WO2022045055A1

WO2022045055A1 - METHOD FOR FORMULATING NON-ENVELOPED VIRAL VECTOR PARTICLES BY CHANGE IN pH

Info

Publication number: WO2022045055A1
Application number: PCT/JP2021/030774
Authority: WO
Inventors: 春子猪瀬; 英人蝶野; 多佳久月原; 優子釜田; 健介安部; 敏和西江; 佳典田中
Original assignee: タカラバイオ株式会社
Priority date: 2020-08-24
Filing date: 2021-08-23
Publication date: 2022-03-03
Also published as: JPWO2022045055A1

Abstract

The present invention provides a method that is for obtaining an AAV formulation from a sample containing AAV vector particles and empty capsids of AAV, and that comprises: (a) a step for applying the sample containing AAV vector particles and empty capsids of AAV to an anionic exchange carrier equilibrated with a buffer so as to cause the AAV vector particles to be bound to the carrier; (b) a step for applying a buffer to the carrier after step (a) so that non-bound substances are eluted from the carrier; (c) a step for applying a buffer having a pH lower than that of the buffer used to equilibrate the carrier to the carrier after step (b) so that the AAV vector particles are eluted from the carrier; and (d) a step for collecting an eluate obtained from the carrier in step (c) to obtain an AAV formulation.

Description

Method of preparing non-enveloped viral vector particles by different pH

The present invention relates to a method for producing non-enveloped virus (hereinafter, non-enveloped virus) particles, preferably adeno-associated virus (hereinafter, AAV) particles. More specifically, the present invention relates to a method for preparing AAV vector particles from a sample containing AAV vector particles and an empty capsid of AAV.

Currently, in the field of gene recombination and medical field, as a method for introducing a gene into mammalian cells including humans, a physical method using electric perforation or metal fine particles, a chemical method using nucleic acid, polycation, or liposome, etc. In addition, as a biological method, there is a method using a vector for introducing a gene derived from a virus (hereinafter referred to as a virus vector). A viral vector is a vector obtained by modifying a naturally occurring virus so that a desired gene or the like can be transferred into a target cell, and technological development has been progressing in recent years. Generally, a virus vector prepared by using gene recombination technology is called a recombinant virus vector, and the virus from which such a recombinant virus vector is derived includes retrovirus, lentivirus, Sendai virus, herpes virus and the like. Non-enveloped viruses such as enveloped viruses, adenoviruses, and AAV are well known.

In particular, AAV can infect a wide variety of cells including humans, it also infects non-dividing cells that have completed differentiation such as blood cells, muscles, and nerve cells, and there is little concern about side effects because it is not pathogenic to humans. Since the virus particles are physicochemically stable, their utility value as a vector for gene transfer used in gene therapy for the treatment of congenital gene diseases as well as cancer and infectious diseases is high. It has been attracting attention in recent years.

As a method for producing a recombinant virus vector, usually, an element essential for virus particle formation is introduced into a cell in the form of a nucleic acid construct to prepare a cell having an ability to produce a virus (hereinafter referred to as a virus-producing cell). It is performed by culturing cells and expressing the elements essential for virus particle formation in the cells. In general, among the essential elements for virus particle formation, those that require cis supply and those that can be trans-supplied are separated and introduced into virus-producing cells to produce wild-type virus and genetic recombination. Measures are taken to prevent self-sustaining replication at the virus-infected destination.

Hereinafter, a recombinant virus (hereinafter referred to as rAAV) vector derived from AAV will be specifically described as an example. Introducing the "rAAV plasmid" loaded on the plasmid, (B) introducing the "rep-cap gene expression plasmid" to supply the Rep protein and Cap protein to the trans, and AAV is a helper for the formation of its infectious virus particles. A method using (C) adenovirus infection has been established because it requires the supply of an auxiliary element from any virus such as adenovirus, herpesvirus, or vaccinia virus, which is called a virus. Furthermore, when the above method is used, adenovirus is theoretically contaminated in the produced vector solution, but in order to avoid it, only the adenovirus-derived elements essential for the formation of AAV virus particles are expressed. A manufacturing method (helper-free system) using the introduction of a "helper plasmid" has been developed.

The virus-producing cells in which virus production has been achieved are then collected, disrupted, and the resulting cell disruption solution containing the rAAV vector is appropriately subjected to a step such as filter filtration, ultracentrifugation, chromatography, or ultrafiltration. The rAAV vector is purified to the final product.

Conventionally, the ultracentrifugation method has been mainly adopted as a method for purifying an rAAV vector. However, the drawbacks of the ultracentrifuge method are (1) special equipment (ultracentrifuge) is required, (2) special work (ultracentrifuge) is required, and (3) AAV particles that can be processed. The amount is limited, and so on.

Currently, as the use of rAAV vectors spreads to the fields of basic research on gene therapy or clinical application, there is a need for a large-scale and easy method for obtaining rAAV vectors with higher titers and higher purity. The improvement method is disclosed. For example, column chromatography is used to purify rAAV vectors on a large scale, but rAAV vectors purified using column chromatography usually contain significant amounts of empty capsids. The presence of large amounts of empty capsids can interfere with clinical application, for example, by eliciting an undesired immune response to the capsid protein or by competing with the rAAV vector for target cell surface binding sites.

In recent years, a method for separating AAV vector particles and empty capsids of AAV using ion exchange chromatography has been developed. That is, Patent Document 1 discloses a method of separating AAV vector particles and an empty capsid of AAV by using an ion exchange chromatography column depending on the difference in the "salt concentration" of the buffer solution added to the column. This is the first document to show that the AAV vector particles and the empty capsids of AAV can be separated by taking advantage of the different charges and / or charge densities.

Patent No. 5166477

As described above, Patent Document 1 discloses a method of separating AAV vector particles and an empty capsid of AAV by using an ion exchange chromatography column depending on the difference in the "salt concentration" of the buffer solution added to the column. do. However, novels that specifically remove non-enveloped virus empty capsid or specifically reduce the number of non-enveloped virus empty capsids from samples containing non-enveloped viral vector particles and non-enveloped virus empty capsids. There is still a need for methods.

As a result of diligent research aimed at providing a method for preparing a non-enveloped virus in which the amount of empty capsid of the non-enveloped virus is reduced, the present inventors apply the anion exchange carrier to the carrier. The present invention was completed by finding that the non-enveloped virus vector particles and the empty capsid of the non-enveloped virus can be separated by the difference in the "pH" of the buffer solution.

That is, the present invention
[1] A method for obtaining an AAV preparation from a sample containing AAV vector particles and an empty capsid of AAV.
(A) A step of applying a sample containing AAV vector particles and an empty capsid of AAV to an anion exchange carrier equilibrated with a buffer and binding the AAV vector particles to the carrier;
(B) A step of applying a buffer solution to the carrier after the step (a) to elute the non-bonded product from the carrier;
(C) A step of applying a buffer solution having a pH lower than that of the buffer solution equilibrated with the carrier to the carrier after the step (b) to elute the AAV vector particles from the carrier; and (d) from the carrier in the step (c). To obtain an AAV preparation by collecting the eluate of
Including, method,
[2] The method according to [1], wherein the empty capsid of AAV bound to the carrier is further eluted in the step (b).
[3] The method according to [1], wherein the difference in pH between the buffer solution in which the carrier is equilibrated and the buffer solution for elution in step (c) is 1.0 or more.
[4] The method according to [1], wherein the difference in pH between the buffer solution in which the carrier is equilibrated and the buffer solution for elution in step (c) is 2.0 or more.
[5] The method according to [1], wherein the functional group of the anion exchange carrier is a quaternary ammonium group.
[6] The method according to [1], wherein the anion exchange carrier packed in the column is used.
[7] The method according to [1], wherein the buffer solution in which the carrier is equilibrated is a buffer solution containing Tris as a buffer component.
[8] The method according to [1], wherein the elution buffer solution in step (c) is a buffer solution containing Tris as a buffer component.
Regarding.

The present invention provides a method for obtaining a non-enveloped virus preparation with a reduced content of empty capsids of non-enveloped virus. In addition, non-enveloped virus particles prepared using this method are provided. The non-enveloped virus particles produced by the method of the present invention can also be applied to a conventional method for producing non-enveloped virus particles.

It is a figure which shows the elution pattern of the empty capsid of AAV in Example 1. FIG. It is a figure which shows the elution pattern of the AAV vector particle in Example 1. FIG. It is a figure in which "the elution pattern of the empty capsid of AAV" and "the elution pattern of AAV vector particles" in Example 1 are superimposed. It is a figure which shows the elution pattern of the mixture containing the AAV vector particle and the empty capsid of AAV in Example 2. FIG. It is a figure which shows SDS-PAGE in Example 2. It is a figure which shows the quantitative real-time PCR in Example 2. FIG.

The present invention will be described in detail below.
<Definition>

In the present specification, the term "non-enveloped virus" refers to a virus other than the enveloped virus. Here, the enveloped virus refers to a virus having a lipid layer or a lipid bilayer on the surface of the virus. Typical non-enveloped viruses include adenovirus, parvovirus, papovavirus, human papillomavirus, etc. for viruses whose genome is DNA, and rotavirus, coxsackie virus, enterovirus, and sapovirus for viruses whose genome is RNA. , Norovirus, poliovirus, echovirus, hepatitis A virus, hepatitis E virus, rhinovirus, astrovirus and the like are exemplified.

The non-enveloped virus to which the production method of the present invention is applied is not particularly limited, and even a non-enveloped virus whose production method is already known is a non-enveloped virus newly obtained from nature or a gene recombination derived from them. It may be a virus vector. The non-enveloped virus produced by the production method of the present invention preferably includes adenovirus or AAV of the family Parvoviridae. The production method of the present invention is applicable to any known serotype of AAV, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13. It can be used to produce at least one AAV selected from the group consisting of. The production method of the present invention is preferably applied to AAV2. When describing the serotype of rAAV in the present specification, the serotype from which the capsid is derived is used as a reference. That is, the serotype of the rAAV shall be determined according to the origin of the cap gene used at the time of rAAV preparation, and shall not depend on the origin of the serotype of the AAV genome encapsulated in the rAAV particles. For example, if the capsid is derived from AAV6 and the ITR in the AAV genome encapsulated in the rAAV particles is derived from AAV2, the rAAV is serotype 6 herein. Furthermore, the production method of the present invention can also be applied to the production of AAV containing a variant of the capsid of AAV of each of the above serotypes.

As used herein, AAV refers to a small virus belonging to the family Parvoviridae, Dependoparvovirus, which infects primate animals including humans and other mammals. AAV has an envelopeless icosahedron outer shell and a single single-stranded DNA inside it. As used herein, AAV includes wild-type viruses and their derivatives, and includes all serotypes and clades unless otherwise noted.

As used herein, the term "non-enveloped virus particle" or "virus particle" means a particle having an outer shell composed of a capsid protein. Here, the "capsid" or "capsid protein" is a protein encoded by a cap gene present in the virus genome, and means a protein constituting the outer shell of the virus.

Taking AAV as an example, the wild-type AAV genome encodes three types of capsid proteins called VP1, VP2, and VP3. As used herein, all of VP1, VP2 and VP3 are included in the capsid protein. In wild-type AAV particles, generally 60 capsid proteins containing VP1, VP2, and VP3 in a ratio of 1: 1:10 aggregate to form one icosahedral outer shell. However, even if the ratios of VP1, VP2 and VP3 are different from those described above, the particles having the outer shell are included in the AAV particles as long as the outer shell can be formed. That is, as used herein, the term "AAV particle" means a particle having an outer shell composed of a capsid protein selected from any one or more of VP1, VP2 and VP3.

In the present specification, the "virus genome" means a nucleic acid contained in a virus particle. That is, the term "viral genome" includes genetic elements contained within non-enveloped viral particles (eg, plasmids, phages, transposons, cosmids, chromosomes, etc.).

In the present specification, the "virus vector particle" means a virus particle having a vector function. That is, a viral vector particle is an infectious particle containing a virus genome in the virus particle. The term "virus vector particle" includes "complete particle", "full particle", "vector particle" and "virion".

As used herein, the term "empty capsid" refers to a non-infectious protein or particle that contains a viral capsid protein but lacks a viral genome. Therefore, empty capsids do not function to transfer the gene of interest into the host cell. The term "empty capsid" includes "hollow particles" and "empty particles".

<Measurement method for non-enveloped virus>
The method for measuring the non-enveloped virus is not particularly limited, but for example, (a) the concentration of the non-enveloped virus vector in a certain amount of sample, for example, the concentration of the virus genome, or (b) the protein constituting the non-enveloped virus. Examples thereof include a method of measuring the concentration, for example, the concentration of the capsid protein.

As the method (a) above, a method of measuring the number of copies of the virus genome in the sample by the PCR method is exemplified. Although not particularly limited, for example, AAVpro (registered trademark) Titration Kit (for Real Time PCR) Ver. 2 (manufactured by Takara Bio Inc.) can be used to calculate the concentration of the AAV genome by the method described in the instruction manual.

Examples of the method (b) above include a method of analyzing the protein by SDS-PAGE or a method of quantifying the protein by an immunological method.

By combining the above (a) and (b), the ratio of the virus vector particles contained in the sample to the empty capsid of the virus can be calculated. That is, the viral vector particles contain both the capsid protein and the viral genome, whereas the empty capsid of the virus contains the capsid protein but not the viral genome. Thus, for example, when a sample is measured, a small amount of viral genome per fixed amount of capsid protein indicates that the sample is high in empty capsids.

Another method for determining the ratio of the virus vector particles contained in the sample to the empty capsid of the virus is (c) a method of observing with an electron microscope.

In addition, as a method for measuring whether or not the viral vector particles contained in the sample are functional, that is, whether or not they have the ability to infect target cells, (d) the ability of the viral vector particles to infect cells experimentally. A method of measuring (infectious titer) can be mentioned. More specifically, for example, a method of infecting an appropriate target cell with a serial diluted solution of a sample containing a virus vector particle to detect a change in cell shape (cell degeneration), a method of detecting the expression of an introductory gene, or a method of detecting the expression of a transgene. Examples thereof include a method for measuring the number of copies of a viral genome introduced into a cell.

<Sample containing virus vector particles and virus empty capsid>
The "sample containing virus vector particles and an empty capsid of virus" used in the method of the present invention is not particularly limited, but is preferably a sample derived from virus-producing cells. The virus-producing cells are not particularly limited, and may be virus-producing cells obtained in the environment or from clinical specimens of patients with infectious diseases, or may be artificially produced virus-producing cells.

The cells for producing the virus-producing cells are not particularly limited, and are mammalian cells such as humans, monkeys, and rodents, preferably 293 cells (ATCC CRL-1573) and 293T cells (ATCC) having high transfection efficiency. CRL-3216), 293T / 17 cells (ATCC CRL-11268), 293F cells, 293FT cells (all manufactured by Life Technologies), G3T-hi cells (International Publication No. 2006/035829), for commercial virus production A cell line, AAV293 cells (manufactured by Stratagene) is exemplified. Further, arthropod cells (insect cells) such as Sf9 cells (ATCC CRL-1711) are exemplified. For example, the 293 cells and the like constitutively express the adenovirus E1 protein, and one or some of the proteins necessary for rAAV production are modified to transiently or constitutively express. It may be a cell. The following elements necessary for virus formation can be introduced into these various cells using a known method or a commercially available kit to obtain virus-producing cells. In addition, the cells can be cultured under known culture conditions. For example, culture at a temperature of 30 to 37 ° C., a humidity of 95% RH, and a CO ₂ concentration of 5 to 10% (v / v) is exemplified, but the present invention is not limited to such conditions. If the desired proliferation of virus-producing cells and the production of virus can be achieved, the temperature, humidity, and CO ₂ concentration other than the above ranges may be used. The culture period is not particularly limited, and is, for example, 12 to 150 hours, preferably 48 to 120 hours.

Taking, for example, rAAV-producing cells as non-enveloped virus-producing cells as an example, as essential elements for rAAV formation, (A) a nucleic acid encapsulated in rAAV particles, (B) an element derived from AAV, for example, Rep protein and Cap proteins and (C) adenovirus-derived elements such as E1a protein, E1b protein, E2 protein, E4 protein and VARNA. By introducing these elements into any cell, rAAV-producing cells can be produced.

The above-mentioned "(A) nucleic acid encapsulated in rAAV particles" is composed of an AAV-derived ITR sequence and a nucleic acid desired to be mounted on rAAV particles. Nucleic acids desired to be loaded on rAAV particles include arbitrary foreign genes such as polypeptides (enzymes, growth factors, cytokines, receptors, structural proteins, etc.), antisense RNA, ribozymes, decoys, RNAs that cause RNA interference, and the like. Nucleic acid to supply is exemplified. In addition, suitable promoters, enhancers, terminators and other transcriptional regulatory elements may be inserted into the nucleic acids to control the expression of foreign genes. For example, the nucleic acid encapsulated in the rAAV particle may contain any foreign gene desired to be loaded into the rAAV vector between the two ITR sequences, or the rAAV particle between the two ITR sequences. It may contain any foreign gene desired to be loaded into and one or more elements for controlling the expression of the foreign gene. The nucleic acid encapsulated in the rAAV particles can be introduced into cells in the form of a plasmid as a nucleic acid construct. The above-mentioned plasmid can be constructed, for example, by a known method using a commercially available or known plasmid. An example of the plasmid is pAAV-CMV Vector (manufactured by Takara Bio Inc.).

The form of the above-mentioned "(B) AAV-derived element, for example, Rep protein and Cap protein" is not limited, and each element can be directly introduced into a cell as a protein, and each element can be supplied 1. Alternatively, it can be loaded into a plasmid or viral vector as a plurality of nucleic acid constructs and introduced into cells. Introduction of these nucleic acids into cells can be performed, for example, by a known method using a commercially available or known plasmid or viral vector. An example of the plasmid is pRC2-mi342 Vector (manufactured by Takara Bio Inc.).

The form of the above-mentioned "(C) adenovirus-derived element, for example, E1a protein, E1b protein, E2 protein, E4 protein and VARNA" is not limited, and each element can be directly introduced into cells as a protein. , Each element can be loaded into a plasmid or viral vector as one or more nucleic acid constructs capable of being introduced into cells. Introduction of these nucleic acids into cells can be performed, for example, by a known method using a commercially available or known plasmid or viral vector. An example of the plasmid is pHelper Vector (manufactured by Takara Bio Inc.). The above-mentioned purpose can also be achieved by directly infecting cells with adenovirus instead of a plasmid or viral vector.

The "sample containing virus vector particles and an empty capsid of virus" used in the method of the present invention is not particularly limited to the present invention, but the virus-producing cells cultured as described above are disrupted or lysed. Can be prepared. In addition, when virus vector particles leak into the medium during culture of virus-producing cells, the present invention can be carried out using the supernatant of the culture solution as a sample.

Crushing and lysis of virus-producing cells can be carried out by known methods such as ultrasonic treatment, freeze-thaw treatment, enzyme treatment, and osmotic pressure treatment. For example, cells can be lysed by adding a surfactant to a culture solution containing virus-producing cells. The surfactant is not particularly limited, and for example, Triton X-100 is exemplified. The concentration of the surfactant is not particularly limited, for example, the final concentration is 0.01 to 1%, preferably the final concentration is 0.05 to 0.5%, and more preferably the final concentration is 0.08 to 0.12%. Is exemplified. The temperature and time during contact with the surfactant are not particularly limited, and the temperature is, for example, 0 to 40 ° C, preferably 4 to 37 ° C, and the time is, for example, 5 minutes to 8 hours. Is exemplified by 10 minutes to 4 hours, more preferably 30 minutes to 2 hours.

The above-mentioned sample obtained by disrupting or lysing virus-producing cells may be directly subjected to the method of the present invention, or may be temporarily subjected to another purification step. Further, the virus vector particles and / or the empty capsid of the virus may be concentrated by the other purification step. Examples of the other purification method include ultracentrifugation, chromatography, ultrafiltration, and other known methods.

Purification by chromatography is performed by an ion exchange column (for example, Mustang Q (manufactured by pool)), an affinity column (for example, AVB Sepharose (registered trademark) (manufactured by Cytiva), a heparin column, etc.), a hydroxyl apatite column, or the like. Can be carried out.

The "sample containing virus vector particles and virus empty capsid" prepared in this way can be stored for a long period of time in an appropriate solution. For example, a sample containing AAV vector particles and an empty capsid of AAV, when replaced with phosphate buffered saline (pH 7.4), under the condition of −80 ° C., for example, for 12 hours or more and 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 1 week or more, 2 weeks or more, 3 weeks or more, 1 month or more, 2 months or more, 3 months or more, 4 months It can be stored for 5 months or more, or 6 months or more.

By carrying out any of the above-mentioned "measurement methods for non-enveloped viruses" for "a sample containing virus vector particles and an empty capsid of virus", "concentration of virus vector" and "concentration of capsid protein" in the sample. , "Ratio of virus vector particles to empty capsids of virus" and the like, desired values can be measured.

<Anion exchange carrier>
Various known anion exchange carriers can be used in the method of the present invention. Examples include, but are not limited to: Macro-Prep (registered trademark) Q (manufactured by Bio-Rad Laboratories), UNOSphere Q (manufactured by Bio-Rad Laboratories), POROS 50HQ (manufactured by Thermo Fisher Scientific), POROS 50 (Thermo Fisher Scientific), SOURCE 30Q (Cytiva), DEAE-Sepharose (Cytiva), Q Sepharose (Cytiva).

Examples of the functional group (also referred to as an ion exchange group) of the anion exchange carrier include, but are not limited to, a quaternary ammonium group, a tertiary amino group, a secondary amino group, and a primary amino group. As the functional group of the anion exchange carrier, a quaternary ammonium group is suitable.

The anion exchange carrier may or may not be packed in the column, but preferably, the anion exchange carrier packed in the column is used. As a commercially available column, CIMmultus QA (manufactured by BIA Separations) is exemplified.

<Equilibration of anion exchange carrier>
The anion exchange carrier is first equilibrated using standard buffer according to the manufacturer's specifications. Here, the buffer solution for equilibrating the anion exchange carrier is referred to as "equilibrium buffer". It is desirable that the composition of the equilibration buffer is such that it can provide conditions under which the AAV vector particles can bind to the carrier. Such a composition can be appropriately determined by those skilled in the art.

The buffer component of the equilibration buffer is not particularly limited, but one based on Tris hydroxymethylaminomethane (hereinafter, Tris) buffer or Bis-Tris propane (hereinafter, BTP) buffer is preferable, and one based on Tris buffer. Is more desirable. When using Tris buffer, the equilibration buffer is, for example, 10-50 mM Tris-HCl, preferably 15-40 (eg, 15, 20, 25, 30, 35, 40, etc.) mM Tris-HCl. The equilibration buffer further comprises salts (eg, NaCl or KCl), eg, 5-50 mM (eg, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 mM, etc., or ranges thereof. It may be contained at any concentration in the above, or may not contain salt. Preferably, the equilibration buffer is salt-free. The pH of the equilibration buffer is, for example, 7-11, preferably 8-10 (eg, pH 8, 8.5, 9, 9.5, 10, etc., or any pH within these ranges). ..

As an equilibration buffer solution, "20 mM Tris-HCl, pH 9.0" is exemplified.

<Step (a): Step of applying the sample to the carrier>
Step (a) is a step of applying a sample containing virus vector particles and an empty capsid of virus to an anion exchange carrier equilibrated with a buffer solution, and binding the virus vector particles to the carrier. The empty capsid of the virus may bind to the anion exchange carrier as long as the virus vector particles bind to the anion exchange carrier. That is, only the viral vector particles may bind to the anion exchange carrier, or both the viral vector particles and the empty capsid of the virus may bind to the anion exchange carrier.

The volume of the sample applied to the anion exchange carrier is appropriately determined depending on the amount of virus contained in the sample and the type and volume of the anion exchange carrier used. In addition, the sample can be diluted or replaced with a suitable solution in advance, and then applied to the anion exchange carrier. For example, the sample can be diluted 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold or 50-fold with equilibration buffer.

<Step (b): Elution of non-bonded product from carrier>
The step (b) is a step of applying a buffer solution to the carrier to which the virus vector particles are bound after the step (a), and elution of the non-bound product from the carrier. The virus vector particles are bound to the carrier after the step (a), but in the step (b), the virus vector particles are not eluted, but only the non-bound product is eluted from the carrier. When both the virus vector particles and the empty capsid of the virus are bound to the carrier after the step (a), the empty capsid of the virus is eluted from the carrier by applying an appropriate buffer solution in this step. ..

The "non-binding substance" means a substance that does not bind to the carrier other than the viral vector particles. Non-bindings include, for example, contaminants in the sample and empty capsids if the empty capsid of the virus does not bind to the carrier in step (a). As described above, when both the viral vector particles and the empty capsid of the virus are bound to the carrier after the step (a), the empty capsid of the virus is eluted from the carrier by applying an appropriate buffer solution. ..

Since the buffer solution used in the step (b) has a higher pH than the buffer solution used in the step (c), which will be described later, it is also called a "high pH buffer". The high pH buffer does not elute the viral vector particles bound to the carrier from the carrier, while the high pH buffer elutes the unbound material from the carrier. If both the viral vector particles and the empty capsid of the virus are bound to the carrier, the viral vector particles bound to the carrier are eluted from the carrier by applying a high pH buffer with a higher salt concentration than the equilibrium buffer. Instead, only the empty capsid of the virus is eluted from the carrier. When only the viral vector particles are bound to the carrier by step (a), the high pH buffer may have the same salt concentration as the equilibration buffer, eg the same buffer as the equilibration buffer. You may.

If both the virus vector particles and the empty capsid of the virus are bound to the carrier, equilibrate after applying a high pH buffer with a salt concentration comparable to that of the equilibration buffer to elute the unbound from the carrier. A high pH buffer solution with a salt concentration higher than that of the conversion buffer solution may be applied to elute the empty capsid of the virus bound to the carrier from the carrier. Alternatively, a high pH buffer solution having a salt concentration higher than that of the equilibration buffer solution may be applied to elute the unbound product and the empty capsid of the virus bound to the carrier from the carrier in one step.

The high pH buffer solution is preferably based on Tris buffer or BTP buffer, and more preferably based on Tris buffer. When using Tris buffer, the high pH buffer is, for example, 10-50 mM Tris-HCl, preferably 15-40 (eg, 15, 20, 25, 30, 35, 40, etc.) mM Tris-HCl. High pH buffers further include salts (eg, NaCl or KCl), such as 10-100 mM, preferably 20-80 (eg, 20, 30, 40, 50, 55, 60, 70, 80, etc., or these. Any concentration within the range of) may be contained in mM. The high pH buffer may further include another salt (eg, MgCl ₂ ), such as 1-10 mM, preferably 2-8 (eg, 2, 3, 4, 5, 5.5, 6, 7, 8 and the like. , Or any concentration within these ranges) may be included in mM. The pH of the high pH buffer is, for example, 7-11, preferably 8-10 (eg, pH 8, 8.5, 9, 9.5, 10, etc., or any pH within these ranges). ..

The high pH buffer has a higher pH than the low pH buffer described later. The difference in pH between the high pH buffer solution and the low pH buffer solution is preferably 1.0 or more, 1.5 or more, 2.0 or more, 2.5 or more, and 3.0 or more. The high pH buffer may have the same pH as the equilibration buffer, or may have a higher or lower pH than the equilibration buffer. Preferably, the high pH buffer has the same pH as the equilibration buffer.

Although not particularly limited, it is desirable that the high pH buffer solution and the low pH buffer solution contain salts of the same type and concentration. That is, it is desirable that the composition of the high pH buffer solution and the low pH buffer solution are the same except that the pH is different.

Examples of the high pH buffer solution include "20 mM Tris-HCl, 55 mM NaCl, 5.5 mM MgCl ₂ , pH 9.0".

<Step (c): Elution of viral vector particles from the carrier>
In the step (c), a buffer solution having a pH lower than that of the equilibrium buffer solution and lower than that of the high pH buffer solution is applied to the carrier to which the virus vector particles are bound after the step (b), and the virus vector particles are used as the carrier. It is a step of elution from.

Since the buffer solution used in the step (c) has a lower pH than the equilibration buffer solution and the buffer solution used in the step (b), it is also called a "low pH buffer". For example, a buffer solution having a pH lower than that of the equilibration buffer solution by 1.0 or more, preferably a buffer solution having a pH lower than that of the equilibration buffer solution by 2.0 or more is used as the low pH buffer solution. The low pH buffer may or may not elute the empty capsid of the virus as long as it elutes the viral vector particles bound to the carrier from the carrier.

The low pH buffer is preferably based on Tris buffer or BTP buffer, and more preferably based on Tris buffer. When using Tris buffer, the low pH buffer is, for example, 10-50 mM Tris-HCl, preferably 15-40 (eg, 15, 20, 25, 30, 35, 40, etc.) mM Tris-HCl. The low pH buffer further comprises salts (eg, NaCl or KCl), eg, 10-100 mM, preferably 20-80 (eg, 20, 30, 40, 50, 55, 60, 70, 80, etc., or these. Any concentration within the range of) may be contained in mM. The low pH buffer may further include another salt (eg, MgCl ₂ ), such as 1-10 mM, preferably 2-8 (eg, 2, 3, 4, 5, 5.5, 6, 7, 8 and the like. , Or any concentration within these ranges) may be included in mM. The pH of the low pH buffer is, for example, 5-9, preferably 6-8 (eg, pH 6, 6.5, 7, 7.5, 8, etc., or any pH within these ranges). ..

The low pH buffer has a lower pH than the high pH buffer. The difference in pH between the high pH buffer solution and the low pH buffer solution is preferably 1.0 or more, 1.5 or more, 2.0 or more, 2.5 or more, and 3.0 or more.

Examples of the low pH buffer solution include "20 mM Tris-HCl, 55 mM NaCl, 5.5 mM MgCl ₂ , pH 7.0".

<Step (d): Step of obtaining virus preparation>
Step (d) is a step of collecting the eluate from the carrier in step (c) to obtain a virus preparation. Here, the eluate from the carrier contains viral vector particles. Therefore, step (d) can also be said to be a step of collecting the eluate containing the virus vector particles from the carrier to obtain a virus preparation. The resulting virus preparation contains viral vector particles. The virus preparation may contain not only the viral vector particles but also the empty capsids of the virus, but the content of the empty capsids is compared to the content in the sample containing the viral vector particles and the empty capsids of the virus. It has decreased much.

The method of collecting the eluate is not particularly limited. For example, when using an anion-exchange carrier packed in a column, it is done by separating the solution eluted from the column when a low pH buffer is applied.

By carrying out any "method for measuring non-enveloped virus" with respect to the "virus preparation" obtained by the above method, "concentration of virus vector", "concentration of capsid protein", "virus vector" in the sample. A desired value such as "ratio of particles to empty capsid of virus" can be measured. In addition, the recovery rate of the viral vector particles can be calculated by comparing these values with the values of the sample before application to the anion exchange carrier.

When a virus preparation is obtained from a sample containing a virus vector particle and a virus empty capsid at a ratio of 1: 1 by the method of the present invention, the virus preparation is based on the total amount of the virus vector particle and the virus empty capsid. , Less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10% or less than 5% containing empty capsids of the virus. Therefore, by the method of the present invention, a virus preparation containing a high concentration of viral vector particles can be obtained.

The recovery rates of viral vector particles in virus preparations are 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more. Therefore, the present invention also provides a method for efficiently recovering viral vector particles from a sample containing viral vector particles and an empty capsid of virus.

INDUSTRIAL APPLICABILITY The present invention provides a method for producing a virus preparation containing non-enveloped viral vector particles, a kit used in the production method, and a preparation containing non-enveloped virus vector particles produced by the production method. The kit includes, for example, a buffer used in each step of the method of the invention, and an anion exchange carrier. The preparation containing the non-enveloped viral vector particles obtained by using the production method of the present invention can be used as an active ingredient of a pharmaceutical composition. The pharmaceutical composition can be used in vitro to cells derived from the patient or administered directly to the patient.

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to these examples.

Preparation Example 1 Preparation of AAV vector particles and empty capsid (1) Production of rAAV2 293T cells (ATCC, CRL-3216) were used as host cells for producing rAAV2. Three plasmids (pRC2-mi342 Vector (manufactured by Takara Bio Inc.), pHelper Vector (manufactured by Takara Bio Inc.), and pAAV-ZsGreen1 Vector (manufactured by Takara Bio Inc.)) were transfected into 293T cells by the lipofection method. After transfection, 293T cells were cultured for 3 days.

(2) Purification of rAAV2 Triton X-100 with a final concentration of 0.1% was added to a culture solution containing 293T cells, and the cells were lysed by treatment at 37 ° C. for 1 hour. The supernatant collected by centrifuging the cytolysate was passed through a filter having a pore size of 0.2 μm to remove cell debris. A sample concentrated about 10-fold using a centrifugal ultrafiltration filter having an exclusion molecular weight of 100 kDa was loaded onto a column packed with AVB Sepharose High Performance (manufactured by Cytiva), and the adsorbate was eluted with a buffer solution containing NaCl. Purified rAAV2 by Purified rAAV2 contains both AAV vector particles and empty capsids.

(3) Separation of AAV vector particles and empty capsids The AAV vector particles and empty capsids contained in rAAV2 obtained in Preparation Example 1- (2) are separated and separated by cesium chloride density gradient centrifugation using an ultracentrifuge. I drew it. Cesium chloride was added to the sample so that the refractive index was 1.371, and ultracentrifugation was performed using Optima XE-90 (manufactured by Beckman Coulter) at 148500 × g at 21 ° C. for 42 hours. A part of each fraction obtained by separating 0.5 ml of the separated sample was subjected to SDS-polyacrylamide gel electrophoresis. The fraction in which the AAV capsid protein was accumulated was determined by Oriole staining with Oriole fluorescent gel stain (manufactured by Bio-Rad Laboratories). Next, the concentration of the AAV genome contained in each fraction was measured by quantitative real-time PCR using AAVpro Titration Kit (manufactured by Takara Bio Inc.) to determine the fraction in which the AAV genome was accumulated. The fraction in which only the AAV capsid protein is accumulated is collected as "empty capsid", and the fraction in which both the AAV capsid protein and the AAV genome are accumulated is collected as "AAV vector particles". It was replaced with (pH 7.4).

Example 1 Differences in Elution Patterns of AAV Vector Particles and Empty Capsids (1) Program Design A program was designed for the purpose of separating AAV vector particles and empty capsids using anion exchange column chromatography. That is, in the program, the sample was first loaded into a CIMmultus QA (quaternary aminoe) -1 column (manufactured by BIA Separations) equilibrated with equilibration buffer (20 mM Tris-HCl, pH 9.0). Run the equilibration buffer and wash the column until the UV280 value drops near baseline. Next, a buffer solution (20 mM Tris-HCl, 55 mM NaCl, 5.5 mM MgCl ₂ , pH 9.0) having the same pH as the equilibration buffer solution and having an increased salt concentration is flowed through the column to elute the empty capsid. Finally, a buffer solution (20 mM Tris-HCl, 55 mM NaCl, 5.5 mM MgCl ₂ , pH 7.0) having the same salt concentration but lower pH is passed through the column to elute the AAV vector particles.
Differences in elution patterns of AAV vector particles and empty capsids were revealed by loading AAV vector particles and empty capsids separately into columns and eluting using the above program.

(2) Elution of empty capsid Equilibration buffer of empty capsid obtained in Preparation Example 1- (3) on a CIMmultus QA-1 column equilibrated with an equilibration buffer (20 mM Tris-HCl, pH 9.0). It was diluted 20-fold with liquid and loaded. After washing the column with equilibration buffer until the value of absorbance (UV280) at 280 nm drops to near baseline, the pH is the same as equilibration buffer and the salt concentration is increased (20 mM Tris-HCl, 55 mM NaCl). 5.5 mM MgCl ₂ , pH 9.0) was flowed through the column, and the peak absorbed by UV280 was recovered (hereinafter referred to as “pH 9.0 elution fraction”). Next, a buffer solution (20 mM Tris-HCl, 55 mM NaCl, 5.5 mM MgCl ₂ , pH 7.0) having the same salt concentration but lowering the pH was flowed through the column, but no peak with absorption in UV280 could be confirmed. rice field. The collected fractions were analyzed by SDS-PAGE and quantitative real-time PCR, and it was confirmed that the "pH 9.0-eluted fraction" contained an empty capsid (data not shoun). The elution pattern of the empty capsid is shown in FIG.

(3) Elution of AAV vector particles In the same manner as in Example 1- (2), the AAV vector particles obtained in Preparation Example 1- (3) were diluted 20-fold with an equilibration buffer solution, and the equilibration buffer solution was used. It was loaded onto a column equilibrated with (20 mM Tris-HCl, pH 9.0). After the column was washed with equilibration buffer and the UV280 value dropped to near the baseline, elution was performed from the column using the same program as in Example 1- (2). The pH was the same as that of the equilibration buffer solution, and a peak with absorption in UV280 could not be confirmed in the buffer solution having an increased salt concentration. On the other hand, with a buffer solution having the same salt concentration and a lowered pH, a peak absorbed by UV280 could be recovered (hereinafter referred to as "pH 7.0 elution fraction"). The collected fractions were analyzed by SDS-PAGE and quantitative real-time PCR. From the obtained data, it was confirmed that the "pH 7.0 elution fraction" contained AAV vector particles (data not shown). The elution pattern of AAV vector particles is shown in FIG.

(4) Comparison of Elution Patterns of AAV Vector Particles and Empty Capsids FIG. 3 shows an overlay of the elution patterns of empty capsids (FIG. 1) and the elution patterns of AAV vector particles (FIG. 2).

Example 2 Separation of AAV Vector Particles and Empty Capsids Based on the findings of Example 1- (4), AAV vector particles and empty capsids were separated using a CIMmultus QA-1 column. That is, the AAV vector particles obtained in Preparation Example 1- (3) and the empty capsid were mixed (about 1: 1 by weight), the mixture was loaded onto a CIMmultus QA-1 column, and Example 1- (1). ) Eluted by the program. Peaks with absorption in UV280 were confirmed in both the pH 9.0 elution fraction and the pH 7.0 elution fraction, and each was recovered. The elution pattern of the mixture of AAV vector particles and empty capsid is shown in FIG.

The collected fractions were analyzed by SDS-PAGE and quantitative real-time PCR. SDS-PAGE and quantitative real-time PCR were performed according to conventional methods. As a result, similar capsid protein bands were confirmed in both fractions by SDS-PAGE (Fig. 5). On the other hand, in quantitative real-time PCR, the AAV genome was detected only in the pH 7.0-eluted fraction (Fig. 6). From these data, it was shown that the pH 9.0 elution fraction contained only empty capsids, whereas the pH 7.0 elution fraction contained AAV vector particles.

By the method of the present invention, a preparation containing high-purity non-enveloped viral vector particles can be obtained. The non-enveloped viral vector particles prepared by the method of the present invention and compositions containing the particles as active ingredients are very useful as gene transfer methods in the field of basic research on gene therapy or clinical application.

Claims

A method for obtaining an AAV preparation from a sample containing AAV vector particles and an empty capsid of AAV.
(A) A step of applying a sample containing AAV vector particles and an empty capsid of AAV to an anion exchange carrier equilibrated with a buffer and binding the AAV vector particles to the carrier;
(B) A step of applying a buffer solution to the carrier after the step (a) to elute the non-bonded product from the carrier;
(C) A step of applying a buffer solution having a pH lower than that of the buffer solution equilibrated with the carrier to the carrier after the step (b) to elute the AAV vector particles from the carrier; and (d) from the carrier in the step (c). To obtain an AAV preparation by collecting the eluate of
A method that embraces.
The method according to claim 1, wherein in the step (b), the empty capsid of AAV bound to the carrier is further eluted.
The method according to claim 1, wherein the difference in pH between the buffer solution in which the carrier is equilibrated and the buffer solution for elution in step (c) is 1.0 or more.
The method according to claim 1, wherein the difference in pH between the buffer solution in which the carrier is equilibrated and the buffer solution for elution in step (c) is 2.0 or more.
The method according to claim 1, wherein the functional group of the anion exchange carrier is a quaternary ammonium group.
The method according to claim 1, wherein an anion exchange carrier packed in a column is used.
The method according to claim 1, wherein the buffer solution in which the carrier is equilibrated is a buffer solution containing Tris as a buffer component.
The method according to claim 1, wherein the elution buffer solution in the step (c) is a buffer solution containing Tris as a buffer component.