dotted line (. Cndot. Cndot.) is UV 260nm, which represents DNA, and the solid line (. Cndot. -) is UV 280nm, which represents protein.

Fig. 20A-20G show that the amount of weak acid (e.g., citric acid) in AEX dilution buffer or AEX equilibration buffer affects peak shape/purity/recovery of AEX purification. FIG. 20A, no citric acid in dilution buffer or equilibration buffer; FIG. 20B, 3mM citric acid in dilution buffer and no citric acid in equilibration buffer; FIG. 20C, 8mM citric acid in dilution buffer and no citric acid in equilibration buffer; FIG. 20D, 5mM citric acid in dilution buffer and 1mM citric acid in equilibration buffer; FIG. 20E, 6mM citric acid in dilution buffer and 1mM citric acid in equilibration buffer; FIG. 20F, 8mM citric acid in dilution buffer and 1mM citric acid in equilibration buffer; and FIG. 20G, 8mM citric acid in dilution buffer and 3mM citric acid in equilibration buffer. In FIGS. 20A-20G, the dotted line (. Cndot. Cndot.) is UV 260nm, which represents DNA, and the solid line (. Cndot. -) is UV 280nm, which represents protein.

Figures 21A-21D show that the amount of weak acid (e.g., citric acid) in the AEX equilibration buffer also affects the peak shape/purity/recovery of AEX purification. FIG. 21A, 6mM citric acid in dilution buffer and 1mM citric acid in equilibration buffer; FIG. 21B, 6mM citric acid in dilution buffer and 3mM citric acid in equilibration buffer; FIG. 21C, 6mM citric acid in dilution buffer and 4mM citric acid in equilibration buffer; and FIG. 21D, 8mM citric acid in dilution buffer and 3mM citric acid in equilibration buffer. In FIGS. 21A-21D, the dotted line (. Cndot. Cndot.) is UV 260nm, which represents DNA, and the solid line (. Cndot. -) is UV 280nm, which represents protein.

FIGS. 22A-22F show that increasing histidine concentration in anion exchange dilution buffer for AEX purification (QA Monolith) from affinity eluate allows UV _260/280 The reversal of the ratio or inflection point is minimized, which indicates an increase in the enrichment of intact capsids in the eluent phase. FIG. 22A, adding 70mM histidine to anion exchange dilution buffer; FIG. 22B, adding 90mM histidine to anion exchange dilution buffer; FIG. 22C dilution to anion exchangeAdding 100mM histidine into the buffer solution; FIG. 22D, 120mM histidine was added to anion exchange dilution buffer; FIG. 22E, 150mM histidine was added to anion exchange dilution buffer; and FIG. 22F, 170mM histidine was added to the anion exchange dilution buffer. In FIGS. 22A-22F, the dotted line (. Cndot. Cndot.) is UV 260nm, which represents DNA, and the solid line (. Cndot. -) is UV 280nm, which represents protein.

Detailed Description

Although methods of purifying viral particles are known in the art, rapid, efficient, scalable and economical methods are not known while maintaining the viral particles in a viable configuration. The methods disclosed herein allow for rapid, efficient, scalable, and economical purification of viral particles (e.g., rAAV) while maintaining the viral particles in a viable configuration. The methods disclosed herein provide several significant advantages. The method is readily adaptable to different viral particles and serotypes, e.g., the method is readily purified for rAAV of different serotypes. For example, the methods disclosed herein can be used to purify/isolate rAAV particles of serotypes selected from the group consisting of: AAV-1, AAV-2i8, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV rh10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, AAV-16, or a chimeric, derivative, modification or pseudotype, or a reasonable polyploid (also known as haploid) AAV thereof. Reasonable polyploid or haploid AAV include VP1, VP2 and VP3, as described in PCT/US18/22725 and US10,550,405, which are incorporated herein by reference.

The methods disclosed herein are scalable and can be applied to efficient and scalable production of recombinant viral particles (e.g., rAAV). In other words, the methods described herein may be used in volumes ranging from a few mL volumes to thousands of liters. Thus, the methods are useful for industrial scale production of therapeutic viral particle compositions, e.g., rAAV compositions. The increased yield of packed recombinant viral particles can provide significant cost savings for the industrial scale production of therapeutic viral particle compositions. Furthermore, as described herein, the methods can be readily adapted to different viruses or serotypes. The selective elimination of empty viral particles as described herein allows for comparable or possibly better transduction of recombinant AAV particles in a target tissue type of a subject compared to recombinant AAV particles purified using a density gradient/ultracentrifugation method. In addition, recombinant AAV formulations with fewer empty AAV particles can be effective in reducing the immune response against AAV particles (or capsids) elicited by subjects receiving recombinant AAV gene therapy, thereby making them a desirable product of AAV gene therapy. The methods described herein may also reduce operator error and/or improve operator safety.

Embodiments of the various aspects disclosed herein are based on the discovery by the inventors that, particularly by equilibrating an anion exchange chromatography medium with a buffer comprising an anionic compound (e.g., a weak acid or salt thereof, such as citric acid or salt thereof) prior to contacting the anion exchange medium with a feed composition comprising viral particles, separation of the packed recombinant viral particles from empty or partially packed recombinant viral particles in the feed composition can be increased in the anion exchange chromatography medium, and wherein the feed composition further comprises a predetermined amount of an anionic compound, e.g., a weak acid or salt thereof.

As used herein, the term "weak acid" refers to an acid that only partially dissociates in an aqueous solution. Generally, a "weak acid" is one having an acid dissociation constant of less than about 10 ^-4 Is a compound of (a). Exemplary weak acids include, but are not limited to, acetic acid, citric acid, succinic acid, acetoacetic acid, adipic acid, tetraoxanic acid, ascorbic acid, aspartic acid, barbituric acid, boric acid, butyric acid, n-butyric acid, carbonic acid, crotonic acid, diglycolic acid, dimethylmalonic acid, formic acid, fumaric acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, glyceric acid, glycolic acid, itaconic acid, lactic acid, maleic acid, malic acid, malonic acid, mesaconic acid, meso-tartaric acid, methylsuccinic acid, methylmalonic acid, oxalic acid, oxaloacetic acid, pentanic acid, valeric acid, phosphoric acid, isophthalic acid, phthalic acid, terephthalic acid, propionic acid, pyruvic acid, salicylic acid, tartaric acid, tartronic acid, terephthalic acid, trans-crotonic acid, trichloroacetic acid, uric acid, alpha-tartaric acid, 2-oxo acid Substituted butyric acid, 2-methylbutyric acid, 2-oxoglutarate, 3-butenoic acid and 3-methylbutyric acid. In some embodiments, the weak acid or salt thereof may be citric acid, acetic acid, succinic acid, or salts thereof.

Recombinant AAV particles can be produced by any means known to those skilled in the art.

In some embodiments, the purified population of recombinant adeno-associated viruses (rAAV) purified by the processes described herein comprises less than about 10% empty viral capsids. The process for purifying or isolating rAAV comprises: (a) Purifying/separating a plurality of recombinantly-expressed viral particles from the harvested medium by affinity chromatography to produce an eluate comprising the plurality of recombinantly-expressed viral particles (affinity chromatography eluate), wherein an elution buffer for affinity chromatography (affinity elution buffer) comprises a predetermined amount of glycine, optionally the affinity elution buffer is substantially free of weak acids or salts thereof; and optionally, the affinity elution buffer comprises an amino acid that is not glycine; (b) Conditioning the affinity chromatography eluate for subsequent purification by anion exchange chromatography, wherein the conditioned eluate comprises a predetermined amount of an anionic compound; (c) Purifying/separating the plurality of recombinantly-expressed viral particles from the conditioned affinity chromatography eluate by anion exchange chromatography to produce a solution comprising the plurality of purified/separated recombinantly-expressed viral particles, wherein an equilibration buffer for anion exchange chromatography comprises a predetermined amount of a weak acid or salt thereof, optionally the weak acid is citric acid, acetic acid or succinic acid. In some embodiments, the purified rAAV population purified by the processes described herein comprises less than about 9.5%, less than about 9%, less than about 8.5%, less than about 8%, less than about 7.5%, less than about 7%, less than about 6.5%, less than about 6%, less than about 5.5%, less than about 5%, less than about 4.5%, less than about 4%, less than about 3.5%, less than about 3%, less than about 2.5%, less than about 2%, less than about 1.5%, less than about 1%, less than about 0.75%, less than about 0.5%, less than about 0.25%, less than about 0.2%, less than about 0.15%, less than about 0.1%, less than about 0.05%, less than about 0.03%, less than about 0.02%, or less than about 0.01% empty viral capsids. In some embodiments, the purified rAAV population purified by the process described herein is substantially free of empty viral capsids.

Anion exchange

In one aspect provided herein is a method of separating packed recombinant viral particles from empty or partially packed recombinant viral particles in a feed composition by anion exchange chromatography. Generally, the method comprises: equilibrating the exchange chromatography medium with an anion exchange equilibration medium (AEX equilibration medium or buffer) comprising a predetermined amount of an anionic compound; contacting a feed composition containing intact recombinant viral particles with empty and partially packed recombinant viral particles with a equilibrated anion exchange medium under conditions allowing binding of the viral particles to the anion exchange chromatography medium; and recovering an eluate comprising intact recombinant toxin particles, wherein the feed composition and/or the conditioned or diluted feed composition comprises a predetermined amount of an anionic compound.

Typically, less than 20% of the empty or partially packed recombinant viral particles in the feed composition are bound to the anion exchange chromatography medium. For example, less than 19.5%, less than 19%, less than 18.5%, less than 18%, less than 17.5%, less than 17%, less than 16.5%, less than 16%, less than 15.5%, less than 15%, less than 14.5%, less than 14%, less than 13.5%, less than 13%, less than 12.5%, less than 12%, less than 11.5%, less than 11%, less than 10.5%, less than 10%, less than 9.5%, less than 9%, less than 8.5%, less than 8%, less than 7.5%, less than 7%, less than 6.5%, less than 6%, less than 5.5%, less than 5%, less than 4.5%, less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than 1.75%, less than 0.5%, less than 0.25%, less than 0.2%, less than 0.15%, less than 0.1%, less than 0.05% of the empty recombinant viral particles in the feed composition bind to the anion exchange chromatography medium. In some embodiments, substantially no empty recombinant viral particles in the feed composition are bound to the anion exchange chromatography medium.

In some embodiments, less than about 50%, or less than about 45%, or less than about 40%, or less than about 35%, or less than about 30%, or less than about 25%, or less than about 21%, or less than about 20%, or even less of the empty viral particles in the feed composition are bound to the anion exchange chromatography medium.

As described herein, the eluate recovered from the anion exchange is enriched in intact recombinant viral particles compared to the feed composition. For example, an isolated or purified recombinant viral particle (e.g., rAAV particle) comprises less than about 40%, or less than about 35%, or less than about 30%, or preferably less than about 25% empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 20% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 19% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 18% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 17% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 16% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 15% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 14% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 13% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 12% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 11% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 10% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 9% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 8% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 7.5% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 7% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 6.5% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 6% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 5.5% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 5% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 4.5% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 4% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 3.5% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 3% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 2.5% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 2% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 1.5% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 1% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 0.75% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 0.5% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 0.25% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 0.2% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 0.15% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 0.1% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 0.05% or less empty viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) are substantially free of empty viral particles.

In some embodiments, the isolated or purified recombinant viral particle (e.g., rAAV particle) comprises less than about 70%, or less than about 65%, or less than about 60%, or less than about 55% of an incomplete intact viral particle. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise less than about 50% of incomplete intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise less than about 45% incomplete intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise less than about 40% of incomplete intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise less than about 35% of incomplete intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise less than about 30% incomplete intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise less than about 25% incomplete intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 20% or less of incompletely intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 19% or less of incompletely intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 18% or less of incompletely intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 17% or less of incompletely intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 16% or less of incompletely intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 15% or less of incompletely intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 14% or less of incompletely intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 13% or less of incompletely intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 12% or less of incompletely intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 11% or less of incompletely intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 10% or less of incompletely intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 9% or less of incompletely intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 8% or less of incompletely intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 7% or less of incompletely intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 6% or less of incompletely intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 5% or less of incompletely intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 4% or less of incompletely intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 3% or less of incompletely intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 9% or less of incompletely intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 2.5% or less of incompletely intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 2% or less of incompletely intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 1.5% or less of incompletely intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 1% or less of incompletely intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 0.75% or less of incompletely intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 0.5% or less of incompletely intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 0.25% or less of incompletely intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 0.2% or less of incompletely intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 0.15% or less of incompletely intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 0.1% or less of incompletely intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) comprise about 0.05% or less of incompletely intact viral particles. In some embodiments, the isolated or purified recombinant viral particles (e.g., rAAV particles) are substantially free of incomplete intact viral particles.

In some embodiments, at least about 45% of the isolated or purified viral particles (e.g., rAAV particles) are intact or whole particles. In some embodiments, at least about 50% of the isolated or purified viral particles (e.g., rAAV particles) are intact or whole particles. In some embodiments, at least about 55% of the isolated or purified viral particles (e.g., rAAV particles) are intact or whole particles. In some embodiments, at least about 60% of the isolated or purified viral particles (e.g., rAAV particles) are intact or whole particles. In some embodiments, at least about 65% of the isolated or purified viral particles (e.g., rAAV particles) are intact or whole particles. In some embodiments, at least about 70% of the isolated or purified viral particles (e.g., rAAV particles) are intact or whole particles. In some embodiments, at least about 75% of the isolated or purified viral particles (e.g., rAAV particles) are intact or whole particles. In some embodiments, at least about 80% of the isolated or purified viral particles (e.g., rAAV particles) are intact or whole particles. In some embodiments, at least about 85% of the isolated or purified viral particles (e.g., rAAV particles) are intact or whole particles. In some embodiments, at least about 90% of the isolated or purified viral particles (e.g., rAAV particles) are intact or whole particles. In some embodiments, at least about 91% of the isolated or purified viral particles (e.g., rAAV particles) are intact or whole particles. In some embodiments, at least about 92% of the isolated or purified viral particles (e.g., rAAV particles) are intact or whole particles. In some embodiments, at least about 93% of the isolated or purified viral particles (e.g., rAAV particles) are intact or whole particles. In some embodiments, at least about 94% of the isolated or purified viral particles (e.g., rAAV particles) are intact or whole particles. In some embodiments, at least about 95% of the isolated or purified viral particles (e.g., rAAV particles) are intact or whole particles. In some embodiments, at least about 96% of the isolated or purified viral particles (e.g., rAAV particles) are intact or whole particles. In some embodiments, at least about 97% of the isolated or purified viral particles (e.g., rAAV particles) are intact or whole particles. In some embodiments, at least about 98% of the isolated or purified viral particles (e.g., rAAV particles) are intact or whole particles. In some embodiments, at least about 99% of the isolated or purified viral particles (e.g., rAAV particles) are intact or whole particles. In some embodiments, substantially all of the isolated or purified viral particles (e.g., rAAV particles) are intact or whole particles.

In some embodiments, the feed composition for anion exchange chromatography is an eluent from an affinity chromatography step.

As disclosed herein, the anion exchange chromatography medium is washed or equilibrated with a buffer prior to contact with the feed composition. The buffer used to wash or equilibrate the anion exchange medium (also referred to as AEX equilibration buffer) contains a predetermined amount of anionic compound. Typically, the AEX equilibration buffer comprises a predetermined amount of an acid or salt thereof. For example, the AEX equilibration buffer contains a predetermined amount of weak acid. In some embodiments of any of the aspects, the AEX equilibration buffer comprises a predetermined amount of citric acid or a salt thereof, acetic acid or a salt thereof, or succinic acid or a salt thereof. For example, the AEX equilibration buffer comprises a predetermined amount of citric acid or a salt thereof, acetic acid or a salt thereof, succinic acid or a salt thereof, propionic acid or a salt thereof, or trimesic acid or a salt thereof. In some embodiments, the AEX equilibration buffer comprises a predetermined amount of citric acid or a salt thereof, i.e., citrate.

The amount of anionic compound (e.g. an acid or a salt thereof, such as a weak acid or a salt thereof, such as citric acid or citrate) in the AEX equilibration buffer may be optimized for the viral particle to be isolated. For example, the AEX equilibration buffer may comprise an anionic compound (e.g., an acid or salt thereof, e.g., a weak acid or salt thereof, such as citric acid or citrate) at a concentration of: at least 0.5mM, 1mM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, 4mM, 4.5mM, 5mM, 5.5mM, 6mM, 6.5mM, 7mM, 8mM, 8.5mM, 9mM, 9.5mM, 10mM or more. For example, the AEX equilibration buffer comprises an anionic compound, e.g., an acid or salt thereof, such as a weak acid or salt thereof, e.g., citric acid or citrate, at a concentration of at least about 3mM, 5mM, 7mM, or more.

In some embodiments of any of the aspects, the AEX equilibration buffer comprises an anionic compound (e.g., an acid or salt thereof, such as a weak acid or salt thereof, such as citric acid or citrate) at a concentration of: from about 0.5mM to about 15mM, from about 1mM to about 10mM, from about 1.5mM to about 7.5mM, from about 2mM to about 7mM, from about 1.5mM to about 9mM, from about 2mM to about 8mM, or from about 2.5mM to about 7.5mM.

In some embodiments of any of the aspects, the AEX equilibration buffer comprises an anionic compound, e.g., an acid or salt thereof, e.g., a weak acid or salt thereof, e.g., citric acid or citrate, at a concentration of about 0.5mM, about 1mM, about 1.5mM, about 2mM, about 2.5mM, about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 8mM, about 8.5mM, about 9mM, about 9.5mM, or about 10 mM. In some embodiments, the AEX equilibration buffer comprises up to 4mM citric acid.

The AEX equilibration buffer may comprise additional components. For example, the AEX equilibration buffer may comprise a predetermined amount of buffer. Exemplary buffers include, but are not limited to, acetate, histidine, phosphate, citrate, propionate, tricine, borate, and tris (hydroxymethyl) aminomethane (tris). In some embodiments of any of the aspects, the AEX equilibration buffer comprises a predetermined amount of bis-tris propane (BTP), tris, borate, and/or tricine.

The amount of buffering agent (e.g., BTP, tris, borate, and/or tricine) in the AEX equilibration buffer can be optimized for the viral particle to be isolated. For example, the AEX equilibration buffer may comprise buffers (e.g., BTP, tris, borate, and/or tricine) at a concentration of at least about 25mM, 50mM, 75mM, 100mM, 125mM, 150mM, or higher. For example, the AEX equilibration buffer comprises buffers (e.g., BTP, tris, borate, and/or tricine) at a concentration from about 5mM to about 125mM, from about 10mM to about 120mM, from about 15mM to about 115mM, from about 20mM to about 110mM, or from about 25mM to about 100 mM. In some embodiments of any of the aspects, the AEX equilibration buffer may comprise a buffer (e.g., BTP, tris, borate, and/or tricine) at a concentration of about 15mM, about 20mM, about 25mM, about 30mM, about 35mMM, about 40mM, about 45mM, about 50mM, about 55mM, about 60mM, about 65mM, about 70mM, about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, or about 110 mM. In some embodiments of any of the aspects, the AEX equilibration buffer comprises BTP, tris, borate and/or tricine at a concentration of 0-100 mM. For example, the AEX equilibration buffer comprises up to 100mM BTP.

In some embodiments of any aspect, the AEX equilibration buffer comprises a predetermined amount of amino acids, e.g., natural or unnatural amino acids. For example, the AEX equilibration buffer comprises a predetermined amount of histidine or arginine. In some embodiments, the equilibration buffer comprises histidine

The amount of amino acids (e.g., histidine or arginine) in the AEX equilibration buffer can be optimized for the viral particle to be isolated. For example, the AEX equilibration buffer may comprise an amino acid, e.g., histidine or arginine, at a concentration of at least about 25mM, 50mM, 75mM, 100mM, 125mM, 150mM, 175mM, 200mM or more. In some embodiments, the AEX equilibration buffer comprises an amino acid, e.g., histidine or arginine, at a concentration from about 25mM to about 200mM, from about 35mM to about 175mM, or from about 50mM to about 150 mM. For example, the AEX equilibration buffer may comprise an amino acid, e.g., histidine or arginine, at a concentration of about 50mM, about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, about 125mM, about 150mM, or about 175 mM. In some embodiments, the AEX equilibration buffer comprises up to 100mM histidine.

The AEX equilibration buffer may comprise a predetermined amount of glycerol. Thus, in some embodiments of any aspect, the AEX equilibration buffer comprises glycerol at a concentration of at least about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10% (w/v, w/w, or v/v) or higher. For example, wherein the AEX equilibration buffer comprises glycerol at a concentration from about 0.5% to about 9.5%, from about 1% to about 9%, from about 2% to about 8.5%, from about 2.5% to about 8%, from about 3% to about 7.5%, from about 3.5% to about 7%, from about 4% to about 6.5%, or from about 4.5% to about 5.5%. In some embodiments of any of the aspects, the AEX equilibration buffer comprises glycerol at a concentration of about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10%. In some embodiments, the AEX equilibration buffer is glycerol at a concentration of 0-5%.

The AEX equilibration buffer may also comprise a surfactant. For example, the AEX equilibration buffer comprises a nonionic surfactant selected from the group consisting of: polyarylphenol polyethoxy ethers; polyalkylphenol polyethoxy ethers; polyethylene glycol ether derivatives of saturated fatty acids; polyethylene glycol ether derivatives of unsaturated fatty acids; polyethylene glycol ether derivatives of fatty alcohols; polyethylene glycol ether derivatives of cycloaliphatic alcohols; fatty acid esters of polyoxyethylene sorbitan; oxyalkylating vegetable oil; alkoxylated acetylenic diols (alkoxylated acetylenic dials); polyalkoxylated alkylphenols; fatty acid alkoxylates; sorbitan alkoxylates; sorbitol esters; c (C) ₈ To C ₂₂ Alkyl or alkenyl polyglycosides; polyalkoxystyryl aryl ethers (polyalkoxy styrylaryl ethers); alkylamine oxides; block copolymer ethers; polyalkoxylated fatty acid glycerides; polyalkylene glycol ethers; linear aliphatic or aromatic polyesters; silicones (organic silicones); a polyarylphenol; sorbitol ester alkoxylates; and monoesters and diesters of ethylene glycol and mixtures thereof; ethoxylated tristyrylphenol; ethoxylated fatty alcohols; ethoxylated lauryl alcohol; ethoxylated castor oil; and ethoxylated nonylphenols; alkoxylated alcohols, amines or acids. In some embodiments of any of the aspects, the AEX equilibration buffer packageA nonionic surfactant, the nonionic surfactant selected from the group consisting of: polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof. Exemplary nonionic surfactants include, but are not limited to, polysorbates, such as polysorbate 20 (TWEEN 20), polysorbate 28, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 81, and polysorbate 85; poloxamers, such as poloxamer 188, poloxamer 407; polyoxyethylene polyoxypropylene ether (polyethylene polypropylene glycol); or polyethylene glycol (PEG).

In some embodiments of any aspect, the AEX equilibration buffer comprises a surfactant that is a nonionic fluorosurfactant. For example, the equilibration buffer comprises a nonionic fluorosurfactant selected from the group consisting of polyethylene glycol polymers, polypropylene glycol polymers, and copolymers thereof. In some embodiments, the equilibration buffer comprises a nonionic fluorosurfactant selected from the group consisting of PF68,Series of fluoropolymers (including +.>FSA, FSP, FSE, UR, FSJ, FSO, FSO-IOO, FS-300, FSN-100 and TBS, available from Sigma-Aldrich), 3M ^TM Novec FC-4434 (from 3m, st. Paul, mn.)), and,Lineplus PDM series (from Maflon)>FS-1400, FS-1900 and FS-2000 (from Mason Chemical Company, arlington H)Light, IL).

The AEX equilibration buffer may comprise at least about 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, about 0.9%, 0.95%, 1% (w/v, w/w, v/v) or higher amounts of nonionic surfactant. For example, the AEX equilibration buffer may comprise a concentration of from about 0.05% to about 0.95%, from about 0.1% to about 0.9%, from about 0.15% to about 0.85%, from about 0.2% to about 0.8%, from about 0.25% to about 0.75%, from about 0.3% to about 0.7%, from about 0.35% to about 0.65%, from about 0.4% to about 0.6%, or from about 0.45% to about 0.55% of the nonionic surfactant. In some embodiments of any of the aspects, the AEX equilibration buffer comprises a concentration of about 0.05%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, or about 0.95% of the nonionic surfactant. In some embodiments, the AEX equilibration buffer comprises a concentration of 0-0.5% nonionic surfactant.

The AEX equilibration buffer may also comprise cations, for example, monovalent or divalent cations. Exemplary monovalent ions for AEX equilibration buffers include, but are not limited to, sodium (Na ⁺ ) Potassium (K) ⁺ ) Ammonium (NH) ₄ ⁺ ) And alkylammons, for example, tetramethylammonium. Exemplary divalent cations of AEX equilibration buffers include, but are not limited to, magnesium (Mg ²⁺ ) Calcium (Ca) ²⁺ ) Copper (Cu) ²⁺ ) Cobalt (Co) ²⁺ ) Manganese (Mn) ² ⁺ ) Nickel (Ni) ²⁺ ) And zinc (Zn) ²⁺ ). The cations may be added in the form of salts. In some embodiments, the AEX equilibration buffer comprises divalent cations, e.g., mg ²⁺ 。

In some embodiments, the AEX equilibration buffer comprises Mg ²⁺ (e.g., mgCl) ₂ ) The concentration of which is at least about 1mM, 5mM, 10mM, 25mM, 50mM, 75mM, 100mM, 125mM, 150mM, 175mM, 200mM, 225mM, 250mM, 275mM, 300mM, 325mM, 35 mM0mM or more. In some embodiments, the AEX buffer comprises Mg at a concentration of 0-22mM ²⁺ For example, mgCl ₂ 。

In some embodiments, the AEX equilibration buffer comprises Na ⁺ (e.g., naCl) at a concentration of at least about 1mM, 5mM, 10mM, 25mM, 50mM, 75mM, 100mM, 125mM, 150mM, 175mM, 200mM, 225mM, 250mM, 275mM, 300mM, 325mM, 350mM, or more.

In some embodiments, the AEX equilibration buffer comprises K ⁺ (e.g., KCl or potassium acetate) at a concentration of at least about 1mM, 5mM, 10mM, 25mM, 50mM, 75mM, 100mM, 125mM, 150mM, 175mM, 200mM, 225mM, 250mM, 275mM, 300mM, 325mM, 350mM or more.

In some embodiments, the AEX equilibration buffer comprises ammonium (e.g., ammonium acetate) at a concentration of at least about 1mM, 5mM, 10mM, 25mM, 50mM, 75mM, 100mM, 125mM, 150mM, 175mM, 200mM, 225mM, 250mM, 275mM, 300mM, 325mM, 350mM, or more.

In some embodiments, the AEX equilibration buffer comprises tetramethylammonium (e.g., tetramethylammonium chloride) at a concentration of at least about 1mM, 5mM, 10mM, 25mM, 50mM, 75mM, 100mM, 125mM, 150mM, 175mM, 200mM, 225mM, 250mM, 275mM, 300mM, 325mM, 350mM, or more.

The AEX equilibration buffer may comprise a polymer. For example, the AEX equilibration buffer may comprise a nonionic polymer, such as hydroxypropyl methylcellulose, polyvinylpyrrolidone, plasmdone, polyvinyl alcohol, poloxamers (also known under the trade name SYNPERONICS ^TM 、PLURONIC ^TM And KOLLIPHOR ^TM ) Polysorbate, polyvinylpyrrolidone/vinyl acetate copolymer (PVP-VA) or polyethylene glycol (PEG).

In some embodiments, the AEX equilibration buffer comprises a poloxamer. For example, the AEX equilibration buffer comprises a poloxamer selected from the group consisting of: poloxamer 188 (P188), F127、F38、F68、F87、F108、10R5、17R2、17R4、25R2、25R4、31R1、F108 Cast Solid Surfacta、F108NF、F108 Pastille、F108NF Prill poloxamer 338, < ->F127NF、F127 NF 500BHT Prill、F127NF Prill poloxamer 407, < >>F38 Pastille、F68LF Pastille、F68 NF、F68 NF Prill、F68Pastille、F77、F77 Micropastille、F87NF、F87 NF Prill poloxamer 237,F 88、F 88Pastille、F 98、FT L 61、L10、L101、L121、L31、L35、L43、L61、L62、L62 LF、L62D、L64、L81、L92、L44 NF INH surfactant poloxamer 124, < >>N3、P103、P104、P105、P123 surfactant, < >>P65、P84、P85, etc. In some embodiments, the AEX equilibration buffer comprises poloxamer P188.

The amount of polymer (e.g., poloxamer) in the AEX equilibration buffer can vary. For example, the amount of polymer (e.g., poloxamer) in the AEX equilibration buffer can be at least about 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5% (w/w, w/v, or v/v) or higher. In some embodiments, the AEX equilibration buffer comprises a polymer (e.g., poloxamer) at a concentration from about 0.1% to about 0.5%, from about 0.15% to about 0.45%, from about 0.2% to about 0.4%, or from about 0.25% to about 0.35%.0.1%. For example, the AEX equilibration buffer comprises a polymer (e.g., poloxamer) at a concentration of about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, or about 0.5%. In some embodiments, the AEX equilibration buffer comprises a polymer, e.g., poloxamer, such as P188, at a concentration of 0-0.5%.

In some embodiments, the AEX equilibration buffer may comprise a predetermined amount of polyethylene glycol (PEG), e.g., PEG-2K, PEG-5K, PEG-6K, PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K, and the like. For example, the AEX equilibration buffer comprises PEG at a concentration of at least about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10% (w/v, w/w, or v/v) or higher. In some embodiments, the AEX equilibration buffer comprises PEG at a concentration from about 0.5% to about 9.5%, from about 1% to about 9%, from about 2% to about 8.5%, from about 2.5% to about 8%, from about 3% to about 7.5%, from about 3.5% to about 7%, from about 4% to about 6.5%, or from about 4.5% to about 5.5%. In some embodiments of any of the aspects, the AEX equilibration buffer comprises PEG at a concentration of about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10%. In some embodiments, the AEX equilibration buffer comprises 0-10% PEG. In some embodiments, the PEG is PEG-6K.

In some embodiments of any aspect, the anion exchange equilibration buffer (also referred to herein as anion exchange conditioning buffer) comprises: a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris) and any combination thereof, or a buffer selected from the group consisting of BTP, tris, borate, tricine and any combination thereof, or a buffer selected from the group consisting of BTP, tris and any combination thereof)A buffer); amino acids (e.g., amino acids selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or amino acids selected from the group consisting of aspartyl, glutamate, and histidine; or amino acids selected from the group consisting of histidine and lysine; or amino acids selected from the group consisting of cysteine and tyrosine); viscosity modifiers (e.g., polyols selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or polyols selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol (dulcitol), sucrose, lactose, maltose, trehalose, dextran (dextran), and any combination thereof, or polyols selected from the group consisting of glycerol, sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or polyols selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, or polyols selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof); nonionic surfactants (e.g., nonionic surfactants selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkyl phenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from polyoxyethylene (12) isooctylphenyl ether (e.g., CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethyleneGlycol stearyl ether), seed oil surfactants (e.g., ecosulf SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338 and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020 and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, GIOL NP-11, GITOL NP-12, TERGITOL NP-13 and any combination thereof); and a salt (for example, a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt and zinc salt; or a salt selected from the group consisting of potassium salt) Salts of the group consisting of ammonium salts, magnesium salts, calcium salts, manganese salts and zinc salts; or a salt selected from the group consisting of potassium, magnesium and calcium salts; or from potassium and magnesium salts (e.g. MgCl) ₂ ) Salts of the group consisting of). For example, the anion exchange equilibration buffer comprises: about 75-125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 75-125mM of an amino acid (e.g., an amino acid selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or an amino acid selected from the group consisting of aspartate, glutamate, and histidine; or an amino acid selected from the group consisting of histidine and lysine; or an amino acid selected from the group consisting of cysteine and tyrosine); about 2.5% -7.5% of a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof); from about 0.3% to about 0.7% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkyl phenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from the group consisting of From polyoxyethylene (12) isooctylphenyl ether (e.g.)>CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combinations thereof Or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combinations thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combinations thereof; and about 0.5 to about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium, potassium, ammonium, magnesium, calcium, copper, cobalt, manganese, nickel, and zinc salts; or a salt selected from the group consisting of potassium, ammonium, magnesium, calcium, manganese, and zinc salts; or a salt selected from the group consisting of potassium, magnesium, and calcium salts; or a salt selected from the group consisting of potassium and magnesium salts (e.g., mgCl) ₂ ) Salts of the group consisting of).

In some embodiments, the anion exchange equilibration buffer comprises: about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof, or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM of amino acids (e.g., amino acids selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or amino acids selected from the group consisting of aspartate, glutamate, and histidine; or amino acids selected from the group consisting of histidine and lysine; or amino acids selected from the group consisting of cysteine and tyrosine); about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, or about 0.7% nonionic surface An active agent (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g.,CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or is selected from poloxamer P188, poloxamer P407, pluronic 10R5, PF68, Ecosurf SA-15, brij S20, tergitol NP-10, IGEPAL CA 720, tween 80 and any combination thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338 and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020 and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, GITOL-64, GINP-7, TOL-8, TERGITOL-9, TERGITOL-NP-10, GINP-4, GINP-14, GIOSUROL-4 and any combination thereof; about 0.5mM, about 0.6mM, about 0.7mM, about 0.8mM, about 0.9mM, about 1mM, about 1.1mM, about 1.2mM, about 1.3mM, about 1.4mM, or about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt, and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt, and zinc salt; or a salt selected from the group consisting of potassium salt, magnesium salt, and calcium salt; or a salt selected from the group consisting of potassium salt and magnesium salt (e.g., mgCl) ₂ ) Salts of the group consisting of; and about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, or about 7% of a viscosity modifier (e.g., a polyol selected from the group consisting of a hydrocarbon, a monosaccharide, a disaccharide, a trisaccharide, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactose, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof).

In some embodiments, the anion exchange equilibration buffer comprises: about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof, or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM of amino acids (e.g., amino acids selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or amino acids selected from the group consisting of aspartate, glutamate, and histidine; or amino acids selected from the group consisting of histidine and lysine; or amino acids selected from the group consisting of cysteine and tyrosine); about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, or about 0.7% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or a nonionic surfactant selected from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g., CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338 and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020 and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, GIOL NP-11, GITOL NP-12, TERGITOL NP-13 and any combination thereof); about 0.5mM, about 0.6mM, about 0.7mM, about 0.8mM, about 0.9mM, about 1mM, about 1.1mM, About 1.2mM, about 1.3mM, about 1.4mM or about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt and zinc salt; or a salt selected from the group consisting of potassium salt, magnesium salt and calcium salt; or a salt selected from the group consisting of potassium salt and magnesium salt (such as MgCl) ₂ ) Salts of the group consisting of; and about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, or about 7% of a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof), or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactose, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, and the buffer has a high pH (e.g., a pH of about 8.5-9.5, e.g., a pH of about 8.5, or about 9.5).

In some embodiments of any of the aspects, the anion exchange equilibration buffer comprises: a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris) and any combination thereof, or a buffer selected from the group consisting of BTP, tris, borate, tricine and any combination thereof, or a buffer selected from the group consisting of BTP, tris and any combination thereof); a weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); nonionic surfactants (e.g. selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates)Pear alcohol ester, polyoxyethylene alkyl phenyl ether), and any combination thereof, or a nonionic surfactant selected from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g.,CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +. >S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surface selected from the group consisting of Pluronic 10R5 and PF68 A non-ionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338 and any combinations thereof, or a non-ionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020 and any combinations thereof, or a non-ionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL-12, TERGITOL NP-13 and any combinations thereof; salts (e.g., salts selected from the group consisting of sodium salts, potassium salts, ammonium salts, magnesium salts, calcium salts, copper salts, cobalt salts, manganese salts, nickel salts, and zinc salts; or salts selected from the group consisting of potassium salts, ammonium salts, magnesium salts, calcium salts, manganese salts, and zinc salts; or salts selected from the group consisting of potassium salts, magnesium salts, and calcium salts; or salts selected from the group consisting of potassium salts and magnesium salts (e.g., mgCl) ₂ ) Salts of the group consisting of; and a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof). For example, the anion exchange equilibration buffer comprises: about 75-125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 0.5-5mM lemon Citric acid; from about 0.3% to about 0.7% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g., jojoba @>CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG block copolymer 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij 0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGL 15-S-9, TERGOL 15-S-12, TERGOL-64, GITONP-7, TERGL-8, TOL-9, TERGL 5, TERGL NP-10, TERGL-10, TONP-12, and any combination thereof, or is selected from the group consisting of poloxacin A nonionic surfactant selected from the group consisting of m P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, tergitol NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL P-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof; about 0.5 mM to about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt, and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt, and zinc salt; or a salt selected from the group consisting of potassium salt, magnesium salt, and calcium salt; or a salt selected from the group consisting of potassium salt and magnesium salt (e.g., mgCl) ₂ ) Salts of the group consisting of; and about 2.5% -7.5% of a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof). In some embodiments, the anion exchange equilibration buffer comprises: about 75-125mM buffer (e.g., selected from the group consisting of acetate, histidine, phosphate, citric acidSalts, propionates, tricine, borates, tris (hydroxymethyl) aminomethane (tris) and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof; about 0.5-5mM citric acid; from about 0.3% to about 0.7% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g., jojoba @ >CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol octadecyl ether), seed oil surfactant (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylate (e.g., TERGITOL NP-10 nonylphenol ethoxylate), or a nonionic surfactant selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij Cl0, brij j 020, osur F9, ecl 7, tos 7-14, and ecl 7, gif 15)SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL SA-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, polysorbate 20 and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80 and any combination thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combinations thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combinations thereof; about 0.5 mM to about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt, and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt, and zinc salt; or a salt selected from the group consisting of potassium salt, magnesium salt, and calcium salt; or a salt selected from the group consisting of potassium salt and magnesium salt (e.g., mgCl) ₂ ) Salts of the group consisting of; and about 3% to about 7% of a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactitol, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof)A polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof), and the buffer has a high pH (e.g., a pH of about 8.5-9.5, e.g., a pH of about 8.5, about 9, or about 9.5).

In some embodiments, the anion exchange equilibration buffer comprises: about 75mM, about 115mM, about 120mM, or about 125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 0.5mM, about 1mM, about 1.5mM, about 2mM, about 2.5mM, 3mM, about 3.5mM, about 4mM, about 4.5mM, or about 5mM citric acid or a salt thereof; about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, or about 0.7% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or a nonionic surfactant selected from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g., CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactant (e.g., ecosurf (tm) SA-15 seed oil surfactant), poloxamer 188 (polyoxyethylene)Copolymers of alkene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL P-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof; about 0.5mM, about 0.6mM, about 0.7mM, about 0.8mM, about 0.9mM, about 1mM, about 1.1mM, about 1.2mM, about 1.3mM, about 1.4mM, or about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt, and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt) Salts of the group consisting of calcium salts, manganese salts and zinc salts; or a salt selected from the group consisting of potassium, magnesium and calcium salts; or from potassium and magnesium salts (e.g. MgCl) ₂ ) Salts of the group consisting of; and about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, or about 7% of a viscosity modifier (e.g., a polyol selected from the group consisting of a hydrocarbon, a monosaccharide, a disaccharide, a trisaccharide, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactose, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof).

In some embodiments, the anion exchange equilibration buffer comprises: about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof, or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 0.5mM, about 1mM, about 1.5mM, about 2mM, about 2.5mM, about 3mM, about 3.5mM, about 4mM, about 4.5mM, or about 5mM citric acid or a salt thereof; about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, or about 0.7% of a nonionic surfactant (e.g., selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof Nonionic surfactants of the group, or from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g.,CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or poloxamer P188, poloxamer P407, poloxamer A nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof; about 0.5mM, about 0.6mM, about 0.7mM, about 0.8mM, about 0.9mM, about 1mM, about 1.1mM, about 1.2mM, about 1.3mM, about 1.4mM, or about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt, and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt, and zinc salt; or a salt selected from the group consisting of potassium salt, magnesium salt, and calcium salt; or a salt selected from the group consisting of potassium salt and magnesium salt (e.g., mgCl) ₂ ) Salts of the group consisting of; and about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, or about 7% of a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof), or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactose, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof), and the buffer has a high pH (e.g., a pH of about 8.5-9.5, e.g., a pH of about 8.5, about 9.5, or about 9.5).

In some embodiments of any of the aspects, the anion exchange equilibration buffer comprises: buffers (e.g. selected from acetate, histidine, phosphorusA buffering agent selected from the group consisting of acid salts, citric acid salts, propionic acid salts, tricine, boric acid salts, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof; amino acids (e.g., amino acids selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or amino acids selected from the group consisting of aspartate, glutamate, and histidine; or amino acids selected from the group consisting of histidine and lysine; or amino acids selected from the group consisting of cysteine and tyrosine); a weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); nonionic surfactants (e.g., nonionic surfactants selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkyl phenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from polyoxyethylene (12) isooctylphenyl ether (e.g., CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactant (e.g., ecosurf (tm) SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylate (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or a nonionic surfactant selected from the group consisting of tween (tm) and combinations thereofN60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brij S0, brij 010, brij Cl0, brij 020, ECOSURF EH-9, ECOSURF EH-14, TERGOL 15-S-7, ECOSURF SA-15, TERGOL 15-S-9, TERGOL 15-S-12, TERGOL-64, GITOP-7, GIOL NP-8, GITOL-9, TERGOL-9, TERGL 5 TERGL-720, TERGL 10, GINP-11, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, briJ 020, and any combinations thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, and combinations thereof, nonionic surfactants of the group consisting of TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof; salts (e.g., salts selected from the group consisting of sodium salts, potassium salts, ammonium salts, magnesium salts, calcium salts, copper salts, cobalt salts, manganese salts, nickel salts, and zinc salts; or salts selected from the group consisting of potassium salts, ammonium salts, magnesium salts, calcium salts, manganese salts, and zinc salts; or salts selected from the group consisting of potassium salts, magnesium salts, and calcium salts; or salts selected from the group consisting of potassium salts and magnesium salts (e.g., mgCl) ₂ ) Salts of the group consisting of; and a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereofOr a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactitol, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof. For example, the anion exchange equilibration buffer comprises: about 75-125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 75-125mM of an amino acid (e.g., an amino acid selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or an amino acid selected from the group consisting of aspartate, glutamate, and histidine; or an amino acid selected from the group consisting of histidine and lysine; or an amino acid selected from the group consisting of cysteine and tyrosine); about 0.5-5mM weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); from about 0.3% to about 0.7% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g., jojoba @ >CA-270 polyoxyethylene (12) isooctylphenyl ether) and polyoxyethyleneSorbitan monooleate (e.g.)>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, briJ 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TE Nonionic surfactants of the group consisting of RGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof; about 0.5 mM to about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt, and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt, and zinc salt; or a salt selected from the group consisting of potassium salt, magnesium salt, and calcium salt; or a salt selected from the group consisting of potassium salt and magnesium salt (e.g., mgCl) ₂ ) Salts of the group consisting of; and about 2.5% -7.5% of a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof). In some embodiments, the anion exchange equilibration buffer comprises: about 75-125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 75-125mM amino acid (e.g., an amino acid selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine, or an amino acid selected from the group consisting of aspartate, glutamate, and histidine, or a group consisting of histidine and lysine) Amino acids of (2); or an amino acid selected from the group consisting of cysteine and tyrosine); about 3-10mM of a weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); from about 0.3% to about 0.7% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g., jojoba @>CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, brij 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIRPOL 15-S-9, TERGOL 15-S-12, TERGL-64, TOGINP-7, GINP-8, TERGOL-9, TERGOL-NP-5 TERGITOL-TERGIN A nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL P-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof; about 0.5 mM to about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt, and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt, and zinc salt; or a salt selected from the group consisting of potassium salt, magnesium salt, and calcium salt; or a salt selected from the group consisting of potassium salt and magnesium salt (e.g., mgCl) ₂ ) Salts of the group consisting of; and about 3% to about 7% of a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactitol, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof)Polyols) and the buffer has a high pH (e.g., a pH of about 8.5-9.5, e.g., a pH of about 8.5, about 9, or about 9.5).

In some embodiments, the anion exchange equilibration buffer comprises: about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof, or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM of amino acids (e.g., amino acids selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or amino acids selected from the group consisting of aspartate, glutamate, and histidine; or amino acids selected from the group consisting of histidine and lysine; or amino acids selected from the group consisting of cysteine and tyrosine); about 0.5mM, about 1mM, about 1.5mM, about 2mM, about 2.5mM, 3mM, about 3.5mM, about 4mM, about 4.5mM, or about 5mM of a weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, or about 0.7% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or a nonionic surfactant selected from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g., CA-270 polyoxyethylene (12) isooctylphenyl ether) and polyoxyethylene dehydrationSorbitol monooleate (e.g.)>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g.,s20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, briJ 020, and any combinations thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S- 9. Nonionic surfactants of the group consisting of TERGITOL 15-S-12, TERGITOL 64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof; about 0.5mM, about 0.6mM, about 0.7mM, about 0.8mM, about 0.9mM, about 1mM, about 1.1mM, about 1.2mM, about 1.3mM, about 1.4mM, or about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt, and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt, and zinc salt; or a salt selected from the group consisting of potassium salt, magnesium salt, and calcium salt; or a salt selected from the group consisting of potassium salt and magnesium salt (e.g., mgCl) ₂ ) Salts of the group consisting of; and about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, or about 7% of a viscosity modifier (e.g., a polyol selected from the group consisting of a hydrocarbon, a monosaccharide, a disaccharide, a trisaccharide, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactose, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof).

In some embodiments, the anion exchange equilibration buffer comprises: about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof, or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof)An agent); about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM of amino acids (e.g., amino acids selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or amino acids selected from the group consisting of aspartate, glutamate, and histidine; or amino acids selected from the group consisting of histidine and lysine; or amino acids selected from the group consisting of cysteine and tyrosine); about 0.5mM, about 1mM, about 1.5mM, about 2mM, about 2.5mM, 3mM, about 3.5mM, about 4mM, about 4.5mM, or about 5mM of a weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, or about 0.7% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or a nonionic surfactant selected from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g., CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g.,s20 polyethylene glycol stearyl ether), seed oil surfactant (e.g., ecosurf (tm) SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylate (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereofAn agent selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGOL 15-S-7, ECOSURF SA-15, TERGOL 15-S-9, TERGOL 15-S-12, TERGL-64, LNP-7, GITOL NP-8, TERGL 9, TERGL NP-10, NP-4, TONP-10, TONP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combinations thereof, or an ECOSURF-9, EH, nonionic surfactants of the group consisting of ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof; about 0.5mM, about 0.6mM, about 0.7mM, about 0.8mM, about 0.9mM, about 1mM, about 1.1mM, about 1.2mM, about 1.3mM, about 1.4mM, or about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt, and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt, and zinc salt; or a salt selected from the group consisting of potassium salt, magnesium salt, and calcium salt; or a salt selected from the group consisting of potassium salt and magnesium salt (e.g., mgCl) ₂ ) Group ofSalts of the group formed); and about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, or about 7% of a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof), or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactose, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, and the buffer has a high pH (e.g., a pH of about 8.5-9.5, e.g., a pH of about 8.5, or about 9.5).

In some embodiments, the anion exchange equilibration buffer comprises: about 100mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof; or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 100mM of amino acids (e.g., an amino acid selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or an amino acid selected from the group consisting of aspartate, glutamate, and histidine; or an amino acid selected from the group consisting of histidine and lysine; or an amino acid selected from the group consisting of cysteine and tyrosine); about 2-4mM weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); about 1mM of a salt (e.g., a salt selected from the group consisting of sodium, potassium, ammonium, magnesium, calcium, copper, cobalt, manganese, nickel, and zinc salts; or a salt selected from the group consisting of potassium, ammonium, magnesium, calcium salts) Salts of the group consisting of manganese salts and zinc salts; or a salt selected from the group consisting of potassium, magnesium and calcium salts; or from potassium and magnesium salts (e.g. MgCl) ₂ ) Salts of the group consisting of; about 0.5% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenolethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g.,CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, brij 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIRPOL 15-S-9, TERGOL 15-S-12, TERGL-64, TOGINP-7, GINP-8, TERGL-9, TERGNP-10, TERGL-10. TERGL-NP-TERGL, and the like GITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, polysorbate 20, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combination thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL P-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof; and about 5% of a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactitol, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof). For example, the anion exchange equilibration buffer comprises: about 100mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof; or a buffer selected from the group consisting of BTP, tris, and any combination thereof) A buffer of the combined group); about 100mM of amino acids (e.g., an amino acid selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or an amino acid selected from the group consisting of aspartate, glutamate, and histidine; or an amino acid selected from the group consisting of histidine and lysine; or an amino acid selected from the group consisting of cysteine and tyrosine); about 2mM, about 2.5mM, about 3mM, about 3.5mM, or about 4mM of a weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); about 1mM of a salt (e.g., a salt selected from the group consisting of sodium, potassium, ammonium, magnesium, calcium, copper, cobalt, manganese, nickel, and zinc salts; or a salt selected from the group consisting of potassium, ammonium, magnesium, calcium, manganese, and zinc salts; or a salt selected from the group consisting of potassium, magnesium, and calcium salts; or a salt selected from the group consisting of potassium and magnesium salts (e.g., mgCl) ₂ ) Salts of the group consisting of; about 0.5% and about 5% of a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, and any combination thereof), and the buffer has a high pH (e.g., a pH of about 8.5-9.5, e.g., a pH of about 8.5, about 9, or about 9.5).

In some embodiments of any of the aspects, the anion exchange equilibration buffer comprises: buffers (e.g., buffers selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) F68); nonionic surfactants (e.g. selected from polyoxyethylene estersFatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or a nonionic surfactant selected from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g.,CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or is selected from poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80 and any combination thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338 and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijS 0, brij 010, brij Cl0, brij 020 and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL-64, TERGITOL-7, TERGITOL-8, TERGITOL NP-9, TOL-9.5, TOOL-10, TERGOL NP-11, TERGITOL NP-12 and any combination thereof, and a non-ionic buffer selected from the group consisting of any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof; amino acids (e.g., amino acids selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or amino acids selected from the group consisting of aspartate, glutamate, and histidine; or amino acids selected from the group consisting of histidine and lysine; or amino acids selected from the group consisting of cysteine and tyrosine); a weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); nonionic surfactants (e.g., nonionic surfactants selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkyl phenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from polyoxyethylene (12) isooctylphenyl ether (e.g., polyoxyethylene) >CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64 Nonionic surfactants of the group consisting of TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combinations thereof; and salts (e.g., salts selected from the group consisting of sodium salts, potassium salts, ammonium salts, magnesium salts, calcium salts, copper salts, cobalt salts, manganese salts, nickel salts, and zinc salts; or salts selected from the group consisting of potassium salts, ammonium salts, magnesium salts, calcium salts, manganese salts, and zinc salts; or salts selected from the group consisting of potassium salts, magnesium salts, and calcium salts; or salts selected from the group consisting of potassium salts and magnesium salts (e.g., mgCl) ₂ ) Salts of the group consisting), and optionally the buffer is substantially free of glycine. For example, the anion exchange equilibration buffer comprises: about 75-125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 75-125mM of an amino acid (e.g., an amino acid selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or an amino acid selected from the group consisting of aspartate, glutamate, and histidine; or an amino acid selected from the group consisting of histidine and lysine; or an amino acid selected from the group consisting of cysteine and tyrosine); about 0.5-5mM weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); from about 0.3% to about 0.7% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkyl phenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from polyoxyethylene (12) isooctylphenyl ether (e.g., CA-270 polyoxygenEthylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g.,80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14 Nonionic surfactants of the group consisting of TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof; and about 0.5 to about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium, potassium, ammonium, magnesium, calcium, copper, cobalt, manganese, nickel, and zinc salts; or a salt selected from the group consisting of potassium, ammonium, magnesium, calcium, manganese, and zinc salts; or a salt selected from the group consisting of potassium, magnesium, and calcium salts; or a salt selected from the group consisting of potassium and magnesium salts (e.g., mgCl) ₂ ) Salts of the group consisting), and optionally the buffer is substantially free of glycine. In some embodiments, the anion exchange equilibration buffer comprises: about 75-125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 75-125mM of an amino acid (e.g., an amino acid selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or an amino acid selected from the group consisting of aspartate, glutamate, and histidine; or an amino acid selected from the group consisting of histidine and lysine; or an amino acid selected from the group consisting of cysteine and tyrosine); about 3-10mM citric acid; from about 0.3% to about 0.7% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g., jojoba @ >CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, and any combinations thereof A sex agent, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof; and about 0.5 to about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium, potassium, ammonium, magnesium, calcium, copper, cobalt, manganese, nickel, and zinc salts; or a salt selected from the group consisting of potassium, ammonium, magnesium, calcium, manganese, and zinc salts; or a salt selected from the group consisting of potassium, magnesium, and calcium salts; or a salt selected from the group consisting of potassium and magnesium salts (e.g., mgCl) ₂ ) Salts of the group consisting) and the buffer has a high pH (e.g., a pH of about 8.5-9.5, e.g., a pH of about 8.5, about 9, or about 9.5), and optionally the buffer is substantially free of glycine.

In some embodiments, the anion exchange equilibration buffer comprises: about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof, or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM of amino acids (e.g., amino acids selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or amino acids selected from the group consisting of aspartate, glutamate, and histidine; or amino acids selected from the group consisting of histidine and lysine; or amino acids selected from the group consisting of cysteine and tyrosine); about 0.5mM, about 1mM, about 1.5mM, about 2mM, about 2.5mM, 3mM, about 3.5mM, about 4mM, about 4.5mM, or about 5mM weak acid or salt thereof (e.g., lemon) An acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, or about 0.7% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or a nonionic surfactant selected from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g.,CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g.,s20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, brij 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOL 15-S-9, TERGOL 15-S-12, TERGL-64, TOGINP-7, GINP-8, TERGL-9, TERGL-NP-10, TONP-TOGINP-10, and the like TOL NP-12, TERGITOL NP-13, polysorbate 20, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, tergitol NP-10, IGEPAL CA 720, tween 80, and any combination thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL P-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof; and about 0.5mM, about 0.6mM, about 0.7mM, about 0.8mM, about 0.9mM, about 1mM, about 1.1mM, about 1.2mM, about 1.3mM, about 1.4mM, or about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt, and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt, and zinc salt; or a salt selected from the group consisting of potassium salt, magnesium salt, and calcium salt; or a salt selected from the group consisting of potassium salt and magnesium salt (e.g., mgCl) ₂ ) Salts of the group consisting), and optionally the buffer is substantially free of glycine.

In some embodiments, the anion exchange equilibration buffer comprises: about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof, or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof)Group buffers); about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM of amino acids (e.g., amino acids selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or amino acids selected from the group consisting of aspartate, glutamate, and histidine; or amino acids selected from the group consisting of histidine and lysine; or amino acids selected from the group consisting of cysteine and tyrosine); about 0.5mM, about 1mM, about 1.5mM, about 2mM, about 2.5mM, 3mM, about 3.5mM, about 4mM, about 4.5mM, or about 5mM of a weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, or about 0.7% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or a nonionic surfactant selected from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g., CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g.,s20 polyethylene glycol stearyl ether), seed oil surfactant (e.g., ecosurf (tm) SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylate (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereofA surfactant selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brij S0, brij 010, brij Cl0, brij 020, ECURF EH-9, ECOSURF EH-14, TERGOL 15-S-7, ECOSURF SA-15, GITOL15-S-9, TERGOL 15-S-12, GITOL-64, GINP-7, TERGL-8, TERGL 9, TERGL-9, TERGL NP-10, GINP-4, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combinations thereof, or an ECOSURF-9, EH, nonionic surfactants of the group consisting of ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof; and about 0.5mM, about 0.6mM, about 0.7mM, about 0.8mM, about 0.9mM, about 1mM, about 1.1mM, about 1.2mM, about 1.3mM, about 1.4mM, or about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt, and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt, and zinc salt; or a salt selected from the group consisting of potassium salt, magnesium salt, and calcium salt; or a salt selected from the group consisting of potassium salt and magnesium salt (e.g., M) gCl ₂ ) Salts of the group consisting) and the buffer has a high pH (e.g., a pH of about 8.5-9.5, e.g., a pH of about 8.5, about 9, or about 9.5), and optionally the buffer is substantially free of glycine.

In some embodiments, the anion exchange equilibration buffer comprises: about 100mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof; or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 100mM of amino acids (e.g., an amino acid selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or an amino acid selected from the group consisting of aspartate, glutamate, and histidine; or an amino acid selected from the group consisting of histidine and lysine; or an amino acid selected from the group consisting of cysteine and tyrosine); about 2-4mM weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); about 1mM of a salt (e.g., a salt selected from the group consisting of sodium, potassium, ammonium, magnesium, calcium, copper, cobalt, manganese, nickel, and zinc salts; or a salt selected from the group consisting of potassium, ammonium, magnesium, calcium, manganese, and zinc salts; or a salt selected from the group consisting of potassium, magnesium, and calcium salts; or a salt selected from the group consisting of potassium and magnesium salts (e.g., mgCl) ₂ ) Salts of the group consisting of; and about 0.4% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g.,CA-270 Polyoxyethylene (1)2) Isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g.)>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, and a combination thereof, A nonionic surfactant of the group consisting of TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof), and optionally the buffer is substantially free of glycine. For example, the anion exchange equilibration buffer comprises: about 100mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof; or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 100mM of amino acids (e.g., an amino acid selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or an amino acid selected from the group consisting of aspartate, glutamate, and histidine; or an amino acid selected from the group consisting of histidine and lysine; or an amino acid selected from the group consisting of cysteine and tyrosine); about 2mM, about 2.5mM, about 3mM, about 3.5mM, or about 4mM of a weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); about 1mM of a salt (e.g., a salt selected from the group consisting of sodium, potassium, ammonium, magnesium, calcium, copper, cobalt, manganese, nickel, and zinc salts; or a salt selected from the group consisting of potassium, ammonium, magnesium, calcium, manganese, and zinc salts; or a salt selected from the group consisting of potassium, magnesium, and calcium salts; or a salt selected from the group consisting of potassium and magnesium salts (e.g., mgCl) ₂ ) Salts of the group consisting of; and about 0.4% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g., jersey)>CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 01 0. Brij Cl0, briJ 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL-64, TERGITOL P-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL LNP-13, and any combination thereof), and the buffer has a high pH (e.g., a pH of about 8.5-9.5, e.g., a pH of about 8.5, about 9, or about 9.5), and optionally the buffer is substantially free of glycine.

In some embodiments, the anion exchange equilibration buffer comprises: about 100mM BTP, about 100mM histidine, about 2-4mM citric acid or salt thereof, about 1mM MgCl ₂ And about 0.4% PF68. For example, the anion exchange equilibration buffer comprises: about 100mM BTP; about 100mM histidine; about 2mM, about 2.5mM, about 3mM, about 3.5mM, or about 4mM citric acid or a salt thereof; about 1mM MgCl ₂ The method comprises the steps of carrying out a first treatment on the surface of the And about 0.4% PF68, and the buffer has a high pH (e.g., a pH of about 8.5-9.5, e.g., a pH of about 8.5, about 9, or about 9.5), and optionally the buffer is substantially free of glycine.

Notably, the anion exchange chromatography media can be equilibrated by washing the media with AEX equilibration buffer. For example, the anion exchange chromatography medium can be washed with an AEX equilibration medium of at least 1 Column Volume (CV). In some embodiments, the anion exchange chromatography medium is equilibrated with at least about 1CV, 1.5CV, 2CV, 2.5CV, 3CV, 3.5CV, 4CV, 4.5CV, 5CV, 5.5CV, 6CV, 6.5CV, 7CV, 7.5CV, 8CV, 8.5CV, 9CV, 9.5CV, 10CV, 10.5CV, 11CV, 11.5CV, 12CV, 12.5CV, 13CV, 13.5CV, 14CV, 14.5CV, 15CV, 15.5CV, 16CV, 16.5CV, 17CV, 17.5CV, 18CV, 18.5CV, 19CV, 19.5CV, 20CV, or more AEX equilibration buffer.

The anion exchange chromatography medium can be equilibrated with AEX equilibration buffer at any desired temperature. Typically, the anion exchange chromatography media can be equilibrated with AEX equilibration buffer at room temperature.

AEX elution buffer

Typically, the AEX elution buffer comprises a buffer. Exemplary buffers include, but are not limited to, acetate, histidine, phosphate, citrate, propionate, tricine, borate, and tris (hydroxymethyl) aminomethane (tris). In some embodiments of any of the aspects, the AEX elution buffer comprises a predetermined amount of bis-tris propane (BTP).

The amount of buffer (e.g., BTP) in the AEX elution buffer can be optimized for the virus particle to be separated. For example, the AEX elution buffer can comprise a buffer, e.g., BTP, tris, borate, and/or tricine, at a concentration of at least about 25mM, 50mM, 75mM, 100mM, 125mM, 150mM, or higher. For example, the AEX elution buffer comprises a buffer, e.g., BTP, at a concentration of from about 50mM to about 150mM, from about 75mM to about 125mM, from about 85mM to about 115mM, or from about 90mM to about 110 mM. In some embodiments of any of the aspects, the AEX elution buffer can comprise a buffer, e.g., BTP, at a concentration of about 15mM, about 20mM, about 25mM, about 30mM, about 35mM, about 40mM, about 45mM, about 50mM, about 55mM, about 60mM, about 65mM, about 70mM, about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125 mM.

The AEX elution buffer may also contain a surfactant. For example, the AEX elution buffer comprises a nonionic surfactant. Exemplary nonionic surfactants include, but are not limited to, polysorbates such as polysorbate 20 (TWEEN 20), polysorbate 28, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 81, and polysorbate 85; poloxamers, such as poloxamer 188 and poloxamer 407; polyoxyethylene polyoxypropylene ether; or polyethylene glycol (PEG). In some embodiments of any of the aspects, the surfactant is a nonionic fluorosurfactant. Exemplary nonionic fluorosurfactants include fluorosurfactants containing polyethylene glycol polymers, polypropylene glycol polymers, and copolymers thereof. Specific exemplary nonionic fluorosurfactants include, but are not limited to, PF68.

The AEX elution buffer can comprise at least about 0.001%, 0.0025%, 0.005%, 0.0075%, 0.01%, 0.0125%, 0.015%, 0.0175%, 0.02%, 0.025%, 0.03%, 0.0325%, 0.035%, 0.0375%, 0.04%, 0.045%, 0.05% (w/v, w/w, v/v) or higher amounts of nonionic surfactant. For example, the AEX elution buffer can include a nonionic surfactant at a concentration of about 0.001% to about 0.02%, from about 0.0025% to about 0.0175%, from about 0.005% to about 0.015%, or from about 0.0075% to about 0.0125%. In some embodiments of any of the aspects, the AEX elution buffer comprises the nonionic surfactant at a concentration of about 0.001%, about 0.0025%, about 0.005%, about 0.0075%, about 0.01%, about 0.0125%, about 0.015%, about 0.0175%, about 0.02%, about 0.025%, about 0.03%, about 0.0325%, about 0.035%, about 0.0375%, about 0.04%, about 0.045%, or about 0.05%. In some embodiments, the AEX elution buffer comprises a nonionic surfactant at a concentration of 0-0.05%.

The AEX equilibration buffer may also comprise cations, for example, monovalent or divalent cations. Exemplary monovalent ions for AEX elution buffers include, but are not limited to, sodium (Na ⁺ ) Potassium (K) ⁺ ) Ammonium (NH) ₄ ⁺ ) And alkylammons, for example, tetramethylammonium. Exemplary divalent cations of AEX elution buffers include, but are not limited to, magnesium (Mg ²⁺ ) Calcium (Ca) ²⁺ ) Copper (Cu) ²⁺ ) Cobalt (Co) ²⁺ ) Manganese (Mn) ²⁺ ) Nickel (Ni) ²⁺ ) And zinc (Zn) ²⁺ ). The cations may be added in the form of salts. In some embodiments, the AEX elution buffer comprises a divalent cation, e.g., mg ²⁺ 。

In some embodiments, the AEX elution buffer comprises Mg ²⁺ For example, mgCl ₂ The concentration thereof is at least about 0.5mM, 1mM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, 4mM, 4.5mM, 5mM or more. In some embodiments, the AEX elution buffer comprises Mg at a concentration of 0-2mM ²⁺ For example, mgCl ₂ 。

In some embodiments, the AEX elution buffer comprises Na ⁺ For example, naCl, at a concentration of at least about 1mM, 5mM, 10mM, 25mM, 50mM, 75mM, 100mM,125mM, 150mM, 175mM, 200mM, 225mM, 250mM, 275mM, 300mM, 325mM, 350mM or more.

In some embodiments, the AEX elution buffer comprises K ⁺ For example, potassium acetate, at a concentration of at least about 1mM, 5mM, 10mM, 25mM, 50mM, 75mM, 100mM, 125mM, 150mM, 175mM, 200mM, 225mM, 250mM, 275mM, 300mM, 325mM, 350mM or more.

In some embodiments, the AEX elution buffer comprises ammonium, e.g., ammonium acetate, at a concentration of at least about 1mM, 5mM, 10mM, 25mM, 50mM, 75mM, 100mM, 125mM, 150mM, 175mM, 200mM, 225mM, 250mM, 275mM, 300mM, 325mM, 350mM, or more.

In some embodiments, the AEX elution buffer comprises tetramethylammonium, e.g., tetramethylammonium chloride, at a concentration of at least about 1mM, 5mM, 10mM, 25mM, 50mM, 75mM, 100mM, 125mM, 150mM, 175mM, 200mM, 225mM, 250mM, 275mM, 300mM, 325mM, 350mM, or more.

In some embodiments, the AEX elution buffer comprises: buffers (e.g., buffers selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris) and any combination thereof, or buffers selected from the group consisting of BTP, tris, borate, tricine and any combination thereof, or buffers selected from the group consisting of BTP, tris and any combination thereof), sodium acetate, and PF68. For example, the AEX elution buffer comprises: about 75-125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 250-350mM sodium acetate and about 0.005% -0.015% of a nonionic surfactant (e.g., selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably) Polysorbate, polyoxyethylene alkylphenyl ethers), and any combination thereof, or a nonionic surfactant selected from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g.,CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g.,80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surface selected from the group consisting of Pluronic10R5 and PF68 A non-ionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338 and any combinations thereof, or a non-ionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020 and any combinations thereof, or a non-ionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL-12, TERGITOL NP-13 and any combinations thereof.

In some embodiments, the AEX elution buffer comprises: about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof, or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 250mM, about 255mM, about 260mM, about 265mM, about 270mM, about 275mM, about 280mM, about 285mM, about 290mM, about 300mM, about 305mM, about 310mM, about 315mM, about 320mM, about 325mM, about 330mM, about 335mM, about 340mM, about 345mM or about 350mM sodium acetate; and about 0.005%, about 0.0075%, about 0.01%, about 0.0125%, or about 0.015% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g., CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (exampleAs an example of the presence of a metal such as,80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TER Nonionic surfactants of the group consisting of GITOL-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof.

In some embodiments, the AEX elution buffer comprises: about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof, or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 250mM, about 255mM, about 260mM, about 265mM, about 270mM, about 275mM, about 280mM, about 285mM, about 290mM, about 300mM, about 305mM, about 310mM, about 315mM, about 320mM, about 325mM, about 330mM, about 335mM, about 340mM, about 345mM or about 350mM sodium acetate; and about 0.005%, about 0.0075%, about 0.01%, about 0.0125%, or about 0.015% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g., CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g.,80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol octadecyl ether) A seed oil surfactant (e.g., ecosurf SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), a nonylphenol ethoxylate (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combinations thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combinations thereof), and the buffer has a high pH (e.g., a pH of about 8.5, about 9.5 or about 9.5.5, about pH 5.5).

The methods described herein can be used with any anion exchange chromatography media known and available to practitioners. Exemplary anion exchange chromatography media include, but are not limited to, MACRO PREP Q (BioRad, hercules, calif.); unoschere Q (BioRad, hercules, calif.); POROS 50HQ (Applied Biosystems, foster City, calif.); POROS 50D (Applied Biosystems, foster City, calif.); POROS 50PI (Applied Biosystems, foster City, calif.); POROS XO (Applied Biosystems, foster City, calif.); SOURCE 30Q (GE Healthcare, n.j.); SOURCE 15Q (GE Healthcare, n.j.); DEAE SEPHAROSE (GE Healthcare, piscataway, n.j.); q SEPHAROSE (GE Healthcare Biosciences, piscataway, n.j.), capto Q and Capto add (GE Healthcare, n.j.); EMPHAZE (3M Corp./Pierce); and multimode chromatography, for example, prima S (BIA Separations-Sartorius; slovenia-Germany).

In some embodiments of any of the aspects, the anion exchange chromatography medium is a monoliths anion exchange chromatography medium. The term "monoliths column" or "monoliths chromatography medium" is a term of art that refers to a column containing a three-dimensional macroporous structure (i.e., monoliths) as the stationary phase. Non-limiting monoliths include CIMmultus ^TM Disposable prefilled chromatographic monolithic column, CIMac ^TM An analytical column,Linear monolithic column,/->Monolith column and->A monolithic HPLC column. Non-limiting monoliths chromatography resins include CIMmultus ^TM QA-1 Advanced Composite column (quaternary amine), CIMmultus ^TM DEAE-l Advanced Composite column (diethylamine),QA Disk (quaternary amine), +.>DEAE、EDA Disk (ethylenediamine), a method for producing the same, and a method for producing the same>A Monolith anion exchange column.

In some embodiments of any of the aspects, the feed composition for anion exchange chromatography comprises a predetermined amount of an ionic compound, e.g., a cationic compound or an anionic compound, e.g., an acid or salt thereof. In some embodiments, the feed composition for anion exchange chromatography comprises a predetermined amount of an anionic compound, e.g., an acid or salt thereof. For example, a feed composition for anion exchange chromatography comprises a predetermined amount of weak acid. As another example, the feed composition is conditioned and/or diluted with a buffer (e.g., an anion exchange dilution buffer comprising a predetermined amount of weak acid or salt thereof). In some embodiments of any of the aspects, the feed composition or the conditioned or diluted feed composition comprises a predetermined amount of citric acid or a salt thereof, acetic acid or a salt thereof, or succinic acid or a salt thereof. For example, the feed composition comprises a predetermined amount of citric acid or a salt thereof, i.e., citrate.

The amount of anionic compound (such as an acid or salt thereof, e.g., citric acid or citrate) in the feed composition for anion exchange chromatography may be optimized for the viral particles to be separated. For example, the feed composition can include an anionic compound (e.g., an acid or salt thereof, e.g., citric acid or citrate) at a concentration of at least 0.5mM, 1mM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, 4mM, 4.5mM, 5mM, 5.5mM, 6mM, 6.5mM, 7mM, 8mM, 8.5mM, 9mM, 9.5mM, 10mM, or more. For example, the feed composition comprises an anionic compound (e.g., an acid or salt thereof, e.g., citric acid or citrate) at a concentration of at least about 3mM, 5mM, 7mM, or more.

In some embodiments of any aspect, the feed composition comprises an anionic compound (e.g., an acid or salt thereof, e.g., citric acid or citrate) at a concentration of from about 0.5mM to about 15mM, from about 1mM to about 10mM, from about 1.5mM to about 7.5mM, from about 2mM to about 7mM, from about 1.5mM to about 9mM, from about 2mM to about 8mM, or from about 2.5mM to about 7.5mM.

In some embodiments of any of the aspects, the feed composition comprises an anionic compound (e.g., an acid or salt thereof, e.g., citric acid or citrate) at a concentration of about 0.5mM, about 1mM, about 1.5mM, about 2mM, about 2.5mM, about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 8mM, about 8.5mM, about 9mM, about 9.5mM, or about 10mM.

In some embodiments, the feed composition for anion exchange chromatography comprises a predetermined amount of a cationic compound, e.g., a monovalent or divalent cation. Exemplary monovalent ions for the feed composition include, but are not limited to, sodium (Na ⁺ ) Potassium (K) ⁺ ) Alkyl ammonia and ammonium. Exemplary divalent cations for dilution buffers include, but are not limited to, magnesium (Mg ²⁺ ) Calcium (Ca) ²⁺ ) Copper (Cu) ²⁺ ) Cobalt (Co) ²⁺ ) Manganese (Mn) ²⁺ ) Nickel (Ni) ²⁺ ) And zinc (Zn) ²⁺ ). The cations may be added in the form of salts. In some embodiments, the feed composition comprises divalent cations, e.g., mg ²⁺ 。

The amount of cationic compound in the feed composition for anion exchange chromatography can be optimized for the viral particles to be separated. For example, the feed composition may comprise cationic compounds at a concentration of at least 0.5mM, 1mM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, 4mM, 4.5mM, 5mM, 5.5mM, 6mM, 6.5mM, 7mM, 8mM, 8.5mM, 9mM, 9.5mM, 10mM, or more. For example, the feed composition comprises a cationic compound at a concentration of at least about 3mM, 5mM, 7mM, or more. In some embodiments of any of the aspects, the feed composition comprises a cationic compound at a concentration of from about 0.5mM to about 15mM, from about 1mM to about 10mM, from about 1.5mM to about 7.5mM, from about 2mM to about 7mM, from about 1.5mM to about 9mM, from about 2mM to about 8mM, or from about 2.5mM to about 7.5 mM. In some embodiments of any of the aspects, the feed composition comprises a cationic compound at a concentration of about 0.5mM, about 1mM, about 1.5mM, about 2mM, about 2.5mM, about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 8mM, about 8.5mM, about 9mM, about 9.5mM, or about 10 mM.

Affinity chromatography

As described herein, the feed composition for anion exchange chromatography may be an eluent from an affinity chromatography step (also referred to as an affinity eluent). For example, an eluate from affinity chromatography of a harvesting medium (e.g., a cell culture medium).

The term "affinity chromatography" or "affinity purification" as used herein refers to any method that uses specific binding interactions between molecules. The specific ligands are chemically immobilized or "coupled" to the solid support such that when a complex mixture is passed through the column, molecules with specific binding affinity for the ligand will bind. After washing away other sample components, the bound molecules are peeled from the support, thereby allowing it to be purified from the original sample. Affinity chromatography includes immunoaffinity chromatography. The term "immunoaffinity chromatography" as used herein refers to any method of using an immobilized antibody or fragment thereof in affinity chromatography.

Exemplary affinity chromatography media include, but are not limited to, affi-Gel (Biorad); affinica Agarose/Polymeric Supports (Schleicher and Schuell); avidGel (BioProbe); bio-Gel (BioRad); fractogel (EM Separations); HEMA-AFC (Alltech); reacti-Gel (Pierce); sephacryl (Pharmacia); sepharose (Pharmacia); superose (Pharmacia); trisacryl (IBF); TSK Gel Toyopearl (TosoHaas); ultragel (IBF); avidGel CPG (BioProbe); hiPAC (ChromatoChem); protein-Pak Affinity Packing (Waters); ultrafiltration-EP (Bodman); CAPTO AAVB (GE Healthcare, N.J.), AAVB Sepharose; and oligonucleotide conjugated (e.g., aptamer conjugated) affinity media. Other affinity chromatography media include affinity monoliths chromatography supports and Affinity chromatography support.

Notably, the affinity eluate can be used as a feed composition for anion exchange purification. Thus, in some embodiments of any of the aspects, the eluate from the affinity chromatography comprises a predetermined amount of the anionic compound. For example, the anionic compound may be added to the eluate from the affinity chromatography prior to the anion exchange chromatography. Typically, an acid or salt thereof is added to the eluate from the affinity chromatography. Thus, in some embodiments of any of the aspects, the affinity eluate comprises a predetermined amount of the weak acid. For example, the affinity eluate or a conditioned/diluted affinity eluate (e.g., a conditioned/diluted feed composition) comprises a predetermined amount of citric acid or a salt thereof, acetic acid or a salt thereof, or succinic acid or a salt thereof. For example, the affinity eluate comprises a predetermined amount of citric acid or a salt thereof, i.e. citrate.

The amount of anionic compound (such as a weak acid or salt thereof, e.g. citric acid or citrate) in the affinity eluate may be optimized for the virus particles to be separated. For example, the affinity eluate may comprise an anionic compound (e.g., an acid or salt thereof, e.g., citric acid or citrate) at a concentration of at least 0.5mM, 1mM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, 4mM, 4.5mM, 5mM, 5.5mM, 6mM, 6.5mM, 7mM, 8mM, 8.5mM, 9mM, 9.5mM, 10mM, or more. For example, the affinity eluate comprises an anionic compound (e.g., a weak acid or salt thereof, e.g., citric acid or citrate) at a concentration of at least about 3mM, 5mM, 7mM, or more.

In some embodiments of any of the aspects, the affinity eluate comprises an anionic compound (e.g., a weak acid or salt thereof, e.g., citric acid or citrate) at a concentration of from about 0.5mM to about 15mM, from about 1mM to about 10mM, from about 1.5mM to about 7.5mM, from about 2mM to about 7mM, from about 1.5mM to about 9mM, from about 2mM to about 8mM, or from about 2.5mM to about 7.5mM.

In some embodiments of any of the aspects, the affinity eluate comprises an anionic compound (e.g., a weak acid or salt thereof, e.g., citric acid or citrate) at a concentration of about 0.5mM, about 1mM, about 1.5mM, about 2mM, about 2.5mM, about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 8mM, about 8.5mM, about 9mM, about 9.5mM, or about 10mM.

In some embodiments of any of the aspects, the affinity eluate may be diluted prior to anion exchange chromatography. For example, a buffer (e.g., a dilution buffer) may be added to the affinity eluate. Notably, the affinity eluate may be diluted by a factor of 1 x to 40 x. For example, the affinity eluate may be diluted by a factor of at least 1×, 1.5×, 2×, 2.5×, 3×, 3.5×, 4×, 4.5×, 5×, 5.5×, 6×, 6.5×, 7×, 7.5×, 8×, 8.5×, 9×, 9.5×, 10×, 10.5×, 11×, 11.5×, 12×, 12.5×, 13×, 13.5×, 14×, 14.5×, 15×, 15.5×, 16×, 16.5×, 17×, 17.5×, 18×, 18.5×, 19×, 19.5×, 20×, or more. Typically, the affinity eluate may be diluted by a factor of 2 x to 6 x, 7 x to 10 x, 11 x to 15 x, or 6 x to 20 x. For example, the affinity eluate may be diluted by a factor of about 1.5 x to about 5 x, about 5 x to about 10 x, about 10 x to about 15 x, or about 15 x to about 20 x. In some embodiments, the affinity eluate may be diluted with a dilution buffer (e.g., an anion exchange dilution buffer) by a factor of about 4.5 x to about 5.5 x. As used herein, the term "anion exchange dilution buffer" refers to a buffer used to dilute an affinity eluate for use as a feed composition for anion exchange chromatography.

In some embodiments, the affinity eluate may be diluted with an affinity eluate buffer and an anion exchange dilution buffer. For example, the affinity eluate may be diluted with an affinity elution buffer (e.g., an affinity buffer for the affinity eluate) by a factor of about 1 x to about 6 x, and then further diluted with an anion exchange buffer by a factor of about 1 x to about 10 x. In some embodiments, the affinity eluate may be diluted with an affinity elution buffer by a factor of about 1×, about 1.5×, about 2×, about 2.5×, about 3×, about 3.5×, about 4×, about 4.5×, about 5×, about 5.5×, or about 6×, and the diluted affinity eluate may be further diluted with an anion exchange dilution buffer. In some embodiments, the affinity eluate may be first diluted with an affinity elution buffer, and the diluted affinity eluate may be further diluted with an anion exchange dilution buffer by a factor of about 1×, about 1.5×, about 2×, about 2.5×, about 3×, about 3.5×, about 4×, about 4.5×, about 5×, about 5.5×, about 6×, about 6.5×, about 7×, about 7.5×, about 8×, about 8.5×, about 9×, about 9.5×, or about 10×. For example, the affinity eluate may be first diluted with an affinity elution buffer by a factor of about 1×, about 1.5×, about 2×, about 2.5×, about 3×, about 3.5×, about 4×, about 4.5×, about 5×, about 5.5×, or about 6×, and the diluted affinity eluate may be further diluted with an anion exchange dilution buffer by a factor of about 1×, about 1.5×, about 2×, about 2.5×, about 3×, about 3.5×, about 4×, about 4.5×, about 5×, about 5.5×, about 6×, about 6.5×, about 7×, about 7.5×, about 8×, about 8.5×, about 9×, about 9.5×, or about 10×.

In some embodiments of any of the aspects, the dilution buffer used to dilute the affinity eluate comprises a predetermined amount of an anionic compound. For example, the dilution buffer comprises a predetermined amount of an acid or salt thereof, e.g., a weak acid or salt thereof. In some embodiments of any of the aspects, the dilution buffer comprises a predetermined amount of weak acid. For example, the dilution buffer comprises a predetermined amount of citric acid or a salt thereof, acetic acid or a salt thereof, or succinic acid or a salt thereof. In some embodiments of any of the aspects, the dilution buffer comprises a predetermined amount of citric acid or a salt thereof, i.e., citrate.

The amount of anionic compound (such as an acid or salt thereof, e.g., citric acid or citrate) in the dilution buffer may be optimized for the virus particles to be isolated. For example, the dilution buffer may comprise an anionic compound (e.g., an acid or salt thereof, e.g., citric acid or citrate) at a concentration of at least 0.5mM, 1mM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, 4mM, 4.5mM, 5mM, 5.5mM, 6mM, 6.5mM, 7mM, 8mM, 8.5mM, 9mM, 9.5mM, 10mM, or more.

In some embodiments of any aspect, the dilution buffer comprises an anionic compound (e.g., a weak acid or salt thereof, e.g., citric acid or citrate) at a concentration of from about 0.5mM to about 15mM, from about 1mM to about 10mM, from about 1.5mM to about 7.5mM, from about 2mM to about 7mM, from about 1.5mM to about 9mM, from about 2mM to about 8mM, or from about 2.5mM to about 7.5mM.

In some embodiments of any of the aspects, the dilution buffer comprises an anionic compound (e.g., a weak acid or salt thereof, e.g., citric acid or citrate) at a concentration of about 0.5mM, about 1mM, about 1.5mM, about 2mM, about 2.5mM, about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 8mM, about 8.5mM, about 9mM, about 9.5mM, or about 10mM. For example, the dilution buffer comprises an anionic compound, e.g., a weak acid or salt thereof, e.g., citric acid or citrate, at a concentration of about 1.5 mM.

In some embodiments, the amount of anionic compound (e.g., weak acid or salt thereof) in the dilution buffer may be at least 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM, 100mM, or more. For example, the amount of anionic compound (e.g., weak acid or salt thereof) in the dilution buffer may be from about 10mM to about 100mM, from about 20mM to about 90mM, from about 30mM to about 80mM, from about 40mM to about 70mM, or from about 50mM to about 60mM.

The dilution buffer may comprise additional components. For example, the dilution buffer may comprise a predetermined amount of buffer. Exemplary buffers include, but are not limited to, acetate, histidine, phosphate, citrate, and propionate. In some embodiments of any of the aspects, the dilution buffer comprises a predetermined amount of bis-tris propane (BTP).

The amount of buffer (e.g., BTP) in the dilution buffer can be optimized for the virus particles to be separated. For example, the dilution buffer can include a buffer (e.g., BTP) at a concentration of at least about 25mM, 50mM, 75mM, 100mM, 125mM, 150mM, or more. For example, the dilution buffer comprises a buffer (e.g., BTP) at a concentration of from about 25mM to about 175mM, from about 50mM to about 150mM, from about 75mM to about 125mM, from about 80mM to about 120mM, from about 85mM to about 115mM, from about 90mM to about 110mM, or from about 95mM to about 105mM. In some embodiments of any of the aspects, the dilution buffer can comprise a buffer (e.g., BTP) at a concentration of about 50mM, about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, about 125mM, about 150mM, or about 175mM. For example, the dilution buffer may comprise a buffer, e.g., BTP, at a concentration of about 100 mM.

In some embodiments of any of the aspects, the dilution buffer comprises a predetermined amount of amino acids, e.g., natural or unnatural amino acids. For example, the dilution buffer comprises a predetermined amount of histidine, arginine, lysine or asparagine.

The amount of amino acids (e.g., histidine, arginine, lysine, or asparagine) in the dilution buffer can be optimized for the virus particle to be isolated. For example, the dilution buffer can include an amino acid (e.g., histidine, arginine, lysine, or asparagine) at a concentration of at least about 25mM, 50mM, 75mM, 100mM, 125mM, 150mM, or more. In some embodiments, the dilution buffer comprises an amino acid (e.g., histidine, arginine, lysine, or asparagine) at a concentration of from about 25mM to about 175mM, from about 50mM to about 150mM, from about 75mM to about 125mM, from about 80mM to about 120mM, from about 85mM to about 115mM, from about 90mM to about 110mM, or from about 95mM to about 105mM. For example, the dilution buffer may comprise an amino acid (e.g., histidine, arginine, lysine, or asparagine) at a concentration of about 50mM, about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, about 125mM, about 150mM, or about 175mM. In some embodiments of any of the aspects, the dilution buffer comprises an amino acid, e.g., histidine, arginine, lysine, or asparagine, at a concentration of about 100 mM.

The dilution buffer may comprise a predetermined amount of glycerol. Thus, in some embodiments of any aspect, the dilution buffer comprises glycerol at a concentration of at least about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% (w/v, w/w or v/v) or higher. For example, wherein the dilution buffer comprises glycerol at a concentration of from about 0.5% to about 9.5%, from about 1% to about 9%, from about 2% to about 8.5%, from about 2.5% to about 8%, from about 3% to about 7.5%, from about 3.5% to about 7%, from about 4% to about 6.5%, or from about 4.5% to about 5.5%. In some embodiments of any of the aspects, the dilution buffer comprises glycerol at a concentration of about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, or about 9.5%. For example, the dilution buffer comprises glycerol at a concentration of about 5%.

The dilution buffer may also contain a surfactant. Exemplary nonionic surfactants include, but are not limited to, polysorbates such as polysorbate 20 (TWEEN 20), polysorbate 28, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 81, and polysorbate 85; poloxamers, such as poloxamer 188 and poloxamer 407; polyoxyethylene polyoxypropylene ether; or polyethylene glycol (PEG).

In some embodiments of any of the aspects, the surfactant is a nonionic fluorosurfactant. Exemplary nonionic fluorosurfactants include fluorosurfactants comprising polyethylene glycol polymers, polypropylene glycol polymers, and copolymers thereof. Specific exemplary nonionic fluorosurfactants include, but are not limited to, PF68.

The dilution buffer may comprise at least about 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, about 0.9%, 0.95% (w/v, w/w, v/v) or higher amounts of nonionic surfactant. For example, the dilution buffer may comprise a nonionic surfactant at a concentration of from about 0.05% to about 0.95%, from about 0.1% to about 0.9%, from about 0.15% to about 0.85%, from about 0.2% to about 0.8%, from about 0.25% to about 0.75%, from about 0.3% to about 7%, from about 0.35% to about 0.65%, from about 0.4% to about 0.6%, or from about 0.45% to about 0.55%. In some embodiments of any of the aspects, the dilution buffer comprises a nonionic surfactant at a concentration of about 0.05%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, or about 0.95%. For example, the dilution buffer comprises a nonionic surfactant at a concentration of about 0.5%.

The dilution buffer may also contain cations, for example, monovalent or divalent cations. Exemplary monovalent ions for the dilution buffer include, but are not limited to, sodium (Na ⁺ ) Potassium (K) ⁺ ) Alkyl ammonia and ammonium. Exemplary divalent cations for dilution buffers include, but are not limited to, magnesium (Mg ²⁺ ) Calcium (Ca) ²⁺ ) Copper (Cu) ²⁺ ) Cobalt (Co) ²⁺ ) Manganese (Mn) ²⁺ ) Nickel (Ni) ²⁺ ) And zinc (Zn) ²⁺ ). The cations may be added in the form of salts. In some embodiments, the dilution buffer comprises a divalent cation, e.g., mg ²⁺ 。

Notably, the dilution buffer can include cations (e.g., divalent cations, such as Mg ²⁺ ) At a concentration of at least about 0.1mM, 0.25mM, 0.5mM, 0.75mM, 1mM, 1.25mM, 1.5mM, 1.75mM, 2mM or more. In some embodiments, the dilution buffer comprises a cation (e.g., a divalent cation, such as Mg ²⁺ ) The concentration is from about 0.1mM to about 2mM, from about 0.25mM to about 1.75mM, from about 0.5mM to about 1.5mM, or from about 0.75mM to about 1.25mM. For example, the dilution buffer comprises cations (e.g., divalent cations, such as Mg ²⁺ ) The concentration is about 0.1mM, about 0.25mM, about 0.5mM, about 0.75mM, about 1mM, about 1.25mM, about 1.5mM, about 1.75mM, or about 2mM. In some embodiments, the dilution buffer comprises a cation, e.g., a divalent cation, such as Mg, at a concentration of about 1mM ²⁺ 。

In some embodiments of any of the aspects, the dilution buffer has a high pH. For example, the pH of the diluted buffer solution is greater than or equal to about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10. In some embodiments, the pH of the dilution buffer is greater than or equal to about 8.5. For example, the pH of the dilution buffer is about 9.

In some embodiments of any of the aspects, the anion exchange dilution buffer comprises: buffers (e.g., selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof)A buffering agent; or a buffer selected from the group consisting of bis-tris propane (BTP), tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); amino acids (e.g., amino acids selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or amino acids selected from the group consisting of aspartate, glutamate, and histidine; or amino acids selected from the group consisting of histidine and lysine; or amino acids selected from the group consisting of cysteine and tyrosine); nonionic surfactants (e.g., nonionic surfactants selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkyl phenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from polyoxyethylene (12) isooctylphenyl ether (e.g., CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol octadecyl ether), seed oil surfactant (e.g., ecosurf (tm) SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylate (e.g., tergitol NP-10 nonylphenol ethoxylate), and any combination thereof, or a nonionic surfactant selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, brij 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, polysorbate 20, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL NP-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, TERGITOL-13, TERGOL 20 and any combination thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-9, TERGITOL SA-15-S-12, TERGITOL NP-7, TERGITOL-9, TERGOL NP-9, TERGOL-7, TERGOL NP-7, and TERGITOL-7, and TERGOL-7, and TERGL-9.5, and TERGL-5, nonionic surfactants of the group consisting of TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof; salts (e.g., salts selected from the group consisting of sodium salts, potassium salts, ammonium salts, magnesium salts, calcium salts, copper salts, cobalt salts, manganese salts, nickel salts, and zinc salts; or salts selected from the group consisting of potassium salts, ammonium salts, magnesium salts, calcium salts, manganese salts, and zinc salts; or salts selected from the group consisting of potassium salts, magnesium salts, and calcium salts; or salts selected from the group consisting of potassium salts and magnesium salts (e.g., mgCl) ₂ ) Salts of the group consisting of; and a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or an alternativeA polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactitol, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof. For example, the anion exchange dilution buffer comprises: about 75-125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 50-250mM of an amino acid (e.g., an amino acid selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or an amino acid selected from the group consisting of aspartate, glutamate, and histidine; or an amino acid selected from the group consisting of histidine and lysine; or an amino acid selected from the group consisting of cysteine and tyrosine); from about 0.3% to about 0.7% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g., jojoba @ >CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g.,s20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338 and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020 and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, GIOL NP-11, GITOL NP-12, TERGITOL NP-13 and any combination thereof); about 0.5 to about 1.5mM of a salt (e.g., selected from the group consisting of sodium, potassium, ammonium, magnesium Salts of the group consisting of salts, calcium salts, copper salts, cobalt salts, manganese salts, nickel salts and zinc salts; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt and zinc salt; or a salt selected from the group consisting of potassium, magnesium and calcium salts; or from potassium and magnesium salts (e.g. MgCl) ₂ ) Salts of the group consisting of; and about 3% -7% of a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactitol, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof). In some embodiments, the anion exchange dilution buffer comprises: about 75-125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 120-170mM of an amino acid (e.g., an amino acid selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or an amino acid selected from the group consisting of aspartate, glutamate, and histidine; or an amino acid selected from the group consisting of histidine and lysine; or an amino acid selected from the group consisting of cysteine and tyrosine); from about 0.3% to about 0.7% of a nonionic surfactant (e.g., selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkyl phenol ethoxylates (preferably polysorbates) Alcohol esters, polyoxyethylene alkylphenyl ethers), and any combination thereof, or a nonionic surfactant selected from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g., +.>CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surface selected from the group consisting of Pluronic 10R5 and PF68 A non-ionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338 and any combinations thereof, or a non-ionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020 and any combinations thereof, or a non-ionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL-12, TERGITOL NP-13 and any combinations thereof; about 0.5 mM to about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt, and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt, and zinc salt; or a salt selected from the group consisting of potassium salt, magnesium salt, and calcium salt; or a salt selected from the group consisting of potassium salt and magnesium salt (e.g., mgCl) ₂ ) Salts of the group consisting of; and about 2.5% -7.5% of a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof). For example, the anion exchange dilution buffer comprises: about 75-125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof; or a buffer selected from the group consisting of BTP, tris, and any combination thereof) A buffer of (a) in a buffer medium; about 120-170mM of an amino acid (e.g., an amino acid selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or an amino acid selected from the group consisting of aspartate, glutamate, and histidine; or an amino acid selected from the group consisting of histidine and lysine; or an amino acid selected from the group consisting of cysteine and tyrosine); from about 0.3% to about 0.7% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g., jojoba @>CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g. +.>S20 polyethylene glycol octadecyl ether), seed oil surfactant (e.g., ecosurf (tm) SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylate (e.g., tergitol NP-10 nonylphenol ethoxylate), and any combination thereof, or a nonionic surfactant selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S2 0. BrijSl0, brij 010, brij Cl0, brij 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL LNP-13, polysorbate 20, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGAL 720, tween 80, and any combination thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10 and the group consisting of Pluronic 10, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combinations thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combinations thereof; about 0.5 mM to about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt, and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt, and zinc salt; or a salt selected from the group consisting of potassium salt, magnesium salt, and calcium salt; or a salt selected from the group consisting of potassium salt and magnesium salt (e.g., mgCl) ₂ ) Salts of the group consisting of; and about 2.5% to about 7.5% of a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactitol, sucrose, lactose, maltose, trehalose, and any combination thereof)Polyols of any combination, or polyols selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, or polyols selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof), and the buffer has a high pH, e.g., a pH of about 8.5-9.5.

In some embodiments, the anion exchange dilution buffer comprises: about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof, or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 120mM, about 125mM, about 130mM, about 135mM, about 140mM, about 145mM, or about 150mM of an amino acid (e.g., an amino acid selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or an amino acid selected from the group consisting of aspartate, glutamate, and histidine; or an amino acid selected from the group consisting of histidine and lysine; or an amino acid selected from the group consisting of cysteine and tyrosine); about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, or about 0.7%; about 0.5mM, about 0.6mM, about 0.7mM, about 0.8mM, about 0.9mM, about 1mM, about 1.1mM, about 1.2mM, about 1.3mM, about 1.4mM, or about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt, and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt, and zinc salt; or a salt selected from the group consisting of potassium salt, magnesium salt, and calcium salt; or a salt selected from the group consisting of potassium salt and magnesium salt (e.g., mgCl) ₂ ) Salts of the group consisting of; about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, or about 7% of a viscosity modifierFor example, a polyol selected from the group consisting of a hydrocarbon, a monosaccharide, a disaccharide, a trisaccharide, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactitol, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof.

In some embodiments, the anion exchange dilution buffer comprises: about 75mM, about 80mM, about 85mM, about 90mM of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenolethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g., CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactant (e.g., ecosurf (tm) SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylate (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereofOr selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGOL 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GITOL-8, TERGL-9, TERGL-NP-10, TERGL-10, and any of the combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combinations thereof, or an ECOSURF-9, EH, nonionic surfactants of the group consisting of ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof; about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 120mM, about 125mM, about 130mM, about 135mM, about 140mM, about 145mM or about 150mM of an amino acid (e.g., an amino acid selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine and tyrosine; or an amino acid selected from the group consisting of aspartate, glutamate and histidine; or an amino acid selected from the group consisting of histidine and lysine; or an amino acid selected from the group consisting of cysteine and tyrosine); about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, or about 0.7% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or a nonionic surfactant selected from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g., polyoxyethylene (12) isooctylphenyl ether>CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +. >S20 polyethylene glycol stearyl ether), seed oil surfactant (e.g., ecosurf (tm) SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylate (e.g., tergitol NP-10 nonylphenol ethoxylate), and any combination thereof, or a nonionic surfactant selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactantA nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, brij 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL LNP-13, polysorbate 20, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer 407, pluronic SA-15, TERGITOL 10, ECURF 15-S-9, TERGITOL 15-S-12, TERGITOL 15-7, TERGITOL NP-9, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL LNP-13, TERGITOL NP-12, TERGOL NP-13, and any combination thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, and combinations thereof, nonionic surfactants of the group consisting of TERGITOL NP-12, TERGITOL NP-13, and any combination thereof; about 0.5mM, about 0.6mM, about 0.7mM, about 0.8mM, about 0.9mM, about 1mM, about 1.1mM, about 1.2mM, about 1.3mM, about 1.4mM, or about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt, and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt, and zinc salt; or a salt selected from the group consisting of potassium salt, magnesium salt, and calcium salt; or a salt selected from the group consisting of potassium salt and magnesium salt (e.g., mgCl) ₂ ) Salts of the group consisting of; and about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, or about 7% of a viscosity modifier (e.g., a polysaccharide selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof)A polyol, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactitol, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof), and the buffer has a high pH (e.g., a pH of about 8.5-9.5, e.g., a pH of about 8.5, about 9, or about 9.5).

In some embodiments of any of the aspects, the anion exchange dilution buffer comprises: a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris) and any combination thereof, or a buffer selected from the group consisting of BTP, tris, borate, tricine and any combination thereof, or a buffer selected from the group consisting of BTP, tris and any combination thereof); a weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); nonionic surfactants (e.g., nonionic surfactants selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkyl phenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from polyoxyethylene (12) isooctylphenyl ether (e.g., CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338 and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020 and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, GIOL NP-11, GITOL NP-12, TERGITOL NP-13 and any combination thereof); salts (e.g. selected from sodium, potassium Salts of the group consisting of salts, ammonium salts, magnesium salts, calcium salts, copper salts, cobalt salts, manganese salts, nickel salts and zinc salts; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt and zinc salt; or a salt selected from the group consisting of potassium, magnesium and calcium salts; or from potassium and magnesium salts (e.g. MgCl) ₂ ) Salts of the group consisting of; and a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof). For example, the anion exchange dilution buffer comprises: about 75-125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 3-12mM of a weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); from about 0.3% to about 0.7% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkyl phenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from polyoxyethylene (12) isooctylphenyl ether (e.g., CA-270 polyoxyethylene%12 Isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g.,80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERG Non-ionic surfactants of the group consisting of ITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof; about 0.5 mM to about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt, and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt, and zinc salt; or a salt selected from the group consisting of potassium salt, magnesium salt, and calcium salt; or a salt selected from the group consisting of potassium salt and magnesium salt (e.g., mgCl) ₂ ) Salts of the group consisting of; and about 2.5% -7.5% of a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof). In some embodiments, the anion exchange dilution buffer comprises: about 75-125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 3-12mM of a weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); from about 0.3% to about 0.7% of a nonionic surfactant (e.g., selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl grape Nonionic surfactants selected from the group consisting of glycosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from polyoxyethylene (12) isooctylphenyl ether (e.g., a->CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or is selected from the group consisting of A nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338 and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020 and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL-12, TERGITOL-13 and any combination thereof; about 0.5 mM to about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt, and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt, and zinc salt; or a salt selected from the group consisting of potassium salt, magnesium salt, and calcium salt; or a salt selected from the group consisting of potassium salt and magnesium salt (e.g., mgCl) ₂ ) Salts of the group consisting of; and about 3% -7% of a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof), and the buffer has a high pH (e.g., a pH of about 8.5-9.5, e.g., a pH of about 8.5, about 9, or about 9.5). / >

In some embodiments, the anion exchange dilution buffer comprises: about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM buffer (e.g., selected from the group consisting of B)A buffering agent of the group consisting of acid salts, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof; about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 7.5mM, about 8mM, about 8.5, about 9mM, about 9.5mM, about 10mM, about 10.5mM, about 11mM, about 11.5mM, or about 12mM weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, or about 0.7% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or a nonionic surfactant selected from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g., CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactant (e.g., ecosurf (tm) SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylate (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or a nonionic surfactant selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brij S0, brij 010, brij Cl0, brij 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGOL L-64, TERGITOL-7, TERGOL-8, GINP-9, GINP-9.5, TERGOL-10, TERGOL-11, TERGOL NP-12, TERGOL NP-13, and combinations thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combination thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, ECOSURF-9, ECOSURF-14, and combinations thereof, nonionic surfactants of the group consisting of TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof; about 0.5mM, about 0.6mM, about 0.7mM, about 0.8mM, about 0.9mM, about 1mM, about 1.1mM, about 1.2mM, about 1.3mM, about 1.4mM, or about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt, and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt, and zinc salt; or a salt selected from the group consisting of potassium salt, magnesium salt, and calcium salt; or a salt selected from the group consisting of potassium salt and magnesium salt (e.g., mgCl) ₂ ) Salts of the group consisting of; about 2.5%, about 3%, about 3.5%, about 4%, about4.5%, about 5%, about 5.5%, about 6%, about 6.5%, or about 7% of a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof).

In some embodiments, the anion exchange dilution buffer comprises: about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof, or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 7.5mM, about 8mM, about 8.5, about 9mM, about 9.5mM, about 10mM, about 10.5mM, about 11mM, about 11.5mM, or about 12mM weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, or about 0.7% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from the group consisting of polyoxyethylene (12) The isooctylphenyl ether (e.g.,CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surface selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combinations thereof An active agent, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL LNP-13, and any combination thereof; about 0.5mM, about 0.6mM, about 0.7mM, about 0.8mM, about 0.9mM, about 1mM, about 1.1mM, about 1.2mM, about 1.3mM, about 1.4mM, or about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt, and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt, and zinc salt; or a salt selected from the group consisting of potassium salt, magnesium salt, and calcium salt; or a salt selected from the group consisting of potassium salt and magnesium salt (e.g., mgCl) ₂ ) Salts of the group consisting of; and about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, or about 7% of a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof), or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactose, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, and the buffer has a high pH (e.g., a pH of about 8.5-9.5, e.g., a pH of about 8.5, or about 9.5).

In some embodiments of any of the aspects, the anion exchange dilution buffer comprises: buffers (e.g. selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxy)A buffer of the group consisting of methyl) aminomethane (tris) and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof; amino acids (e.g., amino acids selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or amino acids selected from the group consisting of aspartate, glutamate, and histidine; or amino acids selected from the group consisting of histidine and lysine; or amino acids selected from the group consisting of cysteine and tyrosine); a weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); nonionic surfactants (e.g., nonionic surfactants selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkyl phenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from polyoxyethylene (12) isooctylphenyl ether (e.g., CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g.,80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactant (e.g., ecosurf SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylate (e.g., tergitol NP-10 nonylphenol ethoxylate), and any combination thereof, or a nonionic surfactant selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic10R5, pluronic F-68 (PF 68),Polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, brij 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP LNP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL-9.5, TERGITOL NP-10, GINP-11, TOL-12, TERGITOL-13, and a combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combination thereof, or a nonionic surfactant selected from the group consisting of Pluronic10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF-9, ECOSURF EH-14, TERGILL 15-S-7, ECOSURF SA-15, GIL 15-S-9, TERGILL 15-S-12 Nonionic surfactants of the group consisting of TERGITOL-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combinations thereof; salts (e.g., salts selected from the group consisting of sodium salts, potassium salts, ammonium salts, magnesium salts, calcium salts, copper salts, cobalt salts, manganese salts, nickel salts, and zinc salts; or salts selected from the group consisting of potassium salts, ammonium salts, magnesium salts, calcium salts, manganese salts, and zinc salts; or salts selected from the group consisting of potassium salts, magnesium salts, and calcium salts; or salts selected from the group consisting of potassium salts and magnesium salts (e.g., mgCl) ₂ ) Salts of the group consisting of; and a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycolA polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactitol, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof. For example, the anion exchange dilution buffer comprises: about 75-125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 75-125mM of an amino acid (e.g., an amino acid selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or an amino acid selected from the group consisting of aspartate, glutamate, and histidine; or an amino acid selected from the group consisting of histidine and lysine; or an amino acid selected from the group consisting of cysteine and tyrosine); about 3-12mM of a weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); from about 0.3% to about 0.7% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g., jojoba @ >CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL LNP-7, TERGITOL NP-8, TERGITOL SA-15-S-7, TERGITOL-12, TERGITOL NP-7, TERGITOL NP-8, TERGITOL-7, TERGITOL NP-7, TERGOL-8, and TERGOL-7, Nonionic surfactants from the group consisting of TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof; about 0.5 mM to about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt, and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt, and zinc salt; or a salt selected from the group consisting of potassium salt, magnesium salt, and calcium salt; or a salt selected from the group consisting of potassium salt and magnesium salt (e.g., mgCl) ₂ ) Salts of the group consisting of; and about 2.5% -7.5% of a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof). In some embodiments, the anion exchange dilution buffer comprises: about 75-125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 75-125mM of an amino acid (e.g., an amino acid selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or an amino acid selected from the group consisting of aspartate, glutamate, and histidine; or an amino acid selected from the group consisting of histidine and lysine; or an amino acid selected from the group consisting of cysteine and tyrosine); About 3-10mM of a weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); from about 0.3% to about 0.7% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g., jojoba @>CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or is selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630 IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, brij 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and Polysorbitol A nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, tergitol NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL P-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof; about 0.5 mM to about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt, and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt, and zinc salt; or a salt selected from the group consisting of potassium salt, magnesium salt, and calcium salt; or a salt selected from the group consisting of potassium salt and magnesium salt (e.g., mgCl) ₂ ) Salts of the group consisting of; and about 3% -7% of a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof), and the buffer has a high pH (e.g., a pH of about 8.5-9.5, e.g., aboutpH of 8.5, about 9, or about 9.5).

In some embodiments, the anion exchange dilution buffer comprises: about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof, or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM of amino acids (e.g., amino acids selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or amino acids selected from the group consisting of aspartate, glutamate, and histidine; or amino acids selected from the group consisting of histidine and lysine; or amino acids selected from the group consisting of cysteine and tyrosine); about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 7.5mM, about 8mM, about 8.5, about 9mM, about 9.5mM, about 10mM, about 10.5mM, about 11mM, about 11.5mM, or about 12mM weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, or about 0.7% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or a nonionic surfactant selected from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g., CA-270 polyoxyethylene (12) isooctylphenyl etherPolyoxyethylene sorbitan monooleate (e.g.)>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOS Nonionic surfactants of the group consisting of URF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof; about 0.5mM, about 0.6mM, about 0.7mM, about 0.8mM, about 0.9mM, about 1mM, about 1.1mM, about 1.2mM, about 1.3mM, about 1.4mM, or about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt, and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt, and zinc salt; or a salt selected from the group consisting of potassium salt, magnesium salt, and calcium salt; or a salt selected from the group consisting of potassium salt and magnesium salt (e.g., mgCl) ₂ ) Salts of the group consisting of; and about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, or about 7% of a viscosity modifier (e.g., a polyol selected from the group consisting of a hydrocarbon, a monosaccharide, a disaccharide, a trisaccharide, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactose, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof).

In some embodiments, the anion exchange dilution buffer comprises: about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof, or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof)A buffer of the combined group); about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM of amino acids (e.g., amino acids selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or amino acids selected from the group consisting of aspartate, glutamate, and histidine; or amino acids selected from the group consisting of histidine and lysine; or amino acids selected from the group consisting of cysteine and tyrosine); about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 7.5mM, about 8mM, about 8.5, about 9mM, about 9.5mM, about 10mM, about 10.5mM, about 11mM, about 11.5mM, or about 12mM weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, or about 0.7% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or a nonionic surfactant selected from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g., CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactant (e.g., ecosurf (tm) SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene)A nonylphenol ethoxylate (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL P-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof; about 0.5mM, about 0.6mM, about 0.7mM, about 0.8mM, about 0.9mM, about 1mM, about 1.1mM, about 1.2mM, about 1.3mM, about 1.4mM, or about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt, and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt, and zinc salt) Salts of the group consisting of; or a salt selected from the group consisting of potassium, magnesium and calcium salts; or from potassium and magnesium salts (e.g. MgCl) ₂ ) Salts of the group consisting of; and about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, or about 7% of a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof), or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactose, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, and the buffer has a high pH (e.g., a pH of about 8.5-9.5, e.g., a pH of about 8.5, or about 9.5).

In some embodiments, the anion exchange dilution buffer comprises: about 100mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof; or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 100mM of amino acids (e.g., an amino acid selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or an amino acid selected from the group consisting of aspartate, glutamate, and histidine; or an amino acid selected from the group consisting of histidine and lysine; or an amino acid selected from the group consisting of cysteine and tyrosine); about 6-8mM weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); about 1mM salt (e.g., selected from sodium, potassium, ammonium Salts of the group consisting of salts, magnesium salts, calcium salts, copper salts, cobalt salts, manganese salts, nickel salts and zinc salts; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt and zinc salt; or a salt selected from the group consisting of potassium, magnesium and calcium salts; or from potassium and magnesium salts (e.g. MgCl) ₂ ) Salts of the group consisting of; about 0.5% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenolethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g.,CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol octadecyl ether), seed oil surfactant (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylate (e.g., TERGITOL NP-10 nonylphenol ethoxylate), or a nonionic surfactant selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij Cl0, brij j 020, osur F9, ecl 7-15, tos 7-15, and ECTeh-15 RGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL SA-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL LNP-13, polysorbate 20, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combination thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combinations thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combinations thereof; and about 5% glycerol. For example, the anion exchange dilution buffer comprises: about 100mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof; or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 100mM of amino acids (e.g., an amino acid selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or an amino acid selected from the group consisting of aspartate, glutamate, and histidine; or an amino acid selected from the group consisting of histidine and lysine; or an amino acid selected from the group consisting of cysteine and tyrosine); about 6mM, about 6.5mM, about 7mM, about 7.5mM or about 8mM of a weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid) Or a salt thereof, or succinic acid or a salt thereof); about 1mM of a salt (e.g., a salt selected from the group consisting of sodium, potassium, ammonium, magnesium, calcium, copper, cobalt, manganese, nickel, and zinc salts; or a salt selected from the group consisting of potassium, ammonium, magnesium, calcium, manganese, and zinc salts; or a salt selected from the group consisting of potassium, magnesium, and calcium salts; or a salt selected from the group consisting of potassium and magnesium salts (e.g., mgCl) ₂ ) Salts of the group consisting of; about 0.5% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from polyoxyethylene (12) isooctylphenyl ether (e.g., polyoxyethylene (12)>CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol octadecyl ether), seed oil surfactant (e.g., ecosurf (tm) SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylate (e.g., tergitol NP-10 nonylphenol ethoxylate), and any combination thereof, or a nonionic surfactant selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, bri j 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL LNP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL LNP-13, polysorbate 20, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, EPAL 720, tween 80, and any combination thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and Pluronic 68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combinations thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combinations thereof; and about 5% of a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, and any combination thereof), and the buffer has a high pH (e.g., a pH of about 8.5-9.5, e.g., a pH of about 8.5, about 9, or about 9.5).

In any aspectIn some embodiments, the anion exchange dilution buffer comprises: a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris) and any combination thereof, or a buffer selected from the group consisting of BTP, tris, borate, tricine and any combination thereof, or a buffer selected from the group consisting of BTP, tris and any combination thereof); amino acids (e.g., amino acids selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or amino acids selected from the group consisting of aspartate, glutamate, and histidine; or amino acids selected from the group consisting of histidine and lysine; or amino acids selected from the group consisting of cysteine and tyrosine); a weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); nonionic surfactants (e.g., nonionic surfactants selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkyl phenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from polyoxyethylene (12) isooctylphenyl ether (e.g., CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g.,80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ecosurf (tm) SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergito)lTM NP-10 nonylphenol ethoxylate), and any combination thereof, or a nonionic surfactant selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brij S0, brij 010, brij Cl0, brij 020, ECOSURF-9, ECOSURF EH-14, TERGL 15-S-7, ECOSURF SA-15, TERGL 15-S-9, TERGL 15-S-12, TERGL-7, TERGL-TERGL 7, TERGL-10, TERGL-NP-10, GINP-10, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL P-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof; salts (e.g., salts selected from the group consisting of sodium salts, potassium salts, ammonium salts, magnesium salts, calcium salts, copper salts, cobalt salts, manganese salts, nickel salts, and zinc salts; or salts selected from the group consisting of potassium salts, ammonium salts, magnesium salts, calcium salts, manganese salts, and zinc salts; or salts selected from the group consisting of potassium salts, magnesium salts, and calcium salts; or salts selected from the group consisting of potassium salts and magnesium salts (e.g., mgCl) ₂ ) Salts of the group consisting of; and viscosity adjustmentA bulking agent (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof). In some embodiments of any of the aspects, the anion exchange dilution buffer comprises: about 75-125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 75-125mM of an amino acid (e.g., an amino acid selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or an amino acid selected from the group consisting of aspartate, glutamate, and histidine; or an amino acid selected from the group consisting of histidine and lysine; or an amino acid selected from the group consisting of cysteine and tyrosine); about 1-10mM of a weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); from about 0.005% to about 0.015% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenolethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from polyoxyethylene (12) isooctylphenyl ether (e.g., CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g.,80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, and any combinations thereof A nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof; about 0.5 mM to about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt, and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt, and zinc salt; or a salt selected from the group consisting of potassium salt, magnesium salt, and calcium salt; or a salt selected from the group consisting of potassium salt and magnesium salt (e.g., mgCl) ₂ ) Salts of the group consisting of; and about 2.5% -7.5% of a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof). For example, the anion exchange dilution buffer comprises: about 75-125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 75-125mM amino acid (e.g., an amino acid selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or selected from the group consisting of aspartyl amine) Amino acids of the group consisting of acid salts, glutamic acid salts and histidine; or an amino acid selected from the group consisting of histidine and lysine; or an amino acid selected from the group consisting of cysteine and tyrosine); about 1-10mM of a weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); from about 0.005% to about 0.015% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenolethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from polyoxyethylene (12) isooctylphenyl ether (e.g.,CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g.,80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, brij 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERG ITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL SA-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL LNP-13, polysorbate 20, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combination thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combinations thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combinations thereof; about 0.5 mM to about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt, and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt, and zinc salt; or a salt selected from the group consisting of potassium salt, magnesium salt, and calcium salt; or a salt selected from the group consisting of potassium salt and magnesium salt (e.g., mgCl) ₂ ) Salts of the group consisting of; and about 2.5% -7.5% of a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof)A polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof), and the buffer has a high pH (e.g., a pH of about 8.5-9.5, e.g., a pH of about 8.5, about 9, or about 9.5).

In some embodiments of any of the aspects described herein, the anion exchange dilution buffer comprises: about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof, or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM of amino acids (e.g., amino acids selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or amino acids selected from the group consisting of aspartate, glutamate, and histidine; or amino acids selected from the group consisting of histidine and lysine; or amino acids selected from the group consisting of cysteine and tyrosine); about 1mM, about 1.5mM, about 2mM, about 2.5mM, 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 7.5mM, about 8mM, about 8.5mM, about 9mM, about 9.5mM, or about 10mM of a weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); about 0.005%, about 0.0075%, about 0.01%, about 0.0125%, or about 0.015% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g., CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combination thereof, or Brij S 20. BrijSl0, brij 010, brij Cl0, brij 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ecoresurf EH-9, ecoresurf EH-14, TERGITOL 15-S-7, ecoresurf SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL p-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof; about 0.5mM, about 0.6mM, about 0.7mM, about 0.8mM, about 0.9mM, about 1mM, about 1.1mM, about 1.2mM, about 1.3mM, about 1.4mM, or about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt, and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt, and zinc salt; or a salt selected from the group consisting of potassium salt, magnesium salt, and calcium salt; or a salt selected from the group consisting of potassium salt and magnesium salt (e.g., mgCl) ₂ ) Salts of the group consisting of; and about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, or about 7% of a viscosity modifier (e.g., a polyol selected from the group consisting of a hydrocarbon, a monosaccharide, a disaccharide, a trisaccharide, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactose, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof).

In some embodiments, the anion exchange dilution buffer comprises: about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM of a buffer (e.g., selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), anda buffer of the group consisting of any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof; about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM of amino acids (e.g., amino acids selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or amino acids selected from the group consisting of aspartate, glutamate, and histidine; or amino acids selected from the group consisting of histidine and lysine; or amino acids selected from the group consisting of cysteine and tyrosine); about 1mM, about 1.5mM, about 2mM, about 2.5mM, 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 7.5mM, about 8mM, about 8.5mM, about 9mM, about 9.5mM, or about 10mM of a weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); about 0.005%, about 0.0075%, about 0.01%, about 0.0125%, or about 0.015% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g., CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol octadecyl ether), seed oil surfaceAn active agent (e.g., ecosulfTM SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), a nonylphenol ethoxylate (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combinations thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combinations thereof; about 0.5mM, about 0.6mM, about 0.7mM, about 0.8mM, about 0.9mM, about 1mM, about 1.1mM, about 1.2mM, about 1.3mM, about 1.4mM, or about 1.5mM of a salt (e.g., selected from the group consisting of sodium, potassium, ammonium, magnesium, calcium, copper, cobalt salts Salts of the group consisting of manganese salts, nickel salts and zinc salts; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt and zinc salt; or a salt selected from the group consisting of potassium, magnesium and calcium salts; or from potassium and magnesium salts (e.g. MgCl) ₂ ) Salts of the group consisting of; and about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, or about 7% of a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof), or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactose, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, and the buffer has a high pH (e.g., a pH of about 8.5-9.5, e.g., a pH of about 8.5, or about 9.5).

In some embodiments, the anion exchange dilution buffer comprises: about 100mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof; or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 100mM of amino acids (e.g., an amino acid selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or an amino acid selected from the group consisting of aspartate, glutamate, and histidine; or an amino acid selected from the group consisting of histidine and lysine; or an amino acid selected from the group consisting of cysteine and tyrosine); about 1-10mM weak acid or salt thereof (e.g., lemonAn acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); about 1mM of a salt (e.g., a salt selected from the group consisting of sodium, potassium, ammonium, magnesium, calcium, copper, cobalt, manganese, nickel, and zinc salts; or a salt selected from the group consisting of potassium, ammonium, magnesium, calcium, manganese, and zinc salts; or a salt selected from the group consisting of potassium, magnesium, and calcium salts; or a salt selected from the group consisting of potassium and magnesium salts (e.g., mgCl) ₂ ) Salts of the group consisting of; about 0.01% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenolethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g.,CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol octadecyl ether), seed oil surfactant (e.g., ecosurf (tm) SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylate (e.g., tergitol NP-10 nonylphenol ethoxylate), and any combination thereof, or a nonionic surfactant selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S2 0. BrijSl0, brij 010, brij Cl0, brij 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL LNP-13, polysorbate 20, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGAL 720, tween 80, and any combination thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10 and the group consisting of Pluronic 10, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combinations thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combinations thereof; and about 5% of a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactitol, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof). For example, the anion exchange dilution buffer comprises: about 100mM buffer (e.g., selected from the group consisting of acetate, histidine, phosphate, citrate Salts, propionates, tricine, borates, tris (hydroxymethyl) aminomethane (tris) and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof; about 100mM of amino acids (e.g., an amino acid selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or an amino acid selected from the group consisting of aspartate, glutamate, and histidine; or an amino acid selected from the group consisting of histidine and lysine; or an amino acid selected from the group consisting of cysteine and tyrosine); about 1mM, about 1.5mM, about 2mM, about 2.5mM, 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 7.5mM, about 8mM, about 8.5mM, about 9mM, about 9.5mM, or about 10mM of a weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); about 1mM of a salt (e.g., a salt selected from the group consisting of sodium, potassium, ammonium, magnesium, calcium, copper, cobalt, manganese, nickel, and zinc salts; or a salt selected from the group consisting of potassium, ammonium, magnesium, calcium, manganese, and zinc salts; or a salt selected from the group consisting of potassium, magnesium, and calcium salts; or a salt selected from the group consisting of potassium and magnesium salts (e.g., mgCl) ₂ ) Salts of the group consisting of; about 0.5% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from polyoxyethylene (12) isooctylphenyl ether (e.g., polyoxyethylene (12)>CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, and any combinations thereof, or is selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-17 12. A nonionic surfactant of the group consisting of TERGITOLNP-13 and any combination thereof); and about 5% of a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, and any combination thereof), and the buffer has a high pH (e.g., a pH of about 8.5-9.5, e.g., a pH of about 8.5, about 9, or about 9.5).

In some embodiments of any of the aspects, the anion exchange dilution buffer comprises: a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris) and any combination thereof, or a buffer selected from the group consisting of BTP, tris, borate, tricine and any combination thereof, or a buffer selected from the group consisting of BTP, tris and any combination thereof); amino acids (e.g., amino acids selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or amino acids selected from the group consisting of aspartate, glutamate, and histidine; or amino acids selected from the group consisting of histidine and lysine; or amino acids selected from the group consisting of cysteine and tyrosine); a weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); nonionic surfactants (e.g. selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers) Class), and any combination thereof, or a nonionic surfactant selected from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g.,CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g.,80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic10R5 and PF68, or a poloxamer P188, Poloxamer P407, poloxamer P338, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combinations thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL P-13, and any combinations thereof; and salts (e.g., salts selected from the group consisting of sodium salts, potassium salts, ammonium salts, magnesium salts, calcium salts, copper salts, cobalt salts, manganese salts, nickel salts, and zinc salts; or salts selected from the group consisting of potassium salts, ammonium salts, magnesium salts, calcium salts, manganese salts, and zinc salts; or salts selected from the group consisting of potassium salts, magnesium salts, and calcium salts; or salts selected from the group consisting of potassium salts and magnesium salts (e.g., mgCl) ₂ ) Salts of the group consisting of), and optionally the buffer is substantially free of glycine. For example, the anion exchange dilution buffer comprises: about 75-125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 75-125mM of an amino acid (e.g., an amino acid selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or an amino acid selected from the group consisting of aspartate, glutamate, and histidine; or an amino acid selected from the group consisting of histidine and lysine; or an amino acid selected from the group consisting of cysteine and tyrosine); about 3-12mM citric acid; from about 0.3% to about 0.7% of a nonionic surfactant (e.g., selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkanes) Phenyl ethers), and any combinations thereof, or a nonionic surfactant selected from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g.,CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g.,80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or from the group consisting of Porro A nonionic surfactant selected from the group consisting of Brij S20, brij Sl0, brij 010, brij Cl0, brij 020, and any combinations thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL-13, and any combinations thereof; and about 0.5 to about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium, potassium, ammonium, magnesium, calcium, copper, cobalt, manganese, nickel, and zinc salts; or a salt selected from the group consisting of potassium, ammonium, magnesium, calcium, manganese, and zinc salts; or a salt selected from the group consisting of potassium, magnesium, and calcium salts; or a salt selected from the group consisting of potassium and magnesium salts (e.g., mgCl) ₂ ) Salts of the group consisting of), and optionally the buffer is substantially free of glycine. In some embodiments, the anion exchange dilution buffer comprises: about 75-125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 75-125mM of an amino acid (e.g., an amino acid selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or an amino acid selected from the group consisting of aspartate, glutamate, and histidine; or an amino acid selected from the group consisting of histidine and lysine; or an amino acid selected from the group consisting of cysteine and tyrosine); about 3-10mM of a weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); from about 0.3% to about 0.7% of a nonionic surfactant (e.g., selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, poly (ethylene oxide) The nonionic surfactant selected from the group consisting of ethylene oxide-polyoxypropylene block copolymers, alkyl glucosides, alkyl phenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from polyoxyethylene (12) isooctylphenyl ether (e.g.,CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g.,80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or selected from the group consisting of poloxamer P188, poloxamer P407, pluronic10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combination thereof A nonionic surfactant, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL P-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, NP-11, TERGITOL-12, TERGITOL-13, and any combination thereof; and about 0.5 to about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium, potassium, ammonium, magnesium, calcium, copper, cobalt, manganese, nickel, and zinc salts; or a salt selected from the group consisting of potassium, ammonium, magnesium, calcium, manganese, and zinc salts; or a salt selected from the group consisting of potassium, magnesium, and calcium salts; or a salt selected from the group consisting of potassium and magnesium salts (e.g., mgCl) ₂ ) Salts of the group consisting), and the buffer has a high pH (e.g., a pH of about 8.5-9.5, e.g., a pH of about 8.5, about 9, or about 9.5), and optionally the buffer is substantially free of glycine.

In some embodiments, the anion exchange dilution buffer comprises: about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof, or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM of amino acids (e.g., selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine)Amino acids; or an amino acid selected from the group consisting of aspartate, glutamate and histidine; or an amino acid selected from the group consisting of histidine and lysine; or an amino acid selected from the group consisting of cysteine and tyrosine); about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 7.5mM, about 8mM, about 8.5mM, about 9mM, about 9.5mM, about 10mM, about 10.5mM, about 11mM, about 11.5mM, or about 12mM weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, or about 0.7% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or a nonionic surfactant selected from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g., CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g.,s20 polyethylene glycol stearyl ether), seed oil surfactant (e.g., ecosurf SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylate (e.g., tergitol NP-10 nonylphenol ethoxylate), and any combination thereof, or a nonionic surfactant selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl etherEthers, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, brij 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, polysorbate 20, and a combination of any of them, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combination thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF-9, ECOSURF EH-14, TERGL 15-S-7, ECOSURF SA-15, GIL 15-S-9, TERGL 15-S-12, TERGL 64, GIRGL-TOL NP-TOL 8, GITOL-TOL 9, GITOL-NPN-TOL 7, GITOL-TOL 4, GITOL-TOL 8 Nonionic surfactants from the group consisting of TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof; and about 0.5mM, about 0.6mM, about 0.7mM, about 0.8mM, about 0.9mM, about 1mM, about 1.1mM, about 1.2mM, about 1.3mM, about 1.4mM, or about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt, and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt, and zinc salt; or a salt selected from the group consisting of potassium salt, magnesium salt, and calcium salt; or a salt selected from the group consisting of potassium salt and magnesium salt (e.g., mgCl) ₂ ) Salts of the group consisting of), and optionally the buffer is substantially free of glycine.

In some embodiments, the anion exchange dilutesThe buffer comprises: about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof, or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof, or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM of amino acids (e.g., amino acids selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or amino acids selected from the group consisting of aspartate, glutamate, and histidine; or amino acids selected from the group consisting of histidine and lysine; or amino acids selected from the group consisting of cysteine and tyrosine); about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 7.5mM, about 8mM, about 8.5mM, about 9mM, about 9.5mM, about 10mM, about 10.5mM, about 11mM, about 11.5mM, or about 12mM weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, or about 0.7% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or a nonionic surfactant selected from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g., CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g.,s20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOSURF 15-S-9, TERGOL 15-S-12, TERGL-64, GINP-7, GINP-8, TERGL-9, TERGL-5, TERGL-NP-10, GINP-12, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TE TERGITOL NP-9, and any combination thereof Nonionic surfactants of the group consisting of RGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof; and about 0.5mM, about 0.6mM, about 0.7mM, about 0.8mM, about 0.9mM, about 1mM, about 1.1mM, about 1.2mM, about 1.3mM, about 1.4mM, or about 1.5mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt, and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt, and zinc salt; or a salt selected from the group consisting of potassium salt, magnesium salt, and calcium salt; or a salt selected from the group consisting of potassium salt and magnesium salt (e.g., mgCl) ₂ ) Salts of the group consisting), and the buffer has a high pH (e.g., a pH of about 8.5-9.5, e.g., a pH of about 8.5, about 9, or about 9.5), and optionally the buffer is substantially free of glycine.

In some embodiments, the anion exchange dilution buffer comprises: about 100mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof; or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 100mM of amino acids (e.g., an amino acid selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or an amino acid selected from the group consisting of aspartate, glutamate, and histidine; or an amino acid selected from the group consisting of histidine and lysine; or an amino acid selected from the group consisting of cysteine and tyrosine); about 6-8mM weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); about 1mM of a salt (e.g., a salt selected from the group consisting of sodium, potassium, ammonium, magnesium, calcium, copper, cobalt, manganese, nickel, and zinc salts; or a salt selected from the group consisting of potassium, ammonium, magnesium, calcium, manganese, and zinc salts; or a salt selected from the group consisting of potassium, magnesium, and calcium salts; or a salt selected from the group consisting of potassium and calcium salts) Magnesium salts (e.g. MgCl) ₂ ) Salts of the group consisting of; and about 0.5% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g.,CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, and nonionic surfactants of the group consisting of Brij 010, brij Cl0, brij 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, polysorbate 20, and any combination thereof A nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, tergitol NP-10, IGEPAL CA 720, tween 80, and any combination thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF-9, ECOSURF EH-14, TERGL 15-S-7, ECOSURF SA-15, GIL 15-S-9, TOL 15-TERGL 12, TERGL-64 TERGL-TERGL 7, TOL-TERGL-10, and any combination thereof, and optionally, and no buffer NP-10, TONP-10, and TONP-10, and no buffer. For example, the anion exchange dilution buffer comprises: about 100mM of a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris), and any combination thereof; or a buffer selected from the group consisting of BTP, tris, borate, tricine, and any combination thereof; or a buffer selected from the group consisting of BTP, tris, and any combination thereof); about 100mM of amino acids (e.g., an amino acid selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or an amino acid selected from the group consisting of aspartate, glutamate, and histidine; or an amino acid selected from the group consisting of histidine and lysine; or an amino acid selected from the group consisting of cysteine and tyrosine); about 6mM, about 6.5mM, about 7mM, about 7.5mM, or about 8mM of a weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); about 1mM of a salt (e.g., a salt selected from the group consisting of sodium, potassium, ammonium, magnesium, calcium, copper, cobalt, manganese, nickel, and zinc salts; or a salt selected from the group consisting of potassium, ammonium, magnesium salts, Salts of the group consisting of calcium salts, manganese salts and zinc salts; or a salt selected from the group consisting of potassium, magnesium and calcium salts; or from potassium and magnesium salts (e.g. MgCl) ₂ ) Salts of the group consisting of; and about 0.5% of a nonionic surfactant (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g., jersey)>CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, brij 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, GIOL 15-S-9, TERGOL 15-S-12, TERGL-64, TONP-7, GINP-8, TERGL-9, TERGL-NP-5, TERGET-TERGL-5, TERGL-NP-TOTERGL-5, and the like OL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, polysorbate 20, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combination thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL P-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL P-13, and any combination thereof, and the buffer has a high pH (e.g., a pH of about 8.5-9.5, e.g., a pH of about 8.5, about 9, or about 9.5), and optionally the buffer is substantially free of glycine.

In some embodiments, the anion exchange dilution buffer comprises: about 100mM BTP, about 100mM histidine, about 6-8mM citric acid or salt thereof, about 1mM MgCl ₂ And about 0.5% PF68, and optionally the buffer is substantially free of glycine. For example, the anion exchange dilution buffer comprises: about 100mM BTP; about 100mM histidine; about 6mM, about 6.5mM, about 7mM, about 7.5mM, or about 8mM citric acid or a salt thereof; about 1mM MgCl ₂ The method comprises the steps of carrying out a first treatment on the surface of the And about 0.5% PF68, and the buffer has a high pH (e.g., a pH of about 8.5-9.5, e.g., a pH of about 8.5, about 9, or about 9.5), and optionally the buffer is substantially free of glycine.

In some embodiments of any of the aspects, the dilution buffer comprises: BTP, histidine, glycerol, PF68, mgCl ₂ And has a high pH. For example, the dilution buffer comprises: about 75-125mM BTP, about75-125mM histidine, about 2.5% -7.5% glycerol, about 0.3% -0.7% PF68, about 0.5-1.5mM MgCl ₂ And has a pH of about 8.5-9.5.

In some embodiments, the dilution buffer comprises: about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM BTP; about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM histidine; about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, or about 0.7% PF68; about 0.5mM, about 0.6mM, about 0.7mM, about 0.8mM, about 0.9mM, about 1mM, about 1.1mM, about 1.2mM, about 1.3mM, about 1.4mM or about 1.5mM MgCl ₂ The method comprises the steps of carrying out a first treatment on the surface of the And about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, or about 7% glycerol.

In some embodiments, the dilution buffer comprises: about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM BTP; about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM histidine; about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, or about 0.7% PF68; about 0.5mM, about 0.6mM, about 0.7mM, about 0.8mM, about 0.9mM, about 1mM, about 1.1mM, about 1.2mM, about 1.3mM, about 1.4mM or about 1.5mM MgCl ₂ The method comprises the steps of carrying out a first treatment on the surface of the And about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, or about 7% glycerol, and the buffer has a high pH, e.g., a pH of about 8.5-9.5, e.g., a pH of about 8.5, about 9, or about 9.5.

In some embodiments of any of the aspects, the dilution buffer comprises: BTP, histidine, PF68, mgCl ₂ And has a high pH. For example, the diluted buffer solution comprises: about 75-125mM BTP, about 75-125mM histidine, about 0.3% -0.7% PF68, about 0.5-1.5mM MgCl ₂ And has a pH of about 8.5-9.5.

In some embodiments, the dilution buffer comprises: about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM BTP; about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM histidine; and about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, or about 0.7% PF68; about 0.5mM, about 0.6mM, about 0.7mM, about 0.8mM, about 0.9mM, about 1mM, about 1.1mM, about 1.2mM, about 1.3mM, about 1.4mM or about 1.5mM MgCl ₂ 。

In some embodiments, the dilution buffer comprises: about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM BTP; about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, or about 125mM histidine; about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, or about 0.7% PF68; about 0.5mM, about 0.6mM, about 0.7mM, about 0.8mM, about 0.9mM, about 1mM, about 1.1mM, about 1.2mM, about 1.3mM, about 1.4mM or about 1.5mM MgCl ₂ And the buffer has a high pH, e.g., a pH of about 8.5-9.5, e.g., a pH of about 8.5, about 9, or about 9.5.

In some embodiments, the anion exchange dilution buffer comprises citric acid or citrate at a concentration of from about 0.5mM to about 15mM, and less than 10% of the empty AAV particles are bound to the anion exchange column. For example, the anion exchange dilution buffer comprises citric acid or citrate at a concentration of from about 1mM to about 10mM, from about 1.5mM to about 7.5mM, from about 2mM to about 7mM, from about 1.5mM to about 9mM, from about 2mM to about 8mM, or from about 2.5mM to about 7.5mM (e.g., the dilution buffer comprises citric acid or citrate at a concentration of about 0.5mM, about 1mM, about 1.5mM, about 2mM, about 2.5mM, about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 8mM, about 8.5mM, about 9mM, about 9.5mM, or about 10 mM), and less than 8%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, less than 0.05%, or even less than 0.02% (e.g., no substantial amount of AAV) bound to an empty column.

In some embodiments, the anion exchange dilution buffer comprises citric acid or citrate at a concentration of from about 0.5mM to about 15mM, and less than 30% of the AAV viral particles in the eluate from anion exchange are empty viral particles. For example, the anion exchange dilution buffer comprises citric acid or citrate at a concentration of from about 1mM to about 10mM, from about 1.5mM to about 7.5mM, from about 2mM to about 7mM, from about 1.5mM to about 9mM, from about 2mM to about 8mM, or from about 2.5mM to about 7.5mM (e.g., the dilution buffer comprises citric acid or citrate at a concentration of about 0.5mM, about 1mM, about 1.5mM, about 2mM, about 2.5mM, about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 8mM, about 8.5mM, about 9mM, about 9.5mM, or about 10 mM), and in the eluate from anion exchange, less than 20%, less than 19.5%, less than 19%, less than 18.5%, less than 18%, less than 17.5%, less than 17%, less than 16.5%, less than 16%, less than 15.5%, less than 15%, less than 14.5%, less than 14%, less than 13.5%, less than 13%, less than 12.5%, less than 12%, less than 11.5%, less than 11%, less than 10.5%, less than 10%, less than 9.5%, less than 9%, less than 8.5%, less than 8%, less than 7.5%, less than 7%, less than 6.5%, less than 6%, less than 5.5%, less than 5%, less than 4.5%, less than 4%, less than 3.5%, less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than 1.75%, less than 0.5%, less than 0.25%, less than 0.2%, less than 0.15%, less than 0.1%, less than 0.05%, less than 0.04%, or less than 0.02% (e.g., substantially no) of the AAV particles are empty viral particles.

In some embodiments, the anion exchange dilution buffer comprises citric acid or citrate at a concentration from about 0.5mM to about 15mM and the amount of empty viral particles in the eluate from anion exchange is reduced 86-fold or more as compared to the amount of empty viral particles in the affinity eluate. For example, the anion exchange dilution buffer comprises citric acid or citrate at a concentration of from about 1mM to about 10mM, from about 1.5mM to about 7.5mM, from about 2mM to about 7mM, from about 1.5mM to about 9mM, from about 2mM to about 8mM, or from about 2.5mM to about 7.5mM (e.g., the dilution buffer comprises citric acid or citrate at a concentration of about 0.5mM, about 1mM, about 1.5mM, about 2mM, about 2.5mM, about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 8.5mM, about 9mM, about 9.5mM, or about 10 mM) and the amount of hollow viral particles in the eluate from anion exchange is reduced by a factor of 90 or more, 95 or more, 99 or more times the amount of hollow viral particles in the affinity eluate.

In some embodiments, the anion exchange dilution buffer comprises citric acid or citrate at a concentration of about 0.5mM to about 15mM, and the ratio of total rAAV viral particles (e.g., intact, incomplete, and empty AAV viral particles) to empty rAAV viral particles in the anion exchange eluate is at least about 1.25× higher than the ratio of total rAAV viral particles to empty particles in the affinity eluate. For example, the anion exchange dilution buffer comprises citric acid or citrate at a concentration of from about 1mM to about 10mM, from about 1.5mM to about 7.5mM, from about 2mM to about 7mM, from about 1.5mM to about 9mM, from about 2mM to about 8mM, or from about 2.5mM to about 7.5mM (e.g., the dilution buffer comprises citric acid or citrate at a concentration of about 0.5mM, about 1mM, about 1.5mM, about 2mM, about 2.5mM, about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 8mM, about 8.5mM, about 9mM, about 9.5mM, or about 10 mM), and the ratio of total rAAV viral particles to empty rAAV viral particles in the anion exchange eluate is at least about 1.5×, at least about 1.6×, at least about 1.7×, at least about 1.8×, at least about 1.9×, at least about 2×, at least about 2.1×, at least about 2.2×, at least about 2.3×, at least about 2.4×, at least about 2.5×, at least about 2.6×, at least about 2.7×, at least about 2.8×, at least about 2.9×, at least about 3×, at least about 3.1×, at least about 3.2×, at least about 3.3×, at least about 3.4×, at least about 3.5×, at least about 3.6× at least about 3.7×, at least about 3.8×, at least about 3.9×, at least about 4×, at least about 4.1×, at least about 4.2×, at least about 4.3×, at least about 4.4×, at least about 4.5×, at least about 4.6×, at least about 4.7×, at least about 4.8×, at least about 4.9×, at least about 5×, at least about 5.1×, at least about 5.2×, at least about 5.3×, at least about 5.4×, at least about 5.5×, at least about 5.6×, at least about 5.7×, at least about 5.8×, at least about 5.9×, at least about 6×, at least about 6.1×, at least about 6.2×, at least about 6.3×, at least about 6.4×, at least about 6.5×, at least about 6.6.6.6.6×, at least about 6.6.6×, at least about 6.7×, at least about 6.8×, at least about 6.9×, at least about 7×, at least about 7.1×, at least about 7.2×, at least about 7.3×, at least about 7.4×, at least about 7.5×, at least about 7.6×, at least about 7.7×, at least about 7.8×, at least about 7.9×, at least about 8×, at least about 8.1×, at least about 8.2×, at least about 8.3×, at least about 8.4×, at least about 8.5×, at least about 8.6×, at least about 8.7×, at least about 8.8×, at least about 8.9×, at least about 9.9×, at least about 9.3×, at least about 9.4×, at least about 9.5×, at least about 9.6×, at least about 9.7, at least about 9.9, or at least about 9.10×.

In some embodiments, the anion exchange dilution buffer comprises citric acid or citrate at a concentration of from about 0.5mM to about 15mM, and the ratio of intact and incompletely intact rAAV particles to empty rAAV viral particles in the anion exchange eluate is at least about 1.25× higher than the ratio of total rAAV viral particles to empty rAAV particles in the affinity eluate. For example, the anion exchange dilution buffer comprises citric acid or citrate at a concentration of from about 1mM to about 10mM, from about 1.5mM to about 7.5mM, from about 2mM to about 7mM, from about 1.5mM to about 9mM, from about 2mM to about 8mM, or from about 2.5mM to about 7.5mM (e.g., the dilution buffer comprises citric acid or citrate at a concentration of about 0.5mM, about 1mM, about 1.5mM, about 2mM, about 2.5mM, about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 8mM, about 8.5mM, about 9mM, about 9.5mM, or about 10 mM), and the ratio of intact and incomplete rAAV particles to intact and incomplete rAAV particles in the null rAAV particles is at least about 1.5×, at least about 1.6×, at least about 1.7×, at least about 1.8×, at least about 2.1×, at least about 2.2×, at least about 2.3×, at least about 2.5×, at least about 2.6×, at least about 2.7×, at least about 2.2.8×, at least about 3.3×, at least about 3.3.2×, at least about 3.3.3×, at least about 3.3.5×, at least about 3.5×, at least about 3.6.5×, at least about 3.6.4×, at least about 4.5×, at least about 4.6×, at least about 4.5×, at least about 3.6× At least about 6.4×, at least about 6.5×, at least about 6.6×, at least about 6.7×, at least about 6.8×, at least about 6.9×, at least about 7×, at least about 7.1×, at least about 7.2×, at least about 7.3×, at least about 7.4×, at least about 7.5×, at least about 7.6×, at least about 7.7×, at least about 7.8×, at least about 7.9×, at least about 8×, at least about 8.1×, at least about 8.2×, at least about 8.3×, at least about 8.4×, at least about 8.5×, at least about 8.6×, at least about 8.7×, at least about 8.8×, at least about 8.9×, at least about 9.1×, at least about 9.2×, at least about 9.3×, at least about 9.4×, at least about 9.9.9×, at least about 9.9, or at least about 9.9.10×.

In some embodiments, the anion exchange dilution buffer comprises citric acid or citrate at a concentration from about 0.5mM to about 15mM, and the UV of the anion exchange eluate ₂₆₀ With UV ₂₈₀ Is at least 1.25 or higher. For example, the anion exchange dilution buffer comprises from about 1mM to about 10mM, from about 1.5mM to about 7.5mM, from about 2mM to about 7mM, from about 1.5mM to about 9mM, from about 2mM to about 8mM, or from about 2.5mM to about 7.5mM of citric acid or citrate (e.g., the dilution buffer)Comprising citric acid or citrate at a concentration of about 0.5mM, about 1mM, about 1.5mM, about 2mM, about 2.5mM, about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 8mM, about 8.5mM, about 9mM, about 9.5mM, or about 10 mM), UV of the anion exchange eluate ₂₆₀ With UV ₂₈₀ At least 1.25, at least 1.26, at least 1.27, at least 1.28, at least 1.29, at least 1.3, at least 1.31, at least 1.32, at least 1.33, at least 1.34, or at least 1.35 or more.

In some embodiments, the anion exchange dilution buffer comprises citric acid or citrate at a concentration from about 0.5mM to about 15mM, and UV in the anion exchange eluate ₂₆₀ With UV ₂₈₀ The ratio of (2) is the UV in the adjusted affinity eluate ₂₆₀ With UV ₂₈₀ At least about 1.15 x or greater. For example, the anion exchange dilution buffer comprises citric acid or citrate at a concentration of from about 1mM to about 10mM, from about 1.5mM to about 7.5mM, from about 2mM to about 7mM, from about 1.5mM to about 9mM, from about 2mM to about 8mM, or from about 2.5mM to about 7.5mM (e.g., the dilution buffer comprises citric acid or citrate at a concentration of about 0.5mM, about 1mM, about 1.5mM, about 2mM, about 2.5mM, about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 8mM, about 8.5mM, about 9mM, about 9.5mM, or about 10 mM), and the UV in the anion exchange eluate ₂₆₀ With UV ₂₈₀ The ratio of (2) is the UV in the adjusted affinity eluate ₂₆₀ With UV ₂₈₀ At least about 1.2×, at least about 1.25×, at least about 1.3× 0, at least about 1.35× 1, at least about 1.4× 2, at least about 1.45× 3, at least about 1.5× 4, at least about 1.55× 5, at least about 1.6× 6, at least about 1.65×, at least about 1.7×, at least about 1.75×, at least about 1.8×, at least about 1.859×, at least about 1.9×, at least about 1.95×, or at least about 2× or higher.

In some embodiments, the anion exchange equilibration buffer comprises a concentration of from about 0.5mM to about 15mM of citric acid or citrate, and less than 10% of empty AAV particles are bound to an anion exchange column. For example, the anion exchange equilibration buffer comprises citric acid or citrate at a concentration from about 1mM to about 10mM, from about 1.5mM to about 7.5mM, from about 2mM to about 7mM, from about 1.5mM to about 9mM, from about 2mM to about 8mM, or from about 2.5mM to about 7.5mM (e.g., the equilibration buffer comprises citric acid or citrate at a concentration of about 0.5mM, about 1mM, about 1.5mM, about 2mM, about 2.5mM, about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 8mM, about 8.5mM, about 9mM, about 9.5mM, or about 10mM, or the equilibration buffer comprises a concentration of up to about 4mM of citric acid or citrate), and less than 8%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, less than 0.1%, less than 0.05%, or less than 0.02% or even less (e.g., substantially none) of the empty AAV particles bind to the anion exchange column.

In some embodiments, the anion exchange equilibration buffer comprises a concentration of from about 0.5mM to about 15mM of citric acid or citrate, and less than 30% of AAV viral particles in the eluate from anion exchange are empty viral particles. For example, the anion exchange equilibration buffer comprises citric acid or citrate at a concentration of from about 1mM to about 10mM, from about 1.5mM to about 7.5mM, from about 2mM to about 7mM, from about 1.5mM to about 9mM, from about 2mM to about 8mM, or from about 2.5mM to about 7.5mM (e.g., the equilibration buffer comprises citric acid or citrate at a concentration of about 0.5mM, about 1mM, about 1.5mM, about 2mM, about 2.5mM, about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 8mM, about 8.5mM, about 9mM, about 9.5mM, or about 10mM, or the equilibration buffer comprises citric acid or citrate at a concentration of up to about 4 mM), and less than 20%, less than 19.5%, less than 19%, less than 18.5%, less than 18%, less than 17.5%, less than 17%, less than 16.5%, less than 16%, less than 15.5%, less than 15%, less than 14.5%, less than 14%, less than 13.5%, less than 13%, less than 12.5%, less than 12%, less than 11.5%, less than 11%, less than 10.5%, less than 10%, less than 9.5%, less than 8.5%, less than 8%, less than 7%, less than 6.5%, less than 6%, less than 5.5%, less than 5%, less than 4.5%, less than 4%, less than 3.5%, less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than 0.75%, less than 0.5%, less than 0.25%, less than 0.2%, less than 0.15%, less than 0.1%, less than 0.05%, less than 0.04%, or less than 0.02% (e.g., substantially none) of the viral particles from the anion exchange eluate are empty viral particles.

In some embodiments, the anion exchange equilibration buffer comprises a concentration of from about 0.5mM to about 15mM of citric acid or citrate and the amount of hollow viral particles from the anion exchange eluate is reduced 86-fold or more compared to the amount of hollow viral particles in the affinity eluate. For example, the anion exchange equilibration buffer comprises citric acid or citrate at a concentration of from about 1mM to about 10mM, from about 1.5mM to about 7.5mM, about 2mM to about 7mM, about 1.5mM to about 9mM, about 2mM to about 8mM, or from about 2.5mM to about 7.5mM (e.g., the equilibration buffer comprises citric acid or citrate at a concentration of about 0.5mM, about 1mM, about 1.5mM, about 2mM, about 2.5mM, about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 8mM, about 8.5mM, about 9mM, about 9.5mM, or about 10 mM), or the equilibration buffer comprises citric acid or citrate at a concentration of up to about 4mM, and the amount of virus particles from the anion exchange is reduced by a factor of about 90 or more, or more than a factor of about 95 or more than the amount of empty particles in the affinity eluate.

In some embodiments, the anion exchange equilibration buffer comprises a concentration of from about 0.5mM to about 15mM of citric acid or citrate, and the ratio of total rAAV viral particles (e.g., intact, incomplete, and empty AAV viral particles) to empty rAAV viral particles in the anion exchange eluate is at least about 1.25× higher than the ratio of total rAAV viral particles to empty particles in the affinity eluate. For example, the anion exchange equilibration buffer comprises citric acid or citrate at a concentration of from about 1mM to about 10mM, from about 1.5mM to about 7.5mM, from about 2mM to about 7mM, from about 1.5mM to about 9mM, from about 2mM to about 8mM, or from about 2.5mM to about 7.5mM (e.g., the equilibration buffer comprises citric acid or citrate at a concentration of about 0.5mM, about 1mM, about 1.5mM, about 2mM, about 2.5mM, about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 8mM, about 8.5mM, about 9mM, about 9.5mM, or about 10mM, or the equilibration buffer comprises citric acid or citrate at a concentration of up to about 4 mM), and the ratio of total rAAV virus particles to empty rAAV virus particles in the anion exchange eluate is at least about 1.5×, at least about 1.6×, at least about 1.7×, at least about 1.8×, at least about 2.1×, at least about 2.2×, at least about 2.3×, at least about 2.5×, at least about 2.6×, at least about 2.7×, at least about 2.8×, at least about 2.3×, at least about 2.2.5×, at least about 2.3×, at least about 3.3.2×, at least about 3.3×, at least about 3.3.6×, at least about 3.3.8×, at least about 3.4, at least about 4.4, at least about 4.5×, at least about 4, at least about 4.5×, at least about 4.6×, at least about 4.5×, at least about 2.6× At least about 6.3×, at least about 6.4×, at least about 6.5×, at least about 6.6×, at least about 6.7×, at least about 6.8×, at least about 6.9×, at least about 7×, at least about 7.1×, at least about 7.2×, at least about 7.3×, at least about 7.4×, at least about 7.5×, at least about 7.6×, at least about 7.7×, at least about 7.8×, at least about 7.9×, at least about 8×, at least about 8.1×, at least about 8.2×, at least about 8.3×, at least about 8.4×, at least about 8.5×, at least about 8.6×, at least about 8.7×, at least about 8.9×, at least about 9.1×, at least about 9.2×, at least about 9.3, at least about 9.9×, at least about 9.9, at least about 9.9.9×, at least about 9.9.10×.

In some embodiments, the anion exchange equilibration buffer comprises a concentration of from about 0.5mM to about 15mM of citric acid or citrate, and the ratio of intact and incompletely intact rAAV particles to empty rAAV viral particles in the anion exchange eluate is at least about 1.25× higher than the ratio of total rAAV viral particles to empty particles in the affinity eluate. For example, the anion exchange equilibration buffer comprises citric acid or citrate at a concentration of from about 1mM to about 10mM, from about 1.5mM to about 7.5mM, from about 2mM to about 7mM, from about 1.5mM to about 9mM, from about 2mM to about 8mM, or from about 2.5mM to about 7.5mM (e.g., the equilibration buffer comprises citric acid or citrate at a concentration of about 0.5mM, about 1mM, about 1.5mM, about 2mM, about 2.5mM, about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 8mM, about 8.5mM, about 9mM, about 9.5mM, or about 10mM, or the equilibration buffer comprises citric acid or citrate at a concentration of up to about 4 mM), and the ratio of intact and incomplete rAAV particles to empty rAAV particles in the anion exchange eluate is at least about 1.5×, at least about 1.6×, at least about 1.7×, at least about 1.8×, at least about 1.9×, at least about 2×, at least about 2.1×, at least about 2.2×, at least about 2.3×, at least about 2.4×, at least about 2.5×, at least about 2.6×, at least about 2.7×, at least about 2.8×, at least about 2.9×, at least about 3×, at least about 3.1× at least about 3.2×, at least about 3.3×, at least about 3.4×, at least about 3.5×, at least about 3.6×, at least about 3.7×, at least about 3.8×, at least about 3.9×, at least about 4×, at least about 4.1×, at least about 4.2×, at least about 4.3×, at least about 4.4×, at least about 4.5×, at least about 4.6×, at least about 4.7×, at least about 4.8×, at least about 4.9×, at least about 5×, at least about 5.1×, at least about 5.2×, at least about 5.3×, at least about 5.4×, at least about 5.5×, at least about 5.6×, at least about 5.7×, at least about 5.8×, at least about 5.9×, at least about 6×, at least about 6.1×, at least about 6.2×, at least about 6.3×, at least about 6.4×, at least about 6.5×, at least about 6.6×, at least about 6.7×, at least about 6.8×, at least about 6.9×, at least about 7×, at least about 7.1×, at least about 7.2×, at least about 7.3×, at least about 7.4×, at least about 7.5×, at least about 7.6×, at least about 7.7×, at least about 7.8×, at least about 7.9×, at least about 8×, at least about 8.1×, at least about 8.2×, at least about 8.3×, at least about 8.4×, at least about 8.5×, at least about 8.6×, at least about 8.7×, at least about 8.8×, at least about 8.9×, at least about 9.9, at least about 9.9.9×.

In some embodiments, the anion exchange equilibration buffer comprises citric acid or citrate at a concentration from about 0.5mM to about 15mM, and the UV of the anion exchange eluate ₂₆₀ With UV ₂₈₀ Is at least 1.25 or higher. For example, the anion exchange equilibration buffer comprises citric acid or citrate at a concentration of from about 1mM to about 10mM, from about 1.5mM to about 7.5mM, from about 2mM to about 7mM, from about 1.5mM to about 9mM, from about 2mM to about 8mM, or from about 2.5mM to about 7.5mM (e.g., the equilibration buffer comprises citric acid or citrate at a concentration of about 0.5mM, about 1mM, about 1.5mM, about 2mM, about 2.5mM, about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 8mM, about 8.5mM, about 9mM, about 9.5mM, or about 10 mM), or the equilibration buffer comprises citric acid or citrate at a concentration of up to about 4 mM), the UV of the anion exchange eluate ₂₆₀ With UV ₂₈₀ At least 1.25, at least 1.26, at least 1.27, at least 1.28, at least 1.29, at least 1.3, at least 1.31, at least 1.32, at least 1.33, at least 1.34, or at least 1.35 or more.

In some embodiments, the anion exchange equilibration buffer comprises citric acid or citrate at a concentration from about 0.5mM to about 15mM, and the UV in the anion exchange eluate ₂₆₀ With UV ₂₈₀ The ratio of (2) is the UV in the adjusted affinity eluate ₂₆₀ With UV ₂₈₀ At least about 1.15 x or greater. For example, the anion exchange equilibration buffer comprises citric acid or citrate at a concentration from about 1mM to about 10mM, from about 1.5mM to about 7.5mM, from about 2mM to about 7mM, from about 1.5mM to about 9mM, from about 2mM to about 8mM, or from about 2.5mM to about 7.5mM (e.g., the equilibration buffer comprises citric acid or citrate at a concentration of about 0.5mM, about 1mM, about 1.5mM, about 2mM5mM, about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 8mM, about 8.5mM, about 9mM, about 9.5mM, or about 10mM citric acid or citrate, or the equilibration buffer contains citric acid or citrate at a concentration up to about 4 mM), and UV in the anion exchange eluate ₂₆₀ With UV ₂₈₀ The ratio of (2) is the UV in the adjusted affinity eluate ₂₆₀ With UV ₂₈₀ At least about 1.2×, at least about 1.25×, at least about 1.3× 0, at least about 1.35× 1, at least about 1.4× 2, at least about 1.45× 3, at least about 1.5× 4, at least about 1.55× 5, at least about 1.6× 6, at least about 1.65×, at least about 1.7×, or at least about 1.75×, at least about 1.8×, at least about 1.859×, at least about 1.9×, at least about 1.95×, or at least about 2× or higher.

In some embodiments of any of the aspects described herein, the affinity eluate comprises a weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof), and the affinity eluate is diluted with an anion exchange dilution buffer comprising: a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris) and any combination thereof, or a buffer selected from the group consisting of BTP, tris, borate, tricine and any combination thereof, or a buffer selected from the group consisting of BTP, tris and any combination thereof); amino acids (e.g., amino acids selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or amino acids selected from the group consisting of aspartate, glutamate, and histidine; or amino acids selected from the group consisting of histidine and lysine; or amino acids selected from the group consisting of cysteine and tyrosine); a weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); salts (e.g., salts selected from the group consisting of sodium salts, potassium salts, ammonium salts, magnesium salts, calcium salts, copper salts, cobalt salts, manganese salts, nickel salts, and zinc salts; or salts selected from the group consisting of potassium salts, ammonium salts, Salts of the group consisting of magnesium, calcium, manganese and zinc salts; or a salt selected from the group consisting of potassium, magnesium and calcium salts; or from potassium and magnesium salts (e.g. MgCl) ₂ ) Salts of the group consisting of; and nonionic surfactants (e.g., nonionic surfactants selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from polyoxyethylene (12) isooctylphenyl ether (e.g.,CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, brij 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL15-S-7, ECOSURF SA-15, GIOL 15-S-9, TERGOL 15-S-12, TERGL-64, TOGINP-7, GINP-8, TERGL-9, TERGL-NP-10, TERGNP-10, TERGITOL-10. NP-TERGL-10, and the like, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, polysorbate 20, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic10R5, PF68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combination thereof, or a nonionic surfactant selected from the group consisting of Pluronic10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL P-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof, optionally the equilibration buffer is substantially free of glycine.

In some embodiments of any of the aspects described herein, the affinity eluate is substantially free of a weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof), and the affinity eluate is diluted with an anion exchange dilution buffer comprising: a buffer (e.g., a buffer selected from the group consisting of acetate, histidine, phosphate, citrate, propionate, tricine, borate, tris (hydroxymethyl) aminomethane (tris) and any combination thereof, or a buffer selected from the group consisting of BTP, tris, borate, tricine and any combination thereof, or a buffer selected from the group consisting of BTP, tris and any combination thereof); amino acids (e.g., amino acids selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine and tyrosine, or amino acids selected from the group consisting of aspartate, glutamate and histidine, or amino acids selected from the group consisting of histidine and lysine)The method comprises the steps of carrying out a first treatment on the surface of the Or an amino acid selected from the group consisting of cysteine and tyrosine); a weak acid or salt thereof (e.g., citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof); salts (e.g., salts selected from the group consisting of sodium salts, potassium salts, ammonium salts, magnesium salts, calcium salts, copper salts, cobalt salts, manganese salts, nickel salts, and zinc salts; or salts selected from the group consisting of potassium salts, ammonium salts, magnesium salts, calcium salts, manganese salts, and zinc salts; or salts selected from the group consisting of potassium salts, magnesium salts, and calcium salts; or salts selected from the group consisting of potassium salts and magnesium salts (e.g., mgCl) ₂ ) Salts of the group consisting of; nonionic surfactants (e.g., nonionic surfactants selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkyl phenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from polyoxyethylene (12) isooctylphenyl ether (e.g.,CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol octadecyl ether), seed oil surfactant (e.g., ecosurf (tm) SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylate (e.g., tergitol NP-10 nonylphenol ethoxylate), and any combination thereof, or a nonionic surfactant selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P40 7. Nonionic surfactants selected from the group consisting of poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, brij 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL P-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL LNP-13, polysorbate 20, and any combination thereof, or nonionic surfactants selected from the group consisting of poloxamer 188, poloxamer 407, pluronic10R5, ecgeneral 10, PF68, ecgeneral 15-S-12, TERGITOL15, TERGITOL NP-9, TERGITOL NP-12, TERGITOL NP-13, TERGITOL 20, TERGOL 20, TERGITOL 9, TERGITOL NP-13, TERGOL 13, TERGITOL-13, TERGITOL 20, TERGOL-13, TERGITOL 20, TERGL-13, TERGITON-13, TERGL-13, TERGITS-13, TEL-13, and any combination thereof, or a nonionic surfactant selected from the group consisting of Pluronic10R5 and PF68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, GIOL NP-11, TERGITOL NP-12, A nonionic surfactant of the group consisting of TERGITOLNP-13 and any combination thereof); and a viscosity modifier (e.g., a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of glycerol, sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of glycerol, sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof).

Typically, the conductivity of the dilution buffer is in the range from about 0.5mS/cm to about 3 mS/cm. For example, the conductivity of the dilution buffer is in the range from about 1mS/cm to about 2.5mS/cm, from about 1.25mS/cm to about 2.25mS/cm, or from about 1.5mS/cm to about 1.75 mS/cm. In some embodiments, the dilution buffer has a conductivity of about 1.67 mS/cm.

The osmotic pressure of the dilution buffer was less than 900mOsm. For example, the dilution buffer has an osmolality of about 200mOsm to about 900mOsm.

As described herein, the feed composition for anion exchange chromatography may be an eluate from affinity chromatography. Thus, another aspect provided herein is a method of obtaining a composition comprising recombinantly expressed viral particles. Typically, the method comprises contacting a harvest medium comprising the recombinant expressed viral particles with an affinity chromatography medium under conditions that allow binding of the viral particles to the affinity chromatography medium, and recovering an eluate comprising the recombinant viral particles (affinity eluate). The recovered eluate may be used as a feed composition for anion exchange chromatography.

In various embodiments, the elution buffer (affinity elution buffer) used to recover recombinant viral particles from affinity chromatography comprises glycine. For example, the affinity elution buffer comprises glycine at a concentration of at least about 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 90mM, 95mM, 100mM or more. In some embodiments of any of the aspects, the affinity elution buffer comprises glycine at a concentration of from about 25mM to about 100mM, from about 30mM to about 95mM, from about 35mM to about 90mM, from about 40mM to about 80mM, or from about 45mM to about 75mM.

In some embodiments of any of the aspects, the affinity elution buffer comprises glycine at a concentration of about 20mM, about 25mM, about 30mM, about 35mM, about 40mM, about 45mM, about 50mM, about 55mM, about 60mM, about 65mM, about 70mM, about 75mM, about 80mM, about 90mM, about 95mM, or about 100mM. For example, the affinity elution buffer comprises glycine at a concentration of about 50 mM. In another non-limiting example, the affinity elution buffer comprises glycine at a concentration of about 75 mM.

Affinity elutionThe buffer may also comprise cations, for example, monovalent or divalent cations. Exemplary monovalent ions for the elution buffer include, but are not limited to, sodium (Na ⁺ ) Lithium (Li) ⁺ ) Potassium (K) ⁺ ) Rubidium (Rb) ⁺ ) Cesium (Cs) ⁺ ) Francium (Fr) ⁺ ) Alkyl ammonia and ammonium. Exemplary divalent cations for elution buffers include, but are not limited to, magnesium (Mg ²⁺ ) Calcium (Ca) ²⁺ ) Copper (Cu) ²⁺ ) Cobalt (Co) ²⁺ ) Manganese (Mn) ²⁺ ) Nickel (Ni) ²⁺ ) And zinc (Zn) ²⁺ ). The cations may be added in the form of salts. In some embodiments, the elution buffer comprises a divalent cation, e.g., mg ²⁺ 。

Notably, the affinity elution buffer can comprise a cation (e.g., a divalent cation, such as Mg) at a concentration of at least about 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 50mM, 55mM, or more ²⁺ ). For example, the affinity elution buffer may comprise a cation (e.g., a divalent cation such as Mg) at a concentration of from about 5mM to about 15mM, from about 6mM to about 14mM, from about 7mM to about 13mM, from about 8mM to about 12mM, or from about 9mM to about 11mM ²⁺ ). In some embodiments, the affinity elution buffer comprises a cation (e.g., a divalent cation, such as Mg) at a concentration of about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, or about 15mM ²⁺ ). For example, the affinity elution buffer comprises a cation (e.g., a divalent cation, such as Mg ²⁺ )。

In some embodiments of any aspect, the affinity elution buffer comprises a magnesium salt, e.g., mgCl ₂ 。

The affinity elution buffer may also comprise a polymer. For example, the affinity elution buffer may comprise a nonionic polymer. Exemplary nonionic polymers include poloxamers (also known under the trade name SYNPERONICS ^TM 、PLURONIC ^TM And KOLLIPHOR ^TM ) And polyethylene glycol (PEG).

In some embodiments, the affinity elution buffer comprises a poloxamer.Exemplary poloxamers include, but are not limited to, poloxamer 188 (P188),F127、F38、F68、F87、F108、10R5、17R2、17R4、25R2、25R4、31R1、F108Cast Solid Surfacta、F108 NF、F108Pastille、F108NF Prill, poloxamer 338,F127 NF、F127 NF 500BHT Prill、F127 NF Prill poloxamer 407,/L- >F38Pastille、F68 LF Pastille、F68 NF、F68 NF Prill、F68 Pastille、F77、F77Micropastille、F87 NF、F87 NF Prill poloxamer 237,F 88、F 88Pastille、F 98、FT L 61、L10、L101、L121、L31、L35、L43、L61、L62、L62LF、L62D、L64、L81、L92、L44NF INH surfactant, poloxamer 124,/i>N3、P103、P104、P105、P123 surfactant, < >>P65、P84、P85, etc.

The amount of polymer (e.g., poloxamer) in the affinity elution buffer can vary. For example, the amount of polymer (e.g., poloxamer) in the affinity elution buffer can be at least about 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5% (w/w, w/v, or v/v) or more. In some embodiments, the affinity elution buffer comprises a polymer, e.g., poloxamer, at a concentration of from about 0.1% to about 0.5%, from about 0.15% to about 0.45%, from about 0.2% to about 0.4%, or from about 0.25% to about 0.35%. 0.1%. For example, the affinity elution buffer comprises a polymer, e.g., poloxamer, at a concentration of about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, or about 0.5%.

Typically, the affinity elution buffer has a low pH. For example, the pH of the affinity elution buffer is less than or equal to about 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.2, 2.0, 1.5 or less. In some embodiments, the pH of the affinity elution buffer is less than or equal to about 4.5. For example, the pH of the affinity elution buffer is from about 2.0 to about 3.0. For example, the pH of the affinity elution buffer is about 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0. In some embodiments, the pH of the affinity elution buffer is from about 2.0 to about 3.0. In some embodiments, the pH of the affinity elution buffer is about 2.2.

The affinity elution buffer may also comprise histidine. When the affinity elution buffer comprises histidine, the concentration of histidine may be at least about 1mM, 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM or more. For example, the affinity elution buffer may comprise histidine at a concentration of from about 1mM to about 50mM, from about 5mM to about 45mM, from about 10mM to about 40mM, from about 15mM to about 35mM, or from about 20mM to about 30mM. In some embodiments, the affinity elution buffer comprises histidine at a concentration of about 1mM, about 5mM, about 10mM, about 15mM, about 20mM, about 25mM, about 30mM, about 35mM, about 40mM, about 45mM, or about 50mM. For example, the affinity elution buffer comprises histidine at a concentration of about 25 mM.

In some embodiments, the affinity elution buffer comprises: about 25-75mM glycine, about 50-100mM histidine, about 2-25mM MgCl ₂ About 0.1% -0.5% P188, and has a pH of about 2.5-3.5. For example, the affinity elution buffer comprises: about 75mM glycine, about 75mM histidine,About 10mM MgCl ₂ About 0.3% P188, and has a pH of about 3.0.

In some embodiments, the affinity elution buffer is substantially free of histidine.

In some embodiments, the affinity elution buffer comprises citric acid or a salt thereof, e.g., citrate. The affinity elution buffer can comprise citric acid or a salt thereof at a concentration of at least about 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM, 100mM, or more. For example, the affinity elution buffer comprises citric acid or a salt thereof at a concentration of from about 50mM to about 100mM, from about 55mM to about 95mM, from about 60mM to about 90mM, from about 65mM to about 85mM, or from about 70mM to about 75mM. In some embodiments, the affinity elution buffer comprises citric acid or a salt thereof at a concentration of about 50mM, about 55mM, about 60mM, about 65mM, about 70mM, about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, or about 100mM. For example, the affinity elution buffer comprises citric acid or a salt thereof at a concentration of about 75mM.

In some embodiments, the affinity elution buffer comprises: about 50-100mM glycine; about 10-50mM of an amino acid (e.g., an amino acid selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or an amino acid selected from the group consisting of aspartate, glutamate, and histidine; or an amino acid selected from the group consisting of histidine and lysine; or an amino acid selected from the group consisting of cysteine and tyrosine); about 2-25mM of a salt (e.g., a salt selected from the group consisting of sodium, potassium, ammonium, magnesium, calcium, copper, cobalt, manganese, nickel, and zinc salts; or a salt selected from the group consisting of potassium, ammonium, magnesium, calcium, manganese, and zinc salts; or a salt selected from the group consisting of potassium, magnesium, and calcium salts; or a salt selected from the group consisting of potassium and magnesium salts (e.g., mgCl) ₂ ) Salts of the group consisting of; about 0.1% to about 0.5% P188. For example, the affinity elution buffer comprises: about 75mM glycine; about 75mM amino acid (e.g., selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosineAmino acids of the group; or an amino acid selected from the group consisting of aspartate, glutamate and histidine; or an amino acid selected from the group consisting of histidine and lysine; or an amino acid selected from the group consisting of cysteine and tyrosine); about 10mM of a salt (e.g., a salt selected from the group consisting of sodium, potassium, ammonium, magnesium, calcium, copper, cobalt, manganese, nickel, and zinc salts; or a salt selected from the group consisting of potassium, ammonium, magnesium, calcium, manganese, and zinc salts; or a salt selected from the group consisting of potassium, magnesium, and calcium salts; or a salt selected from the group consisting of potassium and magnesium salts (e.g., mgCl) ₂ ) Salts of the group consisting of; about 0.3% P188, and has a low pH, e.g., a pH of about 2-5, e.g., a pH of about 2, about 2.5, about 3, about 3.5, or about 4.

In some embodiments, the affinity elution buffer comprises: about 50mM, about 55mM, about 60mM, about 65mM, about 70mM, about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, or about 100mM glycine; about 10mM, about 15mM, about 20mM, about 25mM, about 30mM, about 35mM, about 40mM, about 45mM, or about 50mM of an amino acid (e.g., an amino acid selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or an amino acid selected from the group consisting of aspartate, glutamate, and histidine; or an amino acid selected from the group consisting of histidine and lysine; or an amino acid selected from the group consisting of cysteine and tyrosine); about 2.5mM, about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 7.5mM, about 10mM, about 12.5mM, about 15mM, about 17.5mM, about 20mM, about 22.5mM or about 25mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt and zinc salt; or a salt selected from the group consisting of potassium salt, magnesium salt and calcium salt; or a salt selected from the group consisting of potassium salt and magnesium salt (e.g., mgCl) ₂ ) Salts of the group consisting of; about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, or about 0.5% P188.

In some embodiments, the affinity elution buffer comprises: about 50mM, about 55mM, about 60mM, about 65mM, about 70mM, about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, or about 100mM glycine; about 10mM, about 15mM, about 20mM, about 25mM, about 30mM, about 35mM, about 40mM, about 45mM, or about 50mM of an amino acid (e.g., an amino acid selected from the group consisting of aspartate, glutamate, histidine, arginine, lysine, cysteine, and tyrosine; or an amino acid selected from the group consisting of aspartate, glutamate, and histidine; or an amino acid selected from the group consisting of histidine and lysine; or an amino acid selected from the group consisting of cysteine and tyrosine); about 2.5mM, about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 7.5mM, about 10mM, about 12.5mM, about 15mM, about 17.5mM, about 20mM, about 22.5mM or about 25mM of a salt (e.g., a salt selected from the group consisting of sodium salt, potassium salt, ammonium salt, magnesium salt, calcium salt, copper salt, cobalt salt, manganese salt, nickel salt and zinc salt; or a salt selected from the group consisting of potassium salt, ammonium salt, magnesium salt, calcium salt, manganese salt and zinc salt; or a salt selected from the group consisting of potassium salt, magnesium salt and calcium salt; or a salt selected from the group consisting of potassium salt and magnesium salt (e.g., mgCl) ₂ ) Salts of the group consisting of; about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, or about 0.5% P188, and the buffer has a low pH, e.g., a pH of about 2-5, e.g., a pH of about 2, about 2.5, about 3, about 3.5, or about 4.

In some embodiments, the affinity elution buffer comprises: about 75mM glycine, about 25mM histidine and about 10mM MgCl ₂ . For example, the affinity elution buffer comprises: about 75mM glycine, about 25mM histidine and about 10mM MgCl ₂ And the buffer has a low pH, e.g., a pH of about 2-5, e.g., a pH of about 2, about 2.5, about 3, about 3.5, or about 4.

In some embodiments, the affinity elution buffer comprises: about 50mM glycine, about 75mM citrate, about 10mM MgCl ₂ About 0.3% P188, and has a pH of about 3.0. Typically, the affinity elution buffer is electrokineticThe conductivity is in the range of from about 5mS/cm to about 8mS/cm. For example, the conductivity of the affinity elution buffer is in the range of from about 5.5mS/cm to about 7mS/cm, from about 5.75mS/cm to about 6.75mS/cm, or from about 6.15mS/cm to about 6.25 mS/cm. In some embodiments, the affinity elution buffer has a conductivity of about 6.18mS/cm.

Notably, the conductivity can be measured using methods and equipment standard in the art.

The osmotic pressure of the affinity elution buffer may range from about 100mOsm to about 225 mOsm. For example, the osmotic pressure of the affinity elution buffer may range from about 125mOsm to about 200mOsm, from about 150mOsm to about 175mOsm, or from about 155mOsm to about 165 mOsm. In some embodiments, the affinity elution buffer has an osmotic pressure of about 161mOsm.

Osmotic pressure, a term well known in the art, is defined as the number of solute molecules per kg of water. Osmotic pressure can be measured using techniques standard in the art, for example, using, for example, freezing point depression of an osmometer.

In some embodiments, the affinity elution buffer is substantially free of citric acid or a salt thereof.

Column size

The methods described herein are scalable. Thus, the chromatography steps described herein can be used with a variety of column sizes. Thus, column sizes for affinity and/or anion exchange chromatography may range from mL to thousands of liters. For example, the column may be a 0.5mL column, a 1.5mL column, a 10mL column, a 20mL column, a 30mL column, a 50mL column, a 100mL column, a 200mL column, a 300mL column, a 400mL column, a 500mL column, a 600mL column, a 700mL column, a 800mL column, a 900mL column, a 1000mL (1L) column, a 2000mL (2L) column, a 10L column, a 20L column, a 30L column, a 40L column, a 50L column, a 60L column, a 70L column, an 80L column, a 90L column, a 100L column, or a column having a capacity greater than 100L and any other column having a capacity between the volumes listed above.

Washing step

The methods described herein may include one or more washing steps. For example, the recombinant viral particles bound to the affinity chromatography medium may be washed (e.g., with a buffer) prior to eluting or recovering the recombinant particles from the affinity chromatography medium. Similarly, recombinant viral particles bound to the anion exchange chromatography medium may be washed (e.g., with a buffer) prior to eluting or recovering the recombinant particles from the anion exchange chromatography medium. Notably, the chromatographic medium can be washed with at least about 1CV, 1.5CV, 2CV, 2.5CV, 3CV, 3.5CV, 4CV, 4.5CV, 5CV, 5.5CV, 6CV, 6.5CV, 7CV, 7.5CV, 8CV, 8.5CV, 9CV, 9.5CV, 10CV, 10.5CV, 11CV, 11.5CV, 12CV, 12.5CV, 13CV, 13.5CV, 14CV, 14.5CV, 15CV, 15.5CV, 16CV, 16.5CV, 17CV, 17.5CV, 18CV, 18.5CV, 19CV, 19.5CV, 20CV, or more suitable medium (e.g., wash buffer). In some embodiments, the anion exchange elution gradient length can range from about 2CV to about 100CV, or from about 5CV to about 80CV, or from about 5CV to about 60CV, or from about 5CV to about 50CV, or from about 5CV to about 40CV, or from about 5CV to about 30CV. In some embodiments, the elution flow (CV/min) may range from about 0.05CV/min to about 2CV/min, or from about 0.1CV/min to about 1.5CV/min, or from about 0.1CV/min to about 1CV/min.

Notably, the medium (e.g., wash buffer) used to wash the recombinant viral particles bound to the affinity chromatography medium can be optimized for the viral particles to be separated and/or the elution buffer used. For example, when the affinity eluate comprises histidine, a washing buffer comprising histidine may be used for the washing step. Thus, in some embodiments, the wash buffer for affinity chromatography comprises histidine at a concentration of at least about 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM or more. In some embodiments, the wash buffer for affinity chromatography comprises histidine at a concentration of from about 10mM to about 50mM, from about 15mM to about 40mM, or from about 20mM to about 30 mM.

In various embodiments, the affinity elution buffer comprises citric acid or a salt thereof. In such embodiments, a wash buffer comprising citric acid or a salt thereof (e.g., citrate) may be used for the washing step. Thus, in some embodiments, the wash buffer for affinity chromatography comprises citric acid or a salt thereof at a concentration of at least about 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM, 100mM or more. In some embodiments, the wash buffer for affinity chromatography comprises citric acid or a salt thereof at a concentration of from about 10mM to about 100mM, from about 15mM to about 90mM, or from about 20mM to about 75 mM.

Typically, the wash buffer used for affinity chromatography has a high pH. For example, the pH of the wash buffer used for affinity chromatography is greater than or equal to about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10. In some embodiments, the wash buffer used for affinity chromatography has a pH of greater than or equal to about 8.5. For example, the wash buffer used for affinity chromatography has a pH of about 9.

Additional parameters of chromatography

Typically, the chromatographic steps described herein are performed at ambient temperature. The residence time is from about 1 minute to about 8 minutes. If a washing step is included, the chromatographic medium can be washed with from about 5CV to about 15CV of a suitable wash buffer. If desired, the chromatographic medium can be equilibrated with an equilibration buffer of from about 5CV to 15 CV. Typically, the bound AAV particles may be eluted from the chromatographic medium using an elution medium of from about 5CV to 100 CV.

In one aspect, provided herein are methods of purifying or isolating recombinantly expressed viral particles (e.g., recombinant adeno-associated virus (rAAV), optionally comprising transgenes) from a formulation comprising recombinant vector particles, empty capsids, and host cell impurities to provide a product that is substantially free of empty viral particles. Typically, the method comprises contacting the formulation (e.g., a harvest medium comprising recombinant viral particles) with an affinity chromatography medium under conditions that allow binding of the viral particles to the affinity chromatography medium. The bound virus particles are eluted from the affinity chromatography medium using an elution buffer, and an eluate comprising the eluted virus particles is recovered. In some embodiments, the affinity elution buffer comprises a weak acid or salt thereof. In some embodiments, the affinity elution buffer comprises substantially no weak acid or salt thereof. The eluate from the affinity chromatography is also referred to herein as an affinity eluate. The affinity eluate is contacted with an anion exchange chromatography medium under conditions that allow binding of the virus particles to the anion exchange chromatography medium. The inventors have found in particular that the presence of an ionic compound (e.g., an anionic compound, such as an acid or a salt thereof, such as a weak acid or a salt thereof) in a buffer for equilibration and/or conditioning of chromatography prior to contact with an affinity eluate surprisingly and unexpectedly results in preferential binding of genome-containing viral particles (e.g., adeno-associated viral (AAV) particles having a packaged genomic sequence) (i.e., intact viral particles) compared to genome-defective particles (i.e., empty), thereby producing recombinant viral particles (e.g., recombinant AAV viral particles) having fewer empty particles. In some embodiments, the purified recombinant adeno-associated virus (rAAV) population purified by the methods described herein comprises less than about 10% empty viral capsids. In some embodiments, the purified rAAV population comprises less than about 9.5%, less than about 9%, less than about 8.5%, less than about 8%, less than about 7.5%, less than about 7%, less than about 6.5%, less than about 6%, less than about 5.5%, less than about 5%, less than about 4.5%, less than about 4%, less than about 3.5%, less than about 3%, less than about 2.5%, less than about 2%, less than about 1.5%, less than about 1%, less than about 0.75%, less than about 0.5%, less than about 0.25%, less than about 0.2%, less than about 0.15%, less than about 0.1%, less than about 0.05%, less than about 0.03%, less than about 0.02%, or less than about 0.01% empty viral capsids. In some embodiments, the purified rAAV population is substantially devoid or substantially devoid of empty viral capsids.

In some aspects, provided herein are purified populations of recombinant adeno-associated viruses (rAAV), optionally lacking prokaryotic sequences, wherein the purified viruses have less than 2 x 10 ⁴ Particle to infectivity ratio of vg/TCID50, wherein the purified rAAV population comprises less than about 10% empty viral capsids, and wherein the purified virus is optionally obtained by transfecting a suspension mammalian cell line, wherein the cells are transfected in suspension. In some embodiments, the purified recombinant adeno-associated virus has less than 1.5X10 ⁴ vg/TCID50, less than 1X 10 ⁴ vg/TCID50, less than 9X 10 ³ vg/TCID50, less than 8 x 10 ³ vg/TCID50, less than 6X 10 ³ vg/TCID50, less than 5X 10 ³ vg/TCID50, less than 4X 10 ³ vg/TCID50, less than 3X 10 ³ vg/TCID50, less than 2X 10 ³ vg/TCID50, less than 9X 10 ² vg/TCID50, less than 8 x 10 ² vg/TCID50, less than 7X 10 ² vg/TCID50, less than 6X 10 ² vg/TCID50, less than 5X 10 ² vg/TCID50, less than 4X 10 ² vg/TCID50, less than 3X 10 ² vg/TCID50, less than 2X 10 ² vg/TCID50, or less than 1X 10 ² vg/TCID50, or less than 0.5X10 ² The ratio of vg/TCID50 or even less particles to infectivity. In certain embodiments, the purified rAAV population comprises less than about 9.5%, less than about 9%, less than about 8.5%, less than about 8%, less than about 7.5%, less than about 7%, less than about 6.5%, less than about 6%, less than about 5.5%, less than about 5%, less than about 4.5%, less than about 4%, less than about 3.5%, less than about 3%, less than about 2.5%, less than about 2%, less than about 1.5%, less than about 1%, less than about 0.75%, less than about 0.5%, less than about 0.25%, less than about 0.2%, less than about 0.15%, less than about 0.1%, less than about 0.05%, less than about 0.03%, less than about 0.02%, or less than about 0.01% empty viral capsids. In some preferred embodiments, the purified rAAV population is substantially free of empty viral capsids. In some embodiments, a population of purified rAAV lacking prokaryotic sequences is produced using closed-end linear double-stranded DNA (celDNA or clDNA) as a template. In some embodiments, a purified recombinant adeno-associated virus (rAAV) lacking a prokaryotic sequence is produced by using a) a nucleic acid sequence encoding a helper protein sufficient for rAAV replication; b) Nucleic acid sequences encoding rep and cap genes; and c) a closed-end linear double-stranded rAAV vector nucleic acid comprising at least one ITR and a heterologous transgene operably linked to one or more regulatory elements is produced by transfection of a mammalian cell line in suspension. In certain embodiments, the mammalian cell line is a human embryonic cell line, wherein the human embryonic cell line is an adapted serum-free suspension cell line derived from a human embryonic kidney cell line. In some embodiments, the purified population of recombinant adeno-associated viruses (rAAV) does not lack prokaryotic sequences.

In some embodiments, the purified rAAV is obtained by transfecting a non-adherent human embryonic cell line in suspension. In certain embodiments, the purified virus is obtained by transfecting cells in a suspension of a human embryonic cell line, wherein the human embryonic cell line is a suspension adapted serum-free cell line derived from a human embryonic kidney cell line. In some embodiments, the cells are optionally transfected in suspension. In some embodiments, the purified rAAV lacks bacterial sequences.

In one aspect, provided herein are purified populations of recombinant adeno-associated viruses (rAAV), optionally lacking prokaryotic sequences, wherein the purified viruses have less than 2 x 10 ⁴ Particle to infectivity ratio of vg/TCID 50. In some embodiments, the purified recombinant adeno-associated virus has less than 1.5X10 ⁴ vg/TCID50, less than 1X 10 ⁴ vg/TCID50, less than 9X 10 ³ vg/TCID50, less than 8 x 10 ³ vg/TCID50, less than 6X 10 ³ vg/TCID50, less than 5X 10 ³ vg/TCID50, less than 4X 10 ³ vg/TCID50, less than 3X 10 ³ vg/TCID50, less than 2X 10 ³ vg/TCID50, less than 9X 10 ² vg/TCID50, less than 8 x 10 ² vg/TCID50, less than 7X 10 ² vg/TCID50, less than 6X 10 ² vg/TCID50, less than 5X 10 ² vg/TCID50, less than 4X 10 ² vg/TCID50, less than 3X 10 ² vg/TCID50, less than 2X 10 ² vg/TCID50, or less than 1X 10 ² vg/TCID50, or less than 0.5X10 ² Particle to infectivity ratio of vg/TCID50 or less. In some embodiments, a purified recombinant adeno-associated virus (rAAV) lacking a prokaryotic sequence is produced by using a) a nucleic acid sequence encoding a helper protein sufficient for rAAV replication; b) Nucleic acid sequences encoding rep and cap genes; and c) a closed-end linear double-stranded rAAV vector nucleic acid comprising at least one ITR and a heterologous transgene operably linked to one or more regulatory elements is produced by transfection of a mammalian cell line in suspension. In certain embodiments, the mammalian cell line is a human embryonic cell line, wherein the human embryonic cell line is a suspension adapted serum-free cell line derived from a human embryonic kidney cell line. In some embodiments, the purified recombinant AAV lacks bacterial sequences.

Some implementations of any aspect described hereinIn embodiments, the purified recombinant adeno-associated virus (rAAV) population has about 1×10 ⁵ TCID50/mL (median tissue culture infection dose) to about 1X 10 ¹¹ Infectious particle titer of TCID 50. In certain embodiments, the infectious particle titer is at least 3 x 10 ⁹ TCID50/mL. In several embodiments, the infectious particle titer is at least 2 x 10 ⁵ TCID50/mL, at least 5X 10 ⁵ TCID50/mL, at least 7.5X10 ⁵ TCID50/mL, at least 8X 10 ⁵ TCID50/mL, at least 8.5X10 ⁵ TCID50/mL, at least 9X 10 ⁵ TCID50/mL, at least 9.5X10 ⁵ TCID50/mL, at least 1X 10 ⁶ TCID50/mL, at least 2X 10 ⁶ TCID50/mL, at least 5X 10 ⁶ TCID50/mL, at least 7.5X10 ⁶ TCID50/mL, at least 8X 10 ⁶ TCID50/mL, at least 8.5X10 ⁶ TCID50/mL, at least 9X 10 ⁶ TCID50/mL, at least 9.5X10 ⁶ TCID50/mL, at least 1X 10 ⁷ TCID50/mL, at least 2X 10 ⁷ TCID50/mL, at least 5X 10 ⁷ TCID50/mL, at least 7.5X10 ⁷ TCID50/mL, at least 8X 10 ⁷ TCID50/mL, at least 9X 10 ⁷ TCID50/mL, at least 1X 10 ⁸ TCID50/mL, at least 2.5X10 ⁸ TCID50/mL, at least 5X 10 ⁸ TCID50/mL, at least 7.5X10 ⁸ TCID50/mL, at least 8X 10 ⁸ TCID50/mL, at least 8.5X10 ⁸ TCID50/mL, at least 9X 10 ⁸ TCID50/mL, at least 9.5X10 ⁸ TCID50/mL, at least 0.5X10 ⁹ TCID50/mL, at least 1X 10 ⁹ TCID50/mL, at least 1.5X10 ⁹ TCID50/mL, at least 2X 10 ⁹ TCID50/mL, at least 2.5X10 ⁹ TCID50/mL, at least 3X 10 ⁹ TCID50/mL, at least 3.5X10 ⁹ TCID50/mL, at least 4X 10 ⁹ TCID50/mL, at least 4.5X10 ⁹ TCID50/mL, at least 5X 10 ⁹ TCID50/mL, at least 5.5X10 ⁹ TCID50/mL, at least 6X 10 ⁹ TCID50/mL, at least 6.5X10 ⁹ TCID50/mL, at least 7X 10 ⁹ TCID50/mL, at least 7.5X10 ⁹ TCID50/mL, at least 8X 10 ⁹ TCID50/mL, at least 8.5X10 ⁹ TCID50/mL, at least 9X 10 ⁹ TCID50/mL, at least 9.5X10 ⁹ TCID50/mL, at least 1X10 ¹⁰ TCID50/mL, at least 2X 10 ¹⁰ TCID50/mL, at least 5X 10 ¹⁰ TCID50/mL, at least 7.5X10 ¹⁰ TCID50/mL, at least 8X 10 ¹⁰ TCID50/mL, at least 8.5X10 ¹⁰ TCID50/mL, at least 9X 10 ¹⁰ TCID50/mL, at least 9.5X10 ¹⁰ TCID50/mL, or at least about 10 ¹¹ TCID50/mL. In some embodiments, the infectious titer TCID50/mL is preferably normalized to vg/mL.

In some embodiments of one or more aspects described herein, the method of producing a recombinant AAV comprises transient transfection. In some embodiments, the method of producing a recombinant AAV comprises a stable transfection method. In some embodiments, transfection is performed in suspension.

In certain embodiments, the mammalian cell line is a suspension cell or cell line (i.e., a non-adherent cell or cell line), and the cells are transfected in suspension. In certain embodiments, the cell line is derived from a human embryonic kidney 293 cell line (HEK 293). In certain embodiments, the human embryonic kidney cells lack the SV40 antigen or other transformed antigen. In certain embodiments, the mammalian cell line is a serum-free adapted suspension cell line. In certain embodiments, the cell line is derived from primary blood cells, e.g., lymphocytes, monocytes, macrophages, granulocytes, dendritic cells, erythrocytes. In certain embodiments, the cell lines are derived from a cell biopsy and include, for example, lymph node cells, bone marrow cells, cord blood cells. In certain embodiments, the cell line is derived from a circulating tumor cell. In certain embodiments, the cell line is derived from a blood cell line, e.g., jurkat and Molt 4T cell lines, U937 and THP pre-monocyte cell lines, B cell hybridomas. In certain embodiments, the cell line is derived from a stem cell. In certain embodiments, the cell line used to produce the recombinant AAV is a stable cell line. In some embodiments, the mammalian cell line is an adherent cell line.

In some embodiments, the purified recombinant AAV is obtained by culturing a human embryonic cell line in suspension and transfecting it in suspension.

Cell culture work involving rAAV production (including expansion, seeding, and transfection of adherent cells) is cumbersome and resource intensive. Thus, the use of cells suspended in aqueous liquid medium ("suspension cells") to produce rAAV vectors is desirable because of its scalability and cost effectiveness. Thus, in certain embodiments of any of the aspects described herein, the host cell line may be a suspension adapted cell. For example, host cells can be transfected with nucleic acid vectors in suspension. In certain embodiments, the host cell is a mammalian cell, i.e., the host cell line is a mammalian cell line. For example, the host cell (i.e., host cell line) is a human cell, e.g., a human embryonic cell line. In certain embodiments of any of the aspects described herein, the host cell line is a human embryonic kidney cell line.

Methods of preparing AAV vectors are well known in the art and are described, for example, in U.S. patent nos. US6204059, US5756283, US6258595, US6261551, US6270996, US6281010, US6365394, US6475769, US6482634, US6485966, US6943019, US6953690, US7022519, US7238526, US7291498 and US7491508, US5064764, US6194191, US6566118, US8137948; or International publication Nos. WO1996039530, WO1998010088, WO 1999014354, WO1999/015685, WO1999/047691, WO2000/055342, WO2000/075353 and WO2001/023597; methods In Molecular Biology, ed. Richard, humana Press, NJ (1995); o' Reilly et al Baculovirus Expression Vectors, A Laboratory Manual, oxford Univ. Press (1994); samulski et al, J Fir.63:3822-8 (1989); kajigaya et al, proc.Nat' l.Acad.Sci.USA 88:4646-50 (1991); ruffing et al, J.Vir.66:6922-30 (1992); kimbauer et al, vir.,219:37-44 (1996); zhao et al, vir.272:382-93 (2000); the contents of each of which are incorporated herein by reference. Viral replication cells commonly used to produce recombinant AAV viral particles include, but are not limited to, HEK293 cells, COS cells, heLa cells, KB cells, and other mammalian cell lines.

Components for AAV production (e.g., adenovirus E1a, E1b, E2a, and/or E4ORF6 gene products, rep or fragments thereof, cap, expression cassette, and any other desired helper functions) may be delivered to the packaging host cell alone or in combination in the form of any genetic element that transfers sequences carried thereon. One example of the preparation of AAV is by a triple transfection method, in which a host cell is transfected with three separate nucleic acids; and wherein one nucleic acid encodes a sufficient replication-assisting protein, e.g., an adenovirus-assisting protein that facilitates efficient replication and packaging of the vector but lacks the adenovirus structure and replication genes necessary for adenovirus production, one nucleic acid encodes AAV rep and cap proteins, and the other nucleic acid encodes an AAV ITR flanking transgene or a heterologous transgene. As used herein, genetic elements (vectors) include, for example, naked DNA, plasmids, phages, transposons, cosmids, episomes, proteins in a non-viral delivery medium (e.g., lipid-based vehicles), viruses, and the like, to which sequences carried are transferred. The selected vector may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high speed DNA coated particles (high velocity DNA-coated particles), viral infection, and protoplast fusion. Methods for constructing any of the embodiments of the present invention are known to the skilled artisan of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., sambrook et al Molecular Cloning: A Laboratory Manual, cold Spring Harbor Press, cold Spring Harbor, N.Y. See, e.g., K.Fisher et al, J Virol, 70:520-532 (1993) and U.S. Pat. No. 5,478,745.

In one embodiment, the stable or transient host cell will contain the desired components under the control of an inducible or regulatable promoter. However, the desired components may be under the control of a constitutive promoter or a synthetic promoter.

It will be appreciated that using the methods described herein, the viral expression system will be further modified to include any necessary elements required to supplement a given viral vector during production. For example, in certain embodiments, the nucleic acid cassette is flanked by terminal repeats. In one embodiment, to produce the rAAV vector, the AAV expression system will further comprise at least one of a recombinant AAV plasmid, a Rep-expressing plasmid, a Cap-expressing plasmid, and an adenovirus helper plasmid. The complementing elements of a given viral vector are well known in the art, and the skilled practitioner will be able to modify the viral expression system described herein accordingly.

In some embodiments, the recombinant AAV is produced from plasmid DNA. In some embodiments, the recombinant AAV is produced from closed-end linear double stranded DNA. Blocking linear DNA molecules typically comprise covalently blocked ends, also described as hairpin loops, in which base pairing between complementary DNA strands is not present. The hairpin loop links the ends of the complementary DNA strands. This type of structure is usually formed at the telomere end of the chromosome in order to prevent loss or damage of the chromosomal DNA by blocking the terminal nucleotides in a closed structure. In the examples of blocked linear DNA molecules described herein, hairpin loops flank complementary base-paired DNA strands forming a blocked linear (cl) DNA-shaped structure. The closed linear DNA molecule comprises barbell-shaped DNA.

One or more of the nucleic acids a) to c) may be present on the closed-end linear double-stranded nucleic acid, i.e.: a) Nucleic acid sequences encoding helper proteins sufficient for rAAV replication; b) Nucleic acid sequences encoding rep and cap genes; and c) a closed-end linear double-stranded rAAV vector nucleic acid comprising at least one ITR and a heterologous transgene operably linked to one or more regulatory elements. Closed-end linear double stranded nucleic acids can be produced by a variety of known methods, including in vitro cell-free synthesis and in vivo methods. One method of producing covalently closed terminal linear double stranded nucleic acids is by incorporating a prokaryotic telomerase binding site (protelomerase binding site) in a precursor molecule such that the prokaryotic telomerase binding site is flanked by nucleic acids of interest and exposing the molecule to the prokaryotic telomerase, thereby cleaving and ligating the DNA at that site. The nucleic acid of interest may comprise one or more of a), b and c), i.e. a), b) and c); any combination of a), b) and c); or only a), only b) or only c). Examples of preparing closed-end linear double-stranded DNA are well known in the art, e.g., as described in Nucleic Acids res.2015oct 15;43 (18) e120; antisense & nucleic acid drug development 11:149-153 (2001); U.S. Pat. nos. 9109250, 9499847, 10501782, 10286399; and/or U.S. publication nos. US20190185924, US20190203282; the entire contents of which are incorporated herein by reference in their entirety.

Alternative methods of generating covalently closed-ended linear DNA (e.g., closed-ended linear double-stranded DNA) lacking a prokaryotic or bacterial sequence are known in the art, for example, by forming micro-circular DNA (mini-circle DNA) from a plasmid (e.g., as described in U.S. patent 8,828,726 and U.S. patent 7,897,380, the contents of each of which are incorporated herein by reference in their entirety). For example, one cell-free synthesis method combines the use of two enzymes (Phi 29 DNA polymerase and prokaryotic telomerase) and results in a high-fidelity, covalently-blocked linear DNA construct. The construct does not contain antibiotic resistance markers, thus eliminating packaging of these sequences. This procedure allows for amplification of AAV genomic DNA on a commercial scale over a 2 week period and maintains ITR sequences required for viral production.

In certain embodiments, an in vivo cell system is used to generate a non-viral DNA vector construct for delivering a predetermined nucleic acid sequence into a target cell for sustained expression. The non-viral DNA vector comprises: two DD-ITRs, each containing: inverted terminal repeats with A, A ', B, B ', C, C ' and D regions; a D' region; and wherein the D and D 'regions are complementary palindromic sequences of about 5-20nt in length, located adjacent to the A and A' regions; a predetermined nucleic acid sequence (e.g., a heterologous gene for expression); wherein the two DD-ITRs flank the nucleic acid in the context of covalently blocked non-viral DNA, and wherein the blocked linear vector comprises a 1/2 prokaryotic telomerase binding site on each end, e.g., as described in international publication No. WO 2019246544, which is incorporated herein by reference in its entirety.

Harvesting and clarification

The methods disclosed herein can include the step of producing a harvest medium for contact with an affinity chromatography medium by a method that includes upstream processing (e.g., harvesting a cell culture or cell culture supernatant and/or clarifying the harvested cell culture and cell culture supernatant). Thus, another aspect provided herein is a method of preparing a cell culture or cell culture supernatant for affinity chromatography. Typically, the method comprises harvesting the cell culture or cell culture supernatant and/or clarifying the cell culture or cell culture supernatant to produce a harvesting medium comprising the recombinantly expressed viral particles. The methods described herein produce recombinant AAV particles that are substantially free of empty AAV particles, independent of the cell density of transfected cells. Typically, the transfected cells have a cell density of from about 1E6 cells/mL to about 80E6 cells/mL. Similar effects of substantially eliminating empty particles from recombinant AAV particles can be useful when recombinant AAV is produced using triple transfection methods or stable producer and/or packaging cell lines (e.g., pro 10 cells). In some embodiments, recombinant AAV is produced from a closed-end linear double-stranded nucleic acid (e.g., closed-end linear double-stranded DNA) using the methods described herein. In some embodiments, other forms of nucleic acid (e.g., plasmid DNA) are used to produce recombinant AAV.

In some embodiments, the method of preparing a cell culture or cell culture supernatant for affinity chromatography comprises lysing host cells in the cell culture or cell culture supernatant. A method of lysing host cells in a cell culture or cell culture supernatant. For example, a nonionic surfactant may be added to the cell culture or cell culture supernatant.

The nonionic surfactant is added to the cell culture or cell culture supernatant to a final concentration of at least about 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1% (w/v, w/w or v/v) or higher. For example, the nonionic surfactant is added to the cell culture or cell culture supernatant to a final concentration of from about 0.05% to about 1%, from about 0.1% to about 0.95%, from about 0.15% to about 0.9%, from about 0.2% to about 0.85%, from about 0.25% to about 0.8%, from about 0.3% to about 0.75%, from about 0.35% to about 0.65%, from about 0.4% to about 0.6%, or from about 0.45% to about 0.55%. In some embodiments, the nonionic surfactant is added to the cell culture or cell culture supernatant to a final concentration of about 0.05%, 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, or about 1%. For example, a nonionic surfactant may be added to the cell culture or cell culture supernatant to a final concentration of about 0.5%.

Typically, the nonionic surfactant is allowed to mix with the cell culture or cell culture supernatant for a time sufficient to lyse host cells present in the cell culture or cell culture supernatant. For example, the nonionic surfactant is mixed with the cell culture or cell culture supernatant for a period of time from about 15 minutes to about 2 hours. In some embodiments, the nonionic surfactant is mixed with the cell culture or cell culture supernatant for a period of time from about 30 minutes to about 60 minutes.

The mixing may be performed at ambient or elevated temperatures. For example, the mixing of the nonionic surfactant can be performed at a temperature from about 15 ℃ to about 37 ℃. In some embodiments, the mixing of the nonionic surfactant can be performed at a temperature of about 18 ℃, 19 ℃, 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, or 37 ℃.

Notably, any desired nonionic surfactant can be used to lyse the host cells. Exemplary nonionic surfactants and types of nonionic surfactants for lysing host cells can include: polyarylphenol polyethoxy ethers; polyalkylphenol polyethoxy ethers; polyethylene glycol ether derivatives of saturated fatty acids; polyethylene glycol ether derivatives of unsaturated fatty acids; polyethylene glycol ether derivatives of fatty alcohols; polyethylene glycol ether derivatives of cycloaliphatic alcohols; fatty acid esters of polyoxyethylene sorbitan; oxyalkylating vegetable oil; alkoxylated acetylenic diols (alkoxylated acetylenic dials); polyalkoxylated alkylphenols; fatty acid alkoxylates; sorbitan alkoxylates; sorbitol esters; c (C) ₈ To C ₂₂ Alkyl or alkenyl polyglycosides; polyalkoxystyryl aryl ethers; alkylamine oxides; block copolymer ethers; polyalkoxylated fatty acid glycerides; polyalkylene glycol ethers; linear aliphatic or aromatic polyesters; an organosilicon; a polyarylphenol; sorbitol ester alkoxylates; and monoesters and diesters of ethylene glycol and mixtures thereof; ethoxylated tristyrylphenol; ethoxylated fatty alcohols; ethoxylated lauryl alcohol; ethoxylated castor oil; and ethoxylated nonylphenols; alkoxylated alcohols, amines or acids. In some embodiments of any of the aspects, the nonionic surfactant used to lyse the host cell is selected from the group consisting of: polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof.

Specific exemplary nonionic surfactants for lysing host cells include, but are not limited to, ECOSURF EH-9, polysorbates (such as polysorbate 20 (TWEEN 20), polysorbate 28, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 81 and polysorbate 85), ECOSURF EH-14, TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brij 10, brij 010, brij C10, brij 020, TERGL 15-S-7, ECOSURF SA-15, GITOL15-S-9, GIL 15-S-12, TOL-TERGL 7, TERGL-64, GITERGL-NP-10, GINP-4, GINP-TONP-12, GINP-4, GINP-TONP-14, GINP-TONP-4, and any combination thereof.

Preferably, the nonionic surfactant used to lyse the host cells is not Triton X-100.

In some embodiments, a zwitterionic surfactant may be added to the cell culture or cell culture supernatant to lyse the host cells. Exemplary zwitterionic surfactants include, but are not limited to, sulfonates such as CHAPS (3- [ (3-cholamidopropyl) -dimethylamino ] -1-propanesulfonic acid inner salt), CHAPSO (3- [ (3-cholamidopropyl) dimethylamino ] -2-hydroxy-1-propanesulfonic acid inner salt), 3- (decyldimethylammonium) propanesulfonic acid inner salt, 3- (dodecyldimethylammonium) propanesulfonic acid inner salt, 3- (N, N-dimethyltetradecylammonium) propanesulfonic acid inner salt, 3- (N, N-dimethyloctadecylammonium) propanesulfonic acid inner salt, 3- (N, N-dimethyloctylammonium) propanesulfonic acid inner salt, and 3- (N, N-dimethylhexadecylammonium) propanesulfonic acid inner salt; sultaines, such as cocamidopropyl hydroxysulfobetaine; betaines, such as cocamidopropyl betaine; and phosphates such as lecithin.

In some embodiments, the surfactant (e.g., zwitterionic surfactant) may be an amine oxide surfactant. For example, amine oxide surfactants may be added to the cell culture or cell culture supernatant to lyse the host cells. Amine oxide surfactants useful in the methods described herein may be trialkylamine N-oxides, e.g., of formula R ¹ R ² R ³ Amine oxide of NO, wherein R ¹ Is a substituted or unsubstituted alkyl or alkenyl group containing from about 8 to about 30 carbon atoms; and R is ² And R is ³ Are substituted or unsubstituted alkyl or alkenyl groups each containing from about 1 to about 18 carbon atoms. Non-limiting examples of trialkylamine N-oxides and trialkylamine N-oxide surfactants used are described in WO1998055581, which is incorporated herein by reference in its entirety.

The cell culture or cell culture supernatant may include impurities, for example, host cell DNA (hcna). Thus, a method for preparing a cell culture or cell culture supernatant for affinity chromatography may comprise a post-lysis step of removing or reducing the amount of impurities (e.g., hcna) from the cell culture or cell culture supernatant. Methods and compositions for reducing the amount of host cell DNA in a cell culture or cell culture supernatant are well known in the art. For example, a cationic amine or nuclease may be added to the cell culture or cell culture supernatant.

In some embodiments, the post-lysis step includes the addition of a selective precipitant to reduce or remove impurities, such as hcna, in the cell culture or cell culture supernatant. As used herein, "selective precipitant" refers to any agent, compound, or the like that, when added to a formulation comprising a population of recombinant viral particles and contaminating nucleic acid molecules, will affect the selective precipitation of at least a substantial amount of the contaminating nucleic acid molecules from the recombinant viral particles. Exemplary reagents for addition to the cell culture or cell culture supernatant in a post-lysis step include, but are not limited to, cetyltrimethylammonium bromide, cetylpyridinium chloride, benzethonium chloride, tetradecyltrimethylammonium chloride, polyethylenimine, and combinations thereof.

In some embodiments, a nuclease (e.g., an endonuclease) is added to the cell culture or cell culture supernatant to reduce or remove impurities (e.g., hcna). Exemplary endonucleases include those derived from prokaryotes and eukaryotes. In some embodiments, the nuclease isOr a salt-active nuclease (SAN).

Typically, the nuclease is added to the cell culture or cell culture supernatant to a final concentration of at least about 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1% (w/v, w/w or v/v) or higher. For example, the nuclease is added to the cell culture or cell culture supernatant to a final concentration of from about 0.05% to about 1%, from about 0.1% to about 0.95%, from about 0.15% to about 0.9%, from about 0.2% to about 0.85%, from about 0.25% to about 0.8%, from about 0.3% to about 0.75%, from about 0.35% to about 0.65%, from about 0.4% to about 0.6%, from about 0.45% to about 0.55%, from about 0.05% to about 0.4%, or from about 0.2% to about 0.4%. In some embodiments, the nuclease is added to the cell culture or cell culture supernatant at a final concentration of about 0.05%, 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95% or about 1%. For example, the nuclease may be added to the cell culture or cell culture supernatant to a final concentration of about 0.2%. In some embodiments, the nuclease may be added to the cell culture or cell culture supernatant to a final concentration of about 0.05% to about 0.4%.

Typically, the reagent or nuclease is allowed to mix with the cell culture or cell culture supernatant for a period of about 15, 20, 30, 35, 40, 45, 50, 55 minutes or more. In some embodiments, the reagent or nuclease is allowed to mix with the cell culture or cell culture supernatant for a period of time from about 10 minutes to about 4 hours. For example, the reagent or nuclease is mixed with the cell culture or cell culture supernatant for a period of time from about 15 minutes to about 3 hours. In some embodiments, the reagent or nuclease is mixed with the cell culture or cell culture supernatant for a period of time from about 30 minutes to about 120 minutes. For example, the reagent or nuclease is mixed with the cell culture or cell culture supernatant for a period of about 30 minutes.

In some embodiments, the method of preparing a cell culture or cell culture supernatant for affinity chromatography comprises the step of clarifying the cell culture or cell culture supernatant. For example, the method includes the step of clarifying the cell culture or cell culture supernatant by depth filtration to produce a clarified composition (e.g., a harvest medium) for affinity chromatography. Exemplary depth filters for use in the methods described herein include, but are not limited to ZetaDelipid Filter->Emphize AEX Filter, < > and->30/60ZA filter, < > and->90ZB08A filter, < >>DELI08A Delipid Filter, and +.>DELIP08A Delipid plus Filter (3M, st. Paul, minn.), -A.>grade 60HX、40MS、20MS，HC grade C0HC、D0HC、A1HC、B1HC、X0HC、F0HC；HC Pro grade D0SP, C0SP, and X0SP Millipore filters (EMD Millipore, billerica, mass.) and +.>A double-layer filter element.

In some embodiments, the method further comprises the step of concentrating the clarified cell culture or cell culture supernatant. For example, the method comprises the step of concentrating the clarified cell culture or cell culture supernatant by tangential flow filtration.

Further downstream processing

The eluate from the anion exchange chromatography comprising the isolated recombinant viral particles may be further processed using methods known in the art. Exemplary downstream treatments include, for example, tangential flow filtration, affinity chromatography, size exclusion chromatography, cation exchange chromatography, anion exchange chromatography, hydroxyapatite chromatography, and hydrophobic interaction chromatography. In some embodiments, the downstream process includes a step of tangential flow filtration. In some embodiments, the downstream processing includes a step of sterile filtration.

Recombinant viral particles

As used herein, the term "recombinant" for a viral particle refers to a viral particle that is the product of one or more procedures that result in a viral particle construct that differs from a naturally occurring viral particle. Methods for producing recombinant viral particles are well known in the art and available to those skilled in the art.

"primed particle" or "intact particle" (also alternatively referred to as "intact AAV particle") refers to a viral particle comprising a complete viral particle vector comprising a heterologous polynucleotide (e.g., a transgene, i.e., a polynucleotide other than the wild-type viral genome). "filled" or "completed" particles are also used interchangeably with "packaged particles" or "packaged viruses" or "packaged AAV" or "recombinantly expressed AAV". "empty particle" is also interchangeably referred to as "empty AAV particle" and refers to a viral particle that comprises at least one viral protein but lacks the entire genome (e.g., viral genome or recombinant genome). An "incomplete particle" is also interchangeably referred to as an "incomplete AAV particle" or an "incompletely filled AAV particle," and refers to a viral particle comprising at least one viral protein but a partial genome (e.g., viral genome or recombinant genome) that is deleted. Without being bound by any theory, in the inventions disclosed herein, a population or plurality of "intact" or "recombinantly-expressed AAV particles" may comprise an enriched population or a plurality of "intact" or "recombinantly-expressed AAV particles compared to" incomplete intact AAV particles ". As used herein, "incompletely intact particles" also include particles containing DNA from a host cell or pDNA for transfection. The empty particles do not include, for example, a complete viral particle vector comprising the heterologous polynucleotide. Notably, the terms "particle" and "capsid" are used interchangeably herein.

Recombinant viral particles (rAAV) described herein include recombinant adeno-associated viral (rAAV) particles. The rAAV particle can be AAV-1, AAV-2i8, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, AAV-16, or a chimeric, derivative, modification, or pseudotyped thereof. Thus, in some embodiments of any aspect, a rAAV particle comprises a capsid protein from serotype AAV-1, AAV-2i8, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, or a chimeric, derivative, modification, or pseudotyped thereof. In some embodiments, the rAAV particles include capsids from polyploids (also referred to as haploids) because they can include different combinations of VP1, VP2, and VP3 AAV serotypes in a single AAV capsid, as described in PCT/US18/22725 and US10,550,405, which are incorporated herein by reference. In some embodiments, the rAAV can include rAAV virions. In certain embodiments, the rAAV capsid comprises one or more amino acid substitutions, additions, and/or deletions; for example, a capsid comprising an insertion peptide for targeting.

Notably, the viral particles and capsids are used interchangeably herein.

As used herein, the term "recombinant AAV (rAAV) vector" or "gene delivery vector" refers to a viral particle that functions as a nucleic acid delivery vehicle, including the vector genome packaged within an AAV capsid (e.g., a therapeutic payload packaged into a viral genome). Alternatively, in some instances, the term "vector" may be used alone to refer to a vector genome/therapeutic payload.

An "rAAV vector genome" or "rAAV genome" is an AAV genome (i.e., vDNA) comprising one or more heterologous nucleotide sequences. The production of rAAV vectors typically requires only 145 base terminal repeats (TR (s)) in cis to produce the virus. All other viral sequences are unnecessary and can be provided in trans (Muzyczka, (1992) curr. Topics microbiol. Immunol. 158:97). Typically, the rAAV vector genome will retain only minimal TR sequences in order to maximize the size of the transgene that can be efficiently packaged by the vector. Structural and non-structural protein coding sequences may be provided in trans (e.g., from a vector, such as a plasmid, or by stable integration of the sequence into a packaging cell). The rAAV vector genome comprises at least one TR sequence (e.g., an AAV TR sequence, a synthetic TR sequence, or other parvoviral TR sequence), optionally two TRs (e.g., two AAV TRs), which will typically be located 5 'and 3' of, but not necessarily contiguous with, the heterologous nucleotide sequence. TR may be the same or different from each other.

The term "terminal repeat" or "TR" includes any viral terminal repeat and synthetic sequence that forms a hairpin structure and functions as an Inverted Terminal Repeat (ITR), such as a "double D sequence", described in U.S. Pat. No. 5,478,745, samulski et al. The capsid structure of autonomous parvoviruses and AAV is described in more detail in BERNARD N.FIELDS et al, VIROLOGY, vol.2, chapter 69&70 (4 th edition, lippincott-Raven Press). In addition, see descriptions of the crystal structures of AAV2 (Xie et al, (2002) Proc. Nat. Acad. Sci.99:10405-10), AAV4 (Padron et al, (2005) I. Virol.79:5047-58), AAVS (Walters et al, (2004) I. Virol.78:3361-71) and CPV (Xie et al, (1996) I. Mol. Biol.6:497-520 and Tsao et al, (1991) Science 251:1456-64).

An "AAV terminal repeat" or "AAV TR" may be derived from any AAV, including but not limited to serotypes AAV-1, AAV-2i8, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, or any other AAV now known or later discovered. AAV terminal repeats need not have wild-type terminal repeat sequences (e.g., wild-type sequences can be altered by insertions, deletions, truncations, or missense mutations), so long as at least one of the terminal repeats mediates the desired function, functional TR, e.g., replication, viral packaging, integration, and/or proviral rescue, etc. The skilled person knows to select a Rep protein which is functional for replication of a functional TR. In some embodiments, the methods described herein produce recombinant AAV having 145bp ITR (inverted terminal repeat) sequences or smaller ITR sequences. In some embodiments, the recombinant AAV produced by the method comprises an ITR of 130 bp. In some embodiments, the recombinant AAV produced by the method comprises an ITR of less than 130 bp. In some embodiments, the recombinant AAV produced by the method comprises a synthetic or mutated ITR or a limiting ITR, e.g., as in WO2014143932, US 9447433; WO2011088081, US 9169494; WO 2019143950, which is incorporated herein by reference in its entirety.

In some embodiments of any aspect, the viral particle (e.g., rAAV) comprises a transgene. The transgene may be any transgene. Transgene is used interchangeably herein with heterologous transgene. As used herein, "transgene" refers to a polynucleotide or nucleic acid that is intended to or has been introduced into a cell or organism. Transgenes include any nucleic acid, such as a gene encoding a polypeptide or protein. Suitable transgenes for gene therapy, for example, are well known to those skilled in the art. For example, the rAAV described herein can comprise transgenes and uses including, but not limited to, U.S. patent nos. 6,547,099, 6,506,559, and 4,766,072; published U.S. application nos. 20020006664, 20030153519, 20030139363; and those described in published PCT applications WO01/68836 and WO03/010180, as well as miRNAs and other transgenes such as W02017/152149, each of which is incorporated herein by reference in its entirety.

The terms "host cell" and "producer cell" are used interchangeably herein and refer to any cell capable of producing a recombinant virus, e.g., a recombinant adeno-associated viral vector. For example, a host cell can be transfected with one or more nucleic acids to produce a recombinant virus, e.g., a recombinant adeno-associated viral particle. Notably, the cells can be, for example, from about 1×10 ⁶ Up to about 8X 10 ⁷ Transfection was performed at a density of viable cells/mL. Cells may be cultured in suspension.

The host cell may be a mammalian, bacterial or yeast cell. In some embodiments of any of the aspects, the host cell may be a HeLa cell, COS-1 cell, COS-7 cell, HEK293 cell, A549 cell, BHK cell, BSC-1 cell, BSC-40 cell, vero cell, sf "C9 cell, sf-21 cell, tn-368 cell, BTI-Tn-5B1-4 (High-Five) cell, saos cell, C2C12 cell, L cell, HT1080 cell, hepG2 cell, WEHI cell, 3T3 cell, 10T1/2 cell, MDCK cell, BMT-10 cell, WI38 cell, or primary fibroblast, hepatocyte or myoblast of mammalian origin. The term "cell" or "cell line" is understood to include modified or engineered variants of the indicated cell or cell line, unless otherwise indicated. Recombinant viral particles (e.g., aav) can be produced from a host cell using any suitable method known in the art.

The titer of the recombinant viral particles (e.g., rAAV vector particles) is from about 1 x 10 ⁹ Up to about 1X 10 ¹⁴ Vector genome/mL, or from about 1X 10 ¹⁰ Up to about 5X 10 ¹³ Vector genome/mL or from about 1X 10 ¹⁰ Up to about 1X 10 ¹³ Vector genome/mL, or from about 1X 10 ¹¹ Up to about 5X 10 ¹² Viral particles/mL. In certain embodiments, the titer of the recombinant viral particles (e.g., rAAV vector particles) is from about 1×10 ¹¹ Up to about 1X 10 ¹² Vector genome/mL or viral particle/mL. In some embodiments, the titer may be from about 1 x 10 ⁹ Up to about 1X 10 ¹¹ TCID 50/mL. In some embodiments, the particle to infectivity ratio of a recombinant viral particle (e.g., rAAV) can be from about 10 ² To about 10 ⁵ vg/TCID50, or from about 10 ² Up to about 5X 10 ⁴ vg/TCID50, or from about 10 ² To about 10 ⁴ Range of vg/TCID50. In certain embodiments, the particle to infectivity ratio is from 10 ² To about 10 ³ vg/TCID50. In another embodiment, the particle to infectivity ratio is less than 10 ² vg/TCID50. Notably, the "vector genome" may be used interchangeably with "viral genome".

TCID50 determination: the infection titer (TCID 50) method was used to assess AAV infectivity of the drug product in HeLa RC32 cells in vitro. In this assay HeLa RC32 cells were transduced with adenovirus type 5 helper virus and a series of dilutions of the drug product. Three days after infection, the cells were treated with proteinase K to digest the protein and the replicated AAV vector DNA was quantified using qPCR techniques. The method utilizes a detection system based on DNA primers and fluorescent dyes. The absolute amount of ITR targeting sequence from vector DNA was replaced by an interpolated value from a standard curve prepared with plasmid. Samples containing ITRs were prepared as test samples and used as assay controls. Results are expressed in units of infection per milliliter (IU/mL). Notably, to compare TCID50/mL between different formulations, TCID50/mL is preferably normalized to vg/mL.

"partitioning" or "partitioning effect" is used in the present application to separate "intact" or recombinantly expressed viral particles from empty viral particles. In the present application, the partitioning effect is achieved by means of Anion Exchange (AEX), wherein the conditioning buffer (e.g., dilution buffer and/or equilibration buffer), the empty particles (e.g., empty AAV particles) move to the unbound fraction, thereby producing an enriched population of intact viral particles (e.g., intact AAV particles) in the bound fraction of the AEX chromatograph. Notably, the enriched population of intact or packaged viral particles can include incompletely packaged or incompletely intact viral particles. Without being bound to any theory, the operating window that achieves the best segmentation effect may be established, for example, using a high throughput screening method, as shown in example 1. Thus, it may provide a valuable alternative to bind-elute or standard flow-through chromatography modes. It is not meant to specify any one type of liquid chromatography, e.g. "split" chromatography, which is a term used when exploiting the relative solubilities of the stationary and mobile phases.

Compositions comprising recombinant AAV vector particles

The rAAV vectors disclosed herein can be formulated into compositions. For example, the rAAV vectors disclosed herein can be formulated in a pharmaceutical composition with pharmaceutically acceptable excipients (i.e., one or more pharmaceutically acceptable carrier substances and/or additives, e.g., buffers, carriers, excipients, stabilizers, etc.). The composition (e.g., a pharmaceutical composition) may be provided in the form of a kit.

Thus, in one aspect, provided herein are compositions comprising the recombinant AAV vector particles described herein. Typically, the composition comprises recombinant AAV vector particles described herein at a concentration of from about 1e ⁹ vg/mL to about 1e ¹⁵ vg/mL. In some implementationsIn embodiments, the composition comprises recombinant AAV vector particles described herein at a concentration of from about 1e ¹⁰ vg/mL to about 1e ¹⁴ vg/mL. In some embodiments, the composition comprises recombinant AAV vector particles described herein at a concentration of from about 1e ¹² vg/mL to about 1e ¹⁴ vg/mL. In some embodiments, the composition comprises recombinant AAV vector particles described herein at a concentration of from about 1e ¹² vg/mL to about 1e ¹⁵ vg/mL. For example, the composition comprises recombinant AAV vector particles described herein at a concentration of from about 3e ¹² vg/mL to about 3e ¹³ vg/mL, from about 2.5e ¹² vg/mL to about 1e ¹⁴ vg/mL, from about 3e ¹³ vg/mL to about 1e ¹⁴ vg/mL or from 1e ¹³ vg/mL to about 1e ¹⁴ vg/mL。

In some embodiments, the composition comprises recombinant AAV vector particles described herein at a concentration of about 1e ¹² vg/mL, or about 1.5e ¹² vg/mL, or about 2e ¹² vg/mL, or about 2.5e ¹² vg/mL, or about 3e ¹² vg/mL, or about 3.5e ¹² vg/mL, or about 4e ¹² vg/mL, or about 4.5e ¹² vg/mL, or about 5e ¹² vg/mL, or about 5.5e ¹² vg/mL, or about 6e ¹² vg/mL, or about 6.5e ¹² vg/mL, or about 7e ¹² vg/mL, or about 7.5e ¹² vg/mL, or about 8e ¹² vg/mL, or about 8.5e ¹² vg/mL, or about 9e ¹² vg/mL, or about 9.5e ¹³ vg/mL, or about 1e ¹³ vg/mL, or about 1.5e ¹³ vg/mL, or about 2e ¹³ vg/mL, or about 2.5e ¹³ vg/mL, or about 3e ¹³ vg/mL, or about 3.5e ¹³ vg/mL, or about 4e ¹³ vg/mL, or about 4.5e ¹³ vg/mL, or about 5e ¹³ vg/mL, or about 5.5e ¹³ vg/mL, or about 6e ¹³ vg/mL, or about 6.5e ¹³ vg/mL, or about 7e ¹³ vg/mL, or about 7.5e ¹³ vg/mL, or about 8e ¹³ vg/mL, or about 8.5e ¹³ vg/mL, or about 9e ¹³ vg/mL, or about 9.5e ¹³ vg/mL, or about 1e ¹⁴ vg/mL。

In some embodiments, the composition is substantially free of glycine.

In some embodiments, the composition is substantially free of trehalose. For example, the composition comprises a bulking agent, and the bulking agent is not trehalose, such as trehalose dehydrate.

In some embodiments, the composition is substantially free of sodium chloride.

In some embodiments, the composition is substantially free of nonionic surfactants. For example, the composition is substantially free of polysorbate 80 (PS 80). In some embodiments, the composition comprises a non-ionic surfactant, and wherein the non-ionic surfactant is not a polysorbate, such as PS80.

In some embodiments, the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium, or potassium salts, e.g., naCl).

Typically, the pH of the composition is from about 6.5 to about 8.0. For example, the pH of the composition is from about 6.5 to about 7.5. In some embodiments, the pH of the composition is from about 7 to about 8. For example, the pH of the composition is from about 7.3 to about 7.9. In some other non-limiting examples, the pH of the composition is from about 7.4 to about 7.8 or from about 7.4 to about 7.7. In some embodiments, the pH of the composition is from about 7.3 to about 7.6, for example, from about 7.3 to about 7.55. In some preferred embodiments, the pH of the composition is less than about 7.5. For example, the pH of the composition is about 7.4 or less, about 7.3 or less, about 7.2 or less, about 7.1 or less, about 7.0 or less, about 6.9 or less, about 6.8 or less, about 6.7 or less, about 6.6 or less, or about 6.5 or less. For example, the pH of the composition is about 7.4 or less, about 7.3 or less, about 7.2 or less, about 7.1 or less, or about 7.0 or less. In some embodiments, the pH of the composition is about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8. In some embodiments, the pH of the composition is at least about 7.0, at least about 7.2, at least about 7.3, at least about 7.4, at least about 7.5, at least about 7.6, at least about 7.7, or at least about.

In some embodiments, the composition comprises a buffer. It should be noted that any physiological buffer may be used. Non-limiting examples of buffers include, but are not limited to, PBS, tris.HCl, phosphate, citric acid, histidine, tromethamine, succinic acid, malic acid, alpha-ketoglutaric acid, carbonate (bicarbonate-carbonate buffer), and protein buffers. In some embodiments, the buffer is PBS. In some embodiments, the buffer comprises Tris. In some embodiments, the buffer is tris.hcl. In some embodiments, the buffer is a histidine buffer.

Typically, the salt concentration of the buffer is about 50mM to about 750mM. For example, the salt concentration of the buffer is from about 75mM to about 700mM, from about 100mM to about 650mM, from about 120mM to about 600mM, or from about 140mM to about 550mM. In some embodiments, the salt concentration of the buffer is from about 150mM to about 400mM. In some embodiments, the salt concentration of the buffer is about 150mM, about 175mM, about 200mM, about 225mM, about 250mM, about 275mM, about 300mM, about 325mM, about 350mM, about 375mM, about 400mM, about 425mM, about 450mM, or about 475mM. In some embodiments, the salt concentration of the buffer is about 150mM, about 200mM, or about 365mM.

In some embodiments, the ionic strength of the composition is at least about 100mM. For example, the ionic strength of the composition is from about 125mM to about 750mM, or from about 150mM to about 500mM, or from about 175mM to about 700mM, or from about 200mM to about 600mM, or from about 225mM to about 550mM, or from about 250mM to about 500mM, or from about 275mM to about 450mM, or from about 300mM to about 400mM. In some embodiments, the ionic strength of the composition is at least about 125mM, at least about 150mM, at least about 175mM, at least about 200mM, at least about 225mM, at least about 250mM, at least about 275mM, at least about 300mM, at least about 325mM, at least about 350mM, at least about 375mM, at least about 400mM, at least about 425mM, at least about 450mM, at least about 475mM, or at least about 500mM. In some preferred embodiments, the ionic strength of the composition is about 170mM, about 210mM, or about 380mM. In some embodiments, the ionic strength of the composition is less than 100mM, e.g., about 95mM, about 90mM, about 85mM, about 80mM, about 75mM, about 70mM, about 65mM, about 60mM, about 55mM, about 50mM, or even less.

Typically, the osmotic pressure of the composition is maintained at a near isotonic level. For example, the osmolality of the composition may be from about 100 to about 600mOsm, such as from about 125 to about 500mOsm, or from about 130 to about 350mOsm, or from about 140 to about 400mOsm, or from about 140 to about 350mOsm, or from about 200 to about 400mOsm, or from about 500 to about 600mOsm, or from about 200 to about 600mOsm, or from about 300 to about 600mOsm, or from about 200 to about 500mOsm, or from about 300 to about 400mOsm, or from about 150 to about 350mOsm, or from about 175 to about 300mOsm. In some embodiments, the composition has an osmolality of from about 300mOsm to about 375mOsm, or from about 200mOsm to about 350mOsm, or from about 225mOsm to about 325mOsm, or from about 525mOsm to about 590mOsm. In some embodiments of the present invention, in some embodiments, the composition has an osmolality of about 125mOsm, about 126mOsm, about 127mOsm, about 128mOsm, about 129mOsm, about 130mOsm, about 131mOsm, about 132mOsm, about 133mOsm, about 134mOsm, about 135mOsm, about 136mOsm, about 137mOsm, about 138mOsm, about 139mOsm, about 140mOsm, about 141mOsm, about 142mOsm, about 143mOsm, about 144mOsm, about 145mOsm, about 146mOsm, about 147mOsm, about 148mOsm, about 149mOsm, about 150mOsm, about 151mOsm, about 152mOsm, about 153mOsm, about 154mOsm about 155mOsm, about 160mOsm, about 165mOsm, about 170mOsm, about 175mOsm, about 180mOsm, about 185mOsm, about 190mOsm, about 191mOsm, about 192mOsm, about 193mOsm, about 194mOsm, about 195mOsm, about 200mOsm, about 205mOsm, about 210mOsm, about 215mOsm, about 220mOsm, about 225mOsm, about 250mOsm, about 275mOsm, about 280mOsm, about 285mOsm, about 290mOsm, about 295mOsm, about 300mOsm, about 301mOsm, about 302mOsm, about 303mOsm, about 304mOsm, about 305mOsm, about 210mOsm, about 215mOsm, about 285mOsm, about 290mOsm, about 295mOsm about 155mOsm, about 160mOsm, about 165mOsm, about 170mOsm, about 175mOsm, about 180mOsm, about 185mOsm, about 190mOsm, about 191mOsm, about 192mOsm, about 193mOsm, about 194mOsm, about 195mOsm, about 200mOsm, about 205mOsm, about 210mOsm about 215mOsm, about 220mOsm, about 225mOsm, about 250mOsm, about 275mOsm, about 280mOsm, about 285mOsm, about 290mOsm, about 295mOsm, about 300mOsm, about 301mOsm, about 302mOsm, about 303mOsm, about 304mOsm, about 305mOsm, about. In some embodiments, the composition comprises an isotonic solution.

In some embodiments, the composition has an osmolality of about 500mOsm or less, or about 475mOsm or less, about 450mOsm or less, or about 425mOsm or less, or about 400mOsm or less, or about 375mOsm or less, or about 350mOsm or less, or about 325mOsm or less, or about 300mOsm or less, or about 375mOsm or less, or about 350mOsm or less, or about 340mOsm or less, or about 335mOsm or less, or about 330mOsm or less, or about 325mOsm or less, or about 300mOsm or less, or about 280mOsm or less, or about 260mOsm or less, or about 250mOsm or less, or about 240mOsm or less, or about 230mOsm or less, or about 225mOsm or less, or about 220mOsm or less, or about 335mOsm or less, or about 180mOsm or less, or about 180mOsm or about 150mOsm or less. In some embodiments, the composition comprises one or more ions and/or salts thereof. Exemplary ions include, but are not limited to, sodium, potassium, chlorine, magnesium, ammonium, carbonate, nitrate, chlorate, chlorite, and calcium. The ions may be provided in the form of salts, e.g. sodium, potassium, magnesium And/or calcium halide (F, cl, br, I) salts, non-limiting examples of which include NaCl, KCl, mgCl ₂ 、CaCl ₂ And combinations thereof. Other exemplary salts that may be used include, but are not limited to, carboxylates, e.g., acetate, propionate, pyrrolidone carboxylate (or pidolates), or sorbate; polyhydroxy carboxylates, for example gluconate, heptagluconate, ketogluconate, lactate gluconate, ascorbate or pantothenate; mono-or poly-carboxylic hydroxy acid salts, e.g. citrates or lactates; amino acid salts, for example, aspartate or glutamate; and fulvates (fulvates). The salt concentration is included at a concentration of from about 500. Mu.M to about 500mM, e.g., at a concentration of about 500. Mu.M, about 750. Mu.M, about 1mM, about 1.3mM, about 1.5mM, about 1.7mM, about 2.3mM, about 2.5mM, about 2.7mM, about 3.3mM, about 3.5mM, about 3.7mM, about 4.3mM, about 4.5mM, about 4.7mM, about 5mM, about 10mM, about 25mM, about 50mM, about 75mM, about 100mM, about 125mM, about 150mM, about 175mM, about 200mM, about 225mM, about 250mM, about 275mM, about 300mM, about 325mM, about 350mM, about 375mM, about 400mM, about 425mM, about 450mM, about 475mM, or about 500mM.

In some embodiments, the composition comprises one or more multivalent ions and/or salts thereof. Exemplary multivalent ions include, but are not limited to, calcium, citrate, sulfate, magnesium, and phosphate. Multivalent ions and/or salts thereof may each be included in the composition at the following concentrations: from about 500. Mu.M to about 500mM, for example, at a concentration of about 500. Mu.M, about 750. Mu.M, about 1mM, about 1.3mM, about 1.5mM, about 1.7mM, about 2.3mM, about 2.5mM, about 2.7mM, about 3.3mM, about 3.5mM, about 3.7mM, about 4.3mM, about 4.5mM, about 4.7mM, about 5mM, about 10mM, about 25mM, about 50mM, about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 125mM, about 150mM, about 175mM, about 200mM, about 225mM, about 250mM, about 275mM, about 300mM, about 325mM, about 350mM, about 375mM, about 400mM, about 425mM, about 450mM, about 475mM or about 500mM. Non-limiting examples of salts are NaCl, KCl, caCl ₂ 、CaSO ₄ 、MgSO ₄ 、Na ₃ PO ₄ 、CaCO ₃ 、NaNO ₃ 、Al ₂ (SO ₄ ) ₃ 。

In some embodiments, the composition comprises NaCl. When present, the concentration of NaCl may be from about 100mM to about 500mM, or from about 125mM to about 450mM, or from about 100mM to about 200mM, or from about 150mM to about 200mM. For example, the composition may comprise NaCl at a concentration of from about 150mM to about 425mM, from about 175mM to about 400mM, or from about 175mM to about 375mM. In some embodiments, the composition comprises NaCl at a concentration from about 200mM to about 375mM. For example, the composition may comprise NaCl at a concentration of about 100mM, about 125mM, about 130mM, about 135mM, about 137mM, about 140mM, about 145mM, about 150mM, about 155mM, about 160mM, about 165mM, about 170mM, about 175mM, about 180mM, about 185mM, about 190mM, about 195mM, about 200mM, about 205mM, about 210mM, about 215mM, about 220mM, about 225mM, about 230mM, about 235mM, about 240mM, about 245mM, about 250mM, about 255mM, about 260mM, about 265mM, about 270mM, about 275mM, about 300mM, about 325mM, about 350mM, about 375mM, or about 400mM. In some preferred embodiments, the composition comprises NaCl at a concentration of about 140mM, about 175mM, about 200mM, about 300mM, about 350mM, or about 375mM. In some embodiments, the composition is substantially free of NaCl.

In some embodiments, the composition comprises KCl. When present, the concentration of KCl may be from about 1mM to about 10mM. For example, the composition may comprise KCl at a concentration ranging from about 1.5mM to about 7.5mM. In some embodiments, the composition may comprise KCl at a concentration of from about 2mM to about 5.5mM. For example, the composition can comprise KCl at a concentration of about 2mM, about 2.25mM, about 2.5mM, about 2.7mM, about 2.75mM, about 3mM, about 3.25mM, about 3.5mM, about 3.75mM, about 4mM, about 4.25mM, about 4.5mM, about 4.75mM, about 5mM, or about 5.25mM. In some preferred embodiments, the composition comprises KCl at a concentration of about 2.7mM, about 3mM, about 3.5mM or about 5mM. In some embodiments, the composition is substantially free of KCl.

In some embodiments, the composition comprises CaCl ₂ . CaCl, when present ₂ May be at a concentration of from about 0.1mM to about 2mM. For example, the composition may comprise CaCl ₂ The concentration is from about 0.5mM to about 1.5mM. In some embodiments, the composition comprises CaCl ₂ The concentration is from about 0.75mM to about 1.25mM. For example, the composition may comprise CaCl ₂ The concentration is about 0.1mM, about 0.15mM, about 0.2mM, about 0.25mM, about 0.3mM, about 0.35mM, about 0.4mM, about 0.45mM, about 0.5mM, about 0.55mM, about 0.6mM, about 0.65mM, about 0.7mM, about 0.75mM, about 0.8mM, about 0.85mM, about 0.9mM, about 0.95mM, about 1mM, about 1.1mM, about 1.15mM, about 1.2mM, about 1.25mM, about 1.3mM, about 1.35mM, about 1.4mM, about 1.45mM, about 1.5mM, about 1.55mM, about 1.6mM, about 1.65mM, about 1.7mM, about 1.75mM, about 1.8mM, about 1.85mM, about 1.9mM, about 1.95mM or about 2mM. In some preferred embodiments, the composition comprises CaCl at a concentration of about 0.9mM ₂ . In some embodiments, the composition is substantially free of CaCl ₂ 。

In some embodiments, the composition comprises MgCl ₂ . When present, mgCl ₂ May be at a concentration of from about 0.1mM to about 1.5mM. For example, the composition may comprise MgCl ₂ The concentration is from about 0.25mM to about 1mM. In some embodiments, the composition comprises MgCl ₂ The concentration is from about 0.25mM to about 0.75mM. For example, the composition may comprise MgCl ₂ The concentration is about 0.1mM, about 0.15mM, about 0.2mM, about 0.25mM, about 0.3mM, about 0.35mM, about 0.4mM, about 0.45mM, about 0.5mM, about 0.55mM, about 0.6mM, about 0.65mM, about 0.7mM, about 0.75mM, about 0.8mM, about 0.85mM, about 0.9mM, about 0.95mM, about 1mM, about 1.1mM, about 1.15mM, about 1.2mM, about 1.25mM, about 1.3mM, about 1.35mM, about 1.4mM, about 1.45mM, or about 1.5mM. In some preferred embodiments, the composition comprises MgCl at a concentration of about 0.5mM ₂ . In some embodiments, the composition is substantially free of MgCl ₂ 。

In some embodiments, the composition comprises MgSO ₄ . When present, mgSO ₄ May be from about 5mM to about 150mM. For example, the composition may comprise MgSO ₄ At a concentration of from about 10mM to about 120mM, or from about 10mM to about 50mM, or from about 15mM to about 45mM, or From about 75mM to about 125mM, or from about 80mM to about 100mM, or from about 85mM to about 95mM. In some embodiments, the composition comprises MgSO ₄ The concentration is from about 15mM to about 100mM. For example, the composition may comprise MgSO ₄ The concentration is about 5mM, about 10mM, about 15mM, about 25mM, about 30mM, about 35mM, about 40mM, about 45mM, about 50mM, about 55mM, about 60mM, about 65mM, about 70mM, about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 115mM, or about 120mM. In some embodiments, the composition is substantially free of MgSO ₄ 。

In some embodiments, the composition comprises heptagluconate or a salt thereof. For example, the composition includes calcium heptagluconate, such as alpha-d-calcium heptagluconate. When present, the heptagluconate or salt thereof (e.g., a-d-calcium heptagluconate) may be present at a concentration from about 1% (w/v) to about 20% (w/v). For example, the composition may comprise heptagluconate or a salt thereof (e.g., a-d-calcium heptagluconate) at a concentration of from about 2.5% (w/v) to about 17.5% (w/v), from about 5% (w/v) to about 15% (w/v), or from about 7.5% (w/v) to about 12.5% (w/v). In some embodiments, the composition comprises heptagluconate or a salt thereof (e.g., a-d-calcium heptagluconate) at a concentration of about 1% (w/v), about 2.5% (w/v), about 5% (w/v), about 7.5% (w/v), about 10% (w/v), about 12.5% (w/v), about 15% (w/v), about 17.5% (w/v), or about 20% (w/v). In some embodiments, the composition is substantially free of heptagluconate or a salt thereof, e.g., calcium heptagluconate, such as alpha-d-calcium heptagluconate.

In some embodiments, the composition comprises a phosphate (e.g., a mono-or di-phosphate) or salt thereof. When present, the phosphate (e.g., mono-or di-phosphate) or salt thereof may be at a concentration of from about 5mM to about 30mM. For example, the composition may comprise phosphate (e.g., mono-or di-phosphate) or a salt thereof at a concentration of from about 7.5mM to about 25mM. In some embodiments, the composition comprises phosphate (e.g., mono-or di-phosphate) or a salt thereof at a concentration of from about 10mM to about 20mM. For example, the composition comprises phosphate (e.g., mono-or di-phosphate) or a salt thereof at a concentration of about 5mM, about 7.5mM, about 10mM, about 12.5mM, about 15mM, about 17.5mM, about 20mM, about 22.5mM, about 25mM, about 25.75mM, or about 30mM. In some preferred embodiments, the composition comprises phosphate (e.g., mono-or di-phosphate) or a salt thereof at a concentration of about 10mM, about 15mM, or about 20mM.

In some embodiments, the composition comprises a monobasic phosphate or salt thereof at a concentration of from about 0.25mM to about 3mM. For example, the composition comprises a monophosphate salt or salt thereof at a concentration of from about 0.5mM to about 2.75mM, from about 0.75mM to about 2.5mM, or from about 1mM to about 2.25mM. In some embodiments, the composition comprises a monobasic phosphate or salt thereof at a concentration of from about 1.25mM to about 2.25mM. For example, the composition comprises a monophosphate salt or salt thereof at a concentration of about 0.25mM, about 0.5mM, about 0.75mM, about 1mM, about 1.25mM, about 1.5mM, about 1.75mM, about 2mM, about 2.25mM, about 2.5mM, about 2.75mM, or about 3mM. In some preferred embodiments, the composition comprises a monobasic phosphate or salt thereof at a concentration of about 1mM, about 1.5mM, or about 2mM. In some embodiments, the monobasic phosphate or salt thereof is potassium dihydrogen phosphate. In some embodiments, the composition is substantially free of monobasic phosphate, e.g., monobasic potassium phosphate.

In some embodiments, the composition comprises a dibasic phosphate or salt thereof at a concentration of from about 5mM to about 15mM. For example, the composition comprises a dibasic phosphate or salt thereof at a concentration of from about 7.5mM to about 12.5mM or from about 8mM to about 10mM. In some embodiments, the composition comprises a dibasic phosphate or salt thereof at a concentration of about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 7.5mM, about 8mM, about 8.5mM, about 9mM, about 9.5mM, about 10mM, about 10.5mM, about 11mM, about 11.5mM, about 12mM, about 12.5mM, about 13mM, about 13.5mM, about 14mM, about 14.5mM, or about 15mM. In some preferred embodiments, the composition comprises a dibasic phosphate or salt thereof at a concentration of about 8mM or about 9mM. In some embodiments, the dibasic phosphate or salt thereof is disodium hydrogen phosphate. In some embodiments, the composition is substantially free of dibasic phosphate, e.g., disodium phosphate.

In some embodiments, the composition comprises Tris (e.g., tris.hcl) or a salt thereof at a concentration of from about 1mM to about 50mM. For example, the composition comprises Tris (e.g., tris.hcl) or a salt thereof at a concentration of from about 5mM to about 40mM, from about 7.5mM to about 35mM, from about 10mM to about 30mM, or from about 15mM to about 25mM. In some embodiments, the composition comprises Tris (e.g., tris.hcl) or a salt thereof at a concentration of about 5mM, about 7.5mM, about 10mM, about 12.5mM, about 15mM, about 17.5mM, about 20mM, about 22.5mM, about 25mM, about 27.5mM, about 30mM, about 32.5mM, about 35mM, about 37.5mM, about 40mM, about 42.5mM, about 45mM, about 47.5mM, or about 50mM.

In some embodiments, the composition comprises histidine or a salt thereof at a concentration of from about 1mM to about 50mM. For example, the composition comprises histidine or a salt thereof at a concentration of from about 5mM to about 40mM, from about 7.5mM to about 35mM, from about 10mM to about 30mM, or from about 15mM to about 25mM. In some embodiments, the composition comprises histidine or a salt thereof at a concentration of about 5mM, about 7.5mM, about 10mM, about 12.5mM, about 15mM, about 17.5mM, about 20mM, about 22.5mM, about 25mM, about 27.5mM, about 30mM, about 32.5mM, about 35mM, about 37.5mM, about 40mM, about 42.5mM, about 45mM, about 47.5mM, or about 50mM.

In some embodiments, the composition further comprises a filler. In some embodiments, the filler is a polyol or povidone (PVP K24). Exemplary polyols include, but are not limited to, polyhydroxy hydrocarbons, monosaccharides, disaccharides, and trisaccharides. Some exemplary polyols include, but are not limited to, sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, and dextran. In some embodiments, the polyol is sorbitol, sucrose, or mannitol. In some embodiments, the filler is sorbitol. In some embodiments, the bulking agent is sucrose. In some embodiments, the filler is mannitol. In some embodiments, the bulking agent is not trehalose, e.g., trehalose dehydrate. In some embodiments, the bulking agent is not dextran, e.g., dextran T40 and/or dextran T10.

When present, the filler (e.g., polyol or povidone (PVP K24)) may be present at a concentration of from about 0.5% (w/v) to about 10% (w/v). For example, the composition may comprise a filler (e.g., a polyol or povidone (PVP K24)) at a concentration of from about 1% (w/v) to about 7.5% (w/v), for example, from about 3% (w/v) to about 6% (w/v). In some embodiments, the composition comprises a filler (e.g., polyol or povidone (PVP K24)) at a concentration of about 1% (w/v), about 1.5% (w/v), about 2% (w/v), about 2.5% (w/v), about 3% (w/v), about 3.5% (w/v), about 4% (w/v), about 4.5% (w/v), about 5% (w/v), about 5.5% (w/v), about 6% (w/v), about 6.5% (w/v), about 7% (w/v), about 7.5% (w/v), about 8% (w/v), about 8.5% (w/v), about 9% (w/v), about 9.5% ((w/v), or about 10% (w/v). In some preferred embodiments, the composition comprises a filler (e.g., polyol or povidone (PVP K24) at a concentration of about 1% (w/v), about 3% (w/v), or about 5% (w/v).

In some embodiments, the composition comprises glycerin, sorbitol, sucrose, or mannitol at a concentration of from about 1% (w/v) to about 10% (w/v). In some embodiments, the composition comprises glycerol, sorbitol, sucrose, or mannitol at a concentration of from about 1% (w/v) to about 10% (w/v). In some embodiments, the composition comprises sorbitol at a concentration of from about 3% (w/v) to about 6% (w/v). In some embodiments, the composition comprises sorbitol at a concentration of about 1% (w/v), about 2% (w/v), about 3% (w/v), about 4% (w/v), about 5% (w/v), about 6% (w/v), about 7% (w/v), about 8% (w/v), about 9% (w/v), or about 10% (w/v). In some embodiments, the composition comprises sucrose at a concentration of from about 3% (w/v) to about 6% (w/v). In some embodiments, the composition comprises sucrose at a concentration of about 1% (w/v), about 2% (w/v), about 3% (w/v), about 4% (w/v), about 5% (w/v), about 6% (w/v), about 7% (w/v), about 8% (w/v), about 9% (w/v), or about 10% (w/v). In some embodiments, the composition comprises mannitol at a concentration of from about 3% (w/v) to about 6% (w/v). In some embodiments, the composition comprises mannitol at a concentration of about 1% (w/v), about 2% (w/v), about 3% (w/v), about 4% (w/v), about 5% (w/v), about 6% (w/v), about 7% (w/v), about 8% (w/v), about 9% (w/v), or about 10% (w/v).

In some embodiments, the composition further comprises a nonionic surfactant. The nonionic surfactant may be selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof. Non-limiting examples of suitable nonionic surfactants include polyoxyethylene (12) isooctylphenyl ether (e.g.,CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ecosurf (tm) SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof. In some embodiments, the nonionic surfactant is selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68 (PF 68), polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S -7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, polysorbate 20, and any combination thereof. In some embodiments, the nonionic surfactant is poloxamer P188, poloxamer P407, pluronic F-68, ecosurf SA-15, brij S20, tergitol NP-10, IGEPAL CA 720, or Tween 80. In some embodiments, the composition is substantially free of nonionic surfactants. In some embodiments, the nonionic surfactant is not a polysorbate, e.g., tween 80 (also known as polysorbate 80 or PS 80).

When present, the nonionic surfactant can be present at a concentration of from about 0.0001% (w/v) to about 0.01% (w/v). For example, the composition may comprise a nonionic surfactant at a concentration of from about 0.0005% (w/v) to about 0.0015% (w/v). In some embodiments, the compositions may comprise a nonionic surfactant at a concentration of about 0.0001% (w/v), about 0.0002% (w/v), about 0.0003% (w/v), about 0.0004% (w/v), about 0.0005% (w/v), about 0.0006% (w/v), about 0.0007% (w/v), about 0.0008% (w/v), about 0.0009% (w/v), about 0.001% (w/v), about 0.002% (w/v), about 0.003% (w/v), about 0.004% (w/v), about 0.005% (w/v), about 0.006% (w/v), about 0.007% (w/v), about 0.008% (w/v), about 0.009% (w/v), or about 0.01% (w/v). In some preferred embodiments, the composition comprises a nonionic surfactant at a concentration of about 0.0005% (w/v) or about 0.001% (w/v).

In certain embodiments, the composition comprises poloxamer P188, poloxamer P407, pluronic F-68, or Ecosurf SA-15 at a concentration of about 0.0005% (w/v), about 0.0008% (w/v), about 0.0009% (w/v), about 0.001% (w/v), about 0.002% (w/v), about 0.0025% (w/v), about 0.003% (w/v), about 0.0035% (w/v), about 0.004% (w/v), about 0.0045% (w/v), about 0.005% (w/v), about 0.006% (w/v), about 0.007% (w/v), about 0.008% (w/v), about 0.009% (w/v), or about 0.01% (w/v).

In some embodiments, the composition further comprises a buffer (e.g., PBS, tris.hcl, phosphate, citric acid, histidine, tromethamine, succinic acid, malic acid, alpha-ketoglutaric acid, carbonate buffer), a bulking agent (e.g., a polyol such as sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, and dextran), and a non-ionic surfactant (e.g., poloxamer P188, poloxamer P407, pluronic F-68, ecosurf SA-15, brij S20, tergitol NP-10, IGEPAL CA 720, or tween 80), and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the composition further comprises a buffer (e.g., PBS, tris.hcl, phosphate, citric acid, histidine, tromethamine, succinic acid, malic acid, alpha-ketoglutaric acid, carbonate buffer), a bulking agent (e.g., a polyol such as sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, and dextran), a nonionic surfactant (e.g., poloxamer P188, poloxamer P407, pluronic F-68, ecosurf SA-15, brij S20, tergitol NP-10, IGEPAL CA 720, or tween 80), and a multivalent ion (selected from the group consisting of calcium, citrate, sulfate, and magnesium), and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the composition further comprises a buffer (e.g., PBS, tris.hcl, phosphate, citric acid, histidine, tromethamine, succinic acid, malic acid, alpha-ketoglutaric acid, carbonate buffer), a bulking agent (e.g., a polyol such as sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, and dextran), and a nonionic surfactant (e.g., poloxamer P188, poloxamer P407, pluronic F-68, ecosurf SA-15, brij S20, tergitol NP-10, IGEPAL CA 720, or tween 80), and wherein the composition is substantially free of magnesium sulfate, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the composition further comprises a buffer (e.g., PBS, tris.hcl, phosphate, citric acid, histidine, tromethamine, succinic acid, malic acid, a-ketoglutaric acid, carbonate buffer), a bulking agent (e.g., a polyol such as sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, and dextran), and a multivalent ion (e.g., calcium, citrate, sulfate, or magnesium), and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the composition further comprises a buffer (e.g., PBS, tris.hcl, phosphate, citric acid, histidine, tromethamine, succinic acid, malic acid, a-ketoglutaric acid, carbonate buffer), a bulking agent (e.g., a polyol such as sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, and dextran), a multivalent ion (e.g., calcium, citrate, sulfate = root or magnesium), and a nonionic surfactant (e.g., poloxamer P188, poloxamer P407, pluronic F-68, ecosurf SA-15, brij S20, tergitol NP-10, IGEPAL CA 720, or tween 80), and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the composition further comprises a buffer (e.g., PBS, tris, or histidine), a bulking agent (e.g., sucrose, sorbitol, or mannitol), and a nonionic surfactant (e.g., pluronic F-68), in addition to the rAAV, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the composition further comprises a buffer (e.g., PBS, tris, or histidine), a bulking agent (e.g., sucrose, sorbitol, or mannitol), a nonionic surfactant (e.g., pluronic F-68), and a multivalent ion or salt thereof (e.g., magnesium sulfate), in addition to the rAAV, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the composition further comprises a buffer (e.g., PBS, tris, or histidine), a bulking agent (e.g., sucrose, sorbitol, or mannitol), and a nonionic surfactant (e.g., pluronic F-68) in addition to the rAAV, and the composition is substantially free of magnesium sulfate, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the composition further comprises a buffer (e.g., PBS, tris, or histidine), a bulking agent (e.g., sucrose, sorbitol, or mannitol), and a multivalent ion or salt thereof (e.g., magnesium sulfate), in addition to the rAAV, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the composition further comprises a buffer (e.g., PBS, tris, or histidine), a bulking agent (e.g., sucrose, sorbitol, or mannitol), and a multivalent ion or salt thereof (e.g., magnesium sulfate), in addition to the rAAV, and the composition is substantially free of a nonionic surfactant (e.g., pluronic F-68), and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the composition further comprises a buffer (e.g., selected from the group consisting of PBS, tris.hcl, phosphate, citrate, histidine in addition to rAAVA buffer selected from the group consisting of PBS, tris.hcl, phosphate, citric acid, and any combination thereof, or a buffer selected from the group consisting of citric acid, histidine, succinic acid, malic acid, a-ketoglutaric acid, and any combination thereof, or a buffer selected from the group consisting of PBS, tirs.hcl, histidine, and any combination thereof, or a buffer selected from the group consisting of tris.hcl, phosphate, citric acid, carbonate, and any combination thereof; a filler (e.g., polyol or povidone (PVP K24), or a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactose, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof, or a polyol selected from the group consisting of sucrose, mannitol, sorbitol, glycerol, propylene glycol, polyethylene glycol, dextran, and any combination thereof; and nonionic surfactants (e.g., nonionic surfactants selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from polyoxyethylene (12) isooctylphenyl ether (e.g., CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g.,80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68, polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, brij 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGOL 15-S-9, TERGOL 15-S-12, TERGOL-64, TERGNP-7, GINP-8, TERGOL-9, TERGL-5, TERGNP-10, and combinations thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, pluronic F-68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and Pluronic F-68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338 and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020 and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-64, TERGITOL- A nonionic surfactant of the group consisting of TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL p-13, and any combination thereof), wherein the pH of the composition is from about 7 to about 8, e.g., the pH of the composition is from about 7.3 to about 7.8, or from about 7.4 to about 7.7, or from about 7.3 to about 7.55, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). It is noted that any one of the specific buffers or groups of buffers listed in this paragraph can be used with any one of the specific fillers or groups of fillers listed in this paragraph, as well as any one of the specific nonionic surfactants or groups of nonionic surfactants listed in this paragraph. Similarly, any one of the specific fillers or groups of fillers listed in this paragraph can be used with any one of the specific buffers or groups of buffers listed in this paragraph, as well as any one of the specific nonionic surfactants or groups of nonionic surfactants listed in this paragraph. Likewise, any particular nonionic surfactant or nonionic surfactant group listed in this paragraph can be used with any particular buffer or buffer group listed in this paragraph, and with any particular filler or filler group listed in this paragraph. In other words, all individual specific combinations of buffers, buffer groups, fillers, filler groups, nonionic surfactants and nonionic surfactant groups listed above in this paragraph are specifically contemplated and claimed.

In some embodiments, the composition further comprises a buffer (e.g., a buffer selected from the group consisting of PBS, tris.hcl, phosphate, citric acid, histidine, tromethamine, succinic acid, malic acid, alpha-ketoglutaric acid, carbonate, protein buffer, and any combination thereof, or a buffer selected from the group consisting of PBS, tris.hcl, phosphate, citric acid, and any combination thereof, orA buffer selected from the group consisting of citric acid, histidine, succinic acid, malic acid, alpha-ketoglutaric acid, and any combination thereof, or a buffer selected from the group consisting of PBS, tirs.hcl, histidine, and any combination thereof, or a buffer selected from the group consisting of tris.hcl, phosphate, citric acid, carbonate, and any combination thereof; a filler (e.g., polyol or povidone (PVP K24), or a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactose, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof, or a polyol selected from the group consisting of sucrose, mannitol, sorbitol, glycerol, propylene glycol, polyethylene glycol, dextran, and any combination thereof; nonionic surfactants (e.g., nonionic surfactants selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkyl phenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from polyoxyethylene (12) isooctylphenyl ether (e.g., CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g.,80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68, polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, brij 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGOL 15-S-9, TERGOL 15-S-12, TERGOL-64, TERGNP-7, GINP-8, TERGOL-9, TERGL-5, TERGNP-10, and combinations thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, pluronic F-68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and Pluronic F-68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338 and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020 and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL15-S-12, TERGITOL NP-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL-9.5, GITOL NP-10, TERGITOL-11, TERGITOL-12, TERGITOL-13 and any combination thereof); and multivalent ions or salts thereof (e.g., selected from the group consisting of calcium, citrate, sulfate, magnesium, and phosphate Or a multivalent ion selected from the group consisting of calcium, citrate, sulfate, and magnesium, or a multivalent ion selected from the group consisting of calcium, sulfate, and magnesium, or a multivalent ion selected from the group consisting of citrate, sulfate, and magnesium, or magnesium sulfate), wherein the pH of the composition is from about 7 to about 8, e.g., the pH of the composition is from about 7.3 to about 7.8, or from about 7.4 to about 7.7, or from about 7.3 to about 7.55, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). It is noted that any one of the specific buffers or sets of buffers listed in this paragraph can be used with any one of the specific fillers or sets of fillers listed in this paragraph, as well as any one of the specific nonionic or nonionic surfactants listed in this paragraph, as well as any one of the specific multivalent ions or groups of multivalent ions listed in this paragraph. Similarly, any one of the specific fillers or groups of fillers listed in this paragraph can be used with any one of the specific buffers or groups of buffers listed in this paragraph, as well as any one of the specific nonionic or nonionic surfactants listed in this paragraph, as well as any one of the specific multivalent ions or groups of multivalent ions listed in this paragraph. Likewise, any particular nonionic surfactant or nonionic surfactant group listed in this paragraph can be used with any particular buffer or buffer group listed in this paragraph, and any particular filler or filler group listed in this paragraph, and any particular multivalent ion or multivalent ion group listed in this paragraph. Furthermore, any particular multivalent ion or group of multivalent ions listed in this paragraph can be combined with any particular buffer or group of buffers listed in this paragraph, and any particular filler or group of fillers listed in this paragraph, and any particular nonionic surfactant or nonionic table listed in this paragraph The surfactant sets are used together. In other words, all individual specific combinations of buffers, buffer groups, fillers, bulking agent groups, nonionic surfactants and nonionic surfactant groups, multivalent ions and multivalent ion groups listed above in this paragraph are specifically contemplated and claimed.

In some embodiments, the composition further comprises a buffer (e.g., a buffer selected from the group consisting of PBS, tris.hcl, phosphate, citric acid, histidine, tromethamine, succinic acid, malic acid, alpha-ketoglutaric acid, carbonate, protein buffer, and any combination thereof, or a buffer selected from the group consisting of PBS, tris.hcl, phosphate, citric acid, and any combination thereof, or a buffer selected from the group consisting of citric acid, histidine, succinic acid, malic acid, alpha-ketoglutaric acid, and any combination thereof, or a buffer selected from the group consisting of PBS, tirs.hcl, histidine, and any combination thereof, or a buffer selected from the group consisting of tris.hcl, phosphate, citric acid, carbonate, and any combination thereof) in addition to the rAAV; a filler (e.g., polyol or povidone (PVP K24), or a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactose, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof, or a polyol selected from the group consisting of sucrose, mannitol, sorbitol, glycerol, propylene glycol, polyethylene glycol, dextran, and any combination thereof; and nonionic surfactants (for example, selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably poly) Sorbitol esters, polyoxyethylene alkylphenyl ethers), and any combination thereof, or a nonionic surfactant selected from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g.,CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g.,80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68, polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, brij 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGOL 15-S-9, TERGOL 15-S-12, TERGOL-64, TERGNP-7, GINP-8, TERGOL-9, TERGL-5, TERGNP-10, and combinations thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, pluronic F-68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic selected from the group consisting of Pluronic 10R5 and Pluronic F-68 A subsurface surfactant, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combinations thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combinations thereof, wherein the composition is substantially free of multivalent ions or salts thereof (e.g., multivalent ions selected from the group consisting of calcium, citrate, sulfate, and magnesium, or multivalent ions selected from the group consisting of calcium, sulfate, and magnesium, or multivalent ions selected from the group consisting of citrate, sulfate, and magnesium, or magnesium sulfate), wherein the pH of the composition is from about 7 to about 8, e.g., the pH of the composition is from about 7.3 to about 7.8, or from about 7.4 to about 7.7, or from about 7.3 to about 7.55, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). It is noted that any one of the specific buffers or sets of buffers listed in this paragraph can be used with any one of the specific fillers or sets of fillers listed in this paragraph, as well as any one of the specific nonionic or nonionic surfactants listed in this paragraph, as well as any one of the specific multivalent ions or groups of multivalent ions listed in this paragraph. Similarly, any one of the specific fillers or groups of fillers listed in this paragraph can be combined with any one of the specific buffers or groups of buffers listed in this paragraph, and any one of the specific nonionic surfactants or groups of surfactants listed in this paragraph The surfactant set, as well as any particular multivalent ion or group of multivalent ions listed in this paragraph above, are used together. Likewise, any particular nonionic surfactant or group of surfactants listed in this paragraph can be used with any particular buffer or group of buffers listed in this paragraph, and any particular filler or group of fillers listed in this paragraph, and any particular multivalent ion or group of multivalent ions listed in this paragraph. Furthermore, any of the specific multivalent ions and groups of multivalent ions listed in this paragraph can be used with any of the specific buffers or groups of buffers listed in this paragraph, as well as any of the specific fillers or groups of fillers listed in this paragraph, as well as any of the specific nonionic surfactants or groups of surfactants listed in this paragraph. In other words, all individual specific combinations of buffers, buffer groups, fillers, filler groups, nonionic surfactants, nonionic surfactant groups, multivalent ions, and multivalent ion groups listed above in this paragraph are specifically contemplated and claimed.

In some embodiments, the composition further comprises a buffer (e.g., a buffer selected from the group consisting of PBS, tris.hcl, phosphate, citric acid, histidine, tromethamine, succinic acid, malic acid, alpha-ketoglutaric acid, carbonate, protein buffer, and any combination thereof, or a buffer selected from the group consisting of PBS, tris.hcl, phosphate, citric acid, and any combination thereof, or a buffer selected from the group consisting of citric acid, histidine, succinic acid, malic acid, alpha-ketoglutaric acid, and any combination thereof, or a buffer selected from the group consisting of PBS, tirs.hcl, histidine, and any combination thereof, or a buffer selected from the group consisting of tris.hcl, phosphate, citric acid, carbonate, and any combination thereof) in addition to the rAAV; a filler (e.g., polyol or povidone (PVP K24), or a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactose, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof, or a polyol selected from the group consisting of sucrose, mannitol, sorbitol, glycerol, propylene glycol, polyethylene glycol, dextran, and any combination thereof; and multivalent ions or salts thereof (e.g., multivalent ions selected from the group consisting of calcium, citrate, sulfate, and magnesium, or multivalent ions selected from the group consisting of calcium, sulfate, and magnesium, or multivalent ions selected from the group consisting of citrate, sulfate, and magnesium, or magnesium sulfate), wherein the pH of the composition is from about 7 to about 8, e.g., from about 7.3 to about 7.8, or from about 7.4 to about 7.7, or from about 7.3 to about 7.55, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). It is noted that any one of the specific buffers or groups of buffers listed above in this paragraph can be used with any one of the specific fillers or groups of fillers listed above in this paragraph, as well as any one of the specific multivalent ions and groups of multivalent ions listed above in this paragraph. Similarly, any one of the specific fillers or groups of fillers listed in this paragraph can be used with any one of the specific buffers or groups of buffers listed in this paragraph, as well as any one of the specific multivalent ions and groups of multivalent ions listed in this paragraph. Likewise, any of the specific multivalent ions and groups of multivalent ions listed in this paragraph can be used with any of the specific buffers or groups of buffers listed in this paragraph, as well as any of the specific fillers or groups of fillers listed in this paragraph, as well as any of the specific nonionic surfactants or groups of surfactants listed in this paragraph. In other words, all individual specific combinations of buffers, buffer groups, fillers, filler groups, multivalent ions and multivalent ion groups listed above in this paragraph are specifically contemplated and claimed.

In some embodiments, the composition further comprises a buffer (e.g., a buffer selected from the group consisting of PBS, tris.hcl, phosphate, citric acid, histidine, tromethamine, succinic acid, malic acid, alpha-ketoglutaric acid, carbonate, protein buffer, and any combination thereof, or a buffer selected from the group consisting of PBS, tris.hcl, phosphate, citric acid, and any combination thereof, or a buffer selected from the group consisting of citric acid, histidine, succinic acid, malic acid, alpha-ketoglutaric acid, and any combination thereof, or a buffer selected from the group consisting of PBS, tirs.hcl, histidine, and any combination thereof, or a buffer selected from the group consisting of tris.hcl, phosphate, citric acid, carbonate, and any combination thereof) in addition to the rAAV; a filler (e.g., polyol or povidone (PVP K24), or a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactose, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof, or a polyol selected from the group consisting of sucrose, mannitol, sorbitol, glycerol, propylene glycol, polyethylene glycol, dextran, and any combination thereof; and multivalent ions or salts thereof (e.g., multivalent ions selected from the group consisting of calcium, citrate, sulfate, and magnesium, or multivalent ions selected from the group consisting of calcium, sulfate And magnesium, or a multivalent ion selected from the group consisting of citrate, sulfate, and magnesium, or magnesium sulfate), and the composition is substantially free of nonionic surfactants (e.g., a nonionic surfactant selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or a nonionic surfactant selected from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g.,CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68, polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, brij 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGOL 15-S-9, TERGOL 15-S-12, TERGOL-64, TERGNP-7, GITONP-8, TERGOL-9, TERGNP-10, TERGNP-11, TERGToL-4, TERGNP-10, TERGL-11, and the like 2. A nonionic surfactant selected from the group consisting of TERGITOL P-13, polysorbate 20 and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, pluronic F-68, ecosurf SA-15, brij S20, tergitol NP-10, IGEPAL CA 720, tween 80 and any combination thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and Pluronic F-68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338 and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL P-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof, wherein the composition has a pH of from about 7 to about 8, e.g., from about 7.3 to about 7.8, or from about 7.4 to about 7.7 or about 7.3 to about 7.3, and optionally, 55. (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). It is noted that any one of the specific buffers or sets of buffers listed in this paragraph can be used with any one of the specific fillers or sets of fillers listed in this paragraph, as well as any one of the specific nonionic surfactants or sets of surfactants listed in this paragraph, as well as any one of the specific multivalent ions and sets of multivalent ions listed in this paragraph. In a similar manner to that described above, any one of the specific fillers or groups of fillers listed in this paragraph can be used with any one of the specific buffers or groups of buffers listed in this paragraph, as well as any one of the specific nonionic surfactants or groups of surfactants listed in this paragraph, as well as in this paragraph Any particular multivalent ion listed is used with the multivalent ion set. Likewise, any particular nonionic surfactant or group of surfactants listed in this paragraph can be used with any particular buffer or group of buffers listed in this paragraph, and any particular filler or group of fillers listed in this paragraph, and any particular multivalent ion or group of multivalent ions listed in this paragraph. Furthermore, any of the specific multivalent ions and groups of multivalent ions listed in this paragraph can be used with any of the specific buffers or groups of buffers listed in this paragraph, as well as any of the specific fillers or groups of fillers listed in this paragraph, as well as any of the specific nonionic surfactants or groups of surfactants listed in this paragraph. In other words, all individual specific combinations of buffers, buffer groups, fillers, filler groups, nonionic surfactants, nonionic surfactant groups, multivalent ions, and multivalent ion groups listed above in this paragraph are specifically contemplated and claimed.

In some embodiments, the composition further comprises a buffer (e.g., a buffer selected from the group consisting of PBS, tris.hcl, phosphate, citric acid, histidine, tromethamine, succinic acid, malic acid, alpha-ketoglutaric acid, carbonate, protein buffer, and any combination thereof, or a buffer selected from the group consisting of PBS, tris.hcl, phosphate, citric acid, and any combination thereof, or a buffer selected from the group consisting of citric acid, histidine, succinic acid, malic acid, alpha-ketoglutaric acid, and any combination thereof, or a buffer selected from the group consisting of PBS, tirs.hcl, histidine, and any combination thereof, or a buffer selected from the group consisting of tris.hcl, phosphate, citric acid, carbonate, and any combination thereof) in addition to the rAAV; fillers (e.g., polyol or povidone (PVP K24), or a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of sorbose Alcohols, mannitol, galactitol, sucrose, lactose, maltose, trehalose, and any combination thereof, or polyols selected from the group consisting of sucrose, mannitol, sorbitol, and any combination thereof, or polyols selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof, or polyols selected from the group consisting of sucrose, mannitol, sorbitol, glycerol, propylene glycol, polyethylene glycol, dextran, and any combination thereof; and nonionic surfactants (e.g., nonionic surfactants selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from polyoxyethylene (12) isooctylphenyl ether (e.g.,CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g.,80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol octadecyl ether), seed oil surfactant (e.g., ecosurf (tm) SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylate (e.g., tergitol NP-10 nonylphenol ethoxylate), and any combination thereof, or a nonionic surfactant selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68, polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA- 720. Brij S20, brij Sl0, brij 010, brij Cl0, brij 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, polysorbate 20 and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, pluronic F-68, ecourf SA-15, brij S20, tergol NP-10, IGAL-720, TERGITOL NP-80, and any combination thereof, TERGITOL 4, TERGITOL-7, TERGITOL-8, TERGITOL NP-9, TERGITOL-7, TERGITOL NP-7, TERGITOL-8, TERGITOL-7, TERGITOL-8, TERGOL-9, TERGITOL-7, TERGOL-7, TERGITOL-9, TERGITOL-7, TERGITOL-13, TERGOL-7, TERGITOL-9, TERGITOL-13, TERGOL-9, TERGITOL-7, polysorbate-20 and any combination thereof, or any combination thereof, wherein the composition has an osmolality of from about 100 to about 500mOsm, e.g., an osmolality of from about 125 to about 500mOsm, or from about 200 to about 400mOsm, or from about 200 to about 500mOsm, or from about 300 to about 400mOsm, or from about 150 to about 350mOsm, or from about 175 to about 300mOsm, such as an osmolality of about 500mOsm or less, or about 475mOsm or less, or about 450mOsm or less, or about 425mOsm or less, or about 400mOsm or less, or about 375mOsm or less, or about 350mOsm or less, or about 325mOsm or less, or about 300mOsm or less, or about 350mOsm or less, or about 325mOsm or less, or about 375mOsm or less, and optionally 375mOsm or less: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextrose) Anhydride T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). It is noted that any one of the specific buffers or groups of buffers listed in this paragraph can be used with any one of the specific fillers or groups of fillers listed in this paragraph, as well as any one of the specific nonionic surfactants or groups of surfactants listed in this paragraph. Similarly, any one of the specific fillers or groups of fillers listed in this paragraph can be used with any one of the specific buffers or groups of buffers listed in this paragraph, as well as any one of the specific nonionic surfactants or groups of surfactants listed in this paragraph. Likewise, any particular nonionic surfactant or group of surfactants listed in this paragraph can be used with any particular buffer or group of buffers listed in this paragraph, and with any particular filler or group of fillers listed in this paragraph. In other words, all individual specific combinations of buffers, buffer groups, fillers, filler groups, nonionic surfactants and nonionic surfactant groups listed above in this paragraph are specifically contemplated and claimed.

In some embodiments, the composition further comprises a buffer (e.g., a buffer selected from the group consisting of PBS, tris.hcl, phosphate, citric acid, histidine, tromethamine, succinic acid, malic acid, alpha-ketoglutaric acid, carbonate, protein buffer, and any combination thereof, or a buffer selected from the group consisting of PBS, tris.hcl, phosphate, citric acid, and any combination thereof, or a buffer selected from the group consisting of citric acid, histidine, succinic acid, malic acid, alpha-ketoglutaric acid, and any combination thereof, or a buffer selected from the group consisting of PBS, tirs.hcl, histidine, and any combination thereof, or a buffer selected from the group consisting of tris.hcl, phosphate, citric acid, carbonate, and any combination thereof) in addition to the rAAV; fillers (e.g., polyols or povidone (PVP K24), or polyols selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or fromA polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, galactitol, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof, or a polyol selected from the group consisting of sucrose, mannitol, sorbitol, glycerol, propylene glycol, polyethylene glycol, dextran, and any combination thereof; nonionic surfactants (e.g., nonionic surfactants selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkyl phenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from polyoxyethylene (12) isooctylphenyl ether (e.g., CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g.,80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactant (e.g., ecosurf (tm) SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylate (e.g., tergitol (tm) NP-10 nonylphenol ethoxylate), and any combination thereof, or a nonionic surfactant selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68, polyoxyethylene (18) tridecyl ether, polyoxyethylene(12) Tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, brij 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL LNP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, GITONP-13, polysorbate 20, and combinations of any of them, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, pluronic F-68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and Pluronic F-68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020, and any combinations thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, nonionic surfactants of the group consisting of TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof; and multivalent ions or salts thereof (e.g., multivalent ions selected from the group consisting of calcium, citrate, sulfate, magnesium, and phosphate, or multivalent ions selected from the group consisting of calcium, citrate, sulfate, and magnesium, or multivalent ions selected from the group consisting of calcium, sulfate, and magnesium, or multivalent ions selected from the group consisting of citrate, sulfate, and magnesium, or magnesium sulfate), wherein the composition has an osmolality of from about 100mOsm to about 500mOsm, e.g., an osmolality of from about 125mOsm to about 500mOsm, or from about 200mOsm to about 400mOsm, or from about 200mOsm to about 500mOsm, or from about 300mOsm to about 400mOsm, or from about 400mOsm About 150mOsm to about 350mOsm, or from about 175mOsm to about 300mOsm, such as an osmotic pressure of about 500mOsm or less, or about 475mOsm or less, or about 450mOsm or less, or about 425mOsm or less, or about 400mOsm or less, or about 375mOsm or less, or about 350mOsm or less, or about 325mOsm or less, or about 300mOsm or less, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). It is noted that any one of the specific buffers or sets of buffers listed in this paragraph can be used with any one of the specific fillers or sets of fillers listed in this paragraph, as well as any one of the specific nonionic surfactants or sets of surfactants listed in this paragraph, as well as any one of the specific multivalent ions and sets of multivalent ions listed in this paragraph. Similarly, any one of the specific fillers or groups of fillers listed in this paragraph can be used with any one of the specific buffers or groups of buffers listed in this paragraph, as well as any one of the specific nonionic surfactants or groups of surfactants listed in this paragraph, as well as any one of the specific multivalent ions and groups of multivalent ions listed in this paragraph. Likewise, any particular nonionic surfactant or group of surfactants listed in this paragraph can be used with any particular buffer or group of buffers listed in this paragraph, and any particular filler or group of fillers listed in this paragraph, and any particular multivalent ion or group of multivalent ions listed in this paragraph. Furthermore, any of the specific multivalent ions and groups of multivalent ions listed in this paragraph can be used with any of the specific buffers or groups of buffers listed in this paragraph, as well as any of the specific fillers or groups of fillers listed in this paragraph, as well as any of the specific nonionic surfactants or groups of surfactants listed in this paragraph. In other words, at All individual specific combinations of buffers, buffer groups, fillers, bulking agent groups, nonionic surfactants and nonionic surfactant groups, multivalent ions and multivalent ion groups listed above in this paragraph are specifically contemplated and claimed.

In some embodiments, the composition further comprises a buffer (e.g., a buffer selected from the group consisting of PBS, tris.hcl, phosphate, citric acid, histidine, tromethamine, succinic acid, malic acid, alpha-ketoglutaric acid, carbonate, protein buffer, and any combination thereof, or a buffer selected from the group consisting of PBS, tris.hcl, phosphate, citric acid, and any combination thereof, or a buffer selected from the group consisting of citric acid, histidine, succinic acid, malic acid, alpha-ketoglutaric acid, and any combination thereof, or a buffer selected from the group consisting of PBS, tirs.hcl, histidine, and any combination thereof, or a buffer selected from the group consisting of tris.hcl, phosphate, citric acid, carbonate, and any combination thereof) in addition to the rAAV; a filler (e.g., polyol or povidone (PVP K24), or a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactose, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof, or a polyol selected from the group consisting of sucrose, mannitol, sorbitol, glycerol, propylene glycol, polyethylene glycol, dextran, and any combination thereof; and nonionic surfactants (for example, selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers) And any combination thereof, or a nonionic surfactant selected from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g.,CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g.,80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68, polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, brij 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGOL 15-S-9, TERGOL 15-S-12, TERGOL-64, TERGNP-7, GINP-8, TERGOL-9, TERGL-5, TERGNP-10, and combinations thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, pluronic F-68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and Pluronic F-68, or a poloxamer P188, poloxamer P407, poloxamer P338 and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brij Sl0, brij 010, brij Cl0, brij 020 and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL-13 and any combination thereof, wherein the composition is substantially free of multivalent ions or is a multivalent ion (e.g., a multivalent ion selected from the group consisting of magnesium, calcium and sulfate ions selected from the group consisting of magnesium, magnesium and calcium, or a multivalent ion selected from the group consisting of calcium, sulfate, and magnesium, or a multivalent ion selected from the group consisting of citrate, sulfate, and magnesium, or magnesium sulfate), wherein the composition has an osmotic pressure of from about 100mOsm to about 500mOsm, e.g., from about 125mOsm to about 500mOsm, or from about 200mOsm to about 400mOsm, or from about 200mOsm to about 500mOsm, or from about 300mOsm to about 400mOsm, or from about 150mOsm to about 350mOsm, or from about 175mOsm to about 300mOsm, e.g., an osmotic pressure of about 500mOsm or less, or about 475mOsm or less, or about 450mOsm or less, or about 425mOsm or less, or about 400mOsm or less, or about 350mOsm or less, or about 325mOsm or less, or about 300mOsm or less, or about 375mOsm or less Or about 325mOsm or less, or about 300mOsm or less. It is noted that any one of the specific buffers or sets of buffers listed in this paragraph can be used with any one of the specific fillers or sets of fillers listed in this paragraph, as well as any one of the specific nonionic surfactants or sets of surfactants listed in this paragraph, as well as any one of the specific multivalent ions and sets of multivalent ions listed in this paragraph. Similarly, any one of the specific fillers or groups of fillers listed in this paragraph can be used with any one of the specific buffers or groups of buffers listed in this paragraph, as well as with any one of the specific buffers or groups of buffers listed in this paragraph Any particular nonionic surfactant or group of surfactants, and any particular multivalent ions and group of multivalent ions listed in this paragraph above. Likewise, any particular nonionic surfactant or group of surfactants listed in this paragraph can be used with any particular buffer or group of buffers listed in this paragraph, and any particular filler or group of fillers listed in this paragraph, and any particular multivalent ion or group of multivalent ions listed in this paragraph. Furthermore, any of the specific multivalent ions and groups of multivalent ions listed in this paragraph can be used with any of the specific buffers or groups of buffers listed in this paragraph, as well as any of the specific fillers or groups of fillers listed in this paragraph, as well as any of the specific nonionic surfactants or groups of surfactants listed in this paragraph. In other words, all individual specific combinations of buffers, buffer groups, fillers, filler groups, nonionic surfactants, nonionic surfactant groups, multivalent ions, and multivalent ion groups listed above in this paragraph are specifically contemplated and claimed.

In some embodiments, the composition further comprises a buffer (e.g., a buffer selected from the group consisting of PBS, tris.hcl, phosphate, citric acid, histidine, tromethamine, succinic acid, malic acid, alpha-ketoglutaric acid, carbonate, protein buffer, and any combination thereof, or a buffer selected from the group consisting of PBS, tris.hcl, phosphate, citric acid, and any combination thereof, or a buffer selected from the group consisting of citric acid, histidine, succinic acid, malic acid, alpha-ketoglutaric acid, and any combination thereof, or a buffer selected from the group consisting of PBS, tirs.hcl, histidine, and any combination thereof, or a buffer selected from the group consisting of tris.hcl, phosphate, citric acid, carbonate, and any combination thereof) in addition to the rAAV; a filler (e.g., polyol or povidone (PVP K24), or a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactose, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof, or a polyol selected from the group consisting of sucrose, mannitol, sorbitol, glycerol, propylene glycol, polyethylene glycol, dextran, and any combination thereof; and multivalent ions or salts thereof (e.g., multivalent ions selected from the group consisting of calcium, citrate, sulfate, and magnesium, or multivalent ions selected from the group consisting of calcium, sulfate, and magnesium, or multivalent ions selected from the group consisting of citrate, sulfate, and magnesium, or magnesium sulfate), wherein the osmolality of the composition is from about 100mOsm to about 500mOsm, e.g., an osmolality of from about 125mOsm to about 500mOsm, or from about 200mOsm to about 400mOsm, or from about 200mOsm to about 500mOsm, or from about 300mOsm to about 400mOsm, or from about 150mOsm to about 350mOsm, or from about 175mOsm to about 300mOsm, such as an osmolality of about 500mOsm or less, or about 475mOsm or less, or about 450mOsm or less, or about 425mOsm or less, or about 400mOsm or less, or about 375mOsm or less, or about 350mOsm or less, or about 325mOsm or less, or about 300mOsm or less, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). It is noted that any one of the specific buffers or groups of buffers listed above in this paragraph can be used with any one of the specific fillers or groups of fillers listed above in this paragraph, as well as any one of the specific multivalent ions and groups of multivalent ions listed above in this paragraph. Similarly, any one of the specific fillers or groups of fillers listed in this paragraph can be used with any one of the specific buffers or groups of buffers listed in this paragraph, as well as any one of the specific multivalent ions and groups of multivalent ions listed in this paragraph. Likewise, any of the specific multivalent ions and groups of multivalent ions listed in this paragraph can be used with any of the specific buffers or groups of buffers listed in this paragraph, as well as any of the specific fillers or groups of fillers listed in this paragraph, as well as any of the specific nonionic surfactants or groups of surfactants listed in this paragraph. In other words, all individual specific combinations of buffers, buffer groups, fillers, filler groups, multivalent ions and multivalent ion groups listed above in this paragraph are specifically contemplated and claimed.

In some embodiments, the composition further comprises a buffer (e.g., a buffer selected from the group consisting of PBS, tris.hcl, phosphate, citric acid, histidine, tromethamine, succinic acid, malic acid, alpha-ketoglutaric acid, carbonate, protein buffer, and any combination thereof, or a buffer selected from the group consisting of PBS, tris.hcl, phosphate, citric acid, and any combination thereof, or a buffer selected from the group consisting of citric acid, histidine, succinic acid, malic acid, alpha-ketoglutaric acid, and any combination thereof, or a buffer selected from the group consisting of PBS, tirs.hcl, histidine, and any combination thereof, or a buffer selected from the group consisting of tris.hcl, phosphate, citric acid, carbonate, and any combination thereof) in addition to the rAAV; fillers (e.g., polyol or povidone (PVP K24), or a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, galactitol, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of sucrose, mannitol A polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof, or a polyol selected from the group consisting of sucrose, mannitol, sorbitol, glycerol, propylene glycol, polyethylene glycol, dextran, and any combination thereof; and multivalent ions or salts thereof (e.g., multivalent ions selected from the group consisting of calcium, citrate, sulfate, and magnesium, or multivalent ions selected from the group consisting of citrate, sulfate, and magnesium, or magnesium sulfate), and the composition is substantially free of nonionic surfactants (e.g., nonionic surfactants selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from polyoxyethylene (12) isooctylphenyl ether (e.g., CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactant (e.g., ecosurf (tm) SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylate (e.g., tergitol (tm) NP-10 nonylphenol ethoxylate), and any combination thereof, or a nonionic surfactant selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68, polyoxyethylene(18) Tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, brij 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, GIL NP-11, GITOL NP-12, TERGITOL NP-13, TERGITOL-13, and combinations thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, pluronic F-68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combination thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and Pluronic F-68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brij S0, brij 010, brij Cl0, BRIJ 020, and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSF EH-9, ECOSF EH-14, GITOL 15-S-7, ECOSURF-15, TERGOL 15 SA-9 TERS-TERRE, TERGITOL 15-S-12, TERGITOL 64, TERGITOL P-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof, wherein the osmotic pressure of the composition is from about 100mOsm to about 500mOsm, for example, an osmolality of from about 125mOsm to about 500mOsm, or from about 200mOsm to about 400mOsm, or from about 200mOsm to about 500mOsm, or from about 300mOsm to about 400mOsm, or from about 150mOsm to about 350mOsm, or from about 175mOsm to about 300mOsm, such as an osmolality of about 500mOsm or less, or about 475mOsm or less, or about 450mOsm or less, or about 425mOsm or less, or about 400mOsm or less, or about 375mOsm or less, about 350mOsm or less, or about 325mOsm or less, or about 300mOsm or less, or about 375mOsm or less, or about 350mOsm or less, or about 475mOsm or less mOsm or less, or about 325mOsm or less, or about 300mOsm or less, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). It is noted that any one of the specific buffers or sets of buffers listed in this paragraph can be used with any one of the specific fillers or sets of fillers listed in this paragraph, as well as any one of the specific nonionic surfactants or sets of surfactants listed in this paragraph, as well as any one of the specific multivalent ions and sets of multivalent ions listed in this paragraph. Similarly, any one of the specific fillers or groups of fillers listed in this paragraph can be used with any one of the specific buffers or groups of buffers listed in this paragraph, as well as any one of the specific nonionic surfactants or groups of surfactants listed in this paragraph, as well as any one of the specific multivalent ions and groups of multivalent ions listed in this paragraph. Likewise, any particular nonionic surfactant or group of surfactants listed in this paragraph can be used with any particular buffer or group of buffers listed in this paragraph, and any particular filler or group of fillers listed in this paragraph, and any particular multivalent ion or group of multivalent ions listed in this paragraph. Furthermore, any of the specific multivalent ions and groups of multivalent ions listed in this paragraph can be used with any of the specific buffers or groups of buffers listed in this paragraph, as well as any of the specific fillers or groups of fillers listed in this paragraph, as well as any of the specific nonionic surfactants or groups of surfactants listed in this paragraph. In other words, all individual specific combinations of buffers, buffer groups, fillers, filler groups, nonionic surfactants, nonionic surfactant groups, multivalent ions, and multivalent ion groups listed above in this paragraph are specifically contemplated and claimed.

In some embodiments, the methods further comprise, in addition to a rAAV, a combination ofThe composition further comprises a buffer (e.g., a buffer selected from the group consisting of PBS, tris.hcl, phosphate, citric acid, histidine, tromethamine, succinic acid, malic acid, alpha-ketoglutaric acid, carbonate, protein buffer, and any combination thereof, or a buffer selected from the group consisting of PBS, tris.hcl, phosphate, citric acid, and any combination thereof, or a buffer selected from the group consisting of citric acid, histidine, succinic acid, malic acid, alpha-ketoglutaric acid, and any combination thereof, or a buffer selected from the group consisting of PBS, tirs.hcl, histidine, and any combination thereof, or a buffer selected from the group consisting of tris.hcl, phosphate, citric acid, carbonate, and any combination thereof); a filler (e.g., polyol or povidone (PVP K24), or a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactose, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof, or a polyol selected from the group consisting of sucrose, mannitol, sorbitol, glycerol, propylene glycol, polyethylene glycol, dextran, and any combination thereof; and nonionic surfactants (e.g., nonionic surfactants selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from polyoxyethylene (12) isooctylphenyl ether (e.g., CA-270 polyoxyethylene (12) isooctylphenyl ether) and polyoxyethylene sorbitan monolThe oleic acid esters (for example,80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68, polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, brij 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGOL 15-S-9, TERGOL 15-S-12, TERGOL-64, TERGNP-7, GINP-8, TERGOL-9, TERGL-5, TERGNP-10, and combinations thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, pluronic F-68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and Pluronic F-68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338 and any combination thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020 and any combination thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9 A nonionic surfactant of the group consisting of TERGITOL 15-S-12, TERGITOL L-64, TERGITOL p-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL p-13, and any combination thereof), wherein the composition has a pH of from about 7 to about 8 (e.g., from about 7.3 to about 7.8, or from about 7.4 to about 7.7, or from about 7.3 to about 7.55), and wherein the composition has an osmolarity of from about 100 to about 500mOsm, e.g., from about 125 to about 500mOsm, or from about 200 to about 400mOsm, or from about 200 to about 500mOsm, or from about 300 to about 400mOsm, or from about 150 to about 350, or more or less than 300, and optionally about 350 to about 325, or less than 300, and about 350 to about 325, or less than about 300, or less than about 350 to about 300, or less than about 325, or less than about 300 to about 350, or less than about 325, or less than about 300 to about 300, or less than about 300 to about 35, or less than about 35, or about 35 to about 35, or less of: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). It is noted that any one of the specific buffers or groups of buffers listed in this paragraph can be used with any one of the specific fillers or groups of fillers listed in this paragraph, as well as any one of the specific nonionic surfactants or groups of surfactants listed in this paragraph. Similarly, any one of the specific fillers or groups of fillers listed in this paragraph can be used with any one of the specific buffers or groups of buffers listed in this paragraph, as well as any one of the specific nonionic surfactants or groups of surfactants listed in this paragraph. Also, any particular nonionic surfactant or group of surfactants listed in this paragraph can be used with any particular buffer or group of buffers listed in this paragraph, and any particular filling listed in this paragraph The agent or filler group is used together. In other words, all individual specific combinations of buffers, buffer groups, fillers, filler groups, nonionic surfactants and nonionic surfactant groups listed above in this paragraph are specifically contemplated and claimed.

In some embodiments, the composition further comprises a buffer (e.g., a buffer selected from the group consisting of PBS, tris.hcl, phosphate, citric acid, histidine, tromethamine, succinic acid, malic acid, alpha-ketoglutaric acid, carbonate, protein buffer, and any combination thereof, or a buffer selected from the group consisting of PBS, tris.hcl, phosphate, citric acid, and any combination thereof, or a buffer selected from the group consisting of citric acid, histidine, succinic acid, malic acid, alpha-ketoglutaric acid, and any combination thereof, or a buffer selected from the group consisting of PBS, tirs.hcl, histidine, and any combination thereof, or a buffer selected from the group consisting of tris.hcl, phosphate, citric acid, carbonate, and any combination thereof) in addition to the rAAV; a filler (e.g., polyol or povidone (PVP K24), or a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactose, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof, or a polyol selected from the group consisting of sucrose, mannitol, sorbitol, glycerol, propylene glycol, polyethylene glycol, dextran, and any combination thereof; nonionic surfactants (e.g. selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkyl groups) Phenyl ethers), and any combination thereof, or a nonionic surfactant selected from the group consisting of polyoxyethylene (12) isooctylphenyl ether (e.g.,CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g.,80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol stearyl ether), seed oil surfactants (e.g., ECOSURF SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., tergitolTM NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68, polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, brij 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGOL 15-S-9, TERGOL 15-S-12, TERGOL-64, TERGNP-7, GINP-8, TERGOL-9, TERGL-5, TERGNP-10, and combinations thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, pluronic F-68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and Pluronic F-68, or A nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338 and any combinations thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020 and any combinations thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL P-13 and any combinations thereof; and multivalent ions or salts thereof (e.g., multivalent ions selected from the group consisting of calcium, citrate, sulfate, magnesium, and phosphate, or multivalent ions selected from the group consisting of calcium, citrate, sulfate, and magnesium, or multivalent ions selected from the group consisting of calcium, sulfate, and magnesium, or magnesium sulfate), wherein the composition has a pH of from about 7 to about 8 (e.g., a pH of from about 7.3 to about 7.8, or from about 7.4 to about 7.7, or from about 7.3 to about 7.55), and wherein the composition has an osmolarity of from about 100 to about 500mOsm, e.g., an osmolarity of from about 125 to about 500mOsm, or from about 200 to about 400mOsm, or from about 200 to about 500mOsm, or from about 300 to about 400mOsm, or from about 150 to about 350.3 to about 7.8, or from about 350 to about 350, or more than 350 to about 325 to about or less than 300, and optionally from about 350 to about 325 to about or less than 300 to about 375 to about or less than about 325 to about or less than 300 to about 350 to about 375 to about or less than about 325 to about 35 to reduce the osmolality of the composition. (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). Worth of it It is noted that any one of the specific buffers or groups of buffers listed in this paragraph can be used with any one of the specific fillers or groups of fillers listed in this paragraph, as well as any one of the specific nonionic surfactants or groups of surfactants listed in this paragraph, as well as any one of the specific multivalent ions and groups of multivalent ions listed in this paragraph. Similarly, any one of the specific fillers or groups of fillers listed in this paragraph can be used with any one of the specific buffers or groups of buffers listed in this paragraph, as well as any one of the specific nonionic surfactants or groups of surfactants listed in this paragraph, as well as any one of the specific multivalent ions and groups of multivalent ions listed in this paragraph. Likewise, any particular nonionic surfactant or group of surfactants listed in this paragraph can be used with any particular buffer or group of buffers listed in this paragraph, and any particular filler or group of fillers listed in this paragraph, and any particular multivalent ion or group of multivalent ions listed in this paragraph. Furthermore, any of the specific multivalent ions and groups of multivalent ions listed in this paragraph can be used with any of the specific buffers or groups of buffers listed in this paragraph, as well as any of the specific fillers or groups of fillers listed in this paragraph, as well as any of the specific nonionic surfactants or groups of surfactants listed in this paragraph. In other words, all individual specific combinations of buffers, buffer groups, fillers, filler groups, nonionic surfactants, nonionic surfactant groups, multivalent ions, and multivalent ion groups listed above in this paragraph are specifically contemplated and claimed.

In some embodiments, the composition further comprises a buffer (e.g., a buffer selected from the group consisting of PBS, tris.hcl, phosphate, citric acid, histidine, tromethamine, succinic acid, malic acid, alpha-ketoglutaric acid, carbonate, protein buffer, and any combination thereof, or a buffer selected from the group consisting of PBS, tris.hcl, phosphate, citric acid, and any combination thereof, or a buffer selected from the group consisting of citric acid, histidine, succinic acid, malic acid, alpha-ketoglutaric acid, and any combination thereof) in addition to the rAAVA buffer, or a buffer selected from the group consisting of PBS, tirs.hcl, histidine, and any combination thereof, or a buffer selected from the group consisting of tris.hcl, phosphate, citric acid, carbonate, and any combination thereof; a filler (e.g., polyol or povidone (PVP K24), or a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactose, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof, or a polyol selected from the group consisting of sucrose, mannitol, sorbitol, glycerol, propylene glycol, polyethylene glycol, dextran, and any combination thereof; and nonionic surfactants (e.g., nonionic surfactants selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from polyoxyethylene (12) isooctylphenyl ether (e.g., CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g.,80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +.>S20 polyethylene glycol octadecyl ether), seed oil surfactant (e.g., ecosurf (tm) SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylates (e.g., TERGITOL (tm) NP-10 nonylphenol ethoxylate), and any combination thereof, or selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68, polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, brij 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGOL 15-S-9, TERGOL 15-S-12, TERGOL-64, TERGNP-7, GINP-8, TERGOL-9, TERGL-5, TERGNP-10, and combinations thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, pluronic 10R5, pluronic F-68, ecosurf SA-15, brij S20, TERGITOL NP-10, IGEPAL CA 720, tween 80, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and Pluronic F-68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338 and any combinations thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, brij 020 and any combinations thereof, or a nonionic surfactant selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13 and any combinations thereof, wherein the composition is substantially free of multivalent ions or comprises multivalent ions selected from the group consisting of magnesium, and sulfate, calcium and calcium or sulfate salts thereof Multivalent ions, or multivalent ions selected from the group consisting of calcium, sulfate, and magnesium, or multivalent ions selected from the group consisting of citrate, sulfate, and magnesium, or magnesium sulfate), wherein the pH of the composition is from about 7 to about 8 (e.g., from about 7.3 to about 7.8, or from about 7.4 to about 7.7, or from about 7.3 to about 7.55), and wherein the osmolality of the composition is from about 100 to about 500mOsm, e.g., from about 125 to about 500mOsm, or from about 200 to about 400mOsm, or from about 200 to about 500mOsm, or from about 300 to about 400mOsm, or from about 150 to about 350mOsm, or from about 175 to about 300mOsm, such as an osmolality of about 500 to less, or about 475 to less, or about 450 to less, or less than about 425 to about 350 to about 375 to less than or less than about 300, and optionally about 325 to less than about 300 to about 375 to less than about 300 to about 500 to less than about 35 to reduce the total ion. (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). It is noted that any one of the specific buffers or sets of buffers listed in this paragraph can be used with any one of the specific fillers or sets of fillers listed in this paragraph, as well as any one of the specific nonionic surfactants or sets of surfactants listed in this paragraph, as well as any one of the specific multivalent ions and sets of multivalent ions listed in this paragraph. Similarly, any one of the specific fillers or groups of fillers listed in this paragraph can be used with any one of the specific buffers or groups of buffers listed in this paragraph, as well as any one of the specific nonionic surfactants or groups of surfactants listed in this paragraph, as well as any one of the specific multivalent ions and groups of multivalent ions listed in this paragraph. Also, any particular nonionic surfactant or group of surfactants listed above in this paragraph can be used with any of the above listed in this paragraph A particular buffer or group of buffers, as well as any one of the particular fillers or groups of fillers listed above in this paragraph, as well as any one of the particular multivalent ions and groups of multivalent ions listed above in this paragraph. Furthermore, any of the specific multivalent ions and groups of multivalent ions listed in this paragraph can be used with any of the specific buffers or groups of buffers listed in this paragraph, as well as any of the specific fillers or groups of fillers listed in this paragraph, as well as any of the specific nonionic surfactants or groups of surfactants listed in this paragraph. In other words, all individual specific combinations of buffers, buffer groups, fillers, filler groups, nonionic surfactants, nonionic surfactant groups, multivalent ions, and multivalent ion groups listed above in this paragraph are specifically contemplated and claimed.

In some embodiments, the composition further comprises a buffer (e.g., a buffer selected from the group consisting of PBS, tris.hcl, phosphate, citric acid, histidine, tromethamine, succinic acid, malic acid, alpha-ketoglutaric acid, carbonate, protein buffer, and any combination thereof, or a buffer selected from the group consisting of PBS, tris.hcl, phosphate, citric acid, and any combination thereof, or a buffer selected from the group consisting of citric acid, histidine, succinic acid, malic acid, alpha-ketoglutaric acid, and any combination thereof, or a buffer selected from the group consisting of PBS, tirs.hcl, histidine, and any combination thereof, or a buffer selected from the group consisting of tris.hcl, phosphate, citric acid, carbonate, and any combination thereof) in addition to the rAAV; a filler (e.g., polyol or povidone (PVP K24), or a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactose, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, galactose, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof, or a polyol selected from the group consisting of sucrose, mannitol, sorbitol, glycerol, propylene glycol, polyethylene glycol, dextran, and any combination thereof; and multivalent ions or salts thereof (e.g., multivalent ions selected from the group consisting of calcium, citrate, sulfate, and magnesium, or multivalent ions selected from the group consisting of calcium, sulfate, and magnesium, or magnesium sulfate), wherein the pH of the composition is from about 7 to about 8 (e.g., from about 7.3 to about 7.8, or from about 7.4 to about 7.7, or from about 7.3 to about 7.55), and wherein the osmolality of the composition is from about 100 to about 500mOsm, e.g., from about 125 to about 500mOsm, or from about 200 to about 400mOsm, or from about 200 to about 500mOsm, or from about 300 to about 400mOsm, or from about 150 to about 350, or from about 350 to about 300, or more, or less, and optionally about 350 to about 325, or less than or about 300 to about 300, or less than about 300, and more than about 350 to about 375, or less than about 325, or less than about 300 to about 300, or less than about 300 to about 375, or less than about 35). (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). It is noted that any one of the specific buffers or groups of buffers listed above in this paragraph can be used with any one of the specific fillers or groups of fillers listed above in this paragraph, as well as any one of the specific multivalent ions and groups of multivalent ions listed above in this paragraph. Similarly, any one of the specific fillers or groups of fillers listed in this paragraph can be used with any one of the specific buffers or groups of buffers listed in this paragraph, as well as any one of the specific multivalent ions and groups of multivalent ions listed in this paragraph. Likewise, any of the specific multivalent ions and groups of multivalent ions listed in this paragraph can be used with any of the specific buffers or groups of buffers listed in this paragraph, as well as any of the specific fillers or groups of fillers listed in this paragraph, as well as any of the specific nonionic surfactants or groups of surfactants listed in this paragraph. In other words, all individual specific combinations of buffers, buffer groups, fillers, filler groups, multivalent ions and multivalent ion groups listed above in this paragraph are specifically contemplated and claimed.

In some embodiments, the composition further comprises a buffer (e.g., a buffer selected from the group consisting of PBS, tris.hcl, phosphate, citric acid, histidine, tromethamine, succinic acid, malic acid, alpha-ketoglutaric acid, carbonate, protein buffer, and any combination thereof, or a buffer selected from the group consisting of PBS, tris.hcl, phosphate, citric acid, and any combination thereof, or a buffer selected from the group consisting of citric acid, histidine, succinic acid, malic acid, alpha-ketoglutaric acid, and any combination thereof, or a buffer selected from the group consisting of PBS, tirs.hcl, histidine, and any combination thereof, or a buffer selected from the group consisting of tris.hcl, phosphate, citric acid, carbonate, and any combination thereof) in addition to the rAAV; a filler (e.g., polyol or povidone (PVP K24), or a polyol selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, dextran, and any combination thereof, or a polyol selected from the group consisting of sorbitol, mannitol, galactitol, sucrose, lactose, maltose, trehalose, and any combination thereof, or a polyol selected from the group consisting of sucrose, mannitol, sorbitol, and any combination thereof, or a polyol selected from the group consisting of propylene glycol, polyethylene glycol, dextran, and any combination thereof Polyols of the group, or polyols selected from the group consisting of sucrose, mannitol, sorbitol, glycerol, propylene glycol, polyethylene glycol, dextran, and any combination thereof; and multivalent ions or salts thereof (e.g., multivalent ions selected from the group consisting of calcium, citrate, sulfate, and magnesium, or multivalent ions selected from the group consisting of citrate, sulfate, and magnesium, or magnesium sulfate), and the composition is substantially free of nonionic surfactants (e.g., nonionic surfactants selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof, or from polyoxyethylene (12) isooctylphenyl ether (e.g.,CA-270 polyoxyethylene (12) isooctylphenyl ether), polyoxyethylene sorbitan monooleate (e.g., +.>80 polyoxyethylene sorbitan monooleate), polyethylene glycol stearyl ether (e.g., +. >S20 polyethylene glycol octadecyl ether), seed oil surfactant (e.g., ecosurf (tm) SA-15 seed oil surfactant), poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene), nonylphenol ethoxylate (e.g., tergitol NP-10 nonylphenol ethoxylate), and any combination thereof, or a nonionic surfactant selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68, polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactantA nonionic surfactant selected from the group consisting of poloxamer P188, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL LNP-13, polysorbate 20, and any combination thereof, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer 407, pluronic SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL L-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL LNP-13, polysorbate 20, and any combination thereof, or a combination thereof, or a nonionic surfactant selected from the group consisting of Pluronic 10R5 and Pluronic F-68, or a nonionic surfactant selected from the group consisting of poloxamer P188, poloxamer P407, poloxamer P338, and any combinations thereof, or a nonionic surfactant selected from the group consisting of Brij S20, brijSl0, brij 010, brij Cl0, BRIJ 020, and any combinations thereof, or selected from the group consisting of ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGITOL15-S-9, TERGITOL 15-S-12, TERGITOL-64, TERGITOL NP-7, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-13, and any combination thereof, wherein the pH of the composition is from about 7 to about 8 (e.g., from about 7.3 to about 7.8, or from about 7.4 to about 7.7, or from about 7.3 to about 7.55), and wherein the osmolality of the composition is from about 100mOsm to about 500mOsm, e.g., an osmolality of from about 125mOsm to about 500mOsm, or from about 200mOsm to about 400mOsm, or from about 200mOsm to about 500mOsm, or from about 300mOsm to about 400mOsm, or from about 150mOsm to about 350mOsm, or from about 175mOsm to about 300mOsm, e.g., an osmolality of about 500mOsm or less, or about 475mOsm or less, about 450mOsm or less, or about 425mOsm or less, or about 400mOsm or less, or about 375mOsm or less, about 350mOsm or less, or about 325mOsm or less, or about 300mOsm or less, or about 375mOsm or less, or about 350mOsm or more Low, or about 325mOsm or less, or about 300mOsm or less, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). It is noted that any one of the specific buffers or sets of buffers listed in this paragraph can be used with any one of the specific fillers or sets of fillers listed in this paragraph, as well as any one of the specific nonionic surfactants or sets of surfactants listed in this paragraph, as well as any one of the specific multivalent ions and sets of multivalent ions listed in this paragraph. Similarly, any one of the specific fillers or groups of fillers listed in this paragraph can be used with any one of the specific buffers or groups of buffers listed in this paragraph, as well as any one of the specific nonionic surfactants or groups of surfactants listed in this paragraph, as well as any one of the specific multivalent ions and groups of multivalent ions listed in this paragraph. Likewise, any particular nonionic surfactant or group of surfactants listed in this paragraph can be used with any particular buffer or group of buffers listed in this paragraph, and any particular filler or group of fillers listed in this paragraph, and any particular multivalent ion or group of multivalent ions listed in this paragraph. Furthermore, any of the specific multivalent ions and groups of multivalent ions listed in this paragraph can be used with any of the specific buffers or groups of buffers listed in this paragraph, as well as any of the specific fillers or groups of fillers listed in this paragraph, as well as any of the specific nonionic surfactants or groups of surfactants listed in this paragraph. In other words, all individual specific combinations of buffers, buffer groups, fillers, filler groups, nonionic surfactants, nonionic surfactant groups, multivalent ions, and multivalent ion groups listed above in this paragraph are specifically contemplated and claimed.

In some embodiments, the buffer is PBS, the bulking agent is mannitol, and the nonionic surfactant is Pluronic-F68. For example, the buffer is PBS, the filler is mannitol, the nonionic surfactant is Pluronic-F68, and the pH of the composition is from about 7 to about 8, for example, from about 7.3 to about 7.8, or from about 7.4 to about 7.7, or from about 7.3 to about 7.55.

In some embodiments, the buffer is PBS, the filler is sorbitol, and the nonionic surfactant is Pluronic-F68. For example, the buffer is PBS, the filler is sorbitol, the nonionic surfactant is Pluronic-F68, and the pH of the composition is from about 7 to about 8, for example, from about 7.3 to about 7.8, or from about 7.4 to about 7.7, or from about 7.3 to about 7.55.

In some embodiments, the buffer is PBS, the filler is sorbitol, the nonionic surfactant is Pluronic-F68, and the multivalent ion or salt thereof is magnesium sulfate. For example, the buffer is PBS, the filler is sorbitol, the nonionic surfactant is Pluronic-F68, the multivalent ion or salt thereof is magnesium sulfate, and the pH of the composition is from about 7 to about 8, for example, from about 7.3 to about 7.8, or from about 7.4 to about 7.7, or from about 7.3 to about 7.55.

In some embodiments, the buffer is Tris, the bulking agent is mannitol, and the nonionic surfactant is Pluronic-F68, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). For example, the buffer is PBS, the filler is mannitol, the nonionic surfactant is Pluronic-F68, and the pH of the composition is from about 7 to about 8, for example, from about 7.3 to about 7.8, or from about 7.4 to about 7.7, or from about 7.3 to about 7.55, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the buffer is Tris, the bulking agent is sorbitol, and the nonionic surfactant is Pluronic-F68, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). For example, the buffer is PBS, the filler is sorbitol, the nonionic surfactant is Pluronic-F68, and the pH of the composition is from about 7 to about 8, for example, from about 7.3 to about 7.8, or from about 7.4 to about 7.7, or from about 7.3 to about 7.55, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the buffer is Tris, the filler is sorbitol, the nonionic surfactant is Pluronic-F68, and the multivalent ion or salt thereof is magnesium sulfate, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). For example, the buffer is PBS, the filler is sorbitol, the nonionic surfactant is Pluronic-F68, the multivalent ion or salt thereof is magnesium sulfate, and the pH of the composition is from about 7 to about 8, for example, from about 7.3 to about 7.8, or from about 7.4 to about 7.7, or from about 7.3 to about 7.55, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the buffer is a histidine buffer, the bulking agent is mannitol, and the nonionic surfactant is Pluronic-F68, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). For example, the buffer is PBS, the filler is mannitol, the nonionic surfactant is Pluronic-F68, and the pH of the composition is from about 7 to about 8, for example, from about 7.3 to about 7.8, or from about 7.4 to about 7.7, or from about 7.3 to about 7.55, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the buffer is a histidine buffer, the bulking agent is sorbitol, and the nonionic surfactant is Pluronic-F68, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). For example, the buffer is PBS, the filler is sorbitol, the nonionic surfactant is Pluronic-F68, and the pH of the composition is from about 7 to about 8, for example, from about 7.3 to about 7.8, or from about 7.4 to about 7.7, or from about 7.3 to about 7.55, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the buffer is a histidine buffer, the bulking agent is sorbitol, the nonionic surfactant is Pluronic-F68, and the multivalent ion or salt thereof is magnesium sulfate, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). For example, the buffer is PBS, the filler is sorbitol, the nonionic surfactant is Pluronic-F68, the multivalent ion or salt thereof is magnesium sulfate, and the pH of the composition is from about 7 to about 8, for example, from about 7.3 to about 7.8, or from about 7.4 to about 7.7, or from about 7.3 to about 7.55, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the buffer is PBS, the bulking agent is mannitol, and the nonionic surfactant is Pluronic-F68. For example, the buffer is PBS, the bulking agent is mannitol, the nonionic surfactant is Pluronic-F68, and the osmotic pressure of the composition is from about 125mOsm to about 500mOsm, or from about 200mOsm to about 400mOsm, or from about 200mOsm to about 500mOsm, or from about 300mOsm to about 400mOsm, or from about 150mOsm to about 350mOsm, or from about 175mOsm to about 300mOsm.

In some embodiments, the buffer is PBS, the filler is sorbitol, and the nonionic surfactant is Pluronic-F68. For example, the buffer is PBS, the bulking agent is sorbitol, the nonionic surfactant is Pluronic-F68, and the osmotic pressure of the composition is from about 125mOsm to about 500mOsm, or from about 200mOsm to about 400mOsm, or from about 200mOsm to about 500mOsm, or from about 300mOsm to about 400mOsm, or from about 150mOsm to about 350mOsm, or from about 175mOsm to about 300mOsm.

In some embodiments, the buffer is PBS, the filler is sorbitol, the nonionic surfactant is Pluronic-F68, and the multivalent ion or salt thereof is magnesium sulfate. For example, the buffer is PBS, the bulking agent is sorbitol, the nonionic surfactant is Pluronic-F68, the multivalent ion or salt thereof is magnesium sulfate, and the osmotic pressure of the composition is from about 125mOsm to about 500mOsm, or from about 200mOsm to about 400mOsm, or from about 200mOsm to about 500mOsm, or from about 300mOsm to about 400mOsm, or from about 150mOsm to about 350mOsm, or from about 175mOsm to about 300mOsm.

In some embodiments, the buffer is Tris, the bulking agent is mannitol, and the nonionic surfactant is Pluronic-F68. For example, the buffer is PBS, the bulking agent is mannitol, the nonionic surfactant is Pluronic-F68, and the osmotic pressure of the composition is from about 125mOsm to about 500mOsm, or from about 200mOsm to about 400mOsm, or from about 200mOsm to about 500mOsm, or from about 300mOsm to about 400mOsm, or from about 150mOsm to about 350mOsm, or from about 175mOsm to about 300mOsm.

In some embodiments, the buffer is Tris, the bulking agent is sorbitol, and the nonionic surfactant is Pluronic-F68, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). For example, the buffer is PBS, the bulking agent is sorbitol, the nonionic surfactant is Pluronic-F68, and the osmotic pressure of the composition is from about 125 to about 500, or from about 200 to about 400, or from about 200 to about 500, or from about 300 to about 400, or from about 150 to about 350, or from about 175 to about 300, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the buffer is Tris, the filler is sorbitol, the nonionic surfactant is Pluronic-F68, and the multivalent ion or salt thereof is magnesium sulfate, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). For example, the buffer is PBS, the bulking agent is sorbitol, the nonionic surfactant is Pluronic-F68, the multivalent ion or salt thereof is magnesium sulfate, and the osmotic pressure of the composition is from about 125 to about 500, or from about 200 to about 400, or from about 200 to about 500, or from about 300 to about 400, or from about 150 to about 350, or from about 175 to about 300, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the buffer is a histidine buffer, the bulking agent is mannitol, and the nonionic surfactant is Pluronic-F68, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). For example, the buffer is PBS, the bulking agent is mannitol, the nonionic surfactant is Pluronic-F68, and the osmolality of the composition is from about 125 to about 500, or from about 200 to about 400, or from about 200 to about 500, or from about 300 to about 400, or from about 150 to about 350, or from about 175 to about 300, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the buffer is a histidine buffer, the bulking agent is sorbitol, and the nonionic surfactant is Pluronic-F68, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). For example, the buffer is PBS, the bulking agent is sorbitol, the nonionic surfactant is Pluronic-F68, and the osmotic pressure of the composition is from about 125 to about 500, or from about 200 to about 400, or from about 200 to about 500, or from about 300 to about 400, or from about 150 to about 350, or from about 175 to about 300, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the buffer is a histidine buffer, the bulking agent is sorbitol, the nonionic surfactant is Pluronic-F68, and the multivalent ion or salt thereof is magnesium sulfate, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). For example, the buffer is PBS, the bulking agent is sorbitol, the nonionic surfactant is Pluronic-F68, the multivalent ion or salt thereof is magnesium sulfate, and the osmotic pressure of the composition is from about 125 to about 500, or from about 200 to about 400, or from about 200 to about 500, or from about 300 to about 400, or from about 150 to about 350, or from about 175 to about 300, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the buffer is PBS, the bulking agent is mannitol, and the nonionic surfactant is Pluronic-F68, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). For example, the buffer is PBS, the bulking agent is mannitol, the nonionic surfactant is Pluronic-F68, and the pH of the composition is from about 7 to about 8 (e.g., from about 7.3 to about 7.8, or from about 7.4 to about 7.7, or from about 7.3 to about 7.55), and the osmotic pressure is from about 125 to about 500mOsm (e.g., from about 200 to about 400mOsm, or from about 200 to about 500mOsm, or from about 300 to about 400mOsm, or from about 150 to about 350mOsm, or from about 175 to about 300 mOsm), and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the buffer is PBS, the filler is sorbitol, and the nonionic surfactant is Pluronic-F68, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). For example, the buffer is PBS, the filler is sorbitol, the nonionic surfactant is Pluronic-F68, and the pH of the composition is from about 7 to about 8 (e.g., from about 7.3 to about 7.8, or from about 7.4 to about 7.7, or from about 7.3 to about 7.55), and the osmotic pressure is from about 125 to about 500mOsm (e.g., from about 200 to about 400mOsm, or from about 200 to about 500mOsm, or from about 300 to about 400mOsm, or from about 150 to about 350mOsm, or from about 175 to about 300 mOsm), and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the buffer is PBS, the filler is sorbitol, the nonionic surfactant is Pluronic-F68, and the multivalent ion or salt thereof is magnesium sulfate, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). For example, the buffer is PBS, the filler is sorbitol, the nonionic surfactant is Pluronic-F68, the multivalent ion or salt thereof is magnesium sulfate, and the pH of the composition is from about 7 to about 8 (e.g., from about 7.3 to about 7.8, or from about 7.4 to about 7.7, or from about 7.3 to about 7.55), and the osmotic pressure is from about 125 to about 500mOsm (e.g., from about 200 to about 400mOsm, or from about 200 to about 500mOsm, or from about 300 to about 400mOsm, or from about 150 to about 350mOsm, or from about 175 to about 300 mOsm), and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the buffer is Tris, the bulking agent is mannitol, and the nonionic surfactant is Pluronic-F68, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). For example, the buffer is PBS, the bulking agent is mannitol, the nonionic surfactant is Pluronic-F68, and the pH of the composition is from about 7 to about 8 (e.g., from about 7.3 to about 7.8, or from about 7.4 to about 7.7, or from about 7.3 to about 7.55), and the osmotic pressure is from about 125 to about 500mOsm (e.g., from about 200 to about 400mOsm, or from about 200 to about 500mOsm, or from about 300 to about 400mOsm, or from about 150 to about 350mOsm, or from about 175 to about 300 mOsm), and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the buffer is Tris, the bulking agent is sorbitol, and the nonionic surfactant is Pluronic-F68, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). For example, the buffer is PBS, the filler is sorbitol, the nonionic surfactant is Pluronic-F68, and the pH of the composition is from about 7 to about 8 (e.g., from about 7.3 to about 7.8, or from about 7.4 to about 7.7, or from about 7.3 to about 7.55), and the osmotic pressure is from about 125 to about 500mOsm (e.g., from about 200 to about 400mOsm, or from about 200 to about 500mOsm, or from about 300 to about 400mOsm, or from about 150 to about 350mOsm, or from about 175 to about 300 mOsm), and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the buffer is Tris, the filler is sorbitol, the nonionic surfactant is Pluronic-F68, and the multivalent ion or salt thereof is magnesium sulfate, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). For example, the buffer is PBS, the filler is sorbitol, the nonionic surfactant is Pluronic-F68, the multivalent ion or salt thereof is magnesium sulfate, and the pH of the composition is from about 7 to about 8 (e.g., from about 7.3 to about 7.8, or from about 7.4 to about 7.7, or from about 7.3 to about 7.55), and the osmotic pressure is from about 125 to about 500mOsm (e.g., from about 200 to about 400mOsm, or from about 200 to about 500mOsm, or from about 300 to about 400mOsm, or from about 150 to about 350mOsm, or from about 175 to about 300 mOsm), and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the buffer is a histidine buffer, the bulking agent is mannitol, and the nonionic surfactant is Pluronic-F68, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). For example, the buffer is PBS, the bulking agent is mannitol, the nonionic surfactant is Pluronic-F68, and the pH of the composition is from about 7 to about 8 (e.g., from about 7.3 to about 7.8, or from about 7.4 to about 7.7, or from about 7.3 to about 7.55), and the osmotic pressure is from about 125 to about 500mOsm (e.g., from about 200 to about 400mOsm, or from about 200 to about 500mOsm, or from about 300 to about 400mOsm, or from about 150 to about 350mOsm, or from about 175 to about 300 mOsm), and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the buffer is a histidine buffer, the bulking agent is sorbitol, and the nonionic surfactant is Pluronic-F68, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). For example, the buffer is PBS, the filler is sorbitol, the nonionic surfactant is Pluronic-F68, and the pH of the composition is from about 7 to about 8 (e.g., from about 7.3 to about 7.8, or from about 7.4 to about 7.7, or from about 7.3 to about 7.55), and the osmotic pressure is from about 125 to about 500mOsm (e.g., from about 200 to about 400mOsm, or from about 200 to about 500mOsm, or from about 300 to about 400mOsm, or from about 150 to about 350mOsm, or from about 175 to about 300 mOsm), and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the buffer is a histidine buffer, the bulking agent is sorbitol, the nonionic surfactant is Pluronic-F68, and the multivalent ion or salt thereof is magnesium sulfate, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). For example, the buffer is PBS, the filler is sorbitol, the nonionic surfactant is Pluronic-F68, the multivalent ion or salt thereof is magnesium sulfate, and the pH of the composition is from about 7 to about 8 (e.g., from about 7.3 to about 7.8, or from about 7.4 to about 7.7, or from about 7.3 to about 7.55), and the osmotic pressure is from about 125 to about 500mOsm (e.g., from about 200 to about 400mOsm, or from about 200 to about 500mOsm, or from about 300 to about 400mOsm, or from about 150 to about 350mOsm, or from about 175 to about 300 mOsm), and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the pH of the compositions described herein is from about 7 to about 8. For example, the pH of any of the above compositions may be from about 7.3 to about 7.8 or from about 7.4 to about 7.5. In some embodiments, the pH of any of the above compositions may be about 7.3, or about 7.4, or about 7.5, or about 7.6, or about 7.7, or about 7.8.

In some embodiments, the compositions described herein have an osmolality of from about 100mOsm to about 500mOsm. For example, the osmolality of any of the above compositions is from about 125mOsm to about 500mOsm, or from about 200mOsm to about 400mOsm, or from about 200mOsm to about 500mOsm, or from about 300mOsm to about 400mOsm, or from about 150mOsm to about 350mOsm, or from about 175mOsm to about 300mOsm.

In some embodiments, the pH of the compositions described herein is from about 7 to about 8, and the osmolality is from about 100mOsm to about 500mOsm. For example, the pH of any of the above compositions may be from about 7.3 to about 7.8 or from about 7.4 to about 7.5, and the osmolality may be from about 125 to about 500, or from about 200 to about 400, or from about 200 to about 500, or from about 300 to about 400, or from about 150 to about 350, or from about 175 to about 300mOsm.

Without wishing to be bound by theory, AAV vector particles are stored in the composition without significant aggregation. Aggregation can be assessed by Dynamic Light Scattering (DLS), photon correlation spectroscopy, and visual appearance. In some embodiments, AAV vector particles stored using the compositions described herein do not exhibit significant aggregation when stored for one, two, three, four, five, six, seven, eight, nine, ten, or more days at 4 ℃. In some embodiments, AAV vector particles stored as such compositions do not exhibit significant aggregation after one, two, three, four, five, six, seven, eight, nine, ten or more freeze-thaw cycles at-20 ℃ or at-80 ℃. In some embodiments, the compositions described herein are for multiple AAV serotypes, e.g., AAV2, AAV9, AAVrh10. In some embodiments, the compositions described herein are for a plurality of AAV serotypes, e.g., AAV serotypes for heparin binding and non-heparin binding. In some embodiments, the compositions described herein are for a plurality of AAV serotypes, e.g., AAV serotypes for heparin binding and non-heparin binding, wherein the composition is maintained at or near isotonic strength. In some embodiments, the compositions described herein are for use with a variety of AAV serotypes, e.g., AAV serotypes for heparin binding and non-heparin binding, wherein the composition has a low osmolality, e.g., less than about 400mOsm, or less than about 350mOsm, or less than about 300mOsm, or less than about 250mOsm, or less than about 225mOsm, or less than about 200mOsm, or less than about 180mOsm, or less than about 160mOsm, or less than about 150mOsm, or less than about 145mOsm, or less than about 140mOsm, or even less.

In some embodiments, AAV vectors or purified recombinant AAV particles (rAAV) stored according to the compositions described herein exhibit a low polydispersity index (polydispersity index, PDI) (as measured by dynamic light scattering), indicating that AAV vectors do not significantly aggregate. In some embodiments, AAV vectors or purified rAAV particles stored according to compositions described herein exhibit a PDI of less than 0.1, e.g., about 0.099, about 0.098, about 0.097, about 0.095, about 0.09, about 0.085, about 0.08, about 0.075, about 0.073, about 0.07, about 0.069, about 0.065, about 0.060, about 0.058, about 0.057, about 0.055, about 0.05, about 0.045, about 0.040, about 0.035, or even less. In some embodiments, AAV vectors or purified rAAV particles stored according to compositions described herein exhibit a PDI of less than 0.1 when stored for one, two, three, four, five, six, seven, eight, nine, ten, or more days at 4 ℃, e.g., about 0.099, about 0.098, about 0.097, about 0.095, about 0.09, about 0.085, about 0.08, about 0.075, about 0.073, about 0.07, about 0.069, about 0.065, about 0.060, about 0.058, about 0.057, about 0.055, about 0.05, about 0.045, about 0.040, about 0.035, or even less. In some embodiments, AAV vectors or purified recombinant AAV particles stored according to the compositions described herein exhibit substantially no aggregation. In some embodiments, AAV vectors or purified rAAV particles stored according to compositions described herein exhibit substantially no aggregation, with a PDI value of less than about 0.1. In some embodiments, AAV vectors or purified rAAV particles stored according to compositions described herein exhibit a Polydispersity (PD) value of less than about 30% PD, or less than about 25% PD, or less than about 20% PD, or less than about 15% PD, or even less.

In some embodiments, AAV vectors or purified rAAV particles stored according to compositions described herein exhibit a low polydispersity index (PDI) (as measured by dynamic light scattering), indicating that no significant aggregation of AAV vectors occurs after one, two, three, four, five, six, seven, eight, nine, ten, or more freeze-thaw cycles (wherein each freeze-thaw cycle comprises 24hr at-80 ℃ and then 24hr at room temperature).

In some embodiments, an AAV vector or purified rAAV particle stored according to the compositions described herein exhibits a PDI of less than 0.1 after one, two, three, four, five, six, seven, eight, nine, ten, or more freeze-thaw cycles (wherein each freeze-thaw cycle comprises 24hr at-80 ℃ and then 24hr at room temperature), e.g., about 0.099, about 0.098, about 0.097, about 0.095, about 0.09, about 0.085, about 0.08, about 0.075, about 0.073, about 0.07, about 0.069, about 0.065, about 0.060, about 0.058, about 0.057, about 0.055, about 0.05, about 0.045, about 0.040, about 0.035, or even less.

In some embodiments, AAV vectors or purified rAAV particles stored according to the compositions described herein exhibit substantially no aggregation. In some embodiments, AAV vectors or purified rAAV particles stored according to compositions described herein exhibit substantially no aggregation, with a PDI value of less than 0.1. In some embodiments, the compositions described herein can be used to store an AAV vector, wherein the TCID50/mL of the AAV vector remains at least 50% or more, e.g., at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, after one, two, four, five, six, seven, eight, nine, ten, or more freeze-thaw cycles (wherein each freeze-thaw cycle comprises 24hhr at-80 ℃ and then 24hr at room temperature) as measured before the start of the freeze-thaw cycle. In some embodiments, the compositions described herein can be used to store an AAV vector, wherein the TCID50/mL of the AAV vector is substantially unchanged after one, two, three, four, five, six, seven, eight, nine, ten, or more freeze-thaw cycles (wherein each freeze-thaw cycle comprises 24hr at-80 ℃ and then 24hr at room temperature) as measured before the start of the freeze-thaw cycle.

In some embodiments, AAV vectors stored according to the compositions described herein exhibit an average aggregate particle radius (Rh) (as measured by dynamic light scattering), indicating that no significant aggregation of AAV vectors occurs. In some embodiments, AAV vectors stored according to compositions described herein exhibit an average aggregate particle radius of less than about 35nm when stored for one, two, three, four, five, six, seven, eight, nine, ten, or more days at 4 ℃. For example, AAV vectors stored according to the compositions described herein exhibit an average aggregate particle radius of less than about 30nm, less than about 25nm, less than about 20nm, less than about 15nm, less than about 10nm, or less than about 5nm when stored for one, two, three, four, five, six, seven, eight, nine, ten, or more days at 4 ℃.

In some embodiments, AAV vectors stored according to the compositions described herein exhibit an average aggregate particle radius, as measured by dynamic light scattering, after one, two, three, four, five, six, seven, eight, nine, ten or more freeze-thaw cycles at-20 ℃ or at-80 ℃, indicating that no significant aggregation of AAV vectors occurs. In some embodiments, AAV vectors stored according to the compositions described herein exhibit an average aggregate particle radius of less than 35nm after one, two, three, four, five, six, seven, eight, nine, ten, or more freeze-thaw cycles at-20 ℃ or at-80 ℃. For example, AAV vectors stored according to the compositions described herein exhibit an average aggregate particle radius of less than about 30nm, less than about 25nm, less than about 20nm, less than about 15nm, less than about 10nm, or less than about 5nm after one, two, three, four, five, six, seven, eight, nine, ten, or more freeze-thaw cycles at-20 ℃ or at-80 ℃.

In some embodiments, the composition further comprises about 10mM phosphate (pH 7.4), about 200mM NaCl, about 5mM KCl, about 1% (w/v) mannitol, and about 0.0005% (w/v) IGEPAL CA 720 in addition to the rAAV.

In some embodiments, the composition further comprises about 20mM phosphate (pH 7.4), about 300mM NaCl, about 3mM KCl, about 3% (w/v) mannitol, and about 0.001% (w/v) Brij S20, in addition to the rAAV.

In some embodiments, the composition further comprises, in addition to the rAAV, about 20mM phosphate (pH 7.4), about 300mM NaCl, about 3mM KCl, about 3% (w/v) sorbitol, and about 0.001% (w/v) Ecosurf SA-15.

In some embodiments, the composition further comprises, in addition to the rAAV, about 10mM phosphate (pH 7.4), about 350mM NaCl, about 2.7mM KCl, about 5% (w/v) sorbitol, and about 0.001% (w/v) poloxamer 188.

In some embodiments, the composition comprises, in addition to rAAV, about 10mM phosphate (pH 6.95-7.2), about 137mM NaCl, about 2.7mM KCl, about 0.9mM CaCl ₂ About 0.5mM MgCl ₂ And about 0.001% (w/v) Pluronic F-68.

In some embodiments, the composition further comprises, in addition to the rAAV, about 10mM phosphate (pH 7.3), about 180mM NaCl, about 2.7mM KCl, about 5% (w/v) sorbitol, and about 0.001% (w/v) poloxamer 188.

In some embodiments, the composition comprises, in addition to rAAV, about 15mM phosphate (pH 7.4), about 375mM NaCl, about 3.5mM KCl, about 5% (w/v) sorbitol, and about 0.0005% (w/v) Tergitol NP-10.

In some embodiments, the composition further comprises about 15mM phosphate (pH 7.4), about 375mM NaCl, about 3.5mM KCl, about 3% (w/v) glycerol, and about 0.0005% (w/v) Tween 80 in addition to the rAAV.

In some embodiments, the composition comprises, in addition to rAAV, about 10mM phosphate (pH 7.6), about 137mM NaCl, about 2.7mM KCl, about 5% (w/v) sorbitol, and about 0.01% Pluronic F-68.

In some embodiments, the composition comprises, in addition to rAAV, about 10mM phosphate (pH 7.4), about 137mM NaCl, about 2.7mM KCl, about 5% (w/v) sorbitol, about 0.01% Pluronic F-68, and about 20mM MgSO ₄ 。

In some embodiments, the composition comprises, in addition to rAAV, about 10mM phosphate (pH 7.6), about 137mM NaCl, about 2.7mM KCl, about 5% (w/v) mannitol, and about 0.01% Pluronic F-68.

In some embodiments, the composition comprises, in addition to rAAV, about 10mM phosphate (pH 7.3), about 137mM NaCl, about 2.7mM KCl, about 5% (w/v) mannitol, about 0.01% Pluronic F-68, and about 20mM MgSO ₄ 。

In some embodiments, the composition comprises, in addition to rAAV, about 10mM phosphate (pH 7.4), about 137mM NaCl, about 2.7mM KCl, about 5% (w/v) sorbitol, and about 20mM MgSO ₄ 。

In some embodiments, the composition comprises, in addition to rAAV, about 10mM phosphate (pH 7.4), about 137mM NaCl, about 2.7mM KCl, about 5% (w/v) mannitol, and about 20mM MgSO ₄ 。

In some embodiments, the composition comprises, in addition to rAAV, about 10mM phosphate (pH 6.2), about 137mM NaCl, about 2.7mM KCl, about 5% (w/v) sorbitol, and about 10% (w/v) calcium α -d-heptagluconate.

In some embodiments, the composition further comprises about 10mM phosphate (pH 6.2), about 137mM NaCl, about 2.7mM KCl, about 5% (w/v) mannitol, and about 10% (w/v) calcium α -d-heptagluconate, in addition to the rAAV.

In some embodiments, the composition comprises, in addition to rAAV, about 20mM Tris (pH 7.5), about 5% (w/v) sorbitol, and about 0.01% Pluronic F-68, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the composition comprises, in addition to rAAV, about 20mM Tris (pH 7.4), about 5% (w/v) sorbitol, about 0.01% Pluronic F-68, and about 20mM MgSO ₄ And optionally: (i) The composition is essentiallyThe composition does not contain glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the composition comprises, in addition to rAAV, about 20mM Tris (pH 7.5), about 5% (w/v) mannitol, and about 0.01% Pluronic F-68, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the composition comprises, in addition to rAAV, about 20mM Tris (pH 7.4), about 5% (w/v) mannitol, about 0.01% Pluronic F-68, and about 20mM MgSO ₄ And optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the composition comprises, in addition to rAAV, about 20mM Tris (pH 7.5), about 5% (w/v) sorbitol, and about 20mM MgSO ₄ And optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodimentsIn addition to rAAV, the composition comprises about 20mM Tris (pH 7.5), about 5% (w/v) mannitol, and about 20mM MgSO ₄ And optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the composition comprises, in addition to the rAAV, about 25mM histidine (pH 7.69), about 5% (w/v) sorbitol, and about 0.01% Pluronic F-68, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the composition comprises, in addition to rAAV, about 25mM histidine (pH 7.46), about 5% (w/v) sorbitol, about 0.01% Pluronic F-68, and about 20mM MgSO ₄ And optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the composition comprises, in addition to the rAAV, about 25mM histidine (pH 7.62), about 5% (w/v) mannitol, and about 0.01% Pluronic F-68, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the composition comprises, in addition to rAAV, about 25mM histidine (pH 7.49), about 5% (w/v) mannitol, about 0.01% Pluronic F-68, and about 20mM MgSO ₄ And optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the composition comprises, in addition to a rAAV, about 25mM histidine (pH 7.53), about 5% (w/v) sorbitol, and about 20mM MgSO ₄ And optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the composition comprises, in addition to a rAAV, about 25mM histidine (pH 7.55), about 5% (w/v) mannitol, and about 20mM MgSO ₄ And optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the composition further comprises about 25mM histidine and about 90mM MgSO in addition to the rAAV ₄ And optionally: (i) the composition is substantially free of glycine; and/or (ii) theThe composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the composition further comprises about 25mM histidine, about 90mM MgSO in addition to the rAAV ₄ And about 0.01% Pluronic F-68, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the composition further comprises about 25mM histidine, about 90mM MgSO in addition to the rAAV ₄ And about 5% (w/v) sucrose, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the composition further comprises about 25mM histidine, about 90mM MgSO in addition to the rAAV ₄ About 5% (w/v) sucrose and about 0.01% Pluronic F-68, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the composition further comprises about 20mM in addition to the rAAVTris and about 90mM MgSO ₄ And optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the composition further comprises about 20mM Tris, about 90mM MgSO in addition to the rAAV ₄ And about 0.01% Pluronic F-68, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80).

In some embodiments, the composition further comprises about 20mM Tris, about 90mM MgSO in addition to the rAAV ₄ And about 5% (w/v) sucrose, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose (e.g., trehalose dehydrate) or dextran (e.g., dextran T10 or T40); and/or (iii) the composition is substantially free of pharmaceutically acceptable salts (sodium, ammonium or potassium salts, such as NaCl); and/or (iv) the composition is substantially free of polysorbates (e.g., PS 80). Exemplary compositions:

in some embodiments, the composition (e.g., pharmaceutical composition) comprises a recombinant AAV vector (rAAV) in 10mM phosphate (pH 7.4), 200mM NaCl, 5mM KCl, 1% (w/v) mannitol, 0.0005% (w/v) IGEPAL CA 720, a fill volume of 5mL. In some embodiments, the priming volume is 1mL, 2mL, 3mL, 4mL, 5mL, 6mL, 7mL, 8mL, 9mL, or 10mL.

In some embodiments, the composition (e.g., pharmaceutical composition) comprises a recombinant AAV vector (rAAV) in 20mM phosphate (pH 7.4), 300mM NaCl, 3mM KCl, 3% (w/v) mannitol, 0.001% (w/v) Brij S20, a fill volume of 5mL. In some embodiments, the priming volume is 1mL, 2mL, 3mL, 4mL, 5mL, 6mL, 7mL, 8mL, 9mL, or 10mL.

In some embodiments, the composition (e.g., pharmaceutical composition) comprises a recombinant AAV vector (rAAV) in 20mM phosphate (pH 7.4), 300mM NaCl, 3mM KCl, 3% (w/v) sorbitol, 0.001% (w/v) Ecosurf SA-15, priming volume of 5mL. In some embodiments, the priming volume is 1mL, 2mL, 3mL, 4mL, 5mL, 6mL, 7mL, 8mL, 9mL, or 10mL.

In some embodiments, the composition (e.g., pharmaceutical composition) comprises a recombinant AAV vector (rAAV) in 10mM phosphate (pH 7.4), 350mM NaCl, 2.7mM KCl, 5% (w/v) sorbitol, 0.001% (w/v) poloxamer 188, in a fill volume of 5mL. In some embodiments, the priming volume is 1mL, 2mL, 3mL, 4mL, 5mL, 6mL, 7mL, 8mL, 9mL, or 10mL.

In some embodiments, the composition (e.g., pharmaceutical composition) comprises a recombinant AAV vector (rAAV) in 15mM phosphate (pH 7.4), 375mM NaCl, 3.5mM KCl, 5% (w/v) sorbitol, 0.0005% (w/v) Tergitol NP-10, with a fill volume of 5mL. In some embodiments, the priming volume is 1mL, 2mL, 3mL, 4mL, 5mL, 6mL, 7mL, 8mL, 9mL, or 10mL.

In some embodiments, the composition (e.g., pharmaceutical composition) comprises a recombinant AAV vector (rAAV) in 15mM phosphate (pH 7.4), 375mM NaCl, 3.5mM KCl, 3% (w/v) glycerol, 0.0005% (w/v) tween 80, a fill volume of 5mL. In some embodiments, the priming volume is 1mL, 2mL, 3mL, 4mL, 5mL, 6mL, 7mL, 8mL, 9mL, or 10mL.

HTS assay and kit

In another aspect, provided herein are High Throughput Screening (HTS) assays for determining conditions for purifying or isolating viral particles from a sample (e.g., a harvest medium). For example, provided herein is an HTS method for determining conditions (e.g., buffer component concentration, chromatographic medium, chromatographic size, etc.) for purifying or separating viral particles (e.g., different AAV serotypes) from a sample (e.g., harvest medium) using anion exchange chromatography by HTS.

Typically, the HTS method comprises subjecting a sample (e.g., a harvest medium, such as an affinity eluate obtained from a harvest medium comprising viral particles (e.g., rAAV particles) to a high throughput AEX method, e.g., using Tecan or FPLC in a scout mode, subjecting the harvest medium (e.g., an affinity eluate obtained from a harvest medium comprising viral particles (e.g., rAAV particles)) to a high throughput AEX method (e.g., 0.1 mL.) the buffer for AEX comprises varying amounts of one or more components, e.g., the sample dilution buffer and/or the column equilibration buffer comprises varying amounts of weak acid or salts thereof.

In some embodiments, the sample dilution buffer and/or the column equilibration buffer are substantially free of weak acids or salts thereof, and the sample dilution buffer and/or the column equilibration buffer comprise varying amounts of amino acids. For example, AEX method evaluation includes the addition of 0-150mM amino acid (e.g., histidine) at the sample dilution/column equilibration stage, and optionally, conductivity-mediated isocratic step elution at a pH greater than 8.

AEX flow-through and eluate fractions were then analyzed to evaluate empty capsid to complete capsid separation and capsid recovery. AEX flow-through and eluate fractions were analyzed, for example, using a Tecan microplate reader and/or SEC-HPLC to assess separation of empty capsids from intact capsids and capsid recovery.

In some embodiments, the sample in HTS for high throughput AEX is an affinity eluate from the harvesting medium. Thus, in some embodiments, the method further comprises the step of purifying/isolating the plurality of recombinantly-expressed viral particles from the harvest medium by affinity chromatography to produce an affinity eluate comprising the plurality of recombinantly-expressed viral particles for use in the HTS methods described herein. For example, the harvest medium is subjected to affinity chromatography purification, and the affinity purified material is then transferred to the high throughput AEX method described herein.

In some embodiments, the HTS method comprises subjecting a sample comprising a plurality of recombinantly expressed viral particles to a high throughput AEX method (e.g., 0.1 mL) using Tecan or FPLC in a scout mode. AEX method evaluation included the addition of 0-20mM citric acid (or 0-30mM succinic acid, or 0-60mM acetic acid) at the sample dilution/column equilibration stage, and conductivity-mediated isocratic step elution at pH greater than 8. AEX flow-through and eluate fractions were then analyzed using a Tecan microplate reader and/or SEC-HPLC to assess empty capsid separation from intact capsids and capsid recovery.

In some embodiments, the HTS method comprises purifying/isolating a plurality of recombinantly expressed viral particles from a culture medium (with or without lysis) by affinity chromatography to produce an affinity eluate comprising the plurality of recombinantly expressed viral particles, and subjecting the affinity purified material to a high throughput AEX method (0.1 mL) using Tecan or FPLC in a scout mode. AEX method evaluation included the addition of 0-20mM citric acid (or 0-30mM succinic acid, or 0-60mM acetic acid) at the sample dilution/column equilibration stage, and conductivity-mediated isocratic step elution at pH greater than 8. AEX flow-through and eluate fractions were then analyzed using a Tecan microplate reader or SEC-HPLC to assess separation of empty and intact capsids and capsid recovery.

In some embodiments, the HTS method comprises purifying/separating a plurality of recombinantly-expressed viral particles from a culture medium (with or without lysis) by affinity chromatography using different affinity elution buffers to produce different affinity eluents comprising a plurality of recombinantly-expressed viral particles, and subjecting the affinity-purified material to a high throughput AEX method (0.1 mL) using Tecan or FPLC in a scout mode. AEX method evaluation included the addition of 0-20mM citric acid (or 0-30mM succinic acid, or 0-60mM acetic acid) at the sample dilution/column equilibration stage, and conductivity-mediated isocratic step elution at pH greater than 8. AEX flow-through and eluate fractions were then analyzed using a Tecan microplate reader or SEC-HPLC to assess separation of empty and intact capsids and capsid recovery.

Without wishing to be bound by theory, the HTS methods described herein are capable of identifying various chromatographic conditions (e.g., buffer components and concentrations, chromatographic media, sample preparation, etc.) in a high-throughput manner to remove empty viral particles from a sample. It should be noted that the method can be scaled down or scaled up as required by the production.

In another aspect, provided herein are kits for determining conditions (e.g., buffer component concentration, chromatographic medium, chromatographic column size, etc.) for purifying or isolating viral particles (e.g., different AAV serotypes) from a harvest medium. For example, provided herein are kits for determining conditions (e.g., buffer component concentration, chromatographic medium, chromatographic size, etc.) for purifying or separating viral particles (e.g., different AAV serotypes) from a harvest medium using anion exchange chromatography by HTS. Typically, the kit comprises, for example, one or more of a buffer, a chromatographic medium, a system for HTS, etc., so that one skilled in the art can practice the methods described herein. Furthermore, the kit may comprise instructions for performing the methods described herein.

In some embodiments, the kit comprises a buffer as described herein. For example, the kit comprises a buffer as described herein, e.g., an anion exchange dilution buffer, an anion exchange equilibration buffer, a cation exchange elution buffer, and/or an affinity elution buffer. In some embodiments, the buffers in the kit may have varying amounts of buffer components. For example, the buffer comprises varying amounts of weak acid.

In some embodiments, the kit comprises an anion exchange dilution buffer described herein, wherein the amount of at least one component of the buffer is varied. In some embodiments, the kit comprises an anion exchange dilution buffer described herein, wherein the buffer comprises a weak acid or salt thereof, and wherein the weak acid or salt thereof is present in the buffer in varying amounts. In some embodiments, the kit comprises an anion exchange dilution buffer described herein, wherein the buffer does not comprise a weak acid or salt thereof, and the amount of at least one component (e.g., amino acid) of the buffer is varied.

In some embodiments, the kit comprises an anion exchange equilibration buffer described herein, wherein the amount of at least one component of the buffer is varied. In some embodiments, the kit comprises an anion exchange equilibration buffer described herein, wherein the buffer comprises a weak acid or salt thereof, and wherein the weak acid or salt thereof is present in the buffer in varying amounts. In some embodiments, the kit comprises an anion exchange equilibration buffer described herein, wherein the buffer does not comprise a weak acid or salt thereof, and the amount of at least one component (e.g., amino acid) of the buffer is varied.

The kit may further comprise a chromatographic medium. For example, the kit may comprise an anion exchange chromatography medium and/or an affinity chromatography medium. It should be noted that the chromatographic medium may be in a column used in a chromatographic system (e.g., HTS system). Furthermore, the column may be any desired size for HTS assays. In some embodiments, the kit comprises an anion exchange chromatography medium. In some embodiments, the kit comprises an affinity chromatography medium.

In some embodiments, the kit comprises a high-throughput liquid processor.

In some embodiments, the kit comprises one or more multi-well plates. For example, the kit comprises one or more multi-well plates, and wherein one or more wells comprise a buffer, e.g., a buffer as described herein.

The kit may also include instructions for use. For example, the kit may comprise instructions for practicing the methods described herein. In some embodiments, the kit comprises instructions for determining conditions (e.g., buffer component concentration, chromatographic medium, chromatographic size, etc.) for purifying or isolating viral particles (e.g., different AAV serotypes) from the harvest medium. It is noted that the instructions may be present in the kit in a variety of forms, one or more of which may be present in or on the kit. One form in which these instructions may be present is in the form of printed information on a suitable medium or substrate, for example, one or more sheets of paper on which the information is printed, in or on the packaging of the kit, in a packaging insert, etc. Another way is by a computer readable medium, e.g. a disk, CD, etc., on which the information has been recorded. Another way that may exist is a website address, which can be used by accessing information from a remote website over the internet. Any convenient means may be present in the kit.

In some embodiments, the kit comprises one or more multi-well plates and at least one of an anion exchange dilution buffer, an anion exchange equilibration buffer, an ion exchange elution buffer, or an affinity elution buffer.

In some embodiments, the kit comprises one or more multi-well plates; at least one of an anion exchange dilution buffer, an anion exchange equilibration buffer, an ion exchange elution buffer, or an affinity elution buffer; and chromatographic media, for example, AEX chromatographic media.

In some embodiments, the kit comprises at least two of an anion exchange dilution buffer, an anion exchange equilibration buffer, an anion exchange elution buffer, and an affinity elution buffer.

In some embodiments, the kit comprises a chromatographic medium, e.g., an AEX chromatographic medium and at least two of an anion exchange dilution buffer, an anion exchange equilibration buffer, an anion exchange elution buffer, and an affinity elution buffer.

Exemplary embodiments of the various aspects described herein may be described in terms of the following numbered embodiments:

embodiment 1: a method for purifying or isolating recombinantly expressed adeno-associated virus particles from a harvest medium, the method comprising: (a) Purifying/isolating a plurality of recombinantly-expressed viral particles from a harvesting medium by affinity chromatography to produce an eluate comprising the plurality of recombinantly-expressed viral particles, wherein an elution buffer for affinity chromatography (affinity elution buffer) comprises a predetermined amount of glycine, optionally the affinity elution buffer is substantially free of weak acids or salts thereof; and optionally, the affinity elution buffer comprises an amino acid; (b) Conditioning the affinity chromatography eluate for subsequent purification by anion exchange chromatography, wherein the conditioned eluate comprises a predetermined amount of an anionic compound; and (c) purifying/separating the plurality of recombinantly-expressed viral particles from the conditioned eluate of the affinity chromatography by anion exchange chromatography to produce a solution comprising the plurality of purified/separated recombinantly-expressed viral particles, wherein an equilibration buffer for anion exchange chromatography comprises a predetermined amount of a weak acid or salt thereof, optionally the weak acid is citric acid, acetic acid or succinic acid.

Embodiment 2: the method of embodiment 1, wherein the anionic compound of 1 (b) is an acid or salt thereof, optionally the acid is citric acid, citrate, acetic acid or succinic acid.

Embodiment 3: the method of any of embodiments 1-2, wherein the affinity elution buffer comprises histidine at a concentration of at least about 1mM, 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM or more.

Embodiment 4: the method of any of embodiments 1-3, wherein the affinity elution buffer comprises histidine at a concentration of from about 1mM to about 50mM, from about 5mM to about 45mM, from about 10mM to about 40mM, from about 15mM to about 35mM, or from about 20mM to about 30mM.

Embodiment 5: the method of any of embodiments 1-4, wherein the affinity elution buffer comprises histidine at a concentration of about 1mM, about 5mM, about 10mM, about 15mM, about 20mM, about 25mM, about 30mM, about 35mM, about 40mM, about 45mM, or about 50mM.

Embodiment 6: the method of any one of embodiments 1-5, wherein the affinity elution buffer comprises histidine at a concentration of about 25 mM.

Embodiment 7: the method of any of embodiments 1-6, wherein the affinity elution buffer comprises glycine at a concentration of at least about 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 90mM, 95mM, 100mM or more.

Embodiment 8: the method of any of embodiments 1-7, wherein the affinity elution buffer comprises glycine at a concentration of from about 25mM to about 100mM, from about 30mM to about 95mM, from about 35mM to about 90mM, from about 40mM to about 80mM, or from about 45mM to about 75mM.

Embodiment 9: the method of any of embodiments 1-8, wherein the affinity elution buffer comprises glycine at a concentration of about 20mM, about 25mM, about 30mM, about 35mM, about 40mM, about 45mM, about 50mM, about 55mM, about 60mM, about 65mM, about 70mM, about 75mM, about 80mM, about 90mM, about 95mM, or about 100mM.

Embodiment 10: the method of any one of embodiments 1-9, wherein the affinity elution buffer comprises a salt.

Embodiment 11: the method of any one of embodiments 1-10, wherein the affinity elution buffer comprises a salt at a concentration of at least about 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, or more.

Embodiment 12: the method of any of embodiments 1-11, wherein the affinity elution buffer comprises a salt at a concentration of from about 5mM to about 15mM, from about 6mM to about 14mM, from about 7mM to about 13mM, from about 8mM to about 12mM, or from about 9mM to about 11mM.

Embodiment 13: the method of any of embodiments 1-12, wherein the affinity elution buffer comprises a salt at a concentration of about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, or about 15mM.

Embodiment 14: the method of any of embodiments 10-13, wherein the salt is MgCl ₂ 。

Embodiment 15: the method of any one of embodiments 1-14, wherein the affinity elution buffer comprises a polymer.

Embodiment 16: the method of any one of embodiments 1-15, wherein the affinity elution buffer comprises a polymer at a concentration of at least about 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5% or more.

Embodiment 17: the method of any of embodiments 1-16, wherein the affinity elution buffer comprises a polymer at a concentration of from about 0.1% to about 0.5%, from about 0.15% to about 0.45%, from about 0.2% to about 0.4%, or from about 0.25% to about 0.35%.

Embodiment 18: the method of any one of embodiments 1-17, wherein the affinity elution buffer comprises a polymer at a concentration of about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, or about 0.5%.

Embodiment 19: the method of any of embodiments 15-18, wherein the polymer is a nonionic polymer.

Embodiment 20: the method of any one of embodiments 15-19, wherein the polymer is a poloxamer.

Embodiment 21: the method of any one of embodiments 1-20, wherein the affinity elution buffer has a low pH.

Embodiment 22: the method of any one of embodiments 1-21, wherein the pH of the affinity elution buffer is less than or equal to about 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.2, 2.0, 1.5, or less.

Embodiment 23: the method of any of embodiments 1-22, wherein the pH of the affinity elution buffer is from about 2.0 to about 3.0.

Embodiment 24: the method of any one of embodiments 1-23, wherein the affinity elution buffer comprises: about 75mM glycine, about 25mM histidine, about 10mM MgCl ₂ About 0.3% (w/v) P188, and a pH of about 3.0.

Embodiment 25: the method of any one of embodiments 1, 2 or 7-23, wherein the affinity elution buffer comprises citric acid or a salt thereof.

Embodiment 26: the method of embodiment 25, wherein the affinity elution buffer comprises citric acid or a salt thereof at a concentration of at least about 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM, 100mM, or more.

Embodiment 27: the method of embodiment 25 or 26, wherein the affinity elution buffer comprises citric acid or a salt thereof at a concentration of from about 50mM to about 100mM, from about 55mM to about 95mM, from about 60mM to about 90mM, from about 65mM to about 85mM, or from about 70mM to about 75mM.

Embodiment 28: the method of any one of embodiments 25-27, wherein the affinity elution buffer comprises citric acid or a salt thereof at a concentration of about 50mM, about 55mM, about 60mM, about 65mM, about 70mM, about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, or about 100mM.

Embodiment 29: the method of any one of embodiments 25-28, wherein the affinity elution buffer comprises citric acid or a salt thereof at a concentration of about 75mM.

Embodiment 30: the method of any one of embodiments 1, 2, 7-23 or 25-29, wherein the affinity elution buffer comprises: about 50mM glycine, about 75mM citrate, about 10mM MgCl ₂ About 0.3% (w/v) P188, and a pH of about 3.0.

Embodiment 31: the method of any of embodiments 1-30, wherein the conductivity of the affinity elution buffer ranges from about 5mS/cm to about 8mS/cm, optionally the conductivity of the affinity elution buffer ranges from about 5.5mS/cm to about 7 mS/cm.

Embodiment 32: the method of any of embodiments 1-31, wherein the conductivity of the affinity elution buffer ranges from about 5.75mS/cm to about 6.75mS/cm, optionally the conductivity of the affinity elution buffer ranges from about 6.15mS/cm to about 6.25 mS/cm.

Embodiment 33: the method of any of embodiments 1-32, wherein the affinity elution buffer has an osmolality in the range of from about 100 to about 225mOsm, optionally the affinity elution buffer has an osmolality in the range of from about 125 to about 120 mOsm.

Embodiment 34: the method of any of embodiments 1-33, wherein the affinity elution buffer has an osmolality in the range of from about 150 to about 175mOsm, optionally the affinity elution buffer has an osmolality in the range of from about 155 to about 165 mOsm.

Embodiment 35: the method of any of embodiments 1-34, wherein the equilibration buffer for anion exchange chromatography comprises an acid or salt thereof at a concentration of at least about 0.5mM, 1mM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, 4mM, 4.5mM, 5mM, 5.5mM, 6mM, 6.5mM, 7mM, 8mM, 8.5mM, 9mM, 9.5mM, 10mM, or more.

Embodiment 36: the method of any of embodiments 1-35, wherein the equilibration buffer comprises an acid at a concentration from about 0.5mM to about 15mM, from about 1mM to about 10mM, from about 1.5mM to about 7.5mM, or from about 2mM to about 7mM.

Embodiment 37: the method of any of embodiments 1-36, wherein the equilibration buffer comprises an acid at a concentration of about 0.5mM, about 1mM, about 1.5mM, about 2mM, about 2.5mM, about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 8mM, about 8.5mM, about 9mM, about 9.5mM, or about 10mM.

Embodiment 38: the method of any of embodiments 1-37, wherein the acid is citric acid or a citrate salt.

Embodiment 39: the method of any of embodiments 1-38, wherein conditioning the affinity chromatography eluate for subsequent purification by anion exchange chromatography comprises adding an acid or salt thereof to the eluate, optionally the acid or salt thereof is citric acid, citrate, acetic acid, or succinic acid.

Embodiment 40: the method of embodiment 39, wherein the acid or salt thereof is added to the eluate to a final concentration of at least about 0.5mM, 1mM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, 4mM, 4.5mM, 5mM, 5.5mM, 6mM, 6.5mM, 7mM, 8mM, 8.5mM, 9mM, 9.5mM, 10mM or more.

Embodiment 41: the method of embodiment 39 or 40, wherein the acid or salt thereof is added to the eluate to a final concentration of from about 0.5mM to about 15mM, from about 1mM to about 10mM, from about 1.5mM to about 7.5mM, or from about 2mM to about 7mM.

Embodiment 42: the method of any one of embodiments 39-41, wherein the acid or salt thereof is added to the eluate to a final concentration of about 0.5mM, about 1mM, about 1.5mM, about 2mM, about 2.5mM, about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 8mM, about 8.5mM, about 9mM, about 9.5mM, or about 10mM.

Embodiment 43: the method of any of embodiments 39-42, wherein the acid is citric acid or a salt thereof (e.g., citrate).

Embodiment 44: the method of any of embodiments 1-43, wherein conditioning the affinity chromatography eluate for subsequent purification by anion exchange chromatography comprises diluting the eluate.

Embodiment 45: the method of any of embodiments 1-44, wherein adjusting the affinity eluate for anion exchange chromatography comprises diluting the eluate by at least 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 11×, 12×, 13×, 14×, 15×, 16×, 17×, 18×, 19×, 20×, or more.

Embodiment 46: the method of any one of embodiments 1-41, wherein adjusting the affinity eluate for anion exchange chromatography comprises diluting the eluate with a dilution buffer.

Embodiment 47: the method of embodiment 46, wherein the dilution buffer comprises an acid or salt thereof.

Embodiment 48: the method of embodiment 46 or 47, wherein the dilution buffer comprises an acid or salt thereof at a concentration of at least about 0.5mM, 1mM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, 4mM, 4.5mM, 5mM, 5.5mM, 6mM, 6.5mM, 7mM, 8mM, 8.5mM, 9mM, 9.5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM or more.

Embodiment 49: the method of any of embodiments 46-48, wherein the dilution buffer comprises an acid or salt thereof at a concentration of from about 0.5mM to about 15mM, from about 1mM to about 10mM, from about 1.5mM to about 7.5mM, or from about 2mM to about 7mM.

Embodiment 50: the method of any of embodiments 46-49, wherein the dilution buffer comprises an acid or salt thereof at a concentration of about 0.5mM, about 1mM, about 1.5mM, about 2mM, about 2.5mM, about 3mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 8mM, about 8.5mM, about 9mM, about 9.5mM, or about 10mM.

Embodiment 51: the method of any of embodiments 46-50, wherein the acid is citric acid or a salt thereof, acetic acid or a salt thereof, or succinic acid or a salt thereof.

Embodiment 52: the method of any one of embodiments 46-51, wherein the dilution buffer comprises bis-tris propane (BTP).

Embodiment 53: the method of any one of embodiments 46-52, wherein the dilution buffer comprises BTP at a concentration of at least about 25mM, 50mM, 75mM, 100mM, 125mM, 150mM, or more.

Embodiment 54: the method of any of embodiments 46-53, wherein the dilution buffer comprises BTP at a concentration of from about 25mM to about 175mM, from about 50mM to about 150mM, from about 75mM to about 125mM, from about 80mM to about 120mM, from about 85mM to about 115mM, from about 90mM to about 110mM, or from about 95mM to about 105mM.

Embodiment 55: the method of any of embodiments 46-54, wherein the dilution buffer comprises BTP at a concentration of about 50mM, about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, about 125mM, about 150mM, or about 175mM.

Embodiment 56: the method of any one of embodiments 46-55, wherein the dilution buffer comprises BTP at a concentration of about 100 mM.

Embodiment 57: the method of any one of embodiments 46-56, wherein the dilution buffer comprises amino acids.

Embodiment 58: the method of any one of embodiments 46-57, wherein the dilution buffer comprises amino acids at a concentration of at least about 25mM, 50mM, 75mM, 100mM, 125mM, 150mM, or more.

Embodiment 59: the method of any one of embodiments 46-58, wherein the dilution buffer comprises amino acids at a concentration of from about 25mM to about 175mM, from about 50mM to about 150mM, from about 75mM to about 125mM, from about 80mM to about 120mM, from about 85mM to about 115mM, from about 90mM to about 110mM, or from about 95mM to about 105mM.

Embodiment 60: the method of any one of embodiments 46-59, wherein the dilution buffer comprises an amino acid at a concentration of about 50mM, about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 105mM, about 110mM, about 115mM, about 120mM, about 125mM, about 150mM, or about 175mM.

Embodiment 61: the method of any one of embodiments 46-60, wherein the dilution buffer comprises an amino acid at a concentration of about 100 mM.

Embodiment 62: the method of any one of embodiments 46-61, wherein the amino acid is histidine.

Embodiment 63: the method of any one of embodiments 46-62, wherein the dilution buffer comprises glycerol.

Embodiment 64: the method of any one of embodiments 46-63, wherein the dilution buffer comprises glycerol at a concentration of at least about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% (v/v or w/v) or more.

Embodiment 65: the method of any of embodiments 46-64, wherein the dilution buffer comprises glycerol at a concentration of from about 0.5% to about 9.5%, from about 1% to about 9%, from about 2% to about 8.5%, from about 2.5% to about 8%, from about 3% to about 7.5%, from about 3.5% to about 7%, from about 4% to about 6.5%, or from about 4.5% to about 5.5% (v/v or w/v).

Embodiment 66: the method of any of embodiments 42-65, wherein the dilution buffer comprises glycerol at a concentration of about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, or about 9.5% (v/v or w/v).

Embodiment 67: the method of any one of embodiments 42-66, wherein the dilution buffer comprises glycerol at a concentration of about 5% (v/v or w/v).

Embodiment 68: the method of any one of embodiments 46-67, wherein the dilution buffer comprises a nonionic surfactant.

Embodiment 69: the method of any one of embodiments 46-68, wherein the dilution buffer comprises a nonionic surfactant at a concentration of at least about 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, about 0.9%, 0.95% (v/v or w/v) or more.

Embodiment 70: the method of any of embodiments 46-69, wherein the dilution buffer comprises a nonionic surfactant at a concentration of from about 0.05% to about 0.95%, from about 0.1% to about 0.9%, from about 0.15% to about 0.85%, from about 0.2% to about 0.8%, from about 0.25% to about 0.75%, from about 0.3% to about 7%, from about 0.35% to about 0.65%, from about 0.4% to about 0.6%, or from about 0.45% to about 0.55% (w/v).

Embodiment 71: the method of any of embodiments 46-70, wherein the dilution buffer comprises a nonionic surfactant at a concentration of about 0.05%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, or about 0.95% (w/v).

Embodiment 72: the method of any one of embodiments 46-71, wherein the dilution buffer comprises a nonionic surfactant at a concentration of about 0.5% (w/v).

Embodiment 73: the method of any of embodiments 46-72, wherein the nonionic surfactant is selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof.

Embodiment 74: the method of any one of embodiments 46-73, wherein the dilution buffer comprises a salt.

Embodiment 75: the method of any one of embodiments 46-74, wherein the dilution buffer comprises a salt at a concentration of at least about 0.1mM, 0.25mM, 0.5mM, 0.75mM, 1mM, 1.25mM, 1.5mM, 1.75mM, 2mM, or more.

Embodiment 76: the method of any one of embodiments 46-75, wherein the dilution buffer comprises a salt at a concentration of from about 0.1mM to about 2mM, from about 0.25mM to about 1.75mM, from about 0.5mM to about 1.5mM, or from about 0.75mM to about 1.25mM.

Embodiment 77: the method of any one of embodiments 46-76, wherein the dilution buffer comprises a salt at a concentration of about 0.1mM, about 0.25mM, about 0.5mM, about 0.75mM, about 1mM, about 1.25mM, about 1.5mM, about 1.75mM, or about 2mM.

Embodiment 78: the method of any one of embodiments 46-77, wherein the dilution buffer comprises a salt at a concentration of about 1 mM.

Embodiment 79: the method of any one of embodiments 46-78, wherein the salt comprises MgCl ₂ 。

Embodiment 80: the method of any one of embodiments 46-79, wherein the dilution buffer has a high pH.

Embodiment 81: the method of any of embodiments 46-80, wherein the dilution buffer has a pH greater than or equal to about 8, about 8.5, about 9, about 9.5, or about 10.

Embodiment 82: the method of any one of embodiments 46-81, wherein the dilution buffer has a pH of about 9.

Embodiment 83: the method of any one of embodiments 46-82, wherein the dilution buffer comprises: BTP, histidine, glycerol, PF68, mgCl ₂ And has a high pH.

Embodiment 84: the method of any one of embodiments 14-30 or 35-50, wherein the dilution buffer comprises: BTP, histidine, PF68, mgCl ₂ And has a high pH.

Embodiment 85: the method of any one of embodiments 1-84, wherein the conductivity of the dilution buffer is in the range of from about 0.5mS/cm to about 3mS/cm, optionally the conductivity of the dilution buffer is in the range of from about 1mS/cm to 2.5 mS/cm.

Embodiment 86: the method of any one of embodiments 1-85, wherein the conductivity of the dilution buffer is in the range of from about 1.25mS/cm to about 2.25mS/cm, optionally the conductivity of the dilution buffer is in the range of from about 1.5mS/cm to about 1.75 mS/cm.

Embodiment 87: the method of any one of embodiments 1-86, wherein the dilution buffer has an osmolality of less than 900mOsm.

Embodiment 88: the method of any one of embodiments 1-87, further comprising the step of removing or reducing the amount of impurities (e.g., host cell DNA (hcna)) from the harvest medium prior to affinity purification.

Embodiment 89: the method of embodiment 88, wherein the removing or reducing the amount of impurities comprises adding a cationic amine or nuclease to the harvesting medium.

Embodiment 90: the method of embodiment 88 or 89, wherein the removing or reducing the amount of impurities comprises adding a selective precipitant to the harvest media.

Embodiment 91: the method of any one of embodiments 1-90, further comprising the step of lysing the host cells in the harvest medium with a nonionic surfactant prior to purification/separation by affinity chromatography.

Embodiment 92: the method of embodiment 91, wherein the nonionic surfactant is added to the collection medium to a final concentration of at least about 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1% or more.

Embodiment 93: the method of embodiment 91 or 92, wherein the nonionic surfactant is added to the collection medium to a final concentration of from about 0.05% to about 1%, from about 0.1% to about 0.95%, from about 0.15% to about 0.9%, from about 0.2% to about 0.85%, from about 0.25% to about 0.8%, from about 0.3% to about 0.75%, from about 0.35% to about 0.65%, from about 0.4% to about 0.6%, or from about 0.45% to about 0.55%.

Embodiment 94: the method of any of embodiments 91-93, wherein the nonionic surfactant is added to the collection medium to a final concentration of about 0.05%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, or about 1%.

Embodiment 95: the method of any of embodiments 91-94, wherein the nonionic surfactant is added to the collection medium to a final concentration of about 0.5%.

Embodiment 96: the method of any of embodiments 91-95, wherein the nonionic surfactant is mixed with the harvesting medium for a period of time from about 15 minutes to about 2 hours.

Embodiment 97: the method of any of embodiments 91-96, wherein the nonionic surfactant is mixed with the harvesting medium for a period of time from about 30 minutes to about 60 minutes.

Embodiment 98: the method of any of embodiments 91-97, wherein the nonionic surfactant is not Triton X-100.

Embodiment 99: the method of any of embodiments 91-98, wherein the nonionic surfactant is selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof.

Embodiment 100: the method of any one of embodiments 1-99, wherein less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the empty viral particles in the affinity eluate bind to the anion exchange chromatography medium.

Embodiment 101: the method of any one of embodiments 1-100, wherein substantially no empty viral particles in the affinity eluate are bound to the anion exchange chromatography medium.

Embodiment 102: the method of any one of embodiments 1-101, wherein less than 10%, less than 8%, less than 5% or less than 2% or preferably even less of the viral particles in the eluate from the anion exchange are empty viral particles.

Embodiment 103: the method of any one of embodiments 1-102, wherein the eluate from the anion exchange is substantially free of empty viral particles.

Embodiment 104: the method of any one of embodiments 1-103, wherein the recombinantly expressed viral particle is a recombinant adeno-associated virus (rAAV) particle.

Embodiment 105: the method of any one of embodiments 1-104, wherein the recombinant adeno-associated virus (rAAV) particle comprises a rAAV virion.

Embodiment 106: a population of recombinantly expressed viral particles purified or isolated by the method of any one of embodiments 1-105.

Embodiment 107: a population of purified recombinant adeno-associated viruses (rAAV) lacking prokaryotic sequences, wherein the particle to infectivity ratio of the purified rAAV is less than 2 x 10 ⁴ The vg/TCID50, optionally, the purified rAAV population comprises less than about 10% empty viral capsids, wherein the purified rAAV is obtained by a method comprising transfecting a suspension mammalian cell line, and optionally transfecting the cells in suspension.

Embodiment 108: the purified population of recombinant adeno-associated viruses (rAAV) of embodiment 107, wherein the population comprises less than about 5% empty viral capsids.

Embodiment 109: the purified population of recombinant adeno-associated virus (rAAV) of any one of embodiments 107-108, wherein the population comprises less than about 2% empty viral capsids.

Embodiment 110: the purified population of recombinant adeno-associated virus (rAAV) of any one of embodiments 107-109, wherein the population comprises less than about 1% empty viral capsids.

Embodiment 111: the purified population of recombinant adeno-associated virus (rAAV) of any one of embodiments 107-110, wherein the population comprises less than about 0.5% empty viral capsids.

Embodiment 112: the purified population of recombinant adeno-associated virus (rAAV) of any one of embodiments 107-111, wherein the population comprises less than about 0.2% empty viral capsids.

Embodiment 113: the purified population of recombinant adeno-associated virus (rAAV) of any one of embodiments 107-112, wherein the population comprises less than about 0.05% empty viral capsids.

Embodiment 114: the purified population of recombinant adeno-associated virus (rAAV) of any one of embodiments 107-113, wherein the population comprises less than about 0.03% empty viral capsids.

Embodiment 115: the purified population of recombinant adeno-associated virus (rAAV) of any one of embodiments 107-114, wherein the population is substantially free of empty viral capsids.

Embodiment 116: the purified population of recombinant adeno-associated virus (rAAV) of any one of embodiments 107-115, wherein the mammalian cell line is derived from a human embryonic cell line.

Embodiment 117: the purified population of recombinant adeno-associated virus (rAAV) of any one of embodiments 107-116, wherein the human embryonic cell line is a suspension-adapted serum-free cell line derived from a human embryonic kidney cell line.

Embodiment 118: a population of purified recombinant adeno-associated viruses (rAAV) lacking prokaryotic sequences, wherein the particle to infectivity ratio of the purified rAAV is less than 2 x 10 ⁴ The purified rAAV is obtained by a method comprising transfecting a suspension mammalian cell line, optionally at vg/TCID 50.

Embodiment 119: the purified recombinant adeno-associated virus (rAAV) population of embodiment 118, wherein the nucleic acid sequence encoding a helper protein sufficient for rAAV replication is used a); b) Nucleic acid sequences encoding rep and cap genes; and c) transfecting the mammalian cell line in suspension with a closed-end linear double-stranded rAAV vector nucleic acid comprising at least one ITR and a heterologous transgene operably linked to one or more regulatory elements.

Embodiment 120: the purified population of recombinant adeno-associated virus (rAAV) of any one of embodiments 118-119, wherein the mammalian cell line is derived from a human embryonic cell line.

Embodiment 121: the purified population of recombinant adeno-associated virus (rAAV) of any one of embodiments 118-120, wherein the human embryonic cell line is a suspension-adapted serum-free cell line derived from a human embryonic kidney cell line.

Embodiment 122: a purified population of recombinant adeno-associated viruses (rAAV), wherein the purified population of rAAV comprises less than about 10% empty viral capsids, optionally the population of rAAV is purified by a method comprising:

a. purifying/separating the plurality of recombinantly-expressed viral particles from the harvest medium by affinity chromatography to produce an eluate comprising the plurality of recombinantly-expressed viral particles (affinity chromatography eluate), wherein an elution buffer for affinity chromatography (affinity elution buffer) comprises a predetermined amount of glycine, optionally the affinity elution buffer is substantially free of weak acids or salts thereof; and optionally, the affinity elution buffer comprises an amino acid that is not glycine;

b. conditioning the affinity chromatography eluate for subsequent purification by anion exchange chromatography, wherein the conditioned eluate comprises a predetermined amount of an anionic compound;

c. purifying/separating the plurality of recombinantly-expressed viral particles from the conditioned eluate of the affinity chromatography by anion exchange chromatography to produce a solution comprising the plurality of purified/separated recombinantly-expressed viral particles, wherein an equilibration buffer for anion exchange chromatography comprises a predetermined amount of a weak acid or salt thereof, optionally the weak acid is citric acid, acetic acid or succinic acid,

Thereby producing the purified population of recombinant adeno-associated viruses (rAAV).

Embodiment 123: the purified population of recombinant adeno-associated virus (rAAV) of embodiment 122, wherein the population comprises less than about 5% empty viral capsids.

Embodiment 124: the purified population of recombinant adeno-associated virus (rAAV) of any one of embodiments 122-123, wherein the population comprises less than about 2% empty viral capsids.

Embodiment 125: the purified population of recombinant adeno-associated virus (rAAV) of any one of embodiments 122-124, wherein the population comprises less than about 1% empty viral capsids.

Embodiment 126: the purified population of recombinant adeno-associated virus (rAAV) of any one of embodiments 122-125, wherein the population comprises less than about 0.5% empty viral capsids.

Embodiment 127: the purified population of recombinant adeno-associated virus (rAAV) of any one of embodiments 122-126, wherein the population comprises less than about 0.2% empty viral capsids.

Embodiment 128: the purified population of recombinant adeno-associated virus (rAAV) of any one of embodiments 122-127, wherein the population comprises less than about 0.05% empty viral capsids.

Embodiment 129: the purified population of recombinant adeno-associated virus (rAAV) of any one of embodiments 122-128, wherein the population comprises less than about 0.03% empty viral capsids.

Embodiment 130: the purified population of recombinant adeno-associated virus (rAAV) of any one of embodiments 122-129, wherein the population is substantially free of empty viral capsids.

Embodiment 131: a composition comprising a purified population of recombinant adeno-associated virus particles and having a pH of from about 6.5 to about 8.0, and wherein: (i) The particle to infectivity ratio of the purified rAAV is less than 2X 10 ⁴ vg/TCID50; and/or (ii) the purified rAAV population comprises less than about 10% empty viral capsids.

Embodiment 132: the composition of embodiment 131, wherein the composition comprises the purified rAAV at a concentration of from about 1e9 vg/mL to about 1e15 vg/mL.

Embodiment 133: the composition of embodiment 131 or 132, wherein the composition comprises the purified rAAV at a concentration of from about 1e12 vg/mL to about 1e14vg/mL.

Embodiment 134: the composition of any one of embodiments 131-133, wherein the composition comprises the purified rAAV at a concentration of from about 1e12 vg/mL to about 1e14vg/mL, preferably the purified rAAV at a concentration of from about 1e13 vg/mL to about 1e14vg/mL.

Embodiment 135: the composition of any of embodiments 131-134, wherein the composition has a pH of from about 6.5 to about 8.0.

Embodiment 136: the composition of any of embodiments 131-135, wherein the composition has a pH of from about 6.5 to about 7.5.

Embodiment 137: the composition of any of embodiments 131-136, wherein the composition has a pH of from about 7.0 to about 7.4.

Embodiment 138: the composition of any of embodiments 131-135, wherein the composition has a pH of about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, or about 7.9.

Embodiment 139: the composition of any one of embodiments 131-138, wherein the composition comprises a buffer.

Embodiment 140: the composition of embodiment 139, wherein the salt concentration of the buffer is from about 50mM to about 750mM.

Embodiment 141: the composition of embodiment 139 or 140, wherein the salt concentration of the buffer is from about 100mM to about 650mM.

Embodiment 142: the composition of any of embodiments 139-141, wherein the salt concentration of the buffer is from about 150mM to about 400mM.

Embodiment 143: the composition of any of embodiments 139-142, wherein the salt concentration of the buffer is about 150mM, about 200mM, or about 365mM.

Embodiment 144: the composition of any one of embodiments 131-143, wherein the composition has an ionic strength of at least 100mM.

Embodiment 145: the composition of embodiment 144, wherein the ionic strength of the composition is from about 125mM to about 750mM.

Embodiment 146: the composition of embodiment 144 or 145, wherein the ionic strength of the composition is from about 150mM to about 550mM.

Embodiment 147: the composition of any one of embodiments 144-146, wherein the ionic strength of the composition is about 170mM, about 210mM, or about 380mM.

Embodiment 148: the composition of any of embodiments 131-147, wherein the composition has an osmolality of from about 100mOsm to about 600mOsm.

Embodiment 149: the composition of embodiment 148, wherein the osmolality of the composition is from about 125mOsm to about 500mOsm.

Embodiment 150: the composition of embodiment 148, wherein the composition has an osmolality of from about 200mOsm to about 400mOsm.

Embodiment 151: the composition of embodiment 149, wherein the composition has an osmolality of from about 140mOsm to about 315mOsm.

Embodiment 152: the composition of any of embodiments 131-151, wherein the composition comprises one or more ions and/or salts thereof.

Embodiment 153: the composition of embodiment 152, wherein the ion is selected from the group consisting of sodium, potassium, chloride, ammonium, carbonate, nitrate, chlorate, chlorite, and calcium.

Embodiment 154: the composition of any one of embodiments 131-153, wherein the composition comprises NaCl at a concentration from about 125mM to about 450mM.

Embodiment 155: the composition of embodiment 154, wherein the composition comprises NaCl at a concentration of from about 150mM to about 400mM.

Embodiment 156: the composition of embodiment 155, wherein the composition comprises NaCl at a concentration of from about 175mM to about 375mM.

Embodiment 157: the composition of any one of embodiments 131-156, wherein the composition comprises KCl at a concentration of from about 1mM to about 10mM.

Embodiment 158: the composition of embodiment 157, wherein the composition comprises KCl at a concentration of from about 2mM to about 5.5mM.

Embodiment 159: the composition of any one of embodiments 131-158, wherein the composition comprises CaCl ₂ The concentration is from about 0.1mM to about 2mM.

Embodiment 160: the composition of embodiment 159, wherein the composition comprises CaCl ₂ At a concentration of from about 0.75mM to about 1.25mM。

Embodiment 161: the composition of any one of embodiments 131-160, wherein the composition comprises MgCl ₂ The concentration is from about 0.1mM to about 1.5mM.

Embodiment 162: the composition of embodiment 161, wherein the composition comprises MgCl ₂ The concentration is from about 0.25mM to about 0.75mM.

Embodiment 163: the composition of any one of embodiments 131-162, wherein the composition comprises a monobasic phosphate or salt thereof at a concentration of from about 0.25mM to about 3mM.

Embodiment 164: the composition of embodiment 163, wherein the composition comprises a monobasic phosphate or salt thereof at a concentration of from about 1mM to about 2.25mM.

Embodiment 165: a composition of embodiment 163 or 164, wherein the monobasic phosphate is monobasic potassium phosphate.

Embodiment 166: the composition of any of embodiments 131-165, wherein the composition comprises a dibasic phosphate or salt thereof at a concentration of from about 5mM to about 15mM.

Embodiment 167: the composition of embodiment 166, wherein the composition comprises a dibasic phosphate or salt thereof at a concentration of from about 8mM to about 10mM.

Embodiment 168: the composition of embodiment 166 or 167, wherein the dibasic phosphate or salt thereof is disodium hydrogen phosphate.

Embodiment 169: the composition of any of embodiments 131-168, wherein the composition comprises a filler, e.g., a polyol or povidone (PVP K24).

Embodiment 170: the composition of embodiment 169, wherein the filler is selected from the group consisting of polyhydroxy hydrocarbons, monosaccharides, disaccharides, and trisaccharides.

Embodiment 171: the composition of embodiment 169 or 170, wherein the filler is selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, and dextran.

Embodiment 172: the composition of any of embodiments 166-171, wherein the composition comprises a filler, e.g., a polyol or povidone (PVP K24), at a concentration of from about 0.5% (w/v) to about 10% (w/v).

Embodiment 173: the composition of any of embodiments 166-172, wherein the composition comprises a filler, e.g., a polyol or povidone (PVP K24), at a concentration of from about 1% (w/v) to about 7.5% (w/v).

Embodiment 174: the composition of any of embodiments 166-173, wherein the composition comprises a filler, e.g., a polyol or povidone (PVP K24), at a concentration of about 1% (w/v), about 3% (w/v), or about 5% (w/v).

Embodiment 175: the composition of any of embodiments 131-174, wherein the composition comprises a nonionic surfactant.

Embodiment 176: the composition of embodiment 175, wherein the nonionic surfactant is selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol polyoxyethylene ethers (preferably polysorbates, polyoxyethylene alkylphenyl ethers), and any combination thereof.

Embodiment 177: the composition of embodiment 175 or 176, wherein the nonionic surfactant is selected from the group consisting of TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68, polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, brij 020, ECOSURF EH-9, ECOSURF EH-14, TERGITOL 15-S-7, ECOSURF SA-15, TERGOL 15-S-9, TERGOL 15-S-12, GIL-64, TERGOL-7, TERGOL-8, TERGOL-9, TERGOL-10, and any combination thereof.

Embodiment 178: the composition of any of embodiments 175-177, wherein the composition comprises a nonionic surfactant at a concentration of from about 0.0001% (w/v) to about 0.01% (w/v).

Embodiment 179: the composition of any of embodiments 175-178, wherein the composition comprises a nonionic surfactant at a concentration of from about 0.0005% (w/v) to about 0.0015% (w/v).

Embodiment 180: the composition of any of embodiments 175-179, wherein the composition comprises a nonionic surfactant at a concentration of about 0.001% (w/v).

Embodiment 181: the composition of any one of embodiments 131-180, wherein the composition comprises one or more multivalent ions or salts thereof.

Embodiment 182: the composition of embodiment 181, wherein the multivalent ion is selected from the group consisting of citrate, sulfate, magnesium, and phosphate.

Embodiment 183: the composition of any of embodiments 131-182, wherein the composition comprises MgSO ₄ The concentration is from about 5mM to about 150mM.

Embodiment 184: the composition of embodiment 183, wherein said composition comprises MgSO ₄ The concentration is from about 15mM to about 100mM.

Embodiment 185: the composition of any of embodiments 131-184, wherein the composition comprises alpha-d-calcium heptagluconate in a concentration from about 1% (w/v) to about 20% (w/v).

Embodiment 186: the composition of any one of embodiments 131-185, wherein the purified rAAV population comprises less than about 5%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.2%, less than about 0.15%, less than about 0.1%, less than about 0.05%, less than 0.04%, less than about 0.03%, or less than about 0.025% empty viral capsids.

Embodiment 187: the composition of any one of embodiments 131-186, wherein the purified rAAV population is substantially free of empty viral capsids.

Embodiment 188: the composition of any one of embodiments 131-187, wherein the particles of purified rAAVParticle to infectivity ratio is less than 1.5X10 ⁴ vg/TCID50, less than 1X 10 ⁴ vg/TCID50, less than 9X 10 ³ vg/TCID50, less than 8X 10 ³ vg/TCID50, less than 6X 10 ³ vg/TCID50, less than 5X 10 ³ vg/TCID50, less than 4X 10 ³ vg/TCID50, less than 3X 10 ³ vg/TCID50, less than 2X 10 ³ vg/TCID50, less than 9X 10 ² vg/TCID50, less than 8X 10 ² vg/TCID50, less than 7X 10 ² vg/TCID50, less than 6X 10 ² vg/TCID50, less than 5X 10 ² vg/TCID50, less than 4X 10 ² vg/TCID50, less than 3X 10 ² vg/TCID50, less than 2X 10 ² vg/TCID50, or less than 1X 10 ² vg/TCID50, or less than 0.5X10 ² vg/TCID50。

Embodiment 189: the composition of any one of embodiments 131-188, wherein the purified population of recombinant adeno-associated virus particles is the purified population of rAAV of any one of embodiments 107-130.

Embodiment 190: a pharmaceutical composition comprising a purified population of recombinant adeno-associated virus (rAAV) particles according to any preceding embodiment.

Embodiment 191: the composition of any one of the preceding embodiments, wherein the composition exhibits substantially no aggregation of rAAV particles after two or more freeze-thaw cycles.

Embodiment 192: the composition of any one of the preceding embodiments, wherein the purified rAAV particle retains at least about 80% of its TCID50/mL after two or more freeze-thaw cycles.

Some selected definitions

For the purposes of this specification and the appended claims, unless otherwise indicated, all numbers expressing quantities, dimensions, proportions, shapes, formulations, parameters, percentages, parameters, amounts, characteristics, and other values used in the specification and claims are to be understood as being modified in all instances by the term "about (about)", even though the term "about" may not be expressly shown in connection with its value, amount, or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are not and need not be exact, but may be approximated and/or larger or smaller as desired, reaction tolerances, conversion factors, rounding off, measurement error and the like, as well as other factors known to those of skill in the art, depending on the desired properties sought to be obtained by the presently disclosed subject matter. For example, when referring to a value, the term "about" may be meant to encompass variations from a particular amount of +100% in some embodiments, ±50% in some embodiments, ±20%, in some embodiments, ±10%, in some embodiments, ±5%, in some embodiments, ±1%, in some embodiments, ±0.5% and in some embodiments, ±0.1% as such variations are suitable for performing the disclosed methods or using the disclosed compositions.

Furthermore, when used in conjunction with one or more numbers or numerical ranges, the term "about" should be understood to refer to all such numbers, including all numbers within the range, and to modify the range by extending the boundary above and below the listed numbers. The recitation of numerical ranges by endpoints includes all numbers subsumed within that range, e.g., all integers, including fractions thereof (e.g., the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, and fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, etc.) and any range within that range.

As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, if the terms "comprise/include," have, "" with, "or variants thereof are used in the detailed description and/or claims, these terms are intended to be inclusive in a manner similar to the term" comprise/comprise. It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" is a reference to one or more proteins and includes equivalents thereof known to those skilled in the art, and so forth.

As used herein, when referring to defined or described elements of an article, composition, device, method, process, system, etc., the term "comprising/containing" and variants thereof are intended to include or be open ended, allowing additional elements to be included, thereby indicating that the defined or described article, composition, device, method, process, system, etc., includes those specified elements (or equivalents thereof, as appropriate), and that other elements may be included and still fall within the scope/definition of the defined article, composition, device, method, process, system, etc.

The term "cell culture" refers to cells adhered to or grown in suspensions, bioreactors, roller bottles, hypersacks, microspheres, macrospheres (macrospheres), flasks, etc., as well as components of the supernatant or suspension itself, including but not limited to viral particles (e.g., rAAV particles), cells, cell debris, cell contaminants, colloidal particles, biomolecules, host cell proteins, nucleic acids and lipids, and coagulants. Large scale processes such as bioreactors include suspension cultures and adherent cells grown attached to microcarriers or macrocarriers (macrocarriers) in stirred bioreactors, also included in the term "cell cultures". The present disclosure encompasses cell culture procedures for large-scale and small-scale production of proteins.

The term "cell supernatant" or "cell culture supernatant" refers to the extracellular components and liquid medium in a medium suspension that can be separated from the cell material of an adherent cell culture or cell suspension culture. In general, substances secreted from cells in culture can be purified from cell supernatants. In some cases, the viral particles, whether or not there are lysed cells, can be secreted into the supernatant and purified therefrom.

The terms "purifying", "separating", "isolation", or "separating" as used herein refer to increasing the purity of a recombinant viral particle (e.g., a rAAV particle) from a sample containing the recombinant viral particle of interest (e.g., a rAAV particle and one or more impurities). Generally, the purity of the target product is enhanced by removing (completely or partially) at least one impurity from the sample. In some embodiments, the purity of recombinant viral particles (e.g., rAAV particles) in a sample is increased by removing (completely or partially) one or more impurities from the sample using the methods described herein.

The term "impurity" refers to any foreign or undesirable molecule, including biological macromolecules such as DNA, RNA, one or more host cell proteins, endotoxins, lipids, and one or more additives, which may be present in a sample containing recombinant viral particles. The term "impurities" further includes impurities associated with the product, e.g., inactive carrier forms, empty viral capsids, aggregated viral particles or capsids, misfolded viral capsids, decomposed viral particles. In some embodiments, the impurity comprises empty viral capsids or viral aggregates. Further, such impurities may include any reagent used in a step that may occur prior to one or more of the disclosed methods. The impurities may be soluble or insoluble in nature. Insoluble impurities include any undesired or undesirable entity present in the sample containing the recombinant viral particles, wherein the entity is a suspended particle or a solid. Exemplary insoluble impurities include, but are not limited to, aggregated viral particles or capsids, whole cells, cell fragments, and cell debris. Soluble impurities include any undesired or undesirable entity present in the sample containing the recombinant viral particles, wherein the entity is not an insoluble impurity. Exemplary soluble impurities include, but are not limited to, host cell proteins, DNA, RNA, lipid viruses, endotoxins, and cell culture medium components.

As used herein, the term "helper virus" or "contaminating helper virus" refers to a virus that is used when producing copies of a helper-dependent viral vector (e.g., an adeno-associated virus) and that is not itself replication competent. Helper viruses are used to co-infect cells with viral vectors and provide the necessary proteins for replication of the viral vector genome. The term includes complete viral particles, empty capsids, viral DNA, and the like. Helper viruses commonly used to produce rAAV particles include adenovirus, herpes simplex virus, cytomegalovirus, epstein-Barr virus, and vaccinia virus.

Helper viruses include Adenoviruses (AV) and Herpes Simplex Viruses (HSV), as well as systems exist that use baculoviruses to produce AAV in insect cells and in mammalian cells. Papillomaviruses have also been proposed to provide helper functions to AAV (see, e.g., hermonat et al, molecular Therapy 9,5289-S290 (2004)). Helper viruses include any virus that is capable of producing a virus that allows AAV to replicate. AV is a non-enveloped nuclear DNA virus whose double-stranded DNA genome is about 36kb. AV can rescue potential AAV proviruses in cells by providing the Ela, elb55K, E a, E4orf6 and VA genes, allowing AAV replication and encapsidation. HSV is a family of viruses with a relatively large double-stranded linear DNA genome enclosed in an icosahedral capsid, which is enclosed in a lipid bilayer envelope. HSV is infectious and highly transmissible. The following HSV-1 replicates UL8 and UL52 and the DNA binding protein ICP8 encoded by the UL29 gene, as well as other proteins that enhance helper functions.

The term "non-adherent cell line" or "suspension cell line" as used herein refers to a cell line that is capable of surviving in suspension culture without adhering to a surface (e.g., a tissue culture plastic carrier or microcarrier). Adaptation to non-adherent cell lines is a lengthy process requiring passaging with smaller and smaller amounts of serum to screen for irreversibly modified cell populations. The cell lines can be grown to higher densities than allowed by the adherent conditions and are therefore more suitable for culture on an industrial scale, for example in a bioreactor environment or in stirred culture.

As used in this specification and the appended claims, the term "or" is generally employed in its sense including "and/or" unless the content clearly dictates otherwise.

The term "tropism" as used herein refers to preferential entry of a virus into certain cells or tissues, optionally followed by expression (e.g., transcription, and optionally translation) of sequences carried by the viral genome in the cells, e.g., expression of a heterologous nucleic acid of interest for a recombinant virus.

The term "promoter" as used herein is defined as a DNA sequence recognized by a cellular or introduced synthetic mechanism that is required to initiate specific transcription of a polynucleotide sequence. A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, results in the production of the gene product in a cell under most or all physiological conditions of the cell. An "inducible" promoter is a nucleotide sequence which, when operably linked to a polynucleotide encoding or specifying a gene product, results in the production of the gene product in a cell substantially only when an inducer corresponding to the promoter is present in the cell. A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene, results in the production of a gene product in a cell, essentially only if the cell is a cell of the tissue type corresponding to the promoter.

A "regulatory element" is any transcriptional or non-transcriptional regulatory element that operates at the transcriptional or post-transcriptional level. The regulatory element may be a cis regulatory element or a cis regulatory module, which is a region of non-coding DNA that regulates transcription of an adjacent gene. Non-limiting examples of regulatory elements or cis-regulatory elements are promoters, enhancers, silencers, and operators. The regulatory elements may be synthetic. One example of a synthetic regulatory element is the combination of two or more cis regulatory elements. In some examples, the regulatory element may be a trans regulatory element. The trans-regulatory element is typically a DNA sequence encoding an upstream regulatory factor (i.e., a trans-acting factor) that can modify or regulate the expression of a distant gene. Non-limiting examples of trans-regulatory elements are nucleic acids encoding transcription factors or fragments thereof; non-limiting examples of nucleic acids encoding DNA editing proteins or fragments thereof include nucleic acids encoding RAG1/RAG2, tdT, cas1/Cas 2; non-limiting examples of nucleic acids encoding mRNA processing proteins or fragments thereof include nucleic acids encoding SR proteins, ribonucleoproteins (e.g., hnRNP, snRNP); non-limiting examples of nucleic acids encoding mRNA binding proteins and/or nucleic acids other than RNA sequences include nucleic acid sequences encoding RNA binding proteins or siRNA, shRNA, miRNA or piRNA sequences.

A "prokaryotic telomerase" targeting sequence is any DNA sequence whose presence in a DNA template allows its conversion to closed linear DNA by the enzymatic activity of the prokaryotic telomerase. In other words, the prokaryotic telomerase targeting sequence is necessary for the cleavage and re-ligation of double-stranded DNA by the prokaryotic telomerase to form covalently closed linear DNA. Typically, a prokaryotic telomerase targeting sequence includes any perfect palindromic sequence, i.e., any double-stranded DNA sequence with dual rotational symmetry, also described herein as perfect inverted repeats. The length of the perfect inverted repeat varies from organism to organism. In borrelia burgdorferi (Borrelia burgdorferi), the perfect inverted repeat is 14 base pairs in length. In various mesophilic phages, the perfect inverted repeat is 22 base pairs or more in length. Furthermore, in some cases, e.g., escherichia coli (e.coli) N15, the central perfect reverse palindromic is flanked by inverted repeat sequences, i.e., forming part of a larger imperfect reverse palindromic.

The term "variant" when used with respect to a polynucleotide sequence may include polynucleotide sequences associated with wild-type genes. This definition may also include, for example, "allelic," "splice," "species," or "polymorphic" variants. Splice variants can have significant identity to a reference molecule, but will typically have a greater or lesser number of polynucleotides due to alternative splicing of exons during mRNA processing. The corresponding polypeptide may have additional functional domains or deletions of domains. Species variants are polynucleotide sequences that vary from species to species. Particularly useful in the present invention are variants of the wild-type gene product. Variants may be generated from at least one mutation in the nucleic acid sequence and may result in altered MRNAs or polypeptides whose structure or function may or may not be altered. Any given native or recombinant gene may have zero, one, or multiple allelic forms. Common mutational changes that lead to variants are often due to natural deletions, additions or substitutions of nucleotides. Each of these types of variations may occur alone or in combination with the others, one or more times in a given sequence.

The range is as follows: throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description of the range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges as well as individual values within the range. For example, descriptions of ranges such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within the range, e.g., 1, 2, 2.1, 2.2, 2.7, 3, 4, 5, 5.5, 5.75, 5.8, 5.85, 5.9, 5.95, 5.99, and 6. This applies to any breadth of range.

As used herein, "substantially free" means that no significant amount of the indicated component is present. Typically, the indicated components are not present or are present in only minimal amounts without any substantial effect on the properties of the composition. For example, the indicated component is present in an amount (mol/mol) of about 4.5% or less, about 4% or less, about 3.5% or less, about 3% or less, about 2.5% or less, about 2% or less, about 1.5% or less, about 1% or less, about 0.5% or less, about 0.25% or less, about 0.20% or less, about 0.15% or less, about 0.05% or less, or about 0.01% or less. In some embodiments, the indicated component is present in an amount (v/v, w/v, or w/w) of about 4.5% or less, about 4% or less, about 3.5% or less, about 3% or less, about 2.5% or less, about 2% or less, about 1.5% or less, about 1% or less, about 0.5% or less, about 0.25% or less, about 0.20% or less, about 0.15% or less, about 0.05% or less, about 0.01% or less. In some embodiments, the indicated component is present in an amount of about 10,000ppm or less, 7,500ppm or less, 5,000ppm or less, 2,500ppm or less, 2,000ppm or less, 1,500ppm or less, 1,000ppm or less, 750ppm or less, 500ppm or less, 400ppm or less, 300ppm or less, 200ppm or less, 100ppm or less, 75ppm or less, 50ppm or less, 40ppm or less, 30ppm or less, 20ppm or less, 10ppm or less, or 5ppm or less. In some embodiments, the indicated component is present in an undetectable amount. For example, the indicated component is present in an undetectable amount by HPLC, gas chromatography, mass spectrometry, or other means for detecting the indicated component.

Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the following claims. Further, where not already indicated, one of ordinary skill in the art will appreciate that any of the various embodiments described and illustrated herein may be further modified to incorporate features shown in any of the other embodiments disclosed herein.

The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. Although specific implementations of, and examples for, the disclosure are disclosed herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, although method steps or functions are presented in a given order, alternative embodiments may perform the functions in a different order, or the functions may be performed substantially simultaneously. The teachings of the present disclosure provided herein may be suitably applied to other processes or methods. The various embodiments disclosed herein may be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions, and concepts of the above-described references and applications to provide yet another embodiment of the disclosure.

Certain elements of any of the foregoing embodiments may be combined with or substituted for elements of other embodiments. Moreover, while advantages associated with certain embodiments of the disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments must exhibit such advantages to fall within the scope of the disclosure.

Examples

Example 1: high throughput screening to purify multiple AAV serotypes

In the present invention, several AAV serotypes can be purified simultaneously using a high throughput Ambr system in tandem with the Tecan system. Using the high throughput Ambr system, 24 mini-bioreactors containing 15mL of transfected Pro10 cells were grown for 3 days. In this exemplary method, 12 different AAV serotypes are purified, e.g., AAV2, AAV3b, AAV4, AAV5, AAV6, AAV8, AAV9, AAV2i8, AAVrh10, and AAVrh74. The amount of transfected DNA varies for each serotype depending on the different cell densities used for transfection. After 3 days of cell growth, the culture medium from 24 different bioreactors was purified (with or without lysis) on 48 small-scale (0.1 mL) Affinity RoboColumns. The culture medium (with or without lysis) from each bioreactor was eluted with two different elution buffers containing glycine-histidine or glycine-citrate. The eluate was then analyzed using a microplate reader and a high throughput SEC-HPLC method on a Tecan system to determine the unit productivity (vp/mL culture) and packaging of the% intact capsids. Each sample was evaluated for carrier stability on DLS. The best candidate screened was identified and the affinity column was scaled up (range: 1-2000 mL). The affinity purified material was then sent to a high throughput AEX method (0.1 mL) using Tecan or FPLC in scout mode. AEX method evaluation included the addition of 0-20mM citric acid (or 0-30mM succinic acid or 0-60mM acetic acid) at the sample dilution/column equilibration stage, and 3CV conductivity mediated isocratic step elution at pH greater than 8. AEX flow-through and eluate fractions were then analyzed using a Tecan microplate reader or SEC-HPLC to assess separation of empty and intact capsids and capsid recovery. The method allows the identification of elution buffer conditions and weak acid concentrations for the purification of recombinant expressed (intact) AAV serotypes in a high throughput manner. The method can be scaled down or scaled up according to production requirements.

Example 2: glycine-citrate AEX stream

AEX experiments used 1mL CIMmultus QA monolith (BIA Separations, catalog No. 311.5113-2) with a pore size of 2.0 μm. Chromatography experiments were performed using the AKTA Avant25 system (cytova, forefront GE Healthcare Life Sciences).Chromatographic experiments were performed in the pH range of 8.5 to 9.5 using bis-tris propane as buffer system. CIMmultus QA was first run with 15 column volumes of 100mM BTP, 100mM histidine, 1mM MgCl ₂ The PF68, 0.4%, and variable amounts of citric acid were equilibrated. Affinity purification comprising glycine-citrate was pooled in 100mM BTP, 100mM histidine, 1mM MgCl ₂ Diluted 12-15 times in PF68, 0.4% and variable amount of citric acid, and loaded onto CIMmultus QA column at a loading ratio of 5e13vp/mL to 2e14 vp/mL. The column was then chased with 25 column volumes of equilibration solution and the empty and/or intact particles eluted from the column by applying a linear gradient elution. All chromatographic steps were performed in the downward flow direction at ambient temperature.

Example 3: glycine-histidine AEX streams

AEX experiments used 1mLCIMmultus QA monolith (BIA Separations, catalog No. 311.5113-2) with a pore size of 2.0 μm. Chromatography experiments were performed using the AKTA Avant25 system (cytova, forefront GE Healthcare Life Sciences). Chromatographic experiments were performed in the pH range of 8.5 to 9.5 using bis-tris propane as buffer system. CIMmultus QA was first run with 15 column volumes of 100mM BTP, 100mM histidine, 1mM MgCl ₂ The balance was 5% glycerol, 0.5% PF68 and variable amounts of citric acid. The affinity purification was pooled in 100mM BTP, 100mM histidine, 1mM MgCl ₂ Diluted 5-fold in 5% glycerol, 0.5% PF68, and variable amount of citric acid, and loaded onto a CIMmultus QA column at a loading ratio of 5e13 vp/mL to 2e14 vp/mL. The column was then chased with 25 column volumes of equilibration solution and the empty and/or intact particles eluted from the column by applying a linear gradient elution. All chromatographic steps were performed in the downward flow direction at ambient temperature.

AEX flow-through and eluate fractions were then analyzed using a Tecan microplate reader or SEC-HPLC to assess separation of empty and intact capsids and capsid recovery. AEX eluate as described herein is substantially free of empty particles (e.g., as shown in fig. 7 and 12). Thus, recombinant AAV isolated from AEX eluate is substantially free of empty AAV particles.

Example 4: AAV production using closed linear double stranded (cl) DNA:

table 2. Analysis of the test and specification specifications to complete the 50L scale carrier production.

PRO10T ^M The cell line (AskBio, NC, USA) was used to make recombinant adeno-associated viral vectors (rAAV), which were suspension adapted serum-free cell lines derived from the human embryonic kidney cell line 293 (HEK 293). PRO10 ^TM The production of viral vectors is a batch process carried out at medium to high range cell densities and employs a triple transfection method by agglomeration of linear polyethylenimine MAX and the desired plasmid (pDNA) or closed linear (cl) DNA substrate in a mix of production media. Cell growth and production medium are chemically defined and do not contain components of animal origin. Triple transfection methods include transfection with three DNA molecules or three DNA constructs; each DNA molecule provides a key element for the production of recombinant AAV. The first provides adenovirus helper (Ad helper) proteins for efficient replication and packaging of the vector, but lacks the adenovirus structure and replication genes necessary for adenovirus production. The second is an AAV8 or AAVrh10 trans construct (packaging construct) comprising an AAV2 rep gene and an AAV8 capsid (cap) or AAVrh10 capsid (cap) protein gene. The third construct is an AAV vector construct encoding a therapeutic transgene comprising an adeno-associated virus 2 Inverted Terminal Repeat (ITR) sequence flanking (5 'to 3') the gene of interest. The constructs used for all experiments were dual GFP and luciferase reports. In addition, subsequent studies used two therapeutic transgene cassettes, e.g., comprising the CYP46A1 and GAA transgenes.

In a traditional non-blocking method on a laboratory scale (31.25 mL-2L), preliminary experiments were conducted using a design of an experimental (DoE) methodology to determine and optimize key parameters related to production by simultaneously examining factors such as clDNA concentration, clDNA to transfection reagent ratio, etc. All small scale experiments were controlled by parallel vector production using an optimized triple plasmid transfection system. Other factors to be evaluated include, but are not limited to, medium, cell density, transfection time, transfection volume, temperature, and other cell-dependent or cell-independent factors.

The small-scale transfection cultures were incubated for about 72hr after transfection (hpt) and then harvested by mechanical cell lysis. Total vector yield was quantitatively assessed by vector genome (vg) using an internal qPCR-based dnase resistant particle (DNase Resistant Particle, DRP) approach specific for viral ITRs. As shown in qPCR, the yield is usually from 4X 10 ¹¹ vg/mL to 6X 10 ¹¹ The range of vg/mL. The transgenic targeting qPCR was observed by ELISA and the yield was further assessed per milliliter of total viral particles (capsid) (vp/mL). The relative packing efficiency was also modeled by observing the ratio of A260/280 by SEC-HPLC at the time of harvesting the affinity purified lysate.

The main purpose of small-scale screening experiments was to determine near-optimal transfection conditions for the 50L-scale portion of the experimental plan. For experiments with pDNA and clDNA, cells were thawed, cultured and gradually expanded until seeded into a 50L production bioreactor. The cell culture expansion process is continued in the bioreactor prior to transient transfection. The transfected cell cultures were incubated in a bioreactor for approximately 72-hpt. At harvest, transfected cell cultures were lysed and clarified by depth filtration and membrane filtration, followed by purification. Purification included the steps of capture chromatography, gradient ultracentrifugation, ion exchange chromatography, ultrafiltration/diafiltration (UF/DF) and 0.2 μm filtration.

Detailed process description of 50L SUB upstream operations

To generate a 50L batch, cells were thawed, cultured and gradually expanded until seeded into a 50L production bioreactor. The cell culture expansion process is continued in the bioreactor prior to transient transfection. Currently, seed line growth media is supplemented with L-glutamine to a final concentration of 10mM, which is used to recover frozen cell stock andthe inoculum was amplified to a 5L suspension using a 10L WAVE bag bioreactor. The medium used in the WAVE suspension was supplemented with 0.2% PLURONIC ^TM And (3) acid. The growth medium used after the seeds of the ThermoFisher 50L single use stirred tank bioreactor (SUB, STR) is supplemented with about 1 to 100mM GLUTAMAX ^TM About 0.01% to about 10% PLURONIC ^TM Acid (ThermoFisher, waltham, mass.) and about 0.001% to 1% FOAMAWAY ^TM Seed system growth medium composition of (Gibco, waltham, mass.). GLUTAMAX ^TM Is a stable dipeptide source of L-glutamine, aimed at preventing degradation and reducing toxic accumulation of excess ammonia.

By condensation of three clDNA and linear polyethylenimine MAX (Polysciences inc., warrington, PA) (PEI MAX), at 3.25×10 ⁶ Individual living cells/mL ³ To 4.25X10 ⁶ Individual living cells/mL ³ Transient transfection was performed at cell density to produce AAV. Transfection was performed under suspension conditions. The transfection mix constituted 10% (v/v) of the culture volume (5L). The agglomeration was performed in custom 10L WAVE shake bags equipped with tubing matching 50L SUB. Transfection hybrids were prepared by first adding 4L of medium to the shake-bag at 25℃and gently shaking (8℃angle, 25 RPM). To prevent deflation of the bag, an air blanket was applied at 0.2 LPM. DNA (clDNA is shown in Table 3) was then added and 1L chase was performed with medium.

Table 3: ratio of each clDNA used normalized to cell density at transfection.

After medium chase, PEI was added over 1 minute and chased with 1L of medium. The mixture was incubated for 7 minutes and then transferred to SUB. The transfected cell suspension was incubated for 3 hours and the reaction stopped with 10% (v/v) volume of chemically defined serum-free HEK293 medium supplemented with 10mM L-glutamine.

SUB control parameters

Current mass production platforms use finess G3Pro universal controller equipped with ThermoFisher jacketed 50 LSUB. The single use container was equipped with a 3-blade, 45 ° elevation, axial impeller, dual-ejector (Frit-driven-Hole) design, and a basic Finesse TruFluor pH/DO single use probe protective sheath and a secondary Pall Kleenpak connector for a reusable pH/DO probe insert. The day before the media loading, the bag was installed and inflated with an air blanket at 10 LPM. An optical/reusable DO probe has been connected to the transmitter. On the loading day, the DO probe was calibrated using a 2-pt slope calibration. After addition of the medium, the single use and reusable pH probes were standardized using an off-line sample on a calibrated blood gas analyzer.

The day before inoculation, the SUB temperature was raised to 37 ℃. The media was then conditioned by saturation with a continuous borehole air spray at a flow rate of 0.5LPM (0.025 VVM). Before inoculation, single-use and reusable DO probes were standardized to 100% air saturation using a 1pt calibration.

After inoculation, the controller was set to manage continuous borehole air sprays at a rate of 0.5LPM and sweep the headspace with a 1LPM air blanket. DO through O ₂ Gas cascade control and designed to control by introducing O to the fraction injector ₂ The flow rate was increased from 0.00 to 5.00LPM (0-100% DO output/0-100% MFC-3 output) to maintain the set-point. By introducing CO into the kit injector ₂ Increasing the gas flow from 0.00 to 2.00LPM (0- (-100)% output/0-100% MFC-4 output) to control the pH at the high end (7.0-14); however, no alkali supply is used to control the pH at the low end, but rather to allow for its natural drift.

Results: total vector yield at harvest was assessed by ITR-qPCR (data not shown). The data show a 2-2.5 fold improvement in specific (vg/cell) productivity using clDNA as starting material compared to pDNA as starting material. clDNA for the production of recombinant AAVrh10CYP46A1 suggests that less than 1 μg, e.g., between 0.6 μg and 0.7 μg, of DNA is required to achieve high titer AAV. In addition, PEI was present in a DNA ratio of 2.2 and 2.5 to produce recombinant AAVrh10CYP46A1 and AAV8GAA, and the optimal clDNA was 0.6. Mu.g in view of total yield and packaging efficiency.

Example 5: TCID50/mL and particle to infectivity (vg/TCID 50):

TCID50 determination: the infection titer (TCID 50) method was used to assess AAV infectivity of the drug product in HeLa RC32 cells in vitro. In this assay HeLa RC32 cells were transduced with adenovirus type 5 helper virus and a series of dilutions of the drug product. Three days after infection, the cells were treated with proteinase K to digest the protein and the replicated AAV vector DNA was quantified using qPCR techniques. The method utilizes a detection system based on fluorescent dyes and DNA primers. The absolute amount of ITR targeting sequence from vector DNA was replaced by an interpolated value from a standard curve prepared with plasmid. Samples containing ITRs were prepared as test samples and used as assay controls. Results are expressed in units of infection per milliliter (IU/mL). Notably, to compare TCID50/mL between different formulations, TCID50/mL is preferably normalized to vg/mL.

Without being limited by any limitation, table 4 below shows the particle to infectivity (vg/TCID 50) ratio results and TCID50/mL infection titres for plasmid DNA (pDNA) and closed end linear double strand (clDNA) derived AAV vectors.

Table 4: TCID50/mL and particle to infectivity (vg/TCID 50):

As shown in Table 4, in the DoE and 50L experiments, the clDNA-derived vector showed higher infectivity compared to the pDNA control, as indicated by the lower vg/TCID50 ratio of clDNA than pDNA. In some aspects of the invention described herein, one example of a purified recombinant adeno-associated virus population lacking a prokaryotic sequence is derived from clDNA.

Example 6: use of citric acid for separating empty and complete capsids by AEX

According to some exemplary embodiments of the invention, the present study demonstrates the separation of empty (E) and intact (F) capsids for multiple natural serotypes using weak acid partition patterns. Without wishing to be bound by theory, the chromatographic separation of empty (E) and intact (F) capsids is based on the charge difference between empty and intact capsids, with an empty capsid isoelectric point of 6.3 and an intact capsid isoelectric point of 5.9. At high pH, the intact capsid carries a more negative charge. The anion exchange is positively charged. Thus, the intact capsid later elutes during the salt gradient. The inclusion of a weak acid (e.g., citric acid) may reduce the binding of the empty capsids to the anion exchange resin, thereby partitioning the empty capsids from the intact capsids.

As shown in fig. 14, UV of the sample _260/280 The ratio can be used to determine the% intact capsids in the sample. UV of about 0.6 _260/280 The ratio represents about 0% of the complete capsid in the sample, about 1.30 UV _260/220 The ratio represents about 70% of the complete capsids in the sample.

Sample preparation: the affinity eluate was diluted 5X with AEX dilution buffer containing different amounts of citric acid. The column was conditioned with a buffer similar to the dilution buffer prior to application of the sample to the anion exchange column, except that different amounts of citric acid were included. The results are shown in FIGS. 15A-21D.

As shown in fig. 15A-15C, complete (F) and empty (E) capsids were fully bound and eluted in the absence of weak acid in the dilution buffer. Furthermore, UV of the starting materials (S/M) _260/280 The ratio affects the "empty shoulder" and the consistency of the bioreactor (empty/complete distribution) has a great influence on the chromatographic separation.

As shown in fig. 16A and 16B, inclusion of citric acid as a modulator showed fragmentation of empty capsids and little or no empty capsids were observed by SEC, fractions enriched up to 1.35uv260/280. Since the elution shown in fig. 16A and 16B uses different starting materials, the results demonstrate the reproducibility and robustness of the AEX method described herein.

The effect of citric acid concentration in AEX dilutions was measured and the results are summarized in table 5 and exemplary elution curves are shown in fig. 17A-17C.

Table 5: effect of citric acid on elution

As shown in table 5 and fig. 17A-17C, adjustment of citric acid in the AEX dilution buffer resulted in a reduction of the AEX eluting hollow shoulder. As shown, slight changes in citrate concentration in AEX dilution buffer can adjust the extent of segmentation. HCP was lower than LOQ (< 2 ng/mL) in all experiments.

The effect of column size on AEX purification was studied and the results are shown in table 6 and fig. 18A-18C.

Table 6: influence of column size on AEX purification

As shown in table 6 and fig. 18A-18C, the AEX method was repeatable between the monoliths scale used for segmentation. Although there were small changes in recovery, complete capsid enrichment and capsid partitioning, this change was associated with a change in the monoliths batch.

The effect of starting material packaging on AEX was also investigated and the results are shown in table 7 and fig. 19A-19C.

Table 7: influence of the starting Material Package on AEX separation

UV of the starting materials as shown in Table 7 and FIGS. 19A-19C _260/280 The difference in ratios resulted in similar performance on a 4mL monoliths scale. Between different starting materials by UV _260/280 Ratio of AEX stream to UV of AEX eluate _260/280 The ratios are comparable. Residue from all experimentsHost Cell Proteins (HCPs) are lower than LOQ @ <2ng/mL)。

As shown in fig. 20A-20G, the amount of weak acid (e.g., citric acid) in the AEX dilution buffer or AEX equilibration buffer affects the peak shape/purity/recovery of AEX purification. In the elution shown in FIGS. 20A to 20G, the peak shape was distorted when the concentration of citric acid in the AEX dilution buffer was high (> 6 mM), but the concentration of citric acid in the AEX dilution buffer was low, resulting in poor separation.

As shown in fig. 21A-21D, the partitioning was increased by equilibrating the column with an AEX equilibration buffer containing higher concentration of citric acid while maintaining the concentration of citric acid in the AEX dilution buffer at 6 mM.

Example 7: use of histidine for modulating the separation of empty and complete capsids by AEX

Although citric acid may act as a modulator of many AAV serotypes (AAV 2i8, AAV rh 10), it may not be suitable for certain serotypes because it may readily compete for empty and intact capsids of these AAV serotypes. Thus, the inventors tested histidine, which is similar to weak acids for modulating the separation of empty and intact capsids by AEX, but seems to be less robust.

Sample preparation: the affinity eluate was diluted 5X with AEX dilution buffer containing different amounts of histidine. The column was conditioned with a buffer of histidine composition similar to the diluted sample concentration (+/-10 mM) prior to applying the sample to an anion exchange column (QA Monolith column).

The results are shown in FIGS. 22A-22F and summarized in tables 8 and 9.

Table 8:

table 9:

as shown, the histidine concentration in the AEX dilution buffer was about 120-170mM, showing a good balance between recovery and purity.

Example 8: high throughput rAAV production and downstream purification of recombinant viral particles

Recombinant adeno-associated viral vectors (rAAV) used to develop the HTS AEX purification method were isolated in suspension in a suspension-adapted HEK293 cell line (Pro 10 ^TM ) Produced using a transient triple transfection system. rAAV assessed in the HTS AEX method include both natural serotypes and chimeric serotypes. For process demonstration, these included AAV8 and AAV2i8 serotypes. The harvested cell culture containing the rAAV vector was clarified by depth filtration and further purified by affinity chromatography.

Liquid handling system description: the automated liquid handling system used to conduct these experiments was TECAN Freedom EVO (TECAN, usa). Freecom Evo configurations include a liquid handling arm (LiHa) equipped with a fixed tip for liquid transfer, applications of buffers and samples involve a strong anion exchange chromatography operation using a POROS HQ50 RoboColumns from replingen (replingen GmbH, germany) integrated with a TECAN TeChrom module (TECAN, usa). The freecom Evo 200 system is also equipped with a robotic handling arm (ROMA) for transferring plates, activating and deactivating waste trays associated with the TeChrom module.

Improved HTS AEX RoboColumn method: POROS 50HQ RoboColums (100 μL) were obtained from Repligen (Germany) and tested using the TECAN's Techrome module (TECAN, USA). Buffer and AEX loading materials were transferred from their respective plate sites and applied to RoboColumns using a stationary tip of a liquid handling arm (LiHa). The 96-deep well plate was gradually passed under a robocolumn mount mounted on Te-Chrom by means of Te-shift to collect fractionated flow-through and AEX eluate samples. The number of anion exchange chromatography conditions (dilution buffer and column conditioning buffer combination: 96 conditions maximum) is specified by the user at the beginning of the script and specifies the total number of columns that are subjected to purification cycles and the number of plates required for the experiment.

RoboColumn preparation: the column was washed in place (CIP), removed and conditioned with elution buffer. Robolocolumns are then equilibrated in a buffer with a user-specified weak acid concentration (e.g., A1 column conditioned in a buffer containing X mM weak acid, while B1 is conditioned in a buffer containing Y mM weak acid). Notably, these variable buffers can be rearranged on a TECAN bench to achieve a variable experimental design.

Sample preparation: affinity eluate obtained by bulk purification or by affinity chromatography on TECAN was subjected to 5X dilution (1 part affinity eluate and 4 parts dilution buffer) with a user-specified dilution buffer containing a variable amount of weak acid. The number of samples (conditions) and affinity eluate addition volumes were selected to alter the resin challenge. The dilution buffer containing the concentration of the weakable acid is transferred from the Tecan tank or tube to the sample preparation container. The affinity eluate is then transferred from the single tube on the TECAN bench and applied to the desired amount of sample preparation containers based on the specified number of experimental conditions.

Sample application: the 96 deep well collection vessel was transferred from the rack "hotel" to teshift by robotic arm (RoMA). Te-Shuttle transported the collection vessel under the appropriate RoboColums group in the Te-Chrome module to collect the flow-through generated at sample application and AEX elution fractions at sample elution. Samples were transferred from the sample preparation containers into RoboColumns via fixed needle injection by a liquid handling arm (LiHa). The collection vessel was started by Te-shift and the 1CV fraction was collected. After sample application, roboColumns were subjected to washing to elute any non-specifically bound impurities, and the bound samples were then eluted in a conductivity-mediated step into the corresponding 96-well collection vessel at a 3CV portion. The collection vessel was then returned to the rack "hotel" by robotic handling arm (RoMA) and a new 96 deep-well collection vessel was removed from the hotel and transferred to teshift for subsequent collection of the next designated column conditioning experimental conditions. The sample application, washing and elution process was repeated based on the user specified number of column conditioning experimental conditions. After the last elution was completed, roboColumns were subjected to a post-elution clean-in-place procedure and stored in 20% ethanol solution.

UV absorbance detector: the UV absorbance of AEX flow-through and eluate fractions was measured by a TECAN Infinite 200PRO microplate reader (TECAN, usa). 100 microliter AEX flow-through and eluate fractions were aliquoted from a 96 deep-well collection vessel by a liquid handling arm (LiHa) to a 96-well UV microplate (Greiner 96Flat Transparent[GRE96ft_half area_UV-Star)]) Is a kind of medium. The UV microwell plate was then transferred to an open tray of a TECAN Infinity 200PRO microplate reader by a robotic handling arm (RoMA). UV absorbance of each sample was measured at A260 nm and A280 nm to determine UV _260/280 Ratio. UV (ultraviolet) light _260/280 The ratio was used as a rough estimate of the complete and empty capsid distribution in the sample.

Example 9: exemplary AAV formulations

Materials: trizma base [ 2-amino-2- (hydroxymethyl) -1, 3-propanediol]Potassium chloride, sodium chloride, disodium hydrogen phosphate heptahydrate, potassium dihydrogen phosphate, d-sorbitol, d-mannitol, sucrose, anhydrous magnesium sulfate, alpha-d-heptagluconate dihydrate, proteinase K solution, DNAse enzyme, all purchased from Sigma-Aldrich (St. Louis, MO). Histidine buffer 25mM and 10 XPBS (pH 6.0) were purchased from Bioworld (Dublin, ohio). Pluronic ^TM F-68 nonionic surfactant (100X), low EDTA TE buffer, sheared salmon sperm DNA, sodium deoxycholate detergent, dulbecco's Modified Eagle's Medium (DMEM), 2-propanol and TaqMan Fast Advanced premix available from Thermo Fisher Scientific (Pittsburgh, pa.). Tween 20 was purchased from Millipore Sigma (Burlington, mass.). Unless otherwise specified, all other chemicals were analytical reagent grade and purchased.

Method

Preparation: the formulation is prepared by mixing buffers, excipients and/or surfactants at appropriate concentrations and filtering the 0.2 μm PES needle filter (Corning Life Sciences, corning, NY) for aseptic processing. Stock carriers stored in AskBio final formulation buffer were added to each formulation and buffer exchange was then performed by using an Amicon Ultra-0.5 centrifugal filtration unit. A volume of 0.5mL of the formulation was added to each filtration unit, followed by 15 minutes of spin at 14,000g in a Heraeus Fresco 21 centrifuge (Thermo Fisher Scientific, waltham, mass.). After spinning, the permeate was discarded and the unit was refilled with formulation buffer and spun twice more. After the last rotation, the permeate was discarded and the filter unit was inverted to retrieve the buffer exchanged carrier by now performing a rotation of 1,000g for 2 minutes. The sample was then resuspended to the starting volume of the carrier and analyzed immediately.

Freeze thawing assessment: samples were prepared in respective buffers and stored in Eppendorf tubes. Four freeze-thaw cycles were performed, with one cycle including 24 hours at-80 ℃ followed by 24 hours at room temperature (20 ℃). Analysis was performed after the fourth freeze-thaw cycle.

DLS: for DLS analysis, samples were spun at 21.1×g for 10 minutes in a Heraeus Fresco 21 centrifuge (Thermo Fisher Scientific, waltham, MA), then placed in a transparent container (cuvettes) and measured using a Zetasizer Ultra-Red (Malvern Instruments ltd., worcestershire, uk). The data were analyzed using ZS XPLORER 2.0.0.98 software.

pH and osmotic pressure: using Mettler Toledo Seven Excellence ^TM The pH meter evaluates the pH of the formulation in triplicate. Osmolarity values were collected using PSIMulti osmete 2430 (Precision Systems Inc, washington, DC).

Unclle: the samples were centrifuged at 21.1Xg for 10 minutes in a Heraeus Fresco 21 centrifuge (Thermo Fisher Scientific, waltham, mass.). Then 8 μl was added to each well and the samples were run in unccle (Unchained Labs, plaasanton, CA) for thermal degradation analysis. The starting temperature was set to 25℃and a heating rate of 1℃per minute was used until the ending temperature (95 ℃) was reached. The filter settings were set to 266nm and 473nm. Data Analysis was performed using the un Analysis 5.03 software.

SEC-HPLC: samples were placed neatly into polypropylene HPLC vials (Mfg, part No.) and placed in a multisampler at 6C. The vials were then injected to target a 5e10 vp load on the column (Agilent Bio SEC-5.4.6 mm x 150mm (Agilent P/N5190-2534)). Samples were analyzed at 214nm, 260nm and 280nm wavelengths for 10 minutes at a flow rate of 0.4mL/min on an HPLC system equipped with a pump, multisampler, column heater and DAD detector (Agilent 1260Prime II or equivalent). The data were analyzed using the Empower 3 data acquisition software.

qPCR: the samples were diluted 1:100 in sample dilution buffer (SDB; 100. Mu.g/mL sheared salmon sperm DNA,0.1%Pluronic F68) by 2 series of 1:10 dilutions and treated with DNAse enzyme at 37℃for 30 minutes, followed by proteinase K (0.5 mg/mL) at 55℃for 1 hour and 95℃for 10 minutes. The samples were then diluted again at 1:10 in SDB. Samples were treated simultaneously with the positive control, diluted 1:10 and 1:100 in SDB after DNAse and proteinase K treatment. Premix was prepared using Fast Taqman Advanced premix (Applied Biosystems) with 0.05. Mu.M forward (5'-GGAACCCCTAGTGATGGAGTT-3') and reverse (5'-CGGCCTCAGTGAGCGA-3') primers and 0.25. Mu.M FAM probe (5 '-/56-FAM/CACTCCCTCTCTGCGCGCTCG/3BHQ_1/-3'). mu.L of premix was mixed with 5. Mu.L of treated and diluted sample and control. The extracted single-stranded DNA stock solution of viral vector was heated at 95℃for 3 minutes, then cooled at room temperature, and then used to prepare a 7-point standard curve. The standard curve was also run with sample, positive extraction control, negative extraction control (10 mM phosphate, 350mM NaCl, 2.7mM KCl, 5% sorbitol, 0.001% Pluronic, pH 7.4) and non-template control. Premix containing samples was loaded and run on Quantum studio Flex 6 (Thermo Fisher, pittsburgh, pa.) and held at 95℃for 5 minutes, followed by 40 cycles (including 5 seconds at 95℃and 30 seconds at 60 ℃). The titer of the samples was determined using quantitative interpolation of the extracted viral vector ssDNA standards, which were then adjusted according to dilution during sample preparation and running.

TCID50: samples were serially diluted in an infection medium consisting of DMEM and wild-type adenovirus 5 (ATCC VR-1516) at a concentration of 4E7 IU/mL and used to infect HeLa RC32 cells (ATCC CRL-2972). 72 hours after infection, cells were incubated with lysis buffer (Tween 20, pro K buffer, pro K enzyme, sodium deoxycholate) and the resulting reaction was diluted in sample dilution buffer (low EDTA buffer, 10% Pluronic, sheared salmon sperm DNA). Samples were analyzed by qPCR on quantsudio Flex 6 (Thermo Scientific, pittsburg, PA) to determine the copy number of Inverted Terminal Repeats (ITRs) in each well. The infection titer was then determined using the Spearman-Karber assay.

Exemplary formulations and results are shown in tables 10-24.

Table 10: phosphate-containing formulations

The phosphate-containing formulations were evaluated for vector stability (DLS), genomic titre (ITR qPCR) and% complete capsid packaging (SEC-HPLC). The results are summarized in Table 11.

Table 11: analysis of phosphate-containing formulations

^a Formulation precipitation

From the data summarized in Table 11, there was little change in qPCR titres for formulations 1, 3 and 4. Furthermore, aggregation (DLS) and capsid integrity (SEC) of formulations 1-4 are comparable.

Table 12: tris-containing formulations

Vector stability (DLS), genome titer (transgenic qPCR) and packaging of% intact capsids (SEC-HPLC) of formulations comprising Tris were evaluated. The results are summarized in Table 13.

Table 13: tris formulation assay

As can be seen from the analytical data summarized in table 13, tris buffer resulted in isotonic solution. MgSO was used with or without PF68 ₄ Aggregation was inhibited while qPCR titers were maintained. For buffers containing sorbitol or mannitol, capsid integrity (SEC) is comparable with or without PF 68.

Table 14: histidine-containing formulations

The vector stability (DLS), genome titer (transgenic qPCR) and packaging of% intact capsids (SEC-HPLC) of formulations comprising histidine were evaluated. The results are summarized in table 15.

Table 15: analysis of histidine-containing formulations

From the analytical data summarized in table 15, it can be seen that histidine buffer resulted in isotonic solution and PF68 inhibited aggregation. For sorbitol or mannitol containing buffers, transgene qPCR titers and capsid integrity (SEC) were comparable with or without PF 68. Formulations 15 and 17 did not find aggregation, SEC changes or qPCR changes.

The stability of some exemplary formulations after one or more freeze-thaw cycles was determined. The results are summarized in tables 16 and 17.

Table 16: freeze thawing PBS formulation results

Table 17: freeze thawing study 2 results

Table 18: TCID (TCID) ₅₀ Results

	TCID50/mL	vg/TCID50	% recovery
				Reference formulation ^*	1.12E10
Formulation 1	5.22E9	5114.6	97
				Formulation 15	6.32E9	3826.4	98
Formulation 9	1.12E10	1627.1	100

^* Not frozen and thawed

As shown by the data summarized in table 18, the activity after 4 freeze-thaw cycles was Tris > His > PBS.

Analysis of thermal stability

Thermal stability analysis was performed on some exemplary formulations using a unclcle system (Unchained Labs, plaasanton, CA). The results are summarized in Table 19.

Table 19: stability results

The data in table 19 show the effect of heating to 95 ℃ on the presence of aggregation in the different formulations. As shown, formulations 9 and 15 showed less aggregation than the process control formulation or formulation 1.

Formulation translatability

The formulations shown in table 15 were prepared with different AAV serotypes (AAV 2, AAV9, and AAVrh 10) to determine the translatability of the formulation to the different serotypes.

Table 20: exemplary formulations

Vector stability (DLS) was assessed for formulations 21-28 containing different AAV serotypes. The results are summarized in table 21.

TABLE 21 DLS analysis

The genome titres (ITR qPCR) and the packaging (SEC-HPLC) of% intact capsids for formulations 23 and 26 comprising different AAV serotypes were further assessed. The results are summarized in table 22.

Table 22: qPCR and SEC analysis of formulations 23 and 26 comprising serotype AAV2, AAV9 or AAV rh10

Formulations	Serotypes	ITR qPCR titre (vg/mL)	SEC(vp/mL)
				Control	AAV 2	6.1 E12	1.3E13
Control (buffer exchange)	AAV 2	2.2E12	3.6E12
				Control	AAV 9	6.4E13	9.9E13
Control	AAV rh10	1.1E14	2.7E14
				Formulation 23	AAV 2	9.0E11	7.5E11
Formulation 23	AAV 9	2.1E13	4.0E14
				Formulation 23	AAV rh10	7.9E13	2.2E11
Formulation 26	AAV 2	1.7E12	1.9E12
				Formulation 26	AAV 9	2.7E13	4.3E13
Formulation 26	AAV rh10	8.9E13	2.3E11

The genome titer (ITR-qPCR) and the packaging of% intact capsids (SEC-HPLC) of formulation 26 comprising AAV 2i8 serotype were further assessed. The results are summarized in table 23.

Table 23: qPCR and SEC analysis of formulation 26 comprising serotype AAV 2i8

After 4 freeze-thaw cycles, formulations 23 and 26 containing different AAV serotypes were further evaluated for genomic titres (ITR-qPCR) and packaging of% intact capsids (SEC-HPLC). The results are summarized in table 24.

Table 24: qPCR, SEC and DLS analysis of formulations 23 and 26 containing serotype AAV2, AAV9 or AAV rh10 after 4 freeze-thaw cycles

* Tube rupture during centrifugation step

As can be seen from tables 23 and 24, formulation 26 was able to protect the stability of all 4 AAV serotypes tested from any change with and without freeze-thaw stress. This is important because one of the biggest challenges to AAV vector stability is freeze-thaw agitation.

All patents and other publications identified in the specification and examples are expressly incorporated herein by reference for all purposes. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior application or for any other reason. All statements as to the date or content of these documents is based on the information available to the applicant and does not constitute an admission as to the correctness of the dates or contents of these documents.

Claims

1. A population of purified recombinant expressed adeno-associated virus (rAAV) particles, wherein the recombinant expressed adeno-associated virus particles are purified or isolated from a harvest medium by a method comprising:

a. purifying/separating a plurality of recombinantly-expressed viral particles from the harvest medium by affinity chromatography to produce an eluate comprising the plurality of recombinantly-expressed viral particles (affinity chromatography eluate), wherein an elution buffer for affinity chromatography (affinity elution buffer) comprises a predetermined amount of glycine, optionally the affinity elution buffer is substantially free of weak acids or salts thereof; and optionally, the affinity elution buffer comprises an amino acid that is not glycine;

b. conditioning the affinity chromatography eluate for subsequent purification by anion exchange chromatography, wherein the conditioned affinity eluate comprises a predetermined amount of a weak acid or salt thereof, or the conditioned affinity eluate comprises a predetermined amount of glycine already present in the affinity eluate and a predetermined amount of an amino acid other than glycine;

c. purifying/separating the plurality of recombinantly-expressed viral particles from the conditioned eluate of the affinity chromatography by anion exchange chromatography to produce a solution comprising the plurality of purified/separated recombinantly-expressed viral particles, wherein an equilibration buffer for anion exchange chromatography comprises a predetermined amount of a weak acid or salt thereof; and, optionally,

Wherein: (i) The purified rAAV population comprises less than about 35% empty viral capsids; and/or (ii) the purified rAAV has less than 2X 10 ⁴ Particle to infectivity ratio of vg/TCID 50; and/or (iii) UV in anion exchange eluate ₂₆₀ With UV ₂₈₀ The ratio of (2) is UV in the regulated affinity eluate ₂₆₀ With UV ₂₈₀ At least about 1.15 x or greater.

2. The population of claim 1, wherein less than 5% of the empty viral particles in the affinity eluate are bound to anion exchange chromatography media.

3. The method of claim 2, wherein substantially no empty viral particles in the affinity eluate bind to anion exchange chromatography media.

4. The method of any one of claims 1-3, wherein the purified rAAV population comprises less than about 10% empty viral capsids.

5. The method of claim 4, wherein the purified rAAV population comprises less than about 5% empty viral capsids.

6. The method of claim 5, wherein the purified rAAV population is substantially free of empty capsids.

7. The population of any one of claims 1-6, wherein the weak acid of 1 (b) is citric acid, acetic acid, succinic acid, acetoacetic acid, adipic acid, tetraoxypyrimidic acid, ascorbic acid, aspartic acid, barbituric acid, boric acid, butyric acid, n-butyric acid, carbonic acid, crotonic acid, diglycolic acid, dimethylmalonic acid, formic acid, fumaric acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, glyceric acid, glycolic acid, isocitric acid, itaconic acid, lactic acid, maleic acid, malic acid, malonic acid, mesaconic acid, meso-tartaric acid, methylsuccinic acid, methylmalonic acid, oxalic acid, oxaloacetic acid, pentanic acid, valeric acid, phosphoric acid, isophthalic acid, phthalic acid, terephthalic acid, propionic acid, pyruvic acid, salicylic acid, tartaric acid, tartronic acid, trichloroacetic acid, uric acid, α -tartaric acid, 2-oxobutyric acid, 2-methylbutyric acid, 2-oxoglutaric acid, 3-butenoic acid, or 3-methylbutyric acid, optionally the weak acid is citric acid, citrate, acetic acid or succinic acid.

8. The population of any one of claims 1-7, wherein the affinity elution buffer comprises histidine at a concentration of at least about 1mM or greater.

9. The population of any one of claims 1-8, wherein the affinity elution buffer comprises histidine at a concentration of from about 1mM to about 50 mM.

10. The population of any one of claims 1-9, wherein the affinity elution buffer comprises glycine at a concentration of at least about 20mM or greater.

11. The method of any one of claims 1-10, wherein the affinity elution buffer comprises glycine at a concentration of from about 25mM to about 100 mM.

12. The population of any one of claims 1-11, wherein the affinity elution buffer comprises a salt.

13. The population of any one of claims 1-12, wherein the affinity elution buffer comprises a salt at a concentration of at least about 5mM or greater.

14. The population of any one of claims 1-13, wherein the affinity elution buffer comprises a salt at a concentration of from about 5mM to about 15 mM.

15. The population of any one of claims 12-14, wherein the salt is a magnesium salt, a sodium salt, a potassium salt, an ammonium salt, a calcium salt, a copper salt, a cobalt salt, a manganese salt, a nickel salt, or a zinc salt, optionally the salt is MgCl ₂ 。

16. The population of any one of claims 1-15, wherein the affinity elution buffer comprises a polymer.

17. The population of any one of claims 1-16, wherein the affinity elution buffer comprises a polymer at a concentration of at least about 0.1% or greater.

18. The population of any one of claims 1-17, wherein the affinity elution buffer comprises a polymer at a concentration of from about 0.1% to about 0.5%.

19. The population of any one of claims 16-18, the polymer being a nonionic surfactant.

20. The population of any one of claims 1-19, wherein the affinity elution buffer has a low pH.

21. The population of any one of claims 1-20, wherein the pH of the affinity elution buffer is less than or equal to about 6.5.

22. The method of any one of claims 1-21, wherein the pH of the affinity elution buffer is from about 2.0 to about 3.0.

23. The population of any one of claims 1-22, wherein the affinity elution buffer comprises: about 75mM glycine.

24. The population of any one of claims 1-7 or 10-23, wherein the affinity elution buffer comprises a weak acid or salt thereof.

25. The population of claim 24, wherein the affinity elution buffer comprises a weak acid or salt thereof at a concentration of at least about 50mM or greater.

26. The population of claim 24 or 25, wherein the affinity elution buffer comprises a weak acid or salt thereof at a concentration of from about 50mM to about 100 mM.

27. The population of any one of claims 24-26, wherein the weak acid is citric acid, acetic acid, succinic acid, acetoacetic acid, adipic acid, tetraoxypyrimidic acid, ascorbic acid, aspartic acid, barbituric acid, boric acid, butyric acid, n-butyric acid, carbonic acid, crotonic acid, diglycolic acid, dimethylmalonic acid, formic acid, fumaric acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, glyceric acid, glycolic acid, isocitric acid, itaconic acid, lactic acid, maleic acid, malic acid, malonic acid, mesaconic acid, meso-tartaric acid, methylsuccinic acid, methylmalonic acid, oxalic acid, oxaloacetic acid, pentanic acid, valeric acid, phosphoric acid, isophthalic acid, phthalic acid, terephthalic acid, propionic acid, pyruvic acid, salicylic acid, tartaric acid, tartronic acid, terephthalic acid, uric acid, α -tartaric acid, 2-oxobutyric acid, 2-methylbutyric acid, 2-oxoglutaric acid, 3-butenoic acid or 3-methylbutyric acid, or optionally the weak acid is citric acid, citrate, acetic acid or succinic acid.

28. The population of any one of claims 24-27, wherein the weak acid is citric acid or a salt thereof.

29. The population of any one of claims 1-7, 10-22, or 24-28, wherein the affinity elution buffer comprises: about 50mM glycine, about 75mM citrate, about 10mM MgCl ₂ About 0.3% (w/v) P188, and a pH of about 3.0.

30. The population of any one of claims 1-29, wherein the conductivity of the affinity elution buffer ranges from about 5mS/cm to about 8mS/cm, optionally the conductivity of the affinity elution buffer ranges from about 5.5mS/cm to about 7 mS/cm.

31. The population of any one of claims 1-30, wherein the affinity elution buffer has an osmolality in the range of from about 100mOsm to about 225 mOsm.

32. The population of any one of claims 1-31, wherein an equilibration buffer for anion exchange chromatography comprises a weak acid or salt thereof at a concentration of at least about 0.5 mM.

33. The population of any one of claims 1-32, wherein the equilibration buffer comprises a weak acid or salt thereof at a concentration from about 0.5mM to about 15 mM.

34. The method of any one of claims 1-33, wherein the weak acid in the equilibration buffer is citric acid, acetic acid, succinic acid, acetoacetic acid, adipic acid, tetraoxypyrimidic acid, ascorbic acid, aspartic acid, barbituric acid, boric acid, butyric acid, n-butyric acid, carbonic acid, crotonic acid, diglycolic acid, dimethylmalonic acid, formic acid, fumaric acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, glyceric acid, glycolic acid, isocitric acid, itaconic acid, lactic acid, maleic acid, malic acid, malonic acid, mesaconic acid, meso-tartaric acid, methylsuccinic acid, methylmalonic acid, oxalic acid, oxaloacetic acid, pentanic acid, valeric acid, phosphoric acid, isophthalic acid, phthalic acid, terephthalic acid, propionic acid, pyruvic acid, salicylic acid, tartaric acid, tartronic acid, trichloroacetic acid, uric acid, α -tartaric acid, 2-oxobutyric acid, 2-methylbutyric acid, 2-oxoglutaric acid, 3-butenoic acid, or 3-methylbutyric acid, or optionally the weak acid is citric acid, acetic acid, succinic acid, or succinic acid.

35. The population of any one of claims 1-34, wherein the weak acid or salt thereof in the equilibration buffer is citric acid or citrate.

36. The method of any one of claims 1-35, wherein conditioning the affinity chromatography eluate for subsequent purification by anion exchange chromatography comprises adding the weak acid or salt thereof to the affinity eluate.

37. The population of any one of claims 1-36, wherein the weak acid or salt thereof is added to the affinity eluate to a final concentration of at least about 0.5mM or greater.

38. The population of any one of claims 1-37, wherein the weak acid or salt thereof is added to the affinity eluate to a final concentration of from about 0.5mM to about 15mM.

39. The population of any one of claims 1-38, wherein the weak acid or salt thereof added to the affinity eluate is citric acid or salt thereof, optionally the salt is citrate.

40. The method of any one of claims 1-35, wherein conditioning the affinity chromatography eluate for subsequent purification by anion exchange chromatography comprises adding an amino acid to the affinity eluate.

41. The population of any one of claims 1-35 or 40, wherein the amino acids are added to the affinity eluate to a final concentration of at least about 15mM or greater.

42. The population of any one of claims 1-35 or 40-41, wherein the amino acids are added to the affinity eluate to a final concentration of from about 15mM to about 35mM.

43. The population of any one of claims 1-35 or 40-42, wherein the amino acid added to the affinity eluate is aspartate, glutamate, histidine, arginine, lysine, cysteine, or tyrosine, optionally the amino acid is histidine.

44. The population of any one of claims 1-43, wherein conditioning the affinity chromatography eluate for subsequent purification by anion exchange chromatography comprises diluting the affinity eluate.

45. The population of any one of claims 1-44, wherein conditioning the affinity eluate for anion exchange chromatography comprises diluting the eluate by at least 2 x or more.

46. The population of any one of claims 1-45, wherein conditioning the affinity eluate for anion exchange chromatography comprises diluting the affinity eluate with a dilution buffer (anion exchange dilution buffer).

47. The population of claim 46, wherein the dilution buffer comprises a weak acid or salt thereof.

48. The population of claim 47, wherein the dilution buffer comprises a weak acid or salt thereof at a concentration of at least about 0.5 mM.

49. The population of any one of claims 47 or 48, wherein the dilution buffer comprises a weak acid or salt thereof at a concentration of from about 0.5mM to about 15 mM.

50. The population of any one of claims 47-49, wherein the weak acid in the dilution buffer is citric acid, acetic acid, succinic acid, acetoacetic acid, adipic acid, tetraoxypyrimidic acid, ascorbic acid, aspartic acid, barbituric acid, boric acid, butyric acid, n-butyric acid, carbonic acid, crotonic acid, diglycolic acid, dimethylmalonic acid, formic acid, fumaric acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, glyceric acid, glycolic acid, isocitric acid, itaconic acid, lactic acid, maleic acid, malic acid, malonic acid, mesaconic acid, meso-tartaric acid, methylsuccinic acid, oxalic acid, oxaloacetic acid, pentanic acid, valeric acid, phosphoric acid, isophthalic acid, phthalic acid, terephthalic acid, propionic acid, pyruvic acid, salicylic acid, tartaric acid, tartronic acid, terephthalic acid, trichloroacetic acid, uric acid, alpha-tartaric acid, 2-oxobutyric acid, 2-methylbutyric acid, 2-oxoglutaric acid, 3-butenoic acid, or 3-methylbutyric acid, optionally the weak acid is citric acid, citrate, acetic acid, succinic acid, or succinic acid.

51. The population of any one of claims 47-50, wherein the weak acid or salt thereof in the dilution buffer is citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof, optionally the weak acid is citric acid or salt thereof.

52. The population of any one of claims 46-51, wherein the dilution buffer comprises a buffering agent.

53. The population of claim 52, wherein the buffer is acetate, histidine, phosphate, citrate, propionate, tricine, borate, or tris (hydroxymethyl) aminomethane (tris), optionally the buffer is bris-tris propane (BTP).

54. The population of any one of claims 52-53, wherein the dilution buffer comprises a buffer at a concentration of at least about 25 mM.

55. The population of any one of claims 52-54, wherein the dilution buffer comprises a buffer at a concentration of from about 25mM to about 175 mM.

56. The population of any one of claims 46-55, wherein the dilution buffer comprises amino acids.

57. The population of any one of claims 46-56, wherein the dilution buffer comprises amino acids at a concentration of at least about 25 mM.

58. The population of any one of claims 46-57, wherein the dilution buffer comprises amino acids at a concentration of from about 25mM to about 175 mM.

59. The method of any one of claims 56-58, wherein the amino acid is aspartate, glutamate, histidine, arginine, lysine, cysteine, or tyrosine, optionally the amino acid is histidine.

60. The population of any one of claims 46-59, wherein the dilution buffer comprises a viscosity modifier.

61. The population of claim 60, wherein the viscosity modifier is a polyol, optionally selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof.

62. The population of claim 61, wherein the polyol is sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, dextran, or any combination thereof, optionally glycerol, sorbitol, mannitol, galactitol, sucrose, lactose, maltose, trehalose, and any combination thereof.

63. The population of any one of claims 61-62, wherein the polyol is sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, dextran, or any combination thereof, optionally the polyol is glycerol.

64. The population of any one of claims 60-63, wherein the dilution buffer comprises a viscosity modifier at a concentration of at least about 0.5% (v/v or w/v) or more.

65. The population of any one of claims 60-64, wherein the dilution buffer comprises a viscosity modifier at a concentration of from about 0.5% to about 9.5% (v/v or w/v).

66. The population of any one of claims 46-65, wherein the dilution buffer comprises a nonionic surfactant.

67. The population of any one of claims 46-66, wherein the dilution buffer comprises a nonionic surfactant at a concentration of at least about 0.05% (v/v or w/v) or more.

68. The population of any one of claims 46-67, wherein the dilution buffer comprises a nonionic surfactant at a concentration of from about 0.05% to about 0.95% (v/v or w/v).

69. The population of any of claims 66-68, wherein said nonionic surfactant is selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates, preferably polysorbates, polyoxyethylene alkylphenyl ethers, and any combination thereof.

70. The population of any one of claims 46-69, wherein the dilution buffer comprises a salt.

71. The population of any one of claims 46-70, wherein the dilution buffer comprises a salt at a concentration of at least about 0.1 mM.

72. The population of any one of claims 46-71, wherein the dilution buffer comprises a salt at a concentration of from about 0.1mM to about 2 mM.

73. The population of any of claims 70-72, wherein the salt is a magnesium salt, a sodium salt, a potassium salt, an ammonium salt, a calcium salt, a copper salt, a cobalt salt, a manganese salt, a nickel salt, or a zinc salt, optionally the salt is MgCl ₂ 。

74. The population of any one of claims 46-73, wherein the dilution buffer has a high pH.

75. The population of any one of claims 46-74, wherein the dilution buffer has a pH of greater than or equal to about 8.

76. The population of any one of claims 46 or 52-75, wherein the dilution buffer comprises: BTP, histidine, glycerol and PF68、MgCl ₂ And has a high pH.

77. The population of any one of claims 46-76, wherein the dilution buffer comprises: BTP, histidine, PF68, mgCl ₂ Citric acid, and has a high pH.

78. The population of any one of claims 1-77, wherein the conductivity of the dilution buffer is in the range from about 0.5mS/cm to about 3 mS/cm.

79. The population of any one of claims 1-78, wherein the dilution buffer has an osmolality of less than 900mOsm.

80. The population of any one of claims 1-79, further comprising the step of removing or reducing the amount of impurities (e.g., host cell DNA (hcna)) from the harvest medium prior to affinity purification.

81. The population of claim 80, wherein the removing or reducing the amount of impurities comprises adding a cationic amine or nuclease to the harvest media.

82. The population of claim 80 or 81, wherein the removing or reducing the amount of impurities comprises adding a selective precipitant to the harvest media.

83. The population of any one of claims 1-82, further comprising the step of lysing host cells in the harvest medium with a nonionic surfactant prior to purification/isolation by affinity chromatography.

84. The population of claim 83, wherein a nonionic surfactant is added to the harvest media to a final concentration of at least about 0.05% (v/v or w/v) or higher.

85. The population of claim 83 or 84, wherein a nonionic surfactant is added to the harvest media to a final concentration of from about 0.05% to about 1% (v/v or w/v).

86. The population of any one of claims 83-85, wherein the nonionic surfactant is mixed with the harvesting medium for a period of time from about 15 minutes to about 2 hours.

87. The population of any of claims 83-86, wherein the non-ionic surfactant is not Triton X-100.

88. The population of any of claims 83-87, wherein the nonionic surfactant is selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates, preferably polysorbates, polyoxyethylene alkylphenyl ethers, and any combination thereof.

89. The population of any one of claims 1-88, wherein the recombinant adeno-associated viral particles comprise rAAV viral particles.

90. The population of any one of claims 1-89, wherein the particle to infectivity ratio of the purified rAAV is less than 2 x 10 ⁴ vg/TCID50。

91. The population of any one of claims 1-90, wherein the purified population of rAAV comprises less than about 10% empty viral capsids.

92. The population of any one of claims 1-91, wherein the rAAV is obtained by a method comprising transfecting a suspended mammalian cell line, and wherein the cells are optionally transfected in suspension.

93. The population of claim 92, wherein a) nucleic acid sequences encoding an accessory protein sufficient for rAAV replication are used; b) Nucleic acid sequences encoding rep and cap genes; and c) transfecting the mammalian cell line in suspension with a closed-end linear double-stranded rAAV vector nucleic acid comprising at least one ITR and a heterologous transgene operably linked to one or more regulatory elements.

94. The population of claim 92 or 93, wherein the mammalian cell line is derived from a human embryonic cell line.

95. The population of any of claims 92-94, wherein the human embryonic cell line is a suspension-adapted serum-free cell line derived from a human embryonic kidney cell line.

96. The population of any one of claims 92-95, wherein the purified recombinant adeno-associated virus (rAAV) lacks prokaryotic sequences.

97. The population of any one of claims 1-96, wherein the population is contained in a composition comprising the population, and wherein: (i) Wherein the composition exhibits substantially no aggregation of rAAV particles after two or more freeze-thaw cycles; and/or (ii) the purified rAAV particle retains its TCID50/mL of at least about 80% after two or more freeze-thaw cycles; and/or (iii) a particle to infectivity ratio of the purified rAAV of less than 2X 10 ⁴ vg/TCID ₅₀ The method comprises the steps of carrying out a first treatment on the surface of the And/or (iv) the purified rAAV population comprises less than about 35% empty viral capsids.

98. The population of claim 97, wherein the composition exhibits substantially no aggregation of rAAV particles after two or more freeze-thaw cycles.

99. The population of any one of claims 97-98, wherein the purified rAAV particles retain at least about 80% of their TCID50/mL after two or more freeze-thaw cycles.

100. The population of any one of claims 97-99, wherein the ratio of particles to infectivity of the purified rAAV is less than 2 x 10 ⁴ vg/TCID ₅₀ 。

101. The population of any one of claims 97-100, wherein the purified population of rAAV comprises less than about 10% empty viral capsids.

102. The population of any one of claims 97-101, wherein the composition comprises a concentration of from about 1e ⁹ vg/mL to about 1e ¹⁵ vg/mL of purified rAAV.

103. The population of any one of claims 97-102, wherein the composition comprises a concentration of from about 1e ¹² vg/mL to about 1e ¹⁵ vg/mL of purified rAAV, optionally at a concentration of from about 1e ¹³ vg/mL to about 1e ¹⁴ vg/mL。

104. The population of any of claims 97-103, wherein the pH of the composition is from about 6.5 to about 8, optionally the pH of the composition is from about 7 to about 8.

105. The population of any of claims 97-104, wherein the composition comprises a buffer, a filler, and one or both of a nonionic surfactant and a multivalent salt, and optionally: (i) the composition is substantially free of glycine; and/or (ii) the composition is substantially free of trehalose, trehalose dehydrate, dextran T10 or dextran T40; and/or (iii) the composition is substantially free of sodium, ammonium or potassium salts; and/or (iv) the composition is substantially free of polysorbate-80 (PS 80).

106. The population of claim 105, wherein the buffer is PBS, tris.hcl, phosphate, citric acid, histidine, tromethamine, succinic acid, malic acid, a-ketoglutaric acid, carbonate, protein buffer, or any combination thereof, optionally the buffer is PBS, tris or histidine buffer.

107. The population of claim 105 or 106, wherein the filler is a polyol or povidone (PVP K24).

108. The population of claim 107, wherein the polyol is sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, or maltose, optionally the polyol is sorbitol or mannitol.

109. The population of any of claims 105-108, wherein the nonionic surfactant is selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates, preferably polysorbates, polyoxyethylene alkylphenyl ethers, and any combination thereof.

110. The population of any one of claims 105-109, wherein the nonionic surfactant is selected from the group consisting of Brij 010, briJ 020, brij Cl 0, brij S20, brijSl0, ECOSURF EH-14, ECOSURF EH-9, ECOSURF SA-15, IGEPAL CA-720, IGEPAL CO-630, IGEPAL CO-720, MERPOL HCS surfactant, MERPOL OJ surfactant, MERPOL SH surfactant, pluronic 10R5, pluronic F-68, poloxamer P188, poloxamer P338, poloxamer P188, and poloxamer P188 poloxamer P407, polyoxyethylene (12) tridecyl ether, TERGITOL 15-S-12, TERGITOL 15-S-7, TERGITOL15-S-9, TERGITOL L-64, TERGITOL NP-10, TERGITOL NP-11, TERGITOL NP-12, TERGITOL NP-8, TERGITOL NP-9, TERGITOL NP-9.5, TERGITOL15-S-9, TERGITOL LNP-13, TERGITOL LNP-7, TWEEN 60 nonionic detergents, and any combination thereof.

111. The population of any of claims 105-110, wherein the multivalent salt is a calcium salt, citrate salt, sulfate salt, or magnesium salt.

112. The population of any one of claims 105-111, wherein the composition has an osmolality of less than 750mOsm.

113. The population of any one of claims 105-112, wherein the osmolality of the composition is from about 125mOsm to about 500mOsm.

114. The population of any one of claims 105-113, wherein the composition is a pharmaceutical composition.

115. A method of purifying or isolating recombinantly expressed adeno-associated virus particles from a harvesting medium, the method comprising:

a. purifying/separating a plurality of recombinantly-expressed viral particles from the harvest medium by affinity chromatography to produce an eluate comprising a plurality of recombinantly-expressed viral particles (affinity chromatography eluate), wherein an elution buffer for affinity chromatography (affinity elution buffer) comprises a predetermined amount of glycine, optionally the affinity elution buffer is substantially free of weak acids or salts thereof; and optionally, the affinity elution buffer comprises an amino acid that is not glycine;

b. conditioning the affinity chromatography eluate for subsequent purification by anion exchange chromatography, wherein the conditioned affinity eluate comprises a predetermined amount of a weak acid or salt thereof, or the conditioned affinity eluate comprises a predetermined amount of glycine and a predetermined amount of amino acids already present in the affinity eluate; and

c. Purifying/separating the plurality of recombinantly-expressed viral particles from the conditioned eluate of the affinity chromatography by anion exchange chromatography to produce a solution comprising the plurality of purified/separated recombinantly-expressed viral particles, wherein an equilibration buffer for anion exchange chromatography comprises a predetermined amount of a weak acid or salt thereof, optionally the weak acid is citric acid, acetic acid or succinic acid.

116. The population of claim 115, wherein less than 5% of the empty viral particles in the affinity eluate are bound to anion exchange chromatography media.

117. The method of claim 116, wherein substantially no empty viral particles in the affinity eluate are bound to anion exchange chromatography media.

118. The method of any one of claims 115-117, wherein less than 10% of the viral particles in the eluate from anion exchange are empty viral particles.

119. The method of any one of claims 115-118, wherein less than 5% of the viral particles in the eluate from anion exchange are empty viral particles.

120. The method of any one of claims 1, wherein the eluate from anion exchange is substantially free of empty viral particles.

121. The method of any one of claims 1-103, wherein the recombinantly expressed viral particle is a recombinant adeno-associated virus (rAAV) particle.

122. The method of any one of claims 115-121, wherein the weak acid of 115 (b) is citric acid, acetic acid, succinic acid, acetoacetic acid, adipic acid, tetraoxypyrimidic acid, ascorbic acid, aspartic acid, barbituric acid, boric acid, butyric acid, n-butyric acid, carbonic acid, crotonic acid, diglycolic acid, dimethylmalonic acid, formic acid, fumaric acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, glyceric acid, glycolic acid, isocitric acid, itaconic acid, lactic acid, maleic acid, malic acid, malonic acid, mesaconic acid, meso-tartaric acid, methylsuccinic acid, methylmalonic acid, oxalic acid, oxaloacetic acid, pentanic acid, valeric acid, phosphoric acid, isophthalic acid, phthalic acid, terephthalic acid, propionic acid, pyruvic acid, salicylic acid, tartaric acid, tartronic acid, trichloroacetic acid, uric acid, alpha-tartaric acid, 2-oxobutyric acid, 2-methylbutyric acid, 2-oxoglutaric acid, 3-butenoic acid, or 3-methylbutyric acid, optionally the weak acid is citric acid, citrate, acetic acid, or succinic acid.

123. The method of any one of claims 115-122, wherein the affinity elution buffer comprises histidine at a concentration of at least about 1mM or greater.

124. The method of any one of claims 115-123, wherein the affinity elution buffer comprises histidine at a concentration of from about 1mM to about 50 mM.

125. The method of any one of claims 115-124, wherein the affinity elution buffer comprises glycine at a concentration of at least about 20mM or greater.

126. The method of any one of claims 115-128, wherein the affinity elution buffer comprises glycine at a concentration of from about 25mM to about 100 mM.

127. The method of any one of claims 115-126, wherein the affinity elution buffer comprises a salt.

128. The method of any one of claims 115-127, wherein the affinity elution buffer comprises a salt at a concentration of at least about 5mM or greater.

129. The method of any one of claims 115-128, wherein the affinity elution buffer comprises a salt at a concentration of from about 5mM to about 15 mM.

130. The method of any one of claims 127-129, wherein the salt is a magnesium salt, a sodium salt, a potassium salt Salts, ammonium salts, calcium salts, copper salts, cobalt salts, manganese salts, nickel salts or zinc salts, optionally said salts being MgCl ₂ 。

131. The method of any one of claims 115-130, wherein the affinity elution buffer comprises a polymer.

132. The method of any one of claims 115-131, wherein the affinity elution buffer comprises the polymer at a concentration of at least about 0.1% or greater.

133. The method of any one of claims 115-132, wherein the affinity elution buffer comprises the polymer at a concentration of from about 0.1% to about 0.5%.

134. The method of any of claims 131-133, wherein the polymer is a nonionic surfactant.

135. The method of any one of claims 115-134, wherein the affinity elution buffer has a low pH.

136. The method of any one of claims 115-135, wherein the pH of the affinity elution buffer is less than or equal to about 6.5.

137. The method of any one of claims 115-136, wherein the pH of the affinity elution buffer is from about 2.0 to about 3.0.

138. The method of any one of claims 115-137, wherein the affinity elution buffer comprises: about 75mM glycine.

139. The method of any one of claims 115-122 or 125-138, wherein the affinity elution buffer comprises a weak acid or salt thereof.

140. The method of claim 139, wherein the affinity elution buffer comprises a weak acid or salt thereof at a concentration of at least about 50mM or greater.

141. The method of claim 139 or 140, wherein the affinity elution buffer comprises a weak acid or salt thereof at a concentration of from about 50mM to about 100 mM.

142. The method of any of claims 139-141, wherein the weak acid is citric acid, acetic acid, succinic acid, acetoacetic acid, adipic acid, tetrapyrimidic acid, ascorbic acid, aspartic acid, barbituric acid, boric acid, butyric acid, n-butyric acid, carbonic acid, crotonic acid, diglycolic acid, dimethylmalonic acid, formic acid, fumaric acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, glyceric acid, glycolic acid, isocitric acid, itaconic acid, lactic acid, maleic acid, malic acid, malonic acid, mesaconic acid, meso-tartaric acid, methylsuccinic acid, methylmalonic acid, oxalic acid, oxaloacetic acid, pentanic acid, valeric acid, phosphoric acid, isophthalic acid, phthalic acid, terephthalic acid, propionic acid, pyruvic acid, salicylic acid, tartaric acid, tartronic acid, terephthalic acid, trichloacetic acid, uric acid, α -tartaric acid, 2-oxobutyric acid, 2-methylbutyric acid, 2-oxoglutaric acid, 3-butenoic acid, or 3-methylbutyric acid, optionally the weak acid is citric acid, citrate, acetic acid, or succinic acid.

143. The method of any of claims 139-142, wherein the weak acid is citric acid or a salt thereof.

144. The method of any one of claims 115-122, 125-137, or 139-143, wherein the affinity elution buffer comprises: about 50mM glycine, about 75mM citrate, about 10mM MgCl ₂ About 0.3% (w/v) P188, and a pH of about 3.0.

145. The method of any one of claims 115-144, wherein the affinity elution buffer has a conductivity in the range of from about 5mS/cm to about 8 mS/cm.

146. The method of any one of claims 115-145, wherein the affinity elution buffer has an osmotic pressure ranging from about 100mOsm to about 225 mOsm.

147. The method of any one of claims 115-146, wherein the equilibration buffer for anion exchange chromatography comprises a weak acid or salt thereof at a concentration of at least about 0.5 mM.

148. The method of any one of claims 115-147, wherein the equilibration buffer comprises a weak acid or salt thereof at a concentration from about 0.5mM to about 15 mM.

149. The method of any one of claims 115-149, wherein the weak acid in the equilibration buffer is citric acid, acetic acid, succinic acid, acetoacetic acid, adipic acid, tetraoxypyrimidic acid, ascorbic acid, aspartic acid, barbituric acid, boric acid, butyric acid, n-butyric acid, carbonic acid, crotonic acid, diglycolic acid, dimethylmalonic acid, formic acid, fumaric acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, glyceric acid, glycolic acid, isocitric acid, itaconic acid, lactic acid, maleic acid, malic acid, malonic acid, mesaconic acid, meso-tartaric acid, methylsuccinic acid, methylmalonic acid, oxalic acid, oxaloacetic acid, pentanic acid, valeric acid, phosphoric acid, isophthalic acid, phthalic acid, terephthalic acid, propionic acid, pyruvic acid, salicylic acid, tartaric acid, tartronic acid, terephthalic acid, trichloroacetic acid, uric acid, alpha-tartaric acid, 2-oxobutyric acid, 2-methylbutyric acid, 2-oxoglutaric acid, 3-butenoic acid, or 3-methylbutyric acid, optionally the weak acid is citric acid, citrate, acetic acid, succinic acid, or succinic acid.

150. The method of any one of claims 115-149, wherein the weak acid or salt thereof in the equilibration buffer is citric acid or citrate.

151. The method of any one of claims 115-150, wherein conditioning the affinity chromatography eluate for subsequent purification by anion exchange chromatography comprises adding a weak acid or salt thereof to the affinity eluate.

152. The method of any one of claims 115-151, wherein the weak acid or salt thereof is added to the affinity eluate to a final concentration of at least about 0.5mM or greater.

153. The method of any one of claims 115-152, wherein the weak acid or salt thereof is added to the affinity eluate to a final concentration of from about 0.5mM to about 15mM.

154. The method of any one of claims 115-153, wherein the weak acid or salt thereof added to the affinity eluate is citric acid or a salt thereof, optionally the salt is citrate.

155. The method of any one of claims 115-150, wherein conditioning the affinity chromatography eluate for subsequent purification by anion exchange chromatography comprises adding an amino acid to the affinity eluate.

156. The method of any one of claims 115-150 or 155, wherein the amino acid is added to the affinity eluate to a final concentration of at least about 15mM or greater.

157. The method of any one of claims 115-150 or 155-156, wherein the amino acid is added to the affinity eluate to a final concentration of from about 15mM to about 35mM.

158. The method of any one of claims 115-150 or 155-157, wherein the amino acid added to the affinity eluate is aspartate, glutamate, histidine, arginine, lysine, cysteine, or tyrosine, optionally the amino acid is histidine.

159. The method of any one of claims 115-158, wherein conditioning the affinity chromatography eluate for subsequent purification by anion exchange chromatography comprises diluting the affinity eluate.

160. The method of any one of claims 115-159, wherein conditioning the affinity eluate for anion exchange chromatography comprises diluting the eluate by at least 2 x or more.

161. The method of any one of claims 115-160, wherein conditioning the affinity eluate for anion exchange chromatography comprises diluting the affinity eluate with a dilution buffer (anion exchange dilution buffer).

162. The method of claim 161, wherein the dilution buffer comprises a weak acid or salt thereof.

163. The method of claim 162, wherein the dilution buffer comprises a weak acid or salt thereof at a concentration of at least about 0.5 mM.

164. The method of any one of claims 162 or 163, wherein the dilution buffer comprises a weak acid or salt thereof at a concentration of from about 0.5mM to about 15 mM.

165. The method of any one of claims 162-164, wherein the weak acid in the dilution buffer is citric acid, acetic acid, succinic acid, acetoacetic acid, adipic acid, tetraoxypyrimidic acid, ascorbic acid, aspartic acid, barbituric acid, boric acid, butyric acid, n-butyric acid, carbonic acid, crotonic acid, diglycolic acid, dimethylmalonic acid, formic acid, fumaric acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, glyceric acid, glycolic acid, isocitric acid, itaconic acid, lactic acid, maleic acid, malic acid, malonic acid, mesaconic acid, meso-tartaric acid, methylsuccinic acid, oxalic acid, oxaloacetic acid, pentanic acid, valeric acid, phosphoric acid, isophthalic acid, phthalic acid, terephthalic acid, propionic acid, pyruvic acid, salicylic acid, tartaric acid, tartronic acid, terephthalic acid, trichloroacetic acid, uric acid, alpha-tartaric acid, 2-oxobutyric acid, 2-methylbutyric acid, 2-oxoglutaric acid, 3-butenoic acid, or 3-methylbutyric acid, optionally the weak acid is citric acid, citrate, acetic acid, succinic acid, or succinic acid.

166. The method of any one of claims 162-165, wherein the weak acid or salt thereof in the dilution buffer is citric acid or salt thereof, acetic acid or salt thereof, or succinic acid or salt thereof, optionally the weak acid is citric acid or salt thereof.

167. The method of any one of claims 162-166, wherein the dilution buffer comprises a buffering agent.

168. The method of claim 167, wherein the buffer is acetate, histidine, phosphate, citrate, propionate, tricine, borate, or tris (hydroxymethyl) aminomethane (tris), optionally the buffer is bris-tris propane (BTP).

169. The method of any one of claims 167-168, wherein the dilution buffer comprises a buffer at a concentration of at least about 25 mM.

170. The method of any one of claims 167-169, wherein the dilution buffer comprises a buffer at a concentration of from about 25mM to about 175 mM.

171. The method of any one of claims 161-170, wherein the dilution buffer comprises amino acids.

172. The method of any one of claims 161-171, wherein the dilution buffer comprises an amino acid at a concentration of at least about 25 mM.

173. The method of any one of claims 161-172, wherein the dilution buffer comprises amino acids at a concentration of from about 25mM to about 175 mM.

174. The method of any one of claims 161-173, wherein the amino acid is aspartate, glutamate, histidine, arginine, lysine, cysteine, or tyrosine, optionally the amino acid is histidine.

175. The method of any one of claims 161-174, wherein the dilution buffer comprises a viscosity modifier.

176. The method of claim 175, wherein the viscosity modifier is a polyol, optionally selected from the group consisting of hydrocarbons, monosaccharides, disaccharides, trisaccharides, and any combination thereof.

177. The method of claim 176, wherein the polyol is sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, dextran, or any combination thereof, optionally glycerol, sorbitol, mannitol, galactitol, sucrose, lactose, maltose, trehalose, and any combination thereof.

178. The method of any of claims 176-177, wherein the polyol is sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, dextran, or any combination thereof, optionally the polyol is glycerol.

179. The method of any one of claims 175-178, wherein the dilution buffer comprises a viscosity modifier at a concentration of at least about 0.5% (v/v or w/v) or more.

180. The method of any one of claims 175-179, wherein the dilution buffer comprises a viscosity modifier at a concentration of from about 0.5% to about 9.5% (v/v or w/v).

181. The method of any one of claims 161-180, wherein the dilution buffer comprises a nonionic surfactant.

182. The method of any one of claims 161-181, wherein the dilution buffer comprises a nonionic surfactant at a concentration of at least about 0.05% (v/v or w/v) or higher.

183. The method of any one of claims 161-182, wherein the dilution buffer comprises a nonionic surfactant at a concentration of about 0.05% to about 0.95% (v/v or w/v).

184. The method of any of claims 181-183 wherein the nonionic surfactant is selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates, preferably polysorbates, polyoxyethylene alkylphenyl ethers, and any combination thereof.

185. The method of any one of claims 161-184, wherein the dilution buffer comprises a salt.

186. The method of any one of claims 161-185, wherein the dilution buffer comprises a salt at a concentration of at least about 0.1 mM.

187. The method of any one of claims 161-186, wherein the dilution buffer comprises a salt at a concentration of from about 0.1mM to about 2 mM.

188. The process of any one of claims 185-187, wherein the salt is a magnesium salt, a sodium salt, a potassium salt, an ammonium salt, a calcium salt, a copper salt, a cobalt salt, a manganese salt, a nickel salt, or a zinc salt, optionally the salt is MgCl ₂ 。

189. The method of any one of claims 161-188, wherein the dilution buffer has a high pH.

190. The method of any one of claims 161-189, wherein the dilution buffer has a pH of greater than or equal to about 8.

191. The method of any one of claims 161 or 167-190, wherein the dilution buffer comprises: BTP, histidine, glycerol, PF68, mgCl ₂ And has a high pH.

192. The method of any one of claims 161-191, wherein the dilution buffer comprises: BTP, histidine, PF68, mgCl ₂ Citric acid, and has a high pH.

193. The method of any one of claims 161-192, wherein the conductivity of the dilution buffer is in the range of from about 0.5mS/cm to about 3 mS/cm.

194. The method of any one of claims 161-193, wherein the dilution buffer has an osmolality of less than 900mOsm.

195. The method of any one of claims 115-194, wherein less than 5% of the empty viral particles in the affinity eluate are bound to anion exchange chromatography media.

196. The method of any one of claims 115-195, wherein less than 10% of the viral particles in the eluate from anion exchange are empty viral particles.

197. The method of any one of claims 115-196, wherein the eluate from anion exchange is substantially free of empty viral particles.

198. The method of any one of claims 115-197, wherein the UV in the anion exchange eluate ₂₆₀ With UV ₂₈₀ Ratio of UV in the adjusted affinity eluate ₂₆₀ With UV ₂₈₀ Is at least 1.15 x higher.

199. The method of any one of claims 115-198, wherein the recombinant adeno-associated viral particle comprises a rAAV virion.

200. The method of any one of claims 115-199, wherein the particle to infectivity ratio of the purified rAAV is less than 2 x 10 ⁴ vg/TCID50。

201. A population of recombinantly expressed viral particles purified or isolated by the method of any one of claims 115-200.

202. A composition comprising a population of recombinantly expressed viral particles purified or isolated by the method of any one of claims 115-200.

203. The composition of claim 202, wherein the composition is a pharmaceutical composition.

204. A population of purified recombinant adeno-associated viruses (rAAV) lacking prokaryotic sequences, wherein the particle to infectivity ratio of the purified rAAV is less than 2 x 10 ⁴ vg/TCID50, wherein the purified rAAV population comprises less than about 10% empty viral capsids, and wherein the purified rAAV is optionally obtained by a method comprising transfecting a suspension mammalian cell line, and wherein the cells are optionally transfected in suspension.

205. The purified population of recombinant adeno-associated viruses (rAAV) of claim 204, wherein the population comprises less than about 5% empty viral capsids.

206. The purified population of recombinant adeno-associated virus (rAAV) of any one of claims 204-205, wherein the population is substantially free of empty viral capsids.

207. The purified population of recombinant adeno-associated viruses (rAAV) of any one of claims 204-206, wherein the mammalian cell line is derived from a human embryonic cell line.

208. The purified population of recombinant adeno-associated virus (rAAV) of any of claims 204-207, wherein the human embryonic cell line is a suspension-adapted serum-free cell line derived from a human embryonic kidney cell line.

209. A population of purified recombinant adeno-associated viruses (rAAV) lacking prokaryotic sequences, wherein the particle to infectivity ratio of the purified rAAV is less than 2 x 10 ⁴ vg/TCID50, and wherein the purified rAAV is obtained by a method comprising transfecting a suspension mammalian cell line.

210. The purified population of recombinant adeno-associated viruses (rAAV) of claim 209, wherein the nucleic acid sequence encoding a helper protein sufficient for rAAV replication is used a); b) Nucleic acid sequences encoding rep and cap genes; and c) transfecting the mammalian cell line in suspension with a closed-end linear double-stranded rAAV vector nucleic acid comprising at least one ITR and a heterologous transgene operably linked to one or more regulatory elements.

211. The purified population of recombinant adeno-associated viruses (rAAV) of any one of claims 209-210, wherein the mammalian cell line is derived from a human embryonic cell line.

212. The purified population of recombinant adeno-associated virus (rAAV) of any one of claims 209-211, wherein the human embryonic cell line is a suspension-adapted serum-free cell line derived from a human embryonic kidney cell line.

213. A composition comprising a population of purified recombinant adeno-associated virus particles and a pH of from about 6.5 to about 8.0, and wherein: (i) The particle to infectivity ratio of the purified rAAV is less than 2X 10 ⁴ vg/TCID50; and/or (ii) the purified rAAV population comprises less than about 10% empty viral capsids.

214. The composition of claim 213, wherein the composition comprises a concentration of from about 1e ⁹ vg/mL to about 1e ¹⁵ vg/mL of purified rAAV.

215. The composition of any of claims 213-214, wherein the composition comprises a concentration of from about 1e ¹² vg/mL to about 1e ¹⁴ vg/mL of purified rAAV, optionally at a concentration of from about 1e ¹³ vg/mL to about 1e ¹⁴ vg/mL。

216. The composition of any of claims 213-215, wherein the pH of the composition is from about 6.5 to about 8, optionally the pH of the composition is from about 7 to about 8.

217. The composition of any one of claims 213-216, wherein the composition comprises a buffer.

218. The composition of claim 217, wherein the buffer is selected from the group consisting of PBS, tris.hcl, phosphate, citric acid, histidine, tromethamine, succinic acid, malic acid, alpha-ketoglutaric acid, carbonate, protein buffer, or any combination thereof.

219. The composition of claim 217 or 218, wherein the buffer has a salt concentration of from about 20mM to about 750mM.

220. The composition of any one of claims 214-219, wherein the ionic strength of the composition is at least 100mM.

221. The composition of claim 220, wherein the ionic strength of the composition is from about 125mM to about 750mM.

222. The composition of any one of claims 214-221, wherein the composition has an osmolality of less than about 600mOsm.

223. The composition of claim 222, wherein the osmolality of the composition is from about 125mOsm to about 500mOsm.

224. The composition of any one of claims 214-223, wherein the composition comprises one or more ions and/or salts thereof.

225. The composition according to claim 224, wherein said ion is selected from the group consisting of sodium, potassium, chloride, ammonium, carbonate, nitrate, chlorate, chlorite, and calcium.

226. The composition of any of claims 214-225, wherein the composition comprises a filler.

227. A composition according to claim 226, wherein said filler is a polyol or povidone (PVP K24).

228. The composition of any of claims 226-227 wherein the bulking agent is selected from the group consisting of polyhydroxy hydrocarbons, monosaccharides, disaccharides, and trisaccharides.

229. The composition of any of claims 226-228, wherein the bulking agent is selected from the group consisting of sorbitol, mannitol, glycerol, propylene glycol, polyethylene glycol, galactitol, sucrose, lactose, maltose, trehalose, and dextran.

230. The composition of any one of claims 226-229, wherein the composition comprises a filler in a concentration of from about 0.5% (w/v) to about 10% (w/v).

231. The composition of any one of claims 214-230, wherein the composition comprises a nonionic surfactant.

232. The composition of claim 231, wherein said nonionic surfactant is selected from the group consisting of polyoxyethylene fatty alcohol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene-polyoxypropylene block copolymers, alkyl glucosides, alkylphenol ethoxylates, preferably polysorbates, polyoxyethylene alkylphenyl ethers, and any combination thereof.

233. The composition of claim 231 or 232, wherein the nonionic surfactant is selected from the group consisting of a TWEEN 60 nonionic detergent, PPG-PEG-PPG Pluronic 10R5, pluronic F-68, polyoxyethylene (18) tridecyl ether, polyoxyethylene (12) tridecyl ether, MERPOL SH surfactant, MERPOL OJ surfactant, MERPOL HCS surfactant, poloxamer P188, poloxamer P407, poloxamer P338, IGEPAL CO-720, IGEPAL CO-630, IGEPAL CA-720, brij S20, brijSl0, brij 010, brij Cl0, brij 020, ecoruf EH-9, ecorurf-14, ecorurf 15-S-7, ecorurf SA-15, terton 15-S-9, ginton 15-S-12, ginp-64, terp-7, terp-8, ternp-terp-10, lnnp-10, and any combination thereof, lnnp-10, and any of the group of ginp-10, lnnp-10, and the like.

234. The composition of any one of claims 231-233, wherein the composition comprises a nonionic surfactant at a concentration of from about 0.005% (w/v) to about 0.015% (w/v).

235. The composition of any one of claims 214-234, wherein the composition comprises one or more multivalent ions or salts thereof.

236. The composition of claim 235, wherein said multivalent ion is selected from the group consisting of citrate, sulfate, magnesium, and phosphate.

237. The composition of any one of claims 235-236, wherein the composition comprises multivalent ions at a concentration of from about 5mM to about 150 mM.

238. The composition of claim 237, wherein the composition comprises multivalent ions at a concentration of from about 10mM to about 50 mM.

239. The composition of any one of claims 214-238, wherein the composition comprises calcium α -d-heptagluconate at a concentration of from about 0.0001% (w/v) to about 0.01% (w/v).

240. The composition of any one of claims 214-239, wherein the purified rAAV population comprises less than about 5% empty viral capsids.

241. The composition of any one of claims 214-240, wherein the purified rAAV population is substantially free of empty viral capsids.

242. The composition of any one of claims 214-241, wherein the particle to infectivity ratio of the purified rAAV is less than 1.5 x 10 ⁴ vg/TCID50

243. The composition of any one of claims 214-242, wherein the composition exhibits substantially no aggregation after two or more freeze-thaw cycles.

244. The composition of any one of claims 214-243, wherein the purified rAAV particle of claim retains its TCID after two or more freeze-thaw cycles ₅₀ /mL of at least about 80%.

245. The composition of any one of claims 214-244, wherein the purified population of recombinant adeno-associated virus particles is the purified population of rAAV of any one of claims 115-200.