WO2022136968A1

WO2022136968A1 - Method of separating a virus from a composition using copolymer-grafted nonwoven substrates

Info

Publication number: WO2022136968A1
Application number: PCT/IB2021/060671
Authority: WO
Inventors: Narendranath BHOKISHAM; Minghua Dai; Kristopher E. RICHARDSON; Andrew W. Vail; Daniel J. O'neal; Alexei M. Voloshin; Rebecca A. HOCHSTEIN
Original assignee: 3M Innovative Properties Company
Priority date: 2020-12-23
Filing date: 2021-11-17
Publication date: 2022-06-30

Abstract

The present disclosure provides a method of separating (e.g., purifying, clarifying, etc.) a virus from a composition. The method includes a) obtaining a composition containing a virus and at least one of deoxyribonucleic acid (DNA) or host cell proteins (HCP); b) passing at least a portion of the composition through a copolymer-grafted nonwoven substrate to provide a filtrate; and c) collecting the filtrate, which contains 50% or greater of an amount of the virus present in the composition. The composition has a pH of 6 to 8.5 and a conductivity of 4 or more milliSiemens per centimeter. The grafted copolymer includes interpolymerized monomer units of (i) a quaternary ammonium containing monomer or salt thereof; (ii) a hydrophilic amide containing monomer; and (iii) an epoxy containing monomer. The copolymer-grafted nonwoven substrate assists in retaining impurities such as DNA and HCP while certain negatively-charged viruses pass through the substrate.

Description

METHOD OF SEPARATING A VIRUS FROM A COMPOSITION USING COPOE YMER-GRAFTED NONWOVEN SUBSTRATES

Field

[0001] The present disclosure generally relates to separation (e.g., purification, clarification, etc.) of compositions containing a virus.

Background

[0002] Copolymer-grafted nonwoven substrates have been employed in the clarification stages of the monoclonal antibody (mAb) purification process. These products are unique in the market because the non-woven materials are open, which enables the cell culture media to flow at high flowrates through the media. Quaternary amine chemistry functionalized on the media enables substantial reduction of DNA (up to 4 log removal) and host cell proteins while letting proteins including monoclonal antibodies (mAb) through the filters thereby making these products efficient clarification devices. With regards to viral purification, viruses are an order of magnitude larger in size and more dense than mAbs, providing unique purification challenges. The larger size and density leads to significantly lower mass transfer coefficients than mAbs, making diffusion of these molecules significantly slower than smaller biomolecules such as mAbs. Because most of the chromatography resins used in purification of mAbs rely on diffusion for clarification these tools are not very effective in purification of the larger and more dense viruses.

Summary

[0003] In a first aspect, a method of separating a virus from a composition is provided. The method includes a) obtaining a composition comprising a virus and at least one of deoxyribonucleic acid (DNA) or host cell proteins (HCP); b) passing at least a portion of the composition through a copolymer-grafted nonwoven substrate to provide a filtrate; and c) collecting the filtrate, the filtrate comprising 50% or greater of an amount of the virus present in the composition. The composition has a pH of 6 to 8.5 and a conductivity of 4 or more milliSiemens per centimeter (mS/cm). The grafted copolymer includes interpolymerized monomer units of (i) a quaternary ammonium containing monomer or salt thereof; (ii) a hydrophilic amide containing monomer; and (iii) an epoxy containing monomer. [0004] In a second aspect, a separated product is provided. The separated product includes a virus (e.g., adeno-associated virus (AAV) or Phi-X174) produced by the method according to the first aspect.

Detailed Description

[0005] Glossary

[0006] As used herein:

[0007] “Majority” means greater than 50%.

[0008] “Copolymer” refers to a polymer formed of two or more different monomers.

[0009] “Alkyl” means a linear or branched, cyclic or acyclic, saturated monovalent hydrocarbon having from one to about twelve carbon atoms, e.g., methyl, ethyl, 1 -propyl, 2-propyl, pentyl, and the like.

[0010] Also herein, all numbers are assumed to be modified by the term “about” and preferably by the term “exactly.” As used herein in connection with a measured quantity, the term “about” refers to that variation in the measured quantity as would be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. The recitations of numerical ranges by endpoints include all numbers subsumed within that range as well as the endpoints (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0011] The present disclosure provides a method of separating a virus from a composition. The method comprises:

[0012] a) obtaining a composition comprising a virus and at least one of deoxyribonucleic acid (DNA) or host cell protein (HCP), wherein the composition has a pH of 6 to 8.5 and a conductivity of 4 or more milliSiemens per centimeter (mS/cm);

[0013] b) passing at least a portion of the composition through a copolymer-grafted nonwoven substrate to provide a filtrate, wherein the grafted copolymer comprises interpolymerized monomer units of (i) a quaternary ammonium containing monomer or salt thereof; (ii) a hydrophilic amide containing monomer; and (iii) an epoxy containing monomer; and

[0014] c) collecting the filtrate, the filtrate comprising 50% or greater of an amount of the virus present in the composition. [0015] It has unexpectedly been discovered that various negatively charged viruses behave differently when compositions containing the viruses are each passed through a positively charged copolymer-grafted nonwoven substrate. Although oppositely charged materials and moieties typically have an attraction to each other, it has been discovered that certain negatively charged viruses tend to pass through this positively charged nonwoven substrate. For example, AAVs that are used in gene therapy applications, seem to have low, minimal, or no binding onto the positively charged nonwoven substrate under the pH and salt concentrations of cell culture conditions. This was also found to be the case for the Phi-X174 bacterial virus. Further, more than 50% of each of Lentivirus, T7, and Phi6 bacterial viruses passed through the positively charged nonwoven substrate at certain pH and conductivity conditions outside of typical cell culture conditions.

[0016] Suitable compositions include cell lysates containing the virus, which also contain cells and cellular components such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), HCP, cell debris, oligonucleotides or therapeutic proteins such as mAbs (mAb), nucleotides such as adenosine triphosphate (ATP), or combinations thereof. In some embodiments, the virus is grown in cells and the lysed to form a cell lysate. In other embodiments, the virus is cultured separately and then spiked into a cell lysate. Suitable compositions further include compositions that have already undergone at least one purification or clarification step and contain fewer components than a cell lysate. In an embodiment, the only impurity present in the composition is DNA. In an embodiment, the only impurity present in the composition is HCP. In an embodiment, the composition contains both DNA and HCP. In certain embodiments, the composition contains (intact) cells and/or cell debris.

[0017] This discovery allows for the copolymer-grafted nonwoven substrate to be employed in separating (e.g., purifying or clarifying) such viruses from undesirable components of a composition, for instance under the pH and conductivity of typical cell culture conditions of e.g., pH 7-8 and e.g., 8-15 mS/cm. Unlike the particular negatively charged virus, other negatively charged impurities, such as DNA and HCP are selectively retained by the copolymer-grafted nonwoven substrate.

[0018] The composition has a pH of 6 or greater, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5 or greater; and a pH of 8.5 or less, 8.4, 8.3, 8.2, 8. 1, 8.0, 7.9, 7.8, 7.7, or 7.6 or less. In some embodiments, the composition has a pH of 7 to 8, 6 to 7, 6.5 to 7, or 7.5 to 8.5. If needed, the pH of the composition can be adjusted using a pH buffer or by adding an acid or a base until a desired pH value is reached, as known to the skilled practitioner, with pH measurements made by a digital laboratory pH meter. Some suitable pH buffers include, for instance, Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl) and (4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid) (HEPES). In select embodiments of the present method, the composition comprises a buffer.

[0019] The composition has a conductivity of 4 mS/cm or greater; 5 mS/cm, 6 mS/cm, 7 mS/cm, 8 mS/cm, 9 mS/cm, 10 mS/cm, 11 mS/cm, 12 mS/cm, 13, mS/cm, 14 mS/cm, 15 mS/cm, 16 mS/cm, 17 mS/cm, 18 mS/cm, 19 mS/cm, 20 mS/cm, 21 mS/cm, 22 mS/cm, 23 mS/cm, 24 mS/cm, or 25 mS/cm or greater; and 100 mS/cm or less, 95 mS/cm, 90 mS/cm, 85 mS/cm, 80 mS/cm, 75 mS/cm, 70 mS/cm, 65 mS/cm, 60 mS/cm, 55 mS/cm, 50 mS/cm, 45 mS/cm, 40 mS/cm, 35 mS/cm, or 30 mS/cm or less. In some embodiments, the composition has a conductivity of 5 mS/cm to 15 mS/cm, 5 mS/cm to 30 mS/cm, 8 mS/cm to 12 mS/cm, 8 mS/cm to 20 mS/cm, 15 mS/cm to 25 mS/cm, or 20 mS/cm to 50 mS/cm. The conductivity of the composition can be increased if needed by using a salt, such sodium chloride, potassium chloride, magnesium chloride, magnesium sulphate, or combinations thereof. The conductivity of the composition can be decreased if needed by diluting the composition with a solvent having a lower conductivity. Conductivity can be determined using a conductivity meter, such as the Accumet Excel XL50 commercially available from Fisher Scientific (Hampton, NH).

[0020] A cell lysate may be prepared by any method known to the skilled practitioner, and is typically incomplete; for instance, the cell lysate contains at least 20% lysed cells (in some embodiments 25% or greater, 30%, 35%, 40%, 45%, or 50% or greater lysed cell) and up to 80% intact cells (in some embodiments 75% or less, 70%, 65%, 60%, or 55% or less intact cells). In some embodiments, a cell lysate is prepared using physical or chemical methods. Lysing can occur upon physically lysing the cells. Physical lysing can occur upon vortexing the test sample with glass beads, sonicating, heating and boiling, repeating freezing and thawing, or subjecting the test sample to high pressure, such as occurs upon using a French press, for example. Freeze thawing (e.g., a cyclical freezing and thawing process) and microfluidization may be particularly suitable. Physical lysing can be conducted under conditions, such as, for example, at a temperature of 5°C to 42°C (probably as high as 500°C), often at a temperature of 15°C to 25°C. Lysing can also occur using a lysing agent. Suitable lysing agents include, for example, solubilizing agents (e.g., nonionic surfactants) such as Triton X-100, enzymes (e.g., protease, glycosidases, nucleases, salts (e.g., chaotrophic salts), reducing agents (e.g., beta-mercaptoethanol (BME), dithiothreitol (DTT), dithioerythritol (DTE), tris(2 -carboxyethyl) phosphine hydrochloride (TCEP; Pierce Chemical Company, Rockford, IL), cysteine, n-acetyl cysteine), acids (e.g., HC1), and bases (e.g., NaOH). Various combinations of lysing agents and/or methods can be used if desired. [0021] As mentioned above, following passing of the composition through the copolymer-grafted nonwoven substrate, the filtrate comprises 50% or greater of an amount of the virus (originally) present in the composition. In some embodiments, the filtrate comprises 55% or greater, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% 94%, 95%, 96%, 97%, 98%, or 99% or greater; and 100% or less, of the amount of the virus present in the composition prior to passing through the copolymer-grafted nonwoven substrate. The amount of the virus (e.g., AAV, Phi-X174) is determined using enzyme-linked immunosorbent assay (ELISA) quantitative polymerase chain reaction (qPCR), or multi-angle dynamic light scattering (MA-DLS). Each of these methods is well-known to the skilled practitioner.

[0022] Typically, the virus is an adeno-associated virus (AAV) or Phi-X174. In some embodiments, the virus is Phi-X174. Phi-X174 is a bacteriophage that attacks the bacteria Escherichia coli. Phi-X174 has a single strand of DNA and was the first DNA-based genome to be sequenced.

[0023] In some embodiments, the virus is AAV. AAV is a non-enveloped virus belonging to the parvoviridae family. There are twelve different AAV serotypes. AAV needs the presence of a helper virus for replication and assembly and thus belongs to the genus dependoparvovirus . The AAV optionally comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, or any combination thereof. In select embodiments, the AAV comprises AAV1, AAV2, AAV3, AAV5, AAV6, AAV8, AAV9, or any combination thereof. Often, the virus is a recombinant AAV.

[0024] In some embodiments, the virus is Lentivirus, T7, or Phi6. As mentioned above, each of these bacterial viruses were able to be separated, retaining greater than 50% of their original content when passed through the copolymer-grafted nonwoven substrate at conditions outside of typical cell conditions. For instance, in the Examples below, Lentivirus was retained in an amount greater than 50% at pH 8 and conductivity of 17.35 mS/cm; T7 was retained in an amount greater than 50% at pH 8.5 and conductivities of 15 mS/cm to 85 mS/cm; and Phi6 was retained in an amount greater than 50% at pH 7.4 and conductivities of each 15.8 mS/cm and 19.3 mS/cm as well as at pH 8 and a conductivity of 17.35 mS/cm.

[0025] The copolymer-grafted nonwoven substrate tends to retain DNA. In some embodiments of the present method, the filtrate exhibits a 1 log reduction or more of an amount of the DNA initially present in the composition, a 1.5 log reduction or more, a 2 log reduction or more, a 2.5 log reduction or more, or a 3 log reduction or more, of an amount of the DNA present in the composition prior to being passed through the copolymer-grafted nonwoven substrate. [0026] The copolymer-grafted nonwoven substrate tends to retain HCP. In some embodiments of the present method, the filtrate contains 50% or less of an amount of the HCP initially present in the composition, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, or 10% or less of an amount of the HCP present in the composition prior to being passed through the copolymer-grafted nonwoven substrate.

[0027] In some embodiments, the method provides a reduction in turbidity of the composition, for instance a reduction from an initial turbidity of 90% or greater, 92% or greater, 94% or greater, 96% or greater, or even 98% or greater. Turbidity can be determined, for example, using an Orion AQ4500 turbidity meter (commercially available from Thermo Fisher Scientific), with the turbidity values reported in Nephelometric Turbidity Units (NTU).

[0028] In select embodiments, at least a portion of the (i.e., virus-containing) composition is passed through a copolymer-grafted nonwoven substrate and a microporous membrane to provide a filtrate. This is done, for example, by providing a filtration device comprising one or more layers of copolymer-grafted nonwoven substrate and one or more layers of microporous membrane positioned such that at least a portion of the (virus-containing) composition flows through both types of media. In preferred embodiments, at least one layer of microporous membrane is positioned downstream of at least one layer of copolymer-grafted nonwoven substrate, such that the (virus-containing) composition passes through a layer of copolymer-grafted nonwoven substrate and then subsequently through a layer of microporous membrane.

[0029] The microporous membrane is a porous polymeric substrate (such as sheet or film) comprising micropores with a mean flow pore size, as characterized by ASTM Standard Test Method No. F316-03, “Standard Test Methods for Pore Size Characteristics of Membrane Filters by Bubble Point and Mean Flow Pore Test,” of less than 10 micrometers. In one embodiment, the microporous membrane has a mean flow pore size of at least 0.1, 0.2, 0.5, 0.8, or even 1 micrometer; and at most 10, 5, or even 2 micrometers. The desired pore size may vary depending on the application. The microporous membrane can have a symmetric or asymmetric (e.g., gradient) distribution of pore size in the direction of fluid flow.

[0030] The microporous membrane may be formed from any suitable thermoplastic polymeric material. Suitable polymeric materials include, but are not limited to, polyolefins, poly(isoprenes), poly(butadienes), fluorinated polymers, chlorinated polymers, polyamides, polyimides, polyethers, poly(ether sulfones), poly(sulfones), poly(vinyl acetates), polyesters such as poly(lactic acid), copolymers of vinyl acetate, such as poly(ethylene)-co-poly(vinyl alcohol), poly(phosphazenes), poly(vinyl esters), poly(vinyl ethers), poly (vinyl alcohols), and polycarbonate s). [0031] Suitable polyolefins include, but are not limited to, poly (ethylene), poly(propylene), poly(l -butene), copolymers of ethylene and propylene, alpha olefin copolymers (such as copolymers of ethylene or propylene with 1-butene, 1-hexene, 1-octene, and 1-decene), poly(ethylene-co-l -butene) and poly(ethylene-co-l-butene-co- 1-hexene). Suitable fluorinated polymers include, but are not limited to, poly(vinyl fluoride), poly(vinylidene fluoride), copolymers of vinylidene fluoride (such as poly(vinylidene fluoride-co-hexafluoropropylene)), and copolymers of chlorotrifluoroethylene (such as poly(ethylene-co-chlorotrifluoroethylene).

[0032] Suitable polyamides include, but are not limited to, poly(iminoadipolyliminohexamethylene), poly(iminoadipolyliminodecamethylene), and polycaprolactam. Suitable polyimides include, but are not limited to, poly(pyromellitimide).

[0033] Suitable poly(ether sulfones) include, but are not limited to, poly(diphenylether sulfone) and poly(diphenylsulfoneco-diphenylene oxide sulfone).

[0034] Suitable copolymers of vinyl acetate include, but are not limited to, poly(ethylene-co-vinyl acetate) and such copolymers in which at least some of the acetate groups have been hydrolyzed to afford various poly(vinyl alcohols).

[0035] In one embodiment, the microporous membrane is a solvent-induced phase separation (SIPS) membrane. SIPS membranes are often made by preparing a homogeneous solution of a polymer in first solvent(s), casting the solution into desired shape, e.g. flat sheet or hollow fiber, contacting the cast solution with another second solvent that is a non-solvent for the polymer, but a solvent for the first solvent (i.e., the first solvent is miscible with the second solvent, but the polymer is not). Phase separation is induced by diffusion of the second solvent into the cast polymer solution and diffusion of the first solvent out of the polymer solution and into the second solvent, thus precipitating the polymer. The polymer-lean phase is removed and the polymer is dried to yield the porous structure. SIPS is also called Phase Inversion, or Diffusion-induced Phase Separation, or Nonsolvent-induced Phase Separation, such techniques are commonly known in the art. Microporous SIPS membranes are further disclosed in U.S. Pat. No. 6,056,529 (Meyering et al), U.S. Pat. No. 6,267,916 (Meyering et al), U.S. Pat. No. 6,413,070 (Meyering et al), U.S. Pat. No. 6,776,940 (Meyering et al), U.S. Pat. No. 3,876,738 (Marinacchio et al), U.S. Pat. No. 3,928,517 (Knight et al), U.S. Pat. No. 4,707,265 (Knight et al), and U.S. Pat. No. 5,458,782 (Hou et al).

[0036] In another embodiment, the microporous membrane is a thermally-induced phase separation (TIPS) membrane. TIPS membranes are often prepared by forming a homogenous solution of a thermoplastic material and a second material (such as a diluent), and optionally including a nucleating agent, by mixing at elevated temperatures in plastic compounding equipment, e.g., an extruder. The solution can be shaped by passing through an orifice plate or extrusion die, and upon cooling, the thermoplastic material crystallizes and phase separates from the second material. The crystallized thermoplastic material is often stretched. The second material is optionally removed either before or after stretching, leaving a porous polymeric structure. Microporous TIPS membranes are further disclosed in U.S. Pat. No. 4,529,256 (Shipman); U.S. Pat. No. 4,726,989 (Mrozinski); U.S. Pat. No. 4,867,881 (Kinzer); U.S. Pat. No. 5,120,594 (Mrozinski); U.S. Pat. No. 5,260,360 (Mrozinski); and U.S. Pat. No. 5,962,544 (Waller, Jr.). Some exemplary TIPS membranes comprise poly(vinylidene fluoride) (PVDF), polyolefins such as poly(ethylene) or poly(propylene), vinyl-containing polymers or copolymers such as ethylene-vinyl alcohol copolymers and butadiene-containing polymers or copolymers, and acrylate-containing polymers or copolymers. TIPS membranes comprising PVDF are further described in U.S. Pat. No. 7,338,692 (Smith et al).

[0037] The nonwoven substrate is a nonwoven web which may include nonwoven webs manufactured by any of the commonly known processes for producing nonwoven webs. As used herein, the term “nonwoven web” refers to a fabric that has a structure of individual fibers or filaments which are randomly and/or unidirectionally interlaid in a mat-like fashion. For example, the fibrous nonwoven web can be made by carded, air laid, wet laid, spunlaced, spunbonding, electrospinning or melt-blowing techniques, such as melt-spun or melt-blown, or combinations thereof. Spunbonded fibers are typically small diameter fibers that are formed by extruding molten thermoplastic polymer as filaments from a plurality of fine, usually circular capillaries of a spinneret with the diameter of the extruded fibers being rapidly reduced. Meltblown fibers are typically formed by extruding the molten thermoplastic material through a plurality of fine, usually circular, die capillaries as molten threads or filaments into a high velocity, usually heated gas (e.g. air) stream which attenuates the filaments of molten thermoplastic material to reduce their diameter. Thereafter, the meltblown fibers are carried by the high velocity gas stream and are deposited on a collecting surface to from a web of randomly disbursed meltblown fibers. Any of the non-woven webs may be made from a single type of fiber or two or more fibers that differ in the type of thermoplastic polymer and/or thickness. In select embodiments, the copolymer-grafted nonwoven substrate comprises a spunlaid, a hydroentangled, a meltblown, or an electrospun nonwoven substrate.

[0038] Staple fibers may also be present in the web. The presence of staple fibers generally provides a loftier, less dense web than a web of only melt blown microfibers. Preferably, no more than about 20 weight percent staple fibers are present, more preferably no more than about 10 weight percent. Such webs containing staple fiber are disclosed in U.S. 4,118,531 (Hauser).

[0039] The copolymer-grafted nonwoven substrate may optionally further comprise one or more layers of scrim. For example, either or both major surfaces may each optionally further comprise a scrim layer. The scrim, which is typically a woven or nonwoven reinforcement made from fibers, is included to provide strength to the nonwoven article. Suitable scrim materials include, but are not limited to, nylon, polyester, fiberglass, and the like. The average thickness of the scrim can vary. Typically, the average thickness of the scrim ranges from about 25 to about 100 micrometers, preferably about 25 to about 50 micrometers. The layer of the scrim may optionally be bonded to the nonwoven substrate. A variety of adhesive materials can be used to bond the scrim to the polymeric material. Alternatively, the scrim may be heat-bonded to the nonwoven. In some embodiments, the scrim layer may be grafted as described for the nonwoven substrate.

[0040] The fibers of the copolymer-grafted nonwoven substrate typically have an effective fiber diameter of from about 5 to 35 micrometers, 5 to 30 micrometers, 5 to 25 micrometers, 5 to 20 micrometers, or 5 to 15 micrometers. In some embodiments, the copolymer-grafted nonwoven substrate comprises a plurality of fibers having an average diameter of 1 micrometer or more, 2 micrometers or more, 3 micrometers or more, 4 micrometers or more, 5 micrometers or more, 6 micrometers or more, 7 micrometers or more; and 35 micrometers or less, 30 micrometers or less, 25 micrometers or less, 23 micrometers or less, 20 micrometers or less, 20 micrometers or less, 16 micrometers or less, 14 micrometers or less, 12 micrometers or less, 11 micrometers or less, 10 micrometers or less, 9 micrometers or less, or 8 micrometers or less. In some embodiments, the copolymer-grafted nonwoven substrate comprises a plurality of fibers having an average diameter of 1 micrometer to 12 micrometers, 1 micrometer to 16 micrometers, 1 micrometers to 20 micrometers, 1 micrometers to 25 micrometers, 1 micrometers to 30 micrometers, 1 micrometers to 35 micrometers.

[0041] The fibers of the nonwoven substrate, prior to grafting, typically have an effective fiber diameter of from about 0.5 to 18 micrometers.

[0042] For copolymer-grafted nonwoven substrates, the terms ‘fiber’ and ‘fiber diameter’ refer to fibers that have been functionalized with a grafted copolymer.

[0043] Effective fiber diameter (EFD) is the apparent diameter of the fibers (or diameter of the copolymer grafted fibers) in a nonwoven fibrous web based on an air permeation test in which air at 1 atmosphere and room temperature is passed at a face velocity of 5.3 cm/sec through a web sample of known thickness, and the corresponding pressure drop is measured. Based on the measured pressure drop, the effective fiber diameter is calculated set forth in Davies, C. N., “The Separation of Airborne Dust and Particles”, Institution of Mechanical Engineers, London, Proceedings IB, 1952.

[0044] The copolymer-grafted nonwoven substrate has a basis weight in the range of about 50 to 550 g/m², preferably about 100 to 550 g/m², and more preferably about 100 to 400 g/m². The average thickness of the copolymer-grafted nonwoven substrate is preferably 0.1 to 10 millimeters (mm), and more preferably 1 to 10 mm. The minimum tensile strength of the nonwoven web is about 4.0 Newtons/cm. It is generally recognized that the tensile strength of nonwovens is lower in the machine direction than in the cross-web direction due to better fiber bonding and entanglement in the latter.

[0045] Copolymer-grafted nonwoven web loft is measured by solidity, a parameter that defines the solids fraction in a volume of web. Lower solidity values are indicative of greater web loft. Useful copolymer-grafted nonwoven substrates have a solidity of less than 40%, less than 35%, less than 30%, or less than 25%. Useful copolymer-grafted nonwoven substrates have a solidity of greater than 5%, or greater than 5%. Useful copolymer-grafted nonwoven substrates have a solidity of 40% to 5% 35% to 5%, 30% to 5%, 25% to 5%, or 30% to 10%. Solidity is a unitless fraction typically represented by a: a = n (p_f x L_nOnwoven), where nif is the fiber mass per sample surface area (i.e. basis weight), where pf is the fiber density; and where L_nOnwoven is the nonwoven thickness When a nonwoven substrate contains mixtures of two or more kinds of fibers, the individual solidities are determined for each kind of fiber using the same L_nOnwoven and these individual solidities are added together to obtain the web's solidity, a. Liber density (pf) of copolymer grafted fibers can be determined as in Method A (described below). Fiber density of copolymer grafted fibers can also be determined by a modified version of Method A in which the substrate and copolymer component molar ratios are all obtained from solid state ¹³C NMR measurements and the molar ratios are converted to weight ratios.

[0046] The term “average pore size” (also known as average pore diameter) is related to the arithmetic median fiber diameter and web solidity and can be determined by the following formula: where D is the average pore size, df is arithmetic median fiber diameter, and a is the web solidity.

[0047] The copolymer-grafted nonwoven substrate has a mean pore size of 5-70 micrometers, 5- 60 micrometers, 5-50 micrometers, 5-40 micrometers, 10-60 micrometers, 10-50 micrometers, or 10-40 micrometers.

[0048] The nonwoven substrate, prior to grafting, preferably has a mean pore size of 1-50 micrometers, 5-50 micrometers, or 5-40 micrometers.

[0049] Further details on the manufacturing method of nonwoven webs of this invention may be found in Wente, Superfine Thermoplastic Fibers, 48 INDUS. ENG. CHEM. 1342(1956), or in Wente et al., Manufacture Of Superfine Organic Fibers, (Naval Research Laboratories Report No. 4364, 1954). Useful methods of preparing the nonwoven substrates are described in U.S. RE39,399 (Allen), U.S. 3,849,241 (Butin et al.), U.S. 7,374,416 (Cook et al.), U.S. 4,936,934 (Buehning), and U.S. 6,230,776 (Choi).

[0050] In some embodiments, the nonwoven substrate is calendared using a smooth roll that is nipped against another smooth roll. A calendared or compressed nonwoven web provides for a more uniform substrate and dimensional stability in later washing steps to remove unreacted monomers. Thus, in a preferred embodiment, the nonwoven substrates according to the present invention are thermally calendared with a smooth roll and a solid back-up roll (e.g., a metal, rubber, or cotton cloth covered metal). The nonwoven substrate may be calendered before or after grafting, and may be calendared with or without an additional nonwoven layer, or with or without an additional scrim layer. In a calendaring step, a pattern may be applied to one or both major surfaces using a pattern roll. The patterned imparted to the nonwoven substrate may be any pattern including, for example, intermittent lines, hexagonal cells, diamond cells, square cells, point bonds, patterned point bonds, crosshatched lines, or any other regular or irregular geometric pattern. In particular, it is desirable to impart point bonds to the fibers of the nonwoven matrix and the optional scrim layer to improve the structural integrity.

[0051] Bonding between the fibers of the nonwoven substrate and/or the fibers of an optional scrim layer may be desirable to provide a matrix of desired coherency, making the nonwoven web more easily handled. Bonding fibers themselves means adhering the fibers together firmly, so they generally do not separate when the web is subjected to normal handling. Bonding may be achieved, for example, using thermal bonding, adhesive bonding, powdered adhesive binder, hydroentangling, needlepunching, calendering, or a combination thereof. Conventional bonding techniques using heat and pressure applied in a point-bonding process or by smooth calendar rolls can be used. A useful bonding method is pattern bonding wherein heated calender rolls or ultrasonic bonding equipment are used to bond the fibers together, usually in a localized bond patern. Another technique for bonding fibers or the nonwoven, or to the optional scrim is disclosed in U.S. Patent Application Publication No. 2008/0038976 (Berrigan et al.).

[0052] During calendaring, it is desirable to closely control the temperature and the pressure of the smooth rolls. In general, the fibers are thermally fused at the points of contact without imparting undesirable characteristics to the nonwoven substrate such as forming a film or skin on the surface thereof. For example, when using nylon nonwoven substrates, it is preferred to maintain the temperature of the smooth roll between about 40° C and 100° C, more preferably between about 50° C and 75° C. In addition, the smooth roll should contact the fibrous web at a pressure of from about 10 kilogram-force/cm to about 50 kilogram -force/cm, more preferably from about 15 kilogram-force/cm to about 30 kilogram-force/cm. The average thickness of the calendared nonwoven substrate is preferably about 2/3 the thickness of the starting nonwoven.

[0053] Suitable polyolefins include, but are not limited to, polyethylene, polypropylene, poly(l- butene), copolymers of ethylene and propylene, alpha olefin copolymers (such as copolymers of ethylene or propylene with 1-butene, 1-hexene, 1-octene, and 1-decene), poly(ethylene-co-l- butene), poly( 1 -methylpentene) and poly(ethylene-co- 1 -butene-co- 1 -hexene) . Preferably the nonwoven substrate is a polypropylene.

[0054] The copolymer-grafted article comprises a grafted copolymer made up of interpolymerized monomer units of (i) a quaternary ammonium containing monomer or salt thereof; (ii) a hydrophilic amide containing monomer; and (iii) an epoxy containing monomer. Each of these monomer types are described below.

[0055] The quaternary ammonium containing monomers or salts thereof may include one or more readily polymerizable monomers with quaternary ammonium functional groups. These monomers often include a vinyl or (meth)acrylate or (meth)acrylamide group to participate in the interpolymerization reaction. Useful (meth)acrylates include, for example, trimethylaminoethylmethacrylate, trimethylaminoethylacrylate, triethylaminoethylmethacylate, triethylaminoethylacrylate, trimethylaminopropylmethacrylate, trimethylaminopropylacrylate . Exemplary (meth)acrylamides include, for example, 3 -(trimethylamino) propylmethacrylamide, 3- (triethylamino)propylmethacrylamide, 3 -(ethyldimethylamino)propyhnethacrylamide . Preferred quaternary salts of these (meth)acryloyl monomers include, but are not limited to, (meth)acrylamidoalkyltrimethylammonium salts (e.g., 3- methacrylamidopropyltrimethylammonium chloride (MAPTAC) and 3- acrylamidopropyltrimethylammonium chloride) and (meth)acryloxyalkyltrimethylammonium salts (e.g., 2-acryloxyethyltrimethylammonium chloride, 2-methacryloxyethyltrimethylammonium chloride, 3 -methacryloxy-2 -hydroxypropyltrimethylammonium chloride, 3-acryloxy-2- hydroxypropyltrimethylammonium chloride, and 2-acryloxyethyltrimethylammonium methyl sulfate).

[0056] The hydrophilic amide monomers include those having a water miscibility (water in monomer) of at least 1 wt. %, preferably at least 5 wt. % without reaching a cloud point. The hydrophilic amide monomer units may include (meth)acrylamides and N-vinyl amides of the general formulas la or lb:

la lb

[0057] where:

[0058] R¹ is -H or C1-C4 alkyl;

[0059] Each R² is an H, an alkyl or an aryl group,

[0060] R³ and R⁴ are alkyl groups, or may be taken together to form a 5 or 6-membered ring.

[0061] Useful amide monomers include, for example, n-vinyl caprolactam, N-vinyl acetamide, N- vinyl pyrrolidone (NVP), (meth)acrylamide, mono- or di-N-alkyl substituted acrylamide, and combinations thereof.

[0062] The epoxy containing monomers include readily polymerizable monomers with epoxide functional groups. The epoxy containing monomers often include a vinyl or (meth)acrylate or (meth)acrylamide group to participate in the interpolymerization reaction. The epoxy containing monomers include, for example, glycidyl methacrylate (GMA), thioglycidyl(meth)acrylate, 3-(2,3- epoxypropoxy) phenyl(meth)acrylate, 2-[4-(2,3-epoxypropoxyl)phenyl] -2- (4-(meth)acryloyloxy- phenyl)propane, 4-(2,3- epoxypropoxyl)cyclohexyl(meth)acrylate, 2,3- epoxy cyclohexyl (meth)acrylate, and 3, 4-epoxy cyclohexyl (meth)acrylate. It is believed that the epoxy groups of these monomers in the grafted copolymer may hydrolytically ring open to provide terminal, pendent diol groups on the copolymer. Thus, the original grafted hydrophobic epoxy group hydrolyzes to provide a hydrophilic diol group to the grafted copolymer. [0063] In some embodiments, the copolymer-grafted article comprises a nonwoven substrate, and a grafted copolymer comprising interpolymerized monomer units of 3- (methacrylamido)propyltrimethylammonium chloride (MAPTAC); (ii) N-vinyl pyrrolidone (NVP); and (iii) glycidyl methacrylate (GMA). In certain embodiments, the grafted copolymer comprises interpolymerized monomer units of (i) 10 to 50 parts by weight (or 20 to 40 parts by weight) of the MAPTAC; (ii) 10 to 80 parts by weight (or 30 to 60 parts by weight) of the NVP; and (iii) 10-40 parts by weight (or 15 to 35 parts by weight) of the GMA. In some embodiments, a weight of the grafted copolymer is 0. 1 to 5 times the weight of the nonwoven substrate.

[0064] As the polymer is non-crosslinked, the solution containing the monomer mixture contains no additional thermal- or free-radical crosslinking agents, e.g., no polyethylenically unsaturated monomers. With regard to the grafting monomers supra, the monomers that are grafted to the surface of the nonwoven substrates usually have either an acrylate or other non-acrylate polymerizable functional group for grafting by e-beam. Methacryloyl groups are preferred for grafting of the monomer to the nonwoven substrate surface (using the process described herein) due to the slower, more uniform reactivity and durability of such methacryloyl monomers to nonwovens that have been exposed to e-beam irradiation.

[0065] Functionalized substrates may be prepared using above-described monomers to provide a grafted polymer on the surface of a porous nonwoven base substrate. When the above-described grafting monomers are used, the monomers may be grafted onto the nonwoven base substrate in a single reaction step (i.e., exposure to an ionizing radiation) followed by imbibing with all grafting monomers present or in sequential reaction steps (i.e., a first exposure to ionizing radiation followed by imbibing with one or more grafting monomer, then a second exposure to an ionizing radiation and a second imbibing after the second exposure to the ionizing radiation). It will be further understood that the grafting process will yield a radical species on the surface of the nonwoven substrate. After imbibing with the monomer solution, polymerization will initiate with the formation of a radical on the monomer that may further polymerize with one of more additional monomers, resulting in a grafted polymers having these groups pendent from the polymer chain as simply illustrated below:

[0066] Substrate -(M^Epoxy)_b-(M^Amide)_c-(M^NR4+)_a

[0067] In the formula, the -(M^NR4+)_W- represents the residue of the grafted quaternary ammonium containing monomer or salt thereof having “a” polymerized monomer units where a is at least 1, the -(M^Epoxy)_b- represents the residue of the grafted epoxy containing monomer having “b” polymerized monomer units, where b is at least one, and -(M^Aimde)_c- represents the residue of the grafted hydrophilic amide containing monomer having “c” polymerized monomer units, where c is at least one. Subscripts a to c may alternatively represent the parts by weight of each monomer unit described supra. The monomers are shown in an arbitrary arrangement.

[0068] The process of preparing the copolymer-grafted nonwoven substrate comprises the steps of providing a nonwoven substrate, exposing the nonwoven substrate to electron beam (e-beam) radiation in an inert atmosphere, and subsequently contacting the exposed substrate with a solution or suspension comprising the grafting monomers to graft polymerize the monomers to the surface of the nonwoven substrate. In the first step the nonwoven substrate is exposed to ionizing radiation, such as e-beam radiation, in an inert atmosphere. Generally, the substrate is placed in a chamber purged of oxygen. Typically, the chamber comprises an inert atmosphere such as nitrogen, carbon dioxide, helium, argon, etc. with a minimal amount of oxygen (less than 100 ppm), which is known to inhibit free-radical polymerization. Doses delivered by the ionizing radiation source may happen in a single dose or may be in multiple doses which accumulate to the desired level. One or more layers of nonwoven substrates may be subjected to the ionizing radiation. Ionizing radiation may include gamma, electron-beam, x-ray and other forms of electromagnetic radiation.

[0069] Electron beam is one preferred method of grafting due to the ready availability of commercial sources. Electron beam generators are commercially available from a variety of sources, including the ESI “ELECTROCURE” EB SYSTEM from Energy Sciences, Inc. (Wilmington, MA), and the BROADBEAM EB PROCESSOR from PCT Engineered Systems, LLC (Davenport, IA). For any given piece of equipment and irradiation sample location, the dose delivered can be measured in accordance with ASTM/ISO 5127S entitled “Practice for Use of a Radiochromic Film Dosimetry System.” By altering source type, accelerating voltage, extraction grid voltage, irradiation time, and/or distance to the source, various dose levels can be obtained.

[0070] Generally, the irradiated substrate is contacted with the aqueous monomer solution or suspension subsequent to and not concurrent with, the irradiation step. Often, the irradiated nonwoven substrate is contacted with the monomer solution immediately after the irradiation step. Generally, when using e-beam the irradiated substrate is contacted within an hour, preferably within ten minutes. In this instance “contacted” means bringing the irradiated nonwoven substrate into contact with the monomer solution or suspension. It can also be described as the irradiated nonwoven substrate being saturated with monomer solution or suspension, or the irradiated nonwoven substrate being imbibed with monomer solution or suspension, or the irradiated nonwoven substrate being coated with monomer solution. The monomer solution may only partially fill the void volume of the nonwoven substrate, or much more solution can be contacted to the nonwoven substrate than is necessary to fully fill the void volume. It has been observed that if the substrate is irradiated by ionizing radiation in the presence of the grafting monomers, the grafting yield is lower, extractables are higher, and the biofiltration performance of the grafted nonwoven substrate is inferior to that article prepared by the instant method. Suitable methods of contacting include, but are not limited to, a spray coating, flood coating, knife coating, Meyer bar coating, dip coating, and gravure coating.

[0071] The imbibing solution remains in contact with the nonwoven substrate for a time sufficient for polymerization with the grafting monomers. When contacted with a solution of monomers, grafting reactions are mostly completed after 12 hours exposure; generally about 90+ percent complete. As a result, the nonwoven substrate comprises grafted polymers and/or copolymers attached to the interstitial and outer surfaces of the nonwoven substrate. Once the nonwoven substrate has been saturated for a desired period of time, the nonwoven substrate bearing grafted polymer may be removed from the inert atmosphere. Optionally, once the nonwoven substrate has been saturated for a desired period of time, the nonwoven substrate bearing grafted polymer may be rinsed to remove residual monomer, polymer and/or dried.

[0072] Further details regarding methods of manufacturing the copolymer-grafted nonwoven substrate are described in co-owned U.S. Patent No. 9,821,276 (Berrigan et al.).

[0073] In a second aspect, a separated product is provided. The separated (e.g., purified, clarified, etc.) product comprises a virus, such as AAV or Phi-X174 (e.g., in an amount of 50% or greater of the amount of the virus present in the composition prior to separation), produced by the method according to the first aspect described in detail above. Other suitable viruses contained in the separated product may include Lentivirus, T7, or Phi6. Preferably, the separated product exhibits a decrease in at least one of DNA or HCP from an initial composition. In some embodiments, the separated product exhibits a decrease of cells from an initial composition. Further, the separated product may have a turbidity decrease, such as by 90% or more of an initial turbidity, e.g., to below 100 NTU, 80 NTU, 60 NTU, 40 NTU, or below 20 NTU.

Exemplary Embodiments

[0074] In a first embodiment, the present disclosure provides a method of separating a virus from a composition. The method comprises a) obtaining a composition comprising a virus and at least one of DNA or HCP; b) passing at least a portion of the composition through a copolymer-grafted nonwoven substrate to provide a filtrate; and c) collecting the filtrate, the filtrate comprising 50% or greater of an amount of the virus present in the composition. The composition has a pH of 6 to 8.5 and a conductivity of 4 or more milliSiemens per centimeter (mS/cm). The grafted copolymer comprises interpolymerized monomer units of (i) a quaternary ammonium containing monomer or salt thereof; (ii) a hydrophilic amide containing monomer; and (iii) an epoxy containing monomer.

[0075] In a second embodiment, the present disclosure provides a method according to the first embodiment, wherein the filtrate comprises 60% or greater, 70% or greater, 80% or greater, 90% or greater, 95% or greater, 98% or greater, or 99% or greater, of the amount of the virus present in the composition.

[0076] In a third embodiment, the present disclosure provides a method according to the first embodiment or the second embodiment, wherein the virus is an AAV or Phi-X174.

[0077] In a fourth embodiment, the present disclosure provides a method according to the third embodiment, wherein the virus is AAV and the AAV comprises AAV1, AAV2, AAV3, AAV5, AAV6, AAV8, AAV9, or any combination thereof.

[0078] In a fifth embodiment, the present disclosure provides a method according to the third embodiment or the fourth embodiment, wherein the virus is a recombinant AAV.

[0079] In a sixth embodiment, the present disclosure provides a method according to any of the third through fifth embodiments, wherein the composition comprises a cell lysate.

[0080] In a seventh embodiment, the present disclosure provides a method according to any of the third through sixth embodiments, wherein the filtrate exhibits a 1 log reduction or more, a 2 log reduction or more, a 2.5 log reduction or more, or a 3 log reduction or more, of an amount of the DNA present in the composition.

[0081] In an eighth embodiment, the present disclosure provides a method according to any of the first through seventh embodiments, wherein the filtrate contains 40% or less, 30% or less, or 20% or less or 10% or less of an amount of the HCP present in the composition.

[0082] In a ninth embodiment, the present disclosure provides a method according to any of the first through eighth embodiments, wherein the grafted copolymer comprises interpolymerized monomer units of (i) 10 to 50 parts by weight of 3 -methacrylamidopropyltrimethylammonium chloride (MAPTAC); (ii) 10 to 80 parts by weight of N-vinyl pyrrolidone (NVP); and (iii) 10-40 parts by weight of glycidyl methacrylate (GMA).

[0083] In a tenth embodiment, the present disclosure provides a method according to any of the first through ninth embodiments, wherein the copolymer-grafted nonwoven substrate comprises a spunlaid, a hydroentangled, a meltblown, or an electrospun nonwoven substrate. [0084] In an eleventh embodiment, the present disclosure provides a method according to any of the first through tenth embodiments, wherein the copolymer-grafted nonwoven substrate comprises a plurality of fibers having an average diameter of 2 micrometers or more, 3 micrometers or more, 4 micrometers or more, 5 micrometers or more, 6 micrometers or more, 7 micrometers or more; and 16 or less, 14 or less, 12 micrometers or less, 11 micrometers or less, 10 micrometers or less, 9 micrometers or less, or 8 micrometers or less.

[0085] In a twelfth embodiment, the present disclosure provides a method according to any of the first through eleventh embodiments, wherein the copolymer-grafted nonwoven substrate comprises a plurality of fibers having an average diameter of 2 micrometers to 12 micrometers.

[0086] In a thirteenth embodiment, the present disclosure provides a method according any of the first through twelfth embodiments, wherein the composition comprises cells and cell debris.

[0087] In a fourteenth embodiment, the present disclosure provides a method according to any of the first through thirteenth embodiments, wherein at least a portion of the composition is passed through both a copolymer-grafted nonwoven substrate and a microporous membrane that are used in combination.

[0088] In a fifteenth embodiment, the present disclosure provides a method according to any of the first through fourteenth embodiments, wherein a weight of the grafted copolymer is 0. 1 to 5 times the weight of the nonwoven substrate.

[0089] In a sixteenth embodiment, the present disclosure provides a method according to any of the first through fifteenth embodiments, wherein the composition further comprises a buffer.

[0090] In a seventeenth embodiment, the present disclosure provides a method according to any of the first through sixteenth embodiments, wherein the composition comprises a cell lysate and the cell lysate is prepared using freeze thawing, microfluidization, or using a nonionic surfactant.

[0091] In an eighteenth embodiment, the present disclosure provides a method according to any of the first through seventeenth embodiments, wherein the amount of the virus is determined using enzyme-linked immunosorbent assay (ELISA) quantitative polymerase chain reaction (qPCR), or multi -angle dynamic light scattering (MA-DLS).

[0092] In a nineteenth embodiment, the present disclosure provides a method according to any of the first through eighteenth embodiments, wherein the copolymer-grafted nonwoven substrate has a thickness of 0.1 millimeters (mm) to 10 mm. [0093] In a twentieth embodiment, the present disclosure provides a separated product. The separated product comprises a virus produced according to the method of any of the first through nineteenth embodiments.

[0094] Advantages and embodiments of this invention are further illustrated by the following examples, but the particular materials and amounts thereof recited in these examples, as well as other conditions and details, should not be construed to unduly limit this invention. All parts and percentages are by weight unless otherwise indicated.

Examples

[0095] These examples are merely for illustrative purposes only and are not meant to be limiting on the scope of the appended claims. Unless otherwise noted or readily apparent from the context, all parts, percentages, ratios, etc., in the Examples and the rest of the specification are by weight.

[0096] Materials and Methods

[0097] Sodium chloride, tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), magnesium sulfate, magnesium chloride, polyethylene glycol 8000 (PEG 8000, average MW of 8000), ethylenediaminetetraacetic acid (EDTA), lysogeny agar, tryptone, yeast extract, pancreatic digest of casein, peptic digest of soybean meal, and agar were obtained from the Sigma-Aldrich Company (St. Louis, MO).

[0098] Enveloped Phi6 phage (DSM 21518) and its host strain Pseudomonas syringae strain (DSM 21482) were obtained from the DSMZ -German Collection of Microorganisms and Cell cultures GmbH (Braunschweig Germany). E. coli T7 phage (ATCC BAA-1025-B2) was obtained from ATCC (Manassas, VA). E. coli BL21, Dnase-1, Dnase-1 buffer and proteinase-K were obtained from New England Biolabs (Ipswich, MA). TRITON X-100 nonionic surfactant was obtained from Alfa Aesar (W ard Hill, MA), Polyethyleneimine (PEI) was obtained from Polysciences (Warrington, PA). T-flasks, FREESTYLE HEK293-F cells, FREESTYLE HEK293 media, QUANT-IT PICOGREEN dsDNA detection fluorescent assay kit and SYBR qPCR master mix were obtained from Thermo Fischer Scientific (Waltham, MA).

[0099] AAV2 expressing plasmids (pAAV-GFP, pAAV-RC2, pHelper plasmids), and virus serotypes AAV1, AAV2, AAV3 and AAV6 were obtained from Cell Bio Labs (San Diego, CA). Virus serotypes AAV5, AAV8 and AAV9 were obtained from Vector Biolabs (Malvern, PA). AAV2 ELISA kits were obtained from Progen (Wayne, PA). AAV ITR primers were obtained from IDT Technologies (Coralville, Iowa). HEK-293T adherent cells, LENTI-X Packaging Single Shot mixtures, high-titer one-step lentivirus packaging systems, and LENTI-X p24 Rapid Titer Assay kit were obtained from Takara Bio (Mountain View, CA). HEK host cell protein 3G ELISA kits were obtained from Cygnus Technologies (Southport, NC).

[00100] PEG/NaCl (5X) stock solution was prepared by dissolving 100 g PEG-8000 (100 g) and NaCl (75 g) in deionized water (400 mL) and then sterilizing the solution using an autoclave. Post sterilization, deionized water was added with stirring at room temperature to bring the final volume to 500 mL. The solution was mixed thoroughly by inversion and chilled in an ice bath (or in a refrigerator) for 2 hours.

[00101] Tryptic Soy Broth (TSB) agar was prepared by mixing pancreatic digest of casein (15 g), EDTA of soybean meal (5 g), sodium chloride (5 g), and agar (15 g) in deionized water (IL) at pH 7.3. The agar preparation was autoclaved (instrument setting of 121 °C for 15 minutes) prior to use.

[00102] Soft lysogeny agar plates were prepared by adding tryptone (10 g), yeast extract (5 g), sodium chloride (5 g), magnesium chloride (1 g), and agar (7.5 g) to deionized water (1 L). The resulting product was autoclaved (instrument setting of 121 °C for 15 minutes), separated into 50 mL aliquots, and stored as a solid at room temperature. Prior to use, the soft agar was melted using a micro wave oven.

[00103] Lysogeny agar plates were prepared by adding lysogeny agar powder (35 g) to deionized water (1 L), autoclaving the mixture (instrument setting of 121 °C for 15 minutes), and pouring 15 mL of the molten gel mixture into a plate.

[00104] Lysogeny media was prepared by mixing sodium chloride (10 g), tryptone (10 g), and yeast extract (5 g) in deionized water (1 L) at pH 7. The media was sterilized using an autoclave and then allowed to cool to room temperature.

[00105] Lentivirus Production

[00106] Lentiviral vectors were produced by using the high-titer one-step lentivirus packaging systems as per manufacturer’s protocols. HEK-293T adherent cells were used for transfection. Lentivirus was produced by reconstituting each aliquot of LENTI-X Packaging Single Shot mixtures with the lentiviral transfer vector plasmids in sterile water and the mixtures were added to the 293T adherent HEK cells in T flasks. Lentiviral vectors were harvested 48 hours after infection. Lor virus purification, cultures were centrifuged at 1200 rpm for 5 minutes to remove cell debris. [00107] Phi6 Phage Production

[00108] Enveloped Phi6 phage and its host strain Pseudomonas syringae strain were propagated in lysogeny media. Bacterial cultures were initiated by transferring a single colony from a streak plate into 10 mL of lysogeny media in a sterile 50 mL Erlenmeyer flask. The Pseudomonas syringae culture flasks were incubated with shaking (250 rpm) at 25 °C for 18 hours allowing bacteria to attain stationary phase (cell density of 1 x 10⁸ cells per mL). Phages from a single plaque that was grown in a separate soft lysogeny agar plate were transferred to the bacteria culture by using a disposable plastic loop. Bacterial cultures were continued to be grown until all the host bacteria cells were lysed (about 12 hours). The phages were then harvested from the liquid culture by centrifugation at 5000 rpm for 10 minutes to remove bacterial cell debris. The resulting supernatant containing the phage was precipitated by adding the 5X PEG/NaCl solution. The phage was then pelleted by microcentrifugation for 30 minutes at 13,000 g. Post centrifugation, the supernatant was removed and the pellet containing the phage was resuspended in appropriate buffers as described in each example or stored at 4 °C until used.

[00109] E. coli T7 Phage Production

[00110] E. coli T7 phage and its host strain E. coli BL21 were propagated in lysogeny media. Bacterial cultures were initiated by transferring a single colony from a lysogeny agar streak plate into 10 mL of lysogeny media in a sterile 50 mL Erlenmeyer flask. Culture flasks were incubated with shaking (250 rpm) at 37 °C for 10 hours for /:'. coli BL21 allowing bacteria to attain stationary phase (cell density of 1 x 10⁸ cells per mL). Phages from a single plaque that were grown in a separate soft agar plate were transferred to the bacteria culture by using a disposable plastic loop. Bacterial cultures were continued to be grown until all the host bacteria cells were lysed (3 hours). The phages were then harvested from the liquid culture by centrifugation at 5000 rpm for 10 minutes to remove bacterial cell debris. The resulting supernatant containing the phage was precipitated by adding the 5X PEG/NaCl solution. Phage was then pelleted by microcentrifugation for 30 minutes at 13,000 g. Post centrifugation, the supernatant was removed and the pellet containing the phage was resuspended in appropriate buffers or stored at 4 °C until used. [00111] AAV2 Production

[00112] Method 1.

[00113] Low passage FREESTYLE HEK293-F cells (cell density of 10⁵ cells/mL) were added to 30 mL of FREESTYLE HEK293 media and grown for 2-3 days at 37 °C with 5% CO2 and 110 rpm shaking. Post growth, the cell density of the cell culture was 10⁶ cells/mL.

[00114] Separately, PEI was dissolved at required concentrations (1 microgram/ million cells) into 5% volume/volume phosphate buffered saline (PBS, pH 7.4) (5% volume against total HEK culture volume) and incubated for 5 minutes. Next, AAV expressing plasmids, pAAV-GFP, pAAV-RC2, pHelper at the molar ratio of 1 : 1 : 1 were mixed together and then added to the PEI in PBS solution with mixing. Total plasmid amount was 1 microgram/million HEK cells. The PELplasmid mixture was incubated at room temperature until the solution turned cloudy (incubation for about 30 minutes). The PELplasmid mixture was then added to the HEK293-F cell culture and the cells were grown for 48-72 hours. The resulting culture was spun down at 3000 rpm for 5 minutes and the cell pellet was washed with PBS 2X buffer. The cell pellet was frozen at -80 °C until used. Cell pellets were then re-suspended in the FREESTYLE media and subjected to three freeze\thaw cycles. Each cycle consisted of 30 minutes at -80 °C and 30 minutes at 37 °C. Following the freeze\thaw cycles, samples were spun down at 5000 rpm for 5 minutes. The resulting supernatant was collected and stored at -80 °C until used.

[00115] Method 2.

[00116] Low passage FREESTYLE HEK293-F cells (cell density of 10⁵ cells/mL) were added to 30 mL of FREESTYLE HEK293 media and grown for 2-3 days at 37 °C with 5% CO2 and 110 rpm shaking. Post growth, the cell density of the cell culture was 10⁶ cells/mL. Separately, PEI was dissolved at required concentrations (1 microgram/ million cells) into 5% volume/volume phosphate buffered saline (PBS, pH 7.4) (5% volume against total HEK culture volume) and incubated for 5 minutes. Next, AAV expressing plasmids, pAAV-GFP, pAAV-RC2, pHelper at the molar ratio of 1 : 1 : 1 were mixed together and then added to the PEI in PBS solution with mixing. Total plasmid amount was 1 microgram/million HEK cells. The PEI: plasmid mixture was incubated at room temperature until the solution turned cloudy (incubation for about 30 minutes). The PELplasmid mixture was then added to the HEK293-F cell culture and the cells were grown for 48-72 hours. To the cell culture, Triton X-100 was added to achieve a final concentration of 0.15% and the culture was stirred for 30 minutes. [00117] Determination of T7 Phage Concentration in Assay Samples

[00118] Lysogeny agar was poured into plates and the plates were cooled until the agar solidified. Next, soft lysogeny agar (4 mL, maintained at 50 °C) containing 0.5 mL of E. coli BL21 culture (OD600 = 0.4-0.7) was poured on top of the solidified lysogeny agar plates. Each resulting agar plate was cooled to room temperature. Each phage sample (obtained from either a bacterial culture, a feed solution for purification or the filtrate from purification) was serial diluted (10-fold) in a 96 well plate starting with a set of wells each containing 90 microliters of 0.9% NaCl solution mixed with 10 microliters of the phage containing solution. From each serial dilution, 3 microliters of phage containing solution was spotted onto the top of the soft agar using a pipette. The inoculated plates were incubated overnight at 25 °C. Following incubation, the number of plaque forming units (pfu) were counted. The T7 phage concentration (pfii/mL) was calculated.

[00119] Determination of Phi6 Phage Concentration in Assay Samples

[00120] TSB agar was poured into plates and the plates were cooled till the agar solidified. Next, soft TSB agar (4 mL, maintained at 50 °C) containing 0.5 mL of Pseudomonas syringae culture (OD600 = 0.4-0.7) was poured on top of the solidified TSB agar plate. Each resulting agar plate was cooled to room temperature. Each phage sample (obtained from either a bacterial culture, a feed solution for purification or the filtrate from purification) was serial diluted (10-fold) in a 96 well plate starting with a set of wells each containing 90 microliters of 0.9% NaCl solution mixed with 10 microliters of the phage containing solution. From each serial dilution, 3 microliters of phage containing solution was spotted onto the top of the soft agar using a pipette. The inoculated plates were incubated overnight at 25 °C. Following incubation, the number of plaque forming units (pfu) were counted. The Phi6 phage concentration (pfu/mL) was calculated.

[00121] AAV Quantitation by qPCR

[00122] An aliquot (10 microliters) of a sample containing an AAV serotype was combined in a sample tube with Dnase-1 (10 microliters), Dnase-1 buffer (10 microliters) and water (70 microliters). The sample was incubated overnight at 37 °C. Following incubation, EDTA, pH 7.5 (0.5M, 1 microliter) was added to the tube and the sample was incubated at 75 °C for 10 minutes. Next, Proteinase-K (1 microliter) was added to the tube and the sample was incubated at 37 °C for 90 minutes followed by heat inactivation at 95 °C for 15 minutes. For each qPCR reaction, an aliquot of the treated sample (2.5 microliters) was mixed with AAV ITR primers (2.5 microliters), SYBR qPCR master mix (12.5 microliters), and water (7.5 microliters). Including a negative control and pAAV-GFP as standards, the qPCR plate was prepared and analyzed using an AriaMx Real-Time PCR Instrument with AriaMx software (Agilent Technologies, Santa Clara, CA). The thermal profile was 95 °C for 10 minutes and then 40 repeat cycles of 95 °C for 15 seconds followed by 60 °C for 1 minute.

[00123] Phi-X174 Phage Culture Preparation

[00124] Phi-X174 (ATCC 13706-B1) was obtained from ATCC. The phage culture was produced by growing a 1 liter culture of E. colt (ATCC 13706) in CRITERION Nutrient Broth (Hardy Diagnostics, Santa Maria, CA) plus 5% sodium chloride at 37 °C with shaking to an OD of 0.45. The culture was inoculated with 10¹⁰ plaque forming units (pfu) of Phi-X174 phage. The inoculated culture was grown for an additional 4 hours at 37 °C with shaking.

[00125] Preparation of Chinese Hamster Ovary (CHO) Cell Culture

[00126] Chinese hamster ovary (CHO) cells producing an IgG monoclonal antibody were cultured in suspension from frozen cell stock to a series of flask seeding cultures in a CO2 incubator, followed by a fed-batch culture process using a Wave bioreactor (GE Healthcare, Chicago, IL) and a 50 L disposable cell bag (with pH control and dissolved oxygen monitoring). HYCLONE PF- CHO LS cell culture media (obtained from Cytiva, Marlborough, MA) was used. CHO cell cultures were harvested during stationary phase, typically on day 12. The cell culture was fdtered through a 3M ZETA PLUS 10SP02A depth fdter (obtained from the 3M Company, Maplewood, MN) followed by fdtration through a 0.2 micron PES bottle top fdter (obtained from Thermo Fisher Scientific).

[00127] Determination of Phi -XI 74 Phage Concentration in Assay Samples

[00128] Phi-X174 phage samples were serially diluted (10-fold). Top agar (nutrient broth with 0.9% agar, 2.5 mb) was mixed with 50 microliters of E. coli (ATCC 13706) overnight culture and 100 microliters of diluted Phi-X174 phage. The mixture was poured on top of a standard nutrient agar plate (nutrient broth with 1.5% agar) and incubated for 3-4 hours at 37 °C. Following incubation, the plaque-forming units (pfu) were counted. The phage concentration (pfu/mL) was calculated.

[00129] Grafting Solutions [00130] Table 1. Monomers for Grafting Solutions

[00131] Grafting Solution A was prepared as a monomer solution containing 24.4% NVP, 8.8% GMA, and 19.4% MAPTAC all by weight in deionized water.

[00132] Grafting Solution B was prepared as a monomer solution containing 18.3% NVP, 6.6% GMA, and 14.6% MAPTAC all by weight in deionized water.

[00133] Grafting Solution C was prepared as a monomer solution containing 12.2% NVP, 4.4% GMA, and 9.7% MAPTAC all by weight in deionized water.

[00134] Grafting Solution D was prepared as a monomer solution containing 9.15% NVP, 3.3% GMA, and 7.3% MAPTAC all by weight in deionized water. [00135] Grafting Solution E was prepared as a monomer solution containing 6. 1% NVP, 2.2%

GMA, and 4.85% MAPTAC all by weight in deionized water.

[00136] Method A, Determination of Basis Weight, Effective Fiber Diameter (EFD), Solidity, and Pore Size of Functionalized Nonwovens [00137] Basis weight, EFD, solidity, and pore size measurements of the functionalized (i.e., copolymer-grafted) nonwovens were determined according to the following procedure. Sample discs (13.33 cm diameter) were punched from functionalized nonwoven sheets and then individually rinsed with sufficient deionized water to remove residuals (e.g. unreacted monomers and glycerin) from the discs. Each rinsed disc was dried in an oven at 70 °C for at least 4 hours. During the drying step, a weight (about 100 g) was placed on top of each disc to prevent edge curling. The resulting dried functionalized nonwoven samples were characterized (basis weight, EFD, solidity, pore size) according to the methods and equations described above. For each measurement or calculation, the results were reported as the mean value of three independent trials (n=3) with the calculated standard deviation (SD).

[00138] For the solidity (a) equation, the fiber density (pf) measurement was determined as the sum of the density of the polypropylene substrate (0.91 g/cm³) and the density of the grafted copolymer (1.07 g/cm³) adjusted by the weight ratio of the polypropylene substrate and grafted copolymer of the test sample (Equation 1). The polypropylene substrate and copolymer weight ratios were determined by comparing the basis weight of the nonwoven before the grafting step to the basis weight of the corresponding dried copolymer-grafted nonwoven.

[00139] The density of the grafted copolymer (DGCP) was determined by first using solid state ¹³C NMR (ssNMR) to measure the mol% of monomer components (NVP, MAPTAC, GMA) of the grafted copolymer; converting the mol% values to weight% (wt.%) values. The density value of each monomer component (monomer densities: DNVP = 1.04 g/cm³, DMAPTAC = 1.067 g/cm³, DGMA = 1.07 g/cm³) was adjusted (multiplied) by the corresponding component wt.% value, and the resulting three adjusted density values were summed (Equation 2).

[00140] Equation 1:

[00141] Fiber density (pf) = (0.91 X wt.% polypropylene) + (1.05 X wt.% grafted copolymer)

[00142] Equation 2:

[00143] DGCP = (DNVP X Wt.%NVp) + (DMAPTAC X Wt.%MAPTAc) + (DGMA x Wt.%GMA)

[00144] Preparation of Functionalized Nonwoven A (FNW-A)

[00145] A non-functionalized, melt-blown polypropylene nonwoven web (having an effective fiber diameter of 4.2 micrometers, basis weight of 100 grams per square meter (gsm), solidity of 8.2%, calculated average pore size of 14.2 micrometers) was grafted with nitrogen purged Grafting Solution C. The nonwoven substrate was unwound and passed through an electron beam (Electrocure, from Energy Science, Inc, Wilmington, MA) set to a potential of 300 kV and to deliver a total dose of 7 Mrad. The environment in the electron beam chamber was purged with nitrogen. The web was then conveyed directly into a nitrogen-purged saturation step with the monomer solution. The web was then wound up within the purged atmosphere. The web was left in the purged atmosphere for a minimum of 60 minutes, after which it was exposed to air. The web was then unwound and conveyed into a tank of deionized water for about 8 minutes at a speed of 10 feet per minute. After exiting the tank, the web was flushed multiple times by passing an aqueous salt solution (NaCl) through the web using a vacuum belt. A small amount of glycerin was added to the aqueous salt solution in the final flushing step. The unwound web was dried until the moisture content of the web was less than 14% by mass. The web was then wound up on a spindle. The copolymer-grafted nonwoven article was labeled as Functionalized Nonwoven A (FNW-A). The effective fiber diameter (EFD), basis weight, solidity, and calculated average pore size of FNW-A were determined by Method A and are reported in Table 2. Discs of Functionalized Nonwoven A (2.54 cm diameter) were punched from the web.

[00146] Table 2. Properties of Functionalized Nonwovens A

[00147] Preparation of Functionalized Nonwoven B (FNW-B)

[00148] A non-functionalized, melt-blown polypropylene nonwoven web was prepared having an effective fiber diameter (EFD) of 2.2 micrometers, basis weight of 100 gsm, and solidity of 10%. The nonwoven web was prepared according to the general procedure described in Example 12 of United States Patent Application US20200115833 (Joseph), with the following settings. The extruder temperature was kept at 340 °C and delivered the melt stream to the BMF die (130 cm wide) maintained at 325 °C. The gear pump was adjusted so that a rate of 0.23 Kg/hour/cm (30 Kg/hour) (co)polymer throughput was maintained at the die. The primary air temperature of the air knives adjacent to the die orifices was maintained at approximately 360 °C and the web was collected a die collector distance of about 20 cm.

[00149] A sample of the nonwoven (21.6 cm by 21.6 cm) was placed in a glove box and purged of air under a nitrogen atmosphere. Once the oxygen levels reached <20 ppm, the nonwoven substrate was inserted into a plastic bag and sealed. [00150] Grafting solution E (150 grams) was added to a glass jar. The jar was capped and shaken by hand to mix the contents. The jar was then opened and the solution was sparged with nitrogen for 2 minutes to remove any dissolved oxygen from the solution. The jar was re-capped and transferred into the oxygen depleted glovebox. The jar lid was then removed to flush any residual air from the jar headspace.

[00151] The sealed bag containing nonwoven sample was removed from the glove box and irradiated to a dose level of 40 kGy by passing through a CB-300 electron beam apparatus (Energy Sciences, Inc., Wilmington, MA) in a single pass operation at a speed of approximately 5.5 meters per minute and an accelerating voltage of 300 kV. The bag containing the irradiated nonwoven sample was then returned to the glove box and purged of air as described above.

[00152] The monomer grafting solution was added to the plastic bag containing the nonwoven sample. The bag was sealed and the solution was distributed through the nonwoven sample using a hand roller so that the nonwoven sheet was uniformly covered with the solution. The bag was sealed and the nonwoven sample was maintained flat in the bag for 3 hours. The resulting copolymer-grafted nonwoven sample was removed from the bag and boiled in deionized water for one hour. The sample was removed from the water bath and air dried at room temperature for 24 hours. The copolymer-grafted nonwoven article was labeled as Functionalized Nonwoven B (FNW-B). Discs (25 mm in diameter) were punched from the dried sample.

[00153] Preparation of Functionalized Nonwoven C (FNW-C)

[00154] The same procedure as described for FNW-B was followed with the exception that Grafting Solution C was used, instead of Grafting Solution E.

[00155] Preparation of Functionalized Nonwoven D (FNW-D)

[00156] The same procedure as described for FNW-B was followed with the exception that Grafting Solution B was used, instead of Grafting Solution E.

[00157] Preparation of Functionalized Nonwoven E (FNW-E)

[00158] The same procedure as described for FNW-B was followed with the exception that Grafting Solution A was used, instead of Grafting Solution E. [00159] Reference Example 1. Purification of Lentivims using FNW-A (pH = 7.4)

[00160] Five separate buffer solutions of 20mM Tris-HCl, pH 7.4 with conductivities of 4.3, 9, 11, 15.8, or 19.3 mS/cm were prepared from IM Tris-HCl stock solutions (diluting with deionized water). Each buffer was adjusted for pH with concentrated HC1 and conductivity with 5M NaCl stock solution. The pH was measured using a VWR SYMPHONY benchtop pH meter (VWR International, Radnor, PA). Conductivity was measured using an Accumet Excel XL50 conductivity meter (Fisher Scientific, Hampton, NH). Five feed solutions were prepared by spiking each buffer solution with 1.5 x 10⁷ lentivirus particles/mL.

[00161] Circular discs (8 mm diameter) were punched from the functionalized nonwoven A (FNW-A). A single disc was inserted into each well of a 96-well EMPORE Filter Plate (Model 6015, 3M Corporation, St. Paul, MN) in which the original solid phase extraction material had been previously removed. The filter element discs were held in place with a plastic O-ring. The filter plate was mated with a 96 deep-well collection plate.

[00162] For each buffer solution, three wells in the plate were selected and the discs in each well were washed with 2-8 mb of the selected buffer solution. Feed solution (500 microliters) was added to each well and the assembly was spun at 47 ref (relative centrifugal force) for 5 minutes so that the feed solution flowed through the non-woven disc into the filtrate collection area of the well. The feed solution added to each well was matched to have the same conductivity as the wash solution. Next, the disc in each well was washed using 500 microliters of fresh buffer solution and spinning the plate at 47 ref for 5 minutes. The combined filtrate from each well was analyzed for lentivirus content using a Lenti-X p24 Rapid Titer Assay kit (Takara) according to the manufacturer instructions. The lentivirus content of filtrates is reported in Table 3 as the mean (n = 3) percent of lentivirus recovered from the feed solution.

[00163] Table 3. Percent of Lentivirus recovered from Feed Solution after Filtration (Feed Solution pH = 7.4)

[00164] Reference Example 2, Purification of Lentivirus using FNW -A (pH = 8.0)

[00165] The same procedure and feed solution as described in Reference Example 1 was followed with the exception that buffer and corresponding feed solutions were prepared to have a pH of 8.0 and a conductivity of either 1, 5.2, 9, 10.5, 14, or 17.3 mS/cm. Each feed solution was tested in two wells and the percent of lentivirus recovered from the feed solution for each test well is reported in Table 4.

[00166] Table 4. Lentivirus Content of Filtrate (Feed Solution pH = 8.0)

[00167] Reference Example 3, Purification of Phi6 Phage using FNW-A (pH 7.4) [00168] The same procedure as described in Reference Example 1 was followed with the exception that the feed solutions were prepared by spiking each buffer solution with 2.5 x 10⁶ Phi6 pfii/mL (instead of lentivirus particles). The combined filtrate from each well was analyzed for Phi6 phage titer according to the method described above. Each feed solution was tested in two wells and the measured Phi6 phage content (pfu/mL) of the fdtrate from each test well is reported in Table 5.

[00169] Table 5: Phi6 Phage Content of Filtrate (Feed Solution pH = 7.4)

[00170] Reference Example 4. Purification of Phi6 Phages using FNW-A (pH = 8.0)

[00171] The same procedure and feed solution as described in Reference Example 3 was followed with the exception that buffer and corresponding feed solutions were prepared to have 2.5 x 10⁶ Phi6 pfu/mL, a pH of 8.0 and a conductivity of either 1, 5.2, 9, 10.5, 14, or 17.3 mS/cm. Each feed solution was tested in two wells and the measured Phi6 phage content (pfu/mL) of the filtrate from each test well is reported in Table 6.

[00172] Table 6. Phi6 Phage Content of Filtrate (Feed Solution pH = 8.0)

[00173] Reference Example 5, Purification of T7 Phage using FNW-A (pH = 8.5)

[00174] The same procedure as described in Reference Example 1 was followed with the exception that different buffer and feed solutions were used. Eight buffer solutions of 50mM Tris-HCl, pH 8.5 with conductivities of 0.9, 5, 10, 15, 20, 25, 30, or 85 mS/cm were prepared from IM Tris-HCl stock solutions (diluting with deionized water). Each buffer was adjusted for pH with concentrated HC1 and conductivity with 5M NaCl stock solution. Feed solutions were prepared by spiking each buffer solution with 4 x 10⁹ T7 pfu/mL.

[00175] The combined filtrate from each well was analyzed for T7 phage content according to the method described above. Each feed solution was tested in three wells and the measured T7 phage content (pfu/mL) of the filtrate from each test well is reported in Table 7.

[00176] Table 7. T7 Phage Content of Filtrate (Feed Solution pH = 8.5)

[001771 Reference Example 6, Purification of AAV2 using FNW-A (pH 6,7, and 8)

[00178] The same procedure as described in Reference Example 1 was followed with the exception that different buffer and feed solutions were used. Buffer solutions of 50mM Tris-HCl were prepared to have a pH of either 6, 7, or 8 and a conductivity of either 4.8, 9.6, 15, 21, or 46 mS/cm. Each buffer was adjusted for pH with concentrated HC1 and conductivity with 5M NaCl stock solution. The feed solutions were prepared by spiking each buffer solution with 2 x 10⁹ AAV2 particles/mL. The feed solution added to each well was matched to have the same pH and conductivity as the wash solution. The combined filtrate from each well was analyzed for AAV2 content using an AAV2 ELISA kit according to the manufacturer instructions. Each feed solution was tested in three wells and the measured AAV2 content (particle s/mL) of the fdtrate from each test well is reported in Tables 8-10. [00179] Table 8. AAV2 Content of Filtrate (Feed Solution pH = 6)

[00180] Table 9. AAV2 Content of Filtrate (Feed Solution pH = 7)

[00181] Table 10. AAV2 Content of Filtrate (Feed Solution pH = 8)

[00182] Reference Example 7, Purification of AAV2 using FNW-A (pH = 8.5)

[00183] The same procedure as described in Reference Example 6 was followed with the exception that buffer and corresponding feed solutions were prepared to have a pH of 8.5 and a conductivity of either 0.9, 3, 5.2, 7.5, 10.5,12.5, 15.3, 17.5, 20.4, or 22.5 mS/cm. The feed solutions were prepared by spiking each buffer solution with 2 x 10⁹ AAV2 particles/mL. Each feed solution was tested in two wells and the measured AAV2 content (particles/mL) of the filtrate from each test well is reported in Table 11.

[00184] Table 11. AAV2 Content of Filtrate (Feed Solution pH = 8.5)

[00185] Reference Example 8, Purification of Different AAV Serotypes using FNW-A (pH 7.2)

[00186] The same procedure as described in Reference Example 1 was followed with the exception that different buffer and feed solutions were used. Two separate buffer solutions of 50mM Tris- HC1, pH 7.2 with conductivities of either 10 or 15 mS/cm were prepared from IM Tris-HCl stock solutions (diluting with deionized water). Each buffer was adjusted for pH with concentrated HC1 and conductivity with 5M NaCl stock solution. The feed solutions were prepared by spiking each buffer solution with a single AAV serotype (selected from serotypes 1-3, 5, 6, 8, and 9) at a concentration of AAV genome copies/mL (gc/mL) reported in Table 12. The combined filtrate from each well was analyzed for AAV content using qPCR. Each feed solution was tested in two wells and the measured AAV content (gc/mL) of the fdtrate from each test well is reported in Table 12.

[00187] Table 12. AAV Content of Filtrate (Feed Solution pH = 7.2, Conductivity = 10 or 15 mS/cm)

[00188] Reference Example 9,

[00189] Circular discs (8 mm diameter) were punched from the functionalized nonwovens A-E. A 96-well EMPORE Filter Plate (Model 6015, 3M Corporation,) was modified as described in Example 1 and discs were inserted in the wells with each well having a single disc selected from functionalized nonwovens A-E. The discs were held in place with a plastic O-ring. The filter plate was mated with a 96 deep-well collection plate.

[00190] For each buffer solution, three wells in the plate were selected and the discs in each well were washed with 8 mb of FREESTYLE HEK293 media (pH = 7.3, conductivity = 13.5). The feed solution was FREESTYLE HEK293 media spiked with 2.2 x 10⁹ AAV2 particles/mL. Feed solution (500 microliters) was added to each well and the assembly was spun at 47 ref (relative centrifugal force) for 2 minutes so that the feed solution flowed through the non-woven disc into the fdtrate collection area of the well. The feed solution added to each well was matched to have the same conductivity as the wash solution. Next, the disc in each well was washed using 500 microliters of fresh buffer solution and spinning the plate at 47 ref for 5 minutes. The combined fdtrate from each well was analyzed for AAV2 content using an AAV2 ELISA kit according to the manufacturer instructions. Each functionalized nonwoven (FNW) was tested in three wells and the measured AAV2 content (particles/mL) of the filtrate from each test well is reported in Table 13.

[00191] Table 13. AAV2 Content of Filtrate (FNWs A-E)

[00192] Example 1. Clarification of a Harvested Cell Culture Fluid (HCCF)

[00193] Filter capsules were tested for HCCF clarification with a PendoTech normal flow filtration system (PendoTech Company) connected to the capsule via the Luer lock inlet of the capsule. The plastic filter capsule had an upper housing and a lower housing that were mated together in the final construction by ultrasonic welding. The upper housing had a Luer lock fluid inlet port and a Luer lock vent. The lower housing had a Luer lock fluid outlet port centered in the middle of the lower housing. A disc (2.54 cm diameter) of polypropylene spunbond nonwoven (9 mil thick) was placed in the bottom of the lower housing. A disc (2.54 cm diameter) of a polyethersulfone membrane with a 0.2 micrometer nominal pore size was placed on top of the nonwoven layer. The nonwoven and membrane layers were ultrasonically welded at the margins to the bottom inner surface of the lower housing. A stack of 4 discs of FNW-A (2.54 cm diameter) was then placed on top of the membrane. The upper and lower housings were mated together and ultrasonically welded to form a finished filter capsule. The ultrasonic welding was accomplished by placing the mated assembly in a jig so that the outer surface of the lower housing came into contact with the ultrasonic horn. A Branson 20 kHz Ultrasonic welder (Model 2000xdt, Emerson Electric Company, St. Louis, MO), a black booster, and a horn with a gain of 2.5x was used. An air pressure of 80 psi, down speed of 10%, stepped amplitude of 80%-60%, stepped at 50 Joules, weld time of 2 second, and a trigger force of 200 Ibf to start welding were set as fixed parameters. The weld energy was held constant at 450 Joules to produce samples with consistent compression levels. The housing assembly was positioned below the horn so that housing longitudinal axis was aligned to the axis of the ultrasonic horn. When the welding process was initiated, the horn came down on the lower housing compressing the housing and the internal components until a force of 200 Ibf was reached. The overall outer diameter of the finished capsule was about 3.7 cm and the overall height including inlet, outlet, and vent ports was about 4.8 cm. The frontal surface area for the disc stack was 3.2 cm2.

[00194] The HCCF consisted of human embryonic kidney 293 cells (HEK 293 cells) grown in FREESTYLE 293 media at 37 °C with 5% carbon dioxide. The HEK 293 culture was supplemented with HYCLONE Cell Boost 5 (Cytiva, Marlborough, MA) (2.5 weight% prepared in water and adjusted to pH 7.0) and added 2.5% vol/vol to the FREESTYLE media on day 1 and day 3 of cell culture. On day 5, the cells were harvested and used for experiments. The cell density of the HCCF was determined to be 4.4 x 10⁶ cells/mL using a hemacytometer. The viability of the HCCF was determined to be 98% using trypan blue staining (0.4% trypan blue solution, Thermo Fisher Scientific). Triton X-100 solution (5% stock solution) was added to the HCCF to obtain a final concentration of 0.5% Triton X-100 in the HCCF. Next, the HCCF was spiked with AAV2 and mixed for 30 minutes. The spiked HCCF had an AAV2 concentration of 8.6 x 10⁸ (± 5.0 x 10⁷) particles/mL; HEK 293 host cell protein (HCP) concentration of 381.8 (± 6.7) micrograms/mL; DNA concentration of 3878.6 (± 24) nanograms/mL; pH of 7.3; and conductivity of 13 mS/cm. HCP concentration was determined using HEK 293 Host Cell Protein ELISA kits, 3G (Cygnus Technologies, Southport, NC). DNA concentration was determined using QUANT-IT PICOGREEN dsDNA assay kits (Thermo Fisher Scientific). AAV2 concentration was determined using AAV2 ELISA kits (Progen).

[00195] The AAV2 spiked HCCF was filtered using the filter capsules. For each filtration, the filter capsule headspace was filled with AAV2 spiked HCCF (0.75 mL/minute) by opening the vent and closing the outlet. After the headspace of the capsule was filled, the vent was closed and the outlet was opened to allow for collection of clarified cell culture fluid (CCCF) filtrate. The concentrations of HCP, DNA, and AAV2 in the recovered filtrate are reported in Table 14 as the mean values from two separate filtration experiments with standard deviation (n=2).

[00196] Table 14. HCP, DNA, and AAV2 Concentrations Before and After Filtration by Example 1

[00197] Example 2, Purification of a Phi-X174 Phage Culture

[00198] A plastic filtration capsule was used. The capsule consisted of a sealed, circular housing. The capsule housing was prepared from two halves (upper and lower halves) which were mated and sealed together at the perimeter after the filtration elements were inserted in the internal cavity of the lower housing. Fluid inlet and vent ports were located on the upper portion of the housing and a fluid outlet port was located on the lower portion of the housing. The outlet port was centered in the middle of the lower housing surface. The internal cavity of the filter housing contained a nylon membrane (16 mm diameter) placed in the bottom of the housing and positioned directly over the opening to the outlet port. A stack of 4 discs of FNW -A ( 16 mm diameter) was placed on top of the membrane with a polypropylene ring separating the 2^nd and 3^rd nonwoven layers in the stack. The overall outer diameter of the finished capsule was about 4.3 cm and the overall height including inlet, outlet, and vent ports was about 4.8 cm. The effective filtration area of the capsule was 1.0 cm2 and the bed volume of the nonwoven media was 0.4 mb.

[00199] A Phi-X174 phage culture was prepared as described in the Methods Section. Cells were removed from the culture by centrifuging the culture at 3700 x g and filtering the supernatant through a 0.2 micron PES (polyethersulfone) membrane filter. The resulting filtered culture had a Phi-X174 phage concentration of 1 x 10⁹ pfu/mL and was adjusted to have a pH of 7, and a conductivity of 10 mS/cm.

[00200] The filter capsule was flushed with 7 mb of tris buffer (pH 8, 20 mS/cm) and then 15 mb of the phage culture was filtered through the capsule (from inlet port to outlet port) at 1 mL/minute using a MASSTERFLEX L/S series peristaltic pump (Cole-Parmer, Vernon Hills, IL). The filtrate was collected and analyzed for virus recovery using the Phi-X174 plaque assay. Total protein concentration before and after filtration was measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. DNA was not measured due to interference of the nutrient broth medium with the DNA assay. A virus recovery of 94% was achieved, with a 26% reduction in total protein. The results are summarized in Table 15.

[00201] Table 15. Phage and Protein Content of a Phi-X174 Phage Culture Measured Before and After Filtration thru FNW-A

[00202] Example 3, Purification of Phi-X174 Phage spiked CHO cell culture

[00203] The same type of filter capsule as described in Example 2 was used with the difference being in capsule dimensions. The overall outer diameter of the finished capsule was about 4.3 cm and the overall height including inlet, outlet, and vent ports was about 5.9 cm. The nylon membrane disc and FNW-A discs were all 27 mm in diameter. The effective filtration area of the capsule was 4.0 cm² and the bed volume of the nonwoven material was 1.4 mb.

[00204] The CHO cell culture was prepared as described in the Methods Section and the culture was adjusted to a pH of 7.1 and a conductivity of 15 mS/cm. A Phi-X174 phage culture was prepared as described in Example 2. A 10 mb aliquot of the Phi-X174 culture was spiked into 90 mb of the CHO cell culture resulting in a Phi-X174 phage concentration of 4.6 x 10⁹ pfu/mL.

[00205] The filter cartridge was flushed with 15 mb of 25 mM tris buffer (pH 7.1, 15 mS/cm) and then 100 mb of the phage spiked culture was filtered through the capsule (from inlet port to outlet port) at 3.5 mb/minute using a MASSTERFEEX E/S series peristaltic pump (Cole-Parmer, Vernon Hills, IE). The filtrate was collected and analyzed for virus recovery using the Phi-X174 plaque assay. DNA concentration of the culture before and after filtration was measured using the QUANT-IT PICOGREEN assay according to the manufacturer’s instructions. A virus recovery of 98% was achieved, with a 96% reduction in DNA. The results are summarized in Table 16. [00206] Table 16. Phage and DNA Content of a Phi-X174 Phage Culture Measured Before and After Filtration thru FNW-A

[00207] Example 4, Clarification of a Phi-X174 Phage Culture

[00208] A Phi-X174 culture was prepared as described in the Methods Section. Three filtration capsules were prepared as described in Example 3. The capsules were flushed with 20 mb of 50 mM tris buffer (pH 7.5, 10 mS/cm) at 1 mL/minute using a PendoTech normal flow filtration system (PendoTech Company). The buffer in the headspace was removed by pouring the solution out through the air vent. The capsules were refilled with the Phi-X174 culture to avoid dilution with extra buffer. The culture was pumped through the capsules at 1 mL/minute until a differential pressure of 15 psi was reached, and throughput was calculated using the volume filtered at 15 psi.

[00209] The turbidity of the culture before and after filtration with the filtration capsule was measured using an Orion AQ4500 turbidity meter (Thermo Fisher Scientific) in Nephelometric Turbidity Units (NTU). The turbidity of the Phi-X174 culture before filtration through the filtration capsule was 116 NTU.

[00210] Following the turbidity measurement, the phage titer of the filtrate from each filtration capsule was determined by first filtering each filtrate sample through a 4.9 cm² 0.2 micron PES syringe filter (Pall Corporation, Port Washington, NY) using a syringe and then analyzing the resulting filtrate from the syringe filter using the Phi-X174 plaque assay described in the Methods Section.

[00211] To determine phage recovery, the phage titer of filtered samples from the capsule was compared to the phage titer of a 43 mb sample of the starting phage culture that was filtered through a 26.5 cm² 0.2 micron PES filter unit (Fisherbrand, from Thermo Fisher Scientific ) using vacuum filtration.

[00212] Filtration of the Phi-X174 phage culture through the filtration capsule showed a 111.6 ± 20.9% phage recovery (n=3) and a 97.0 ± 0.17% reduction in turbidity. [00213] The results for turbidity of the culture before and after filtration through a filtration capsule are reported in Table 17. The results for filter cartridge throughput are reported in Table 18. The phage concentration measurements are reported in Table 18. [00214] Table 17. Turbidity of Starting and Filtered Phi-X174 Phage Culture

[00215] Table 18.

n/a = not applicable

[00216] Foreseeable modifications and alterations of this invention will be apparent to those skilled in the art without departing from the scope and spirit of this invention. This invention should not be restricted to the embodiments that are set forth in this application for illustrative purposes.

Claims

What is claimed is:

1. A method of separating a virus from a composition, the method comprising: a) obtaining a composition comprising a virus and at least one of deoxyribonucleic acid (DNA) or host cell proteins (HCP), wherein the composition has a pH of 6 to 8.5 and a conductivity of 4 or more milliSiemens per centimeter (mS/cm); b) passing at least a portion of the composition through a copolymer-grafted nonwoven substrate to provide a fdtrate, wherein the grafted copolymer comprises interpolymerized monomer units of (i) a quaternary ammonium containing monomer or salt thereof; (ii) a hydrophilic amide containing monomer; and (iii) an epoxy containing monomer; and c) collecting the filtrate, the filtrate comprising 50% or greater of an amount of the virus present in the composition.

2. The method of claim 1, wherein the filtrate comprises 60% or greater, 70% or greater, 80% or greater, 90% or greater, 95% or greater, 98% or greater, or 99% or greater, of the amount of the virus present in the composition.

3. The method of claim 1 or claim 2, wherein the virus is an adeno-associated virus (AAV) or Phi-X174.

4. The method of claim 3, wherein the virus is AAV and the AAV comprises AAV1, AAV2, AAV3, AAV5, AAV6, AAV8, AAV9, or any combination thereof.

5. The method of claim 3 or claim 4, wherein the virus is a recombinant AAV.

6. The method of any of claims 3 to 5, wherein the composition comprises a cell lysate.

7. The method of any of claims 1 to 6, wherein the filtrate exhibits a 1 log reduction or more, a 2 log reduction or more, a 2.5 log reduction or more, or a 3 log reduction or more, of an amount of the DNA present in the composition.

8. The method of any of claims 1 to 7, wherein the filtrate contains 40% or less, 30% or less, 20% or less, or 10% or less of an amount of the HCP present in the composition.

9. The method of any of claims 1 to 8, wherein the (i) quaternary ammonium containing monomer or salt thereof comprises 3-methacrylamidopropyltrimethylammonium chloride (MAPTAC); wherein the (ii) hydrophilic amide containing monomer comprises N-vinyl

-42- pyrrolidone (NVP); and wherein the (iii) epoxy containing monomer comprises glycidyl methacrylate (GMA). The method of any of claims 1 to 9, wherein the copolymer-grafted nonwoven substrate comprises a plurality of fibers having an average diameter of 2 micrometers or more, 3 micrometers or more, 4 micrometers or more, 5 micrometers or more, 6 micrometers or more, 7 micrometers or more; and 16 micrometers or less, 14 micrometers or less, 12 micrometers or less, 11 micrometers or less, 10 micrometers or less, 9 micrometers or less, or 8 micrometers or less. The method of any of claims 1 to 10, wherein the copolymer-grafted nonwoven substrate comprises a plurality of fibers having an average diameter of 2 micrometers to 12 micrometers. The method of any of claims 1 to 11, wherein at least a portion of the composition is passed through both a copolymer-grafted nonwoven substrate and a microporous membrane that are used in combination. The method of any of claims 1 to 12, wherein a weight of the grafted copolymer is 0. 1 to 5 times the weight of the nonwoven substrate. The method of any of claims 1 to 13, wherein the amount of the AAV is determined using enzyme-linked immunosorbent assay (ELISA) quantitative polymerase chain reaction (qPCR), or multi -angle dynamic light scattering (MA-DLS). The method of any of claims 1 to 14, wherein a turbidity of the filtrate exhibits a reduction from a turbidity of the composition of 90% or greater. A separated product comprising a virus produced by the method of any of claims 1 to 15. The separated product of claim 16, wherein the virus comprises AAV or Phi-X174.

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