Virus purification method

Abstract

To provide a method for producing viruses that is more efficient than conventional methods and for obtaining highly purified virus particles.SOLUTION: The present invention is a method for obtaining a virus, which comprises treating a sample containing the virus with a surfactant. The surfactant may be one or more selected from the group consisting of amphoteric surfactants, anionic surfactants, cationic surfactants, and nonionic surfactants. Furthermore, the method for obtaining a virus according to the present invention may comprise purifying the virus by performing tangential flow filtration (TFF).SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for producing or purifying a virus.

Background Art

[0002] For the purpose of treating diseases, gene therapy that administers genes or cells into which genes have been introduced into the human body is one of the important treatment methods for treating intractable diseases. Currently, as a method for introducing genes into mammalian cells for the purpose of gene therapy, a biological method using a viral vector has become mainstream. A viral vector is a carrier for incorporating a gene to be introduced for treatment into a virus strain that has lost or partially lost its replication ability and proliferation ability, and for efficiently introducing and expressing the gene into cells. Known viruses from which viral vectors are derived include enveloped viruses (viruses with an envelope) such as retroviruses, lentiviruses, herpesviruses, and Sendai viruses, and non-enveloped viruses (viruses without an envelope) such as adenoviruses and adeno-associated viruses (AAV). Among them, AAV is used for gene therapy for the treatment of various diseases because it can infect many types of cells, has no pathogenicity to humans, and the virus particles are physically stable.

[0003] By the way, in order to promote highly safe and effective treatment in the implementation of gene therapy, it is necessary to prepare the virus used as a vector with high efficiency and purification degree. In recent years, harmful events associated with systemic large-dose administration of AAV vectors have occurred frequently. In order to ensure safety and efficacy, the importance of an AAV vector production / purification method with higher biological activity and less contamination of hollow particles has been increasing.

[0004] To date, methods for obtaining high-purity virus particles have been known, including a method of culturing virus-producing cells under stress conditions by increasing the pH of the culture medium to increase the rate of virus particle release into the culture supernatant (Patent Document 1), and a method of obtaining high-purity virus by contacting virus-producing cells with an acidic solution (Non-Patent Document 2). These methods are reports on improvements to the step of obtaining virus particles from virus-producing cells.

[0005] On the other hand, as mentioned above, it is also extremely important to reduce impurities as much as possible and prepare highly pure and concentrated virus particles, and improvements to the virus particle purification process are necessary. Conventional methods for concentrating and purifying virus particles are cumbersome for large-scale preparation and have a significant impact on biological activity, posing significant challenges for industrial application. For example, for AAV purification, an affinity purification system using VHH antibodies with high affinity for AAV capsids has been put into practical use, but the low pH conditions during elution after adsorption affect biological activity, making it difficult to purify while maintaining high biological activity. Thus, currently, no satisfactory method for purifying virus particles has been reported, and further improvements are needed. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] WO2000 / 14205 [Patent Document 2] WO2015 / 06272 [Overview of the project] [Problems that the invention aims to solve]

[0007] In view of the above circumstances, the object of the present invention is to provide a method for obtaining viruses that is more efficient than conventional methods and for obtaining high-purity virus particles. More specifically, the present invention relates to a method for producing or obtaining virus particles, which includes a step of efficiently and highly purifying virus particles produced from virus-producing cells. [Means for solving the problem]

[0008] The inventors have discovered conditions for purifying high-purity virus by culturing virus-producing cells, treating the culture supernatant containing AAV with CHAPS, a surfactant that forms amphoteric ionic micelles from the cells (amphoteric surfactant), and deoxycholic acid, an anionic surfactant, and introducing a filtration operation by tangential flow filtration (TFF, also known as cross-flow filtration).

[0009] The AAV purified by the above method retains its original biological activity. Furthermore, electron microscopy observations revealed that, surprisingly, the hollow particles of AAV are preferentially destroyed when this method is performed. Therefore, this method efficiently reduces hollow particles, which have been difficult to separate and remove using column chromatography, which has been used to purify AAV, making it possible to prepare extremely high-quality AAV.

[0010] In other words, the present invention is as follows (1) to (8). (1) A method for obtaining a virus, comprising the step of treating a sample containing a virus with a surfactant. (2) The method according to (1), wherein the surfactant is one or more selected from the group consisting of amphoteric surfactants, anionic surfactants, cationic surfactants, and nonionic surfactants. (3) The method according to (1) or (2) above, further comprising the step of purifying the virus by performing tangential flow filtration (TFF). (4) The method according to any one of (1) to (3) above, wherein the amphoteric surfactant is one or more selected from the group consisting of CHAPS, CHAPSO, NDSB-211 and NDSB-201. (5) The method according to any one of (1) to (4) above, wherein the anionic surfactant is sodium cholate and / or sodium deoxycholate. (6) The method according to any one of (1) to (5) above, characterized in that the virus is a non-enveloped virus. (7) The method according to (6) above, wherein the non-enveloped virus is an adeno-associated virus vector. (8) A method for producing a virus, (a) A step of culturing virus-producing cells, (b) A step of preparing a sample containing the virus from the virus-producing cells or cell culture medium, (c) A method comprising the step of obtaining a virus from a sample containing the virus prepared in step (b) by the method according to any one of claims 1 to 5. In this specification, the symbol "~" indicates a numerical range that includes the values ​​to its left and right. [Effects of the Invention]

[0011] This invention provides a method for purifying and obtaining highly biologically active and high-purity viruses. This makes it possible to improve the safety and efficacy of gene therapy using viral vectors. [Brief explanation of the drawing]

[0012] [Figure 1] Results of the investigation of filtration conditions using the TFF method. The results of SDS-PAGE are shown for samples a (lane 1), b (lane 2), c (lane 3), d (lane 4), e (lane 5), f (lane 6), g (lane 7), and h (lane 8) shown in the example. M indicates the electrophoresis result of the marker. [Figure 2]Results of examining the effect of surfactants in the virus purification process. The results of subjecting Sample i (lane 1), Sample j (lane 2), Sample k (lane 3), Sample l (lane 4), and Sample m (lane 5) shown in the examples to SDS-PAGE are presented. SM represents the sample before TFF filtration, and M represents the electrophoresis results of the marker. [Figure 3] Results of purifying AAV9 by treating it with a surfactant and filtering it by the TFF method. The results of subjecting Sample n (lane 1) and Sample o (lane 2) shown in the examples to SDS-PAGE are presented. SM represents the sample before TFF filtration, and M represents the electrophoresis results of the marker. [Figure 4] Results of examining the infectivity of the purified virus. The percentage of HEK cells infected by the virus (AAV1) in each sample is shown. Sup is the culture supernatant of ZsGreen-expressing AAV1, TFF is the sample obtained by treating the culture supernatant with a surfactant and then filtering it by TFF, CsCl is the sample obtained by adding a CsCl solution to the culture supernatant and storing it for 72 hours, and affinity represents the infection rate of the virus in the sample eluted with glycine hydrochloride (pH 2.0) after subjecting the culture supernatant to affinity chromatography. [Figure 5] Observation results of the purified AAV1 by transmission electron microscopy (negative staining image). [Figure 6] Observation results of the purified AAV9 by transmission electron microscopy (negative staining image).

Mode for Carrying Out the Invention

[0013] Hereinafter, the mode for carrying out the present invention will be described. The first embodiment is a method for obtaining a virus, which includes a step of treating a sample containing the virus with a surfactant. More specifically, it includes a step of treating with one or more surfactants selected from the group consisting of amphoteric surfactants, anionic surfactants, cationic surfactants, and nonionic surfactants. That is, the first embodiment is a method for obtaining a target virus with high purity by purifying the virus through a method that includes a step of treating a sample containing the virus, such as a suspension of virus-producing cells, a cell culture solution containing the virus, or a sample obtained by roughly purifying the virus from these suspensions or cell culture solutions (a sample containing impurities other than the virus), with a surfactant.

[0014] The viruses in this embodiment include, in addition to wild-type viruses, inactivated viruses (e.g., inactivated vaccine antigens, etc.), virus-like particles (VLPs) without genetic information, viruses that carry foreign genes used as vectors (also referred to as viral vectors), etc., but are not limited to these. Also, the type of virus is not particularly limited and includes both enveloped viruses and non-enveloped viruses.

[0015] An enveloped virus is a virus in which the protein shell called the viral genome and capsid is covered by a membranous structure (envelope), and a non-enveloped virus is a virus without an envelope. Known enveloped viruses include, for example, DNA viruses such as herpesvirus, poxvirus, and hepadnavirus, and RNA viruses such as flavivirus, togavirus, coronavirus, orthomyxovirus, paramyxovirus, rhabdovirus, bunyavirus, and retrovirus. Known non-enveloped viruses include DNA viruses such as adenovirus, adeno-associated virus (AAV), and papillomavirus, and RNA viruses such as picornavirus, calicivirus, norovirus, and rotavirus.

[0016] Surfactants are a general term for substances that have hydrophilic groups and lipophilic / hydrophobic groups within their molecules. They have the function of uniformly mixing polar and nonpolar substances by forming micelles or lamellar structures. Generally, surfactants are classified into amphoteric surfactants, anionic surfactants, cationic surfactants, and nonionic surfactants. Amphoteric surfactants are surfactants that possess both anionic and cationic sites within their molecule, existing as cations, amphoters, or anions depending on the pH of the solution. Anionic surfactants are surfactants that become anions when dissociated in water, and are known to have carboxylic acid, sulfonic acid, or phosphate structures as their hydrophilic group. Cationic surfactants are surfactants that become cations when dissociated in water, and are known to have tetraalkylammonium as their hydrophilic group. Nonionic surfactants are surfactants that have a hydrophilic portion that does not ionize, and are known to include low-molecular-weight systems such as alkyl glycosides, or high-molecular-weight systems such as polyethylene glycol and polyvinyl alcohol.

[0017] The surfactant used in this embodiment is not particularly limited and can be appropriately selected by those skilled in the art. Examples of amphoteric surfactants include CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate), CHAPSO (3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate), and NDSB-211. Examples include (3-[(2-Hydroxyethyl)dimethylammonio]propane-1-sulfonate, 3-[(2-hydroxyethyl)dimethylammonio]propane-1-sulfonate), NDSB-201 (3-(1-Pyridinio)propanesulfonate, 3-(1-pyridino)propanesulfonic acid), and NDSB-256-4T (3-(4-tert-Butyl-1-pyridinio)propanesulfonate, 3-(4-tert-butyl-1-pyridinio)propanesulfonic acid). Examples of anionic surfactants include sodium cholate and sodium deoxycholate. Examples of cationic surfactants include benzalkonium chloride and benzethonium chloride. Other examples of nonionic surfactants include octylphenol ethoxylate (Triton X series (trade name)), polyoxyethylene sorbitan monolaurate (Tween (trade name)), and octyl glucoside.

[0018] The treatment of a virus-containing sample with a surfactant can be carried out by adding the surfactant to the sample and allowing it to react until the final concentration of the surfactant reaches the desired concentration. The final concentration of the surfactant varies depending on the surfactant used, but those skilled in the art can determine the optimal final concentration through preliminary experiments. For example, when using CHAPS as the amphoteric surfactant and sodium deoxycholate as the anionic surfactant to obtain AAV, it is preferable to add them so that the final concentrations are, for example, about 0.1% to 5.0% and 0.2% to 1.5%, preferably about 0.4% to 1.0%, respectively. The conditions for adding the surfactant to the virus sample and allowing it to react should be conditions that allow the surfactant to exert its effect. There are no particular limitations, but the temperature conditions are, for example, 25°C to 45°C, 30°C to 40°C, preferably about 35°C to 38°C, and the reaction time is, for example, a few minutes to a few hours, 10 minutes to 1 hour, preferably about 30 minutes.

[0019] This embodiment may include a step of filtering a virus-containing sample by tangential flow filtration (TFF) to remove impurities and / or concentrate the virus. TFF is a method of filtering by horizontally feeding a liquid containing the target substance (virus in this embodiment) along the surface of a filtration membrane. This differs from normal flow filtration (NFF), in which the liquid is fed perpendicularly to the surface of the filtration membrane for filtering.

[0020] When performing filtration by the TFF method, it is necessary to select a filtration membrane with an appropriate pore size. However, since filtration membranes (hollow fiber membranes) made of various materials and with different pore sizes are commercially available, those skilled in the art can easily obtain a suitable one. Generally, the particle size of viruses is about 10 nm to 300 nm, for example, AAV is said to be about 18 to 25 nm. It is desirable to select an appropriate filtration membrane considering such virus particle sizes. When removing contaminants smaller than virus particles, filtration by the TFF method may be performed using hollow fiber membranes with pores smaller than the virus particle size, and the circulating liquid containing the virus particles may be recovered. Alternatively, when removing contaminants larger than virus particles, the TFF method may be performed using hollow fiber membranes with pores larger than the virus particle size, and the filtrate containing the virus particles may be recovered.

[0021] In this embodiment, the filtration of the sample by the TFF method may be performed multiple times as needed, and the sample liquid may be dispensed under pressure or without pressure. Those skilled in the art can select appropriate conditions for the TFF method (such as the pore size of the filtration membrane, the liquid dispensing speed, and the temperature) by conducting preliminary experiments.

[0022] Treatment of virus-containing samples with surfactants and filtration by TFF are preferable, but it is preferable to treat low-purity samples (e.g., culture medium of virus-producing cells) with surfactants and then filter them by TFF. However, surfactant treatment and filtration by TFF may each be performed multiple times until the virus is finally obtained, and the order in which they are performed does not matter.

[0023] A second embodiment is a method for producing a virus, (a) A step of culturing virus-producing cells, (b) A step of preparing a sample containing the virus from the virus-producing cells or cell culture medium, (c) A method comprising the step of obtaining a virus from a sample containing the virus prepared in step (b) using the method according to the first embodiment.

[0024] In this embodiment, "virus-producing cell" refers to a cell that produces the elements necessary for forming a virus particle and has the ability to produce a virus. The virus-producing cell may be a cell artificially created to produce a virus, or it may be a cell that has been infected with a virus in the natural environment and has become capable of producing the virus. In this embodiment, the virus-producing cell is preferably an artificially created virus-producing cell, and particularly preferably, the virus is a non-enveloped virus.

[0025] The methods for artificially creating virus-producing cells vary depending on the virus, and detailed information has already been provided in numerous reviews; please refer to those reviews. Here, we will only provide a brief overview of the creation of cells that produce viral vectors. To produce viral particles that function as vectors, virus-producing cells can be created by introducing plasmids into any cell type. These plasmids contain deletions in the regions encoding non-structural proteins (proteins involved in viral replication, etc.) and structural proteins (proteins such as the capsid) of the virus, and instead insert the target gene; plasmids encoding both non-structural and structural proteins of the virus; or plasmids encoding other necessary genes depending on the type of viral vector. For example, in the case of AAV vectors, AAV vector-producing cells can be created by introducing a plasmid containing the target gene, a plasmid containing genes encoding Rep protein (a protein necessary for viral replication) or Cap protein (a protein that makes up the capsid), or a plasmid containing genes encoding adenovirus-derived E1a, E1b, E2, and E4 proteins into HEK293 cells or HEK293T cells.

[0026] The culture conditions for virus-producing cells are already known and can be appropriately selected by those skilled in the art depending on the type of virus. There are no particular limitations, but for example, the cells may be cultured for several days to about 20 days in a medium such as DMEM or IMDM containing necessary supplements (growth factors, amino acids, etc.) and serum at a temperature of about 30-38°C and a CO2 concentration of about 5-10%.

[0027] A sample containing a virus may be an extract obtained by extracting the virus from virus-producing cells, or a crudely purified version of said extract. For example, in the case of viruses released into the culture medium, the culture medium after culturing virus-producing cells may be collected and used as a sample. In the case of viruses accumulated inside cells, the collected virus-producing cells may be disrupted by methods such as freeze-thawing or sonication, and debris may be removed before using the resulting sample. Furthermore, since many reagents and kits for preparing virus-containing samples from virus-producing cells are commercially available, samples may also be prepared using these reagents and kits.

[0028] Furthermore, the method according to the first embodiment can be used to obtain the target virus from a virus-containing sample. The virus produced by the method according to the second embodiment can be used for various purposes as a highly pure and biologically active virus.

[0029] Where this specification is translated into English and contains the singular forms of "a," "an," and "the," it shall be understood to include both singular and plural forms unless the context clearly indicates otherwise. The present invention will be further explained below with reference to examples, but these examples are merely illustrative of embodiments of the present invention and do not limit the scope of the present invention. [Examples]

[0030] 1. Preparation of culture supernatant for ZsGreen-expressing AAV1 or ZsGreen-expressing AAV9 HEK293 cells were added to a hyperflask (Corning) at a rate of 40,000 / cm³. 2Cells were seeded at a density and cultured in 10% FBS / DMEM. Three days after culturing, pHelper (TaKaRa), pAAVZsGreen (TaKaRa), and pR2C1 (plasmid containing the Rep gene for AAV1 and the Cap gene for the AAV1 serotype, TaKaRa) or pR2C9 (plasmid containing the Rep gene for AAV9 and the Cap gene for the AAV9 serotype, TaKaRa) were transfected, and the cells were cultured for 11 days. After that, the culture supernatant containing AAV1 or AAV9 was collected. The collected culture supernatant was centrifuged at 10,000 × g for 15 minutes, and the resulting supernatant was passed through a 0.45 μm bottle-top filter (Thermo Fisher) and used as the starting sample (sample containing AAV1 or AAV9) for the next experiment.

[0031] 2. Examination of experimental conditions for filtration using the TFF method Unless otherwise specified, TFF filtration was performed using the KrosFlo (registered trademark) KR2i TFF system (Repligen) with a UF membrane (Sterile ReadyToProcess Hollow Fiber Cartridge, 500 kD, 0.5 mm id fiber, size 4 M housing, AdvantaPure tubing, Cytiva). Condition 1 The sample (200 mL) (Sample a) after passing through a bottle-top filter was filtered through a TFF (without applying pressure to the hollow fiber primary outlet) and concentrated fourfold (volume after TFF: 50 mL) (Sample b). The buffer of the concentrated sample was then replaced with PBS through a TFF (using 18 times the volume of PBS after concentration). Immediately after the buffer change, no precipitates were observed, but after standing at room temperature, precipitates gradually became visible. Next, the sample after buffer replacement was ultrafiltered using an Amicon Ultra-15 filter (fraction size: 100 K, Merck) and concentrated 60-fold (sample d), at which point the filter became clogged.

[0032] Condition 2 The sample (200 mL) (Sample b) after passing through a bottle-top filter was filtered through a TFF (pressure was applied to the hollow fiber primary outlet (14.5 psi)) and concentrated fourfold (volume after TFF: 50 mL) (Sample c). The buffer of the concentrated sample was then replaced with PBS through a TFF (18 times the volume of the concentrated sample was used in PBS). Precipitates were observed immediately after the buffer was changed. Next, the sample after buffer replacement was ultrafiltered using an Amicon Ultra-15 filter (fraction size: 100 K, Merck) and concentrated 60-fold (sample e), at which point the filter became clogged.

[0033] Condition 3 The sample (4.8 L) after passing through a bottle-top filter was filtered through a TFF (without applying pressure to the primary outlet of the air fiber) and concentrated (volume after TFF: 125 mL). The buffer of the concentrated sample was then replaced with HNM buffer (50 mM HEPES, 150 mM NaCl, 1 mM MgCl2 pH 7.4) through a TFF (replacement with 200 times the volume of the concentrated sample with HNM buffer). Precipitates were observed immediately after the buffer was changed.

[0034] Condition 4 The sample (220 mL) after passing through the bottle-top filter was filtered through a TFF (without applying pressure to the hollow fiber primary outlet) and concentrated to 2 times its original volume (volume after TFF: 110 mL). The buffer of the concentrated sample was then replaced with HNM buffer through a TFF (replaced with 100 times the volume of the concentrated sample with HNM buffer). After the buffer was replaced, almost no precipitates were observed. Next, the sample after buffer exchange was ultrafiltered using an Amicon Ultra-15 filter (fraction size: 100 K, Merck) to concentrate it 120-fold, and then the buffer was changed to PBS, with almost no clogging occurring.

[0035] The above samples a, b, c, d, and e were subjected to SDS-PAGE to check for residual proteins. Lanes 1, 2, 3, 4, and 5 in Figure 1 correspond to samples a, b, c, d, and e, respectively. By filtering the culture supernatant containing AAV1 through TFF, it was confirmed that other contaminants besides the virus were removed, and the AAV1 capsid proteins, VP1, VP2, and VP3, were relatively concentrated (lanes 4 and 5 in Figure 1). For SDS-PAGE, 30 μL of samples a, b, c, d, and e are prepared using NuPAGE. TM After adding 10 μL of LDS Sample Buffer (4×), the sample was heated at 90°C for 10 minutes to prepare it. Electrophoresis gel (NuPAGE) TM (4 to 12%, Bis-Tris, 1.0 mm, Mini Protein Gel, 10-well, Invitrogen) Heat-treated sample 35 μL / well, Marker (PageRuler) TM Unstained Protein Ladder (Thermo Fisher) 3 μL / well was loaded. Electrophoresis was performed at CV 200V for 50 minutes, after which the gel was immersed in Oriole fluorescent gel stain (BioRad) and shaken for 90 minutes. The staining solution was then replaced with water and shaken for 10 minutes, after which images were acquired using the ChemiDoc MP Imaging System (BioRad).

[0036] 3. Examination of the effects of surfactants 3-1. Examination using AAV1 To investigate the effect of surfactants on the purification efficiency (purity) of virus particles, we first examined the effect of sodium deoxycholate, an anionic surfactant. The culture supernatant containing AAV1 was centrifuged at 10,000 × g for 15 minutes. The obtained supernatant was passed through a 0.45 μm bottle-top filter. Sodium deoxycholate was added to the supernatant after passing through the bottle-top filter to a final concentration of 0.5%, and the mixture was stirred at 37°C for 30 minutes (sample f). The supernatant treated with sodium deoxycholate (220 mL) was filtered through a TFF (no pressure was applied to the hollow fiber primary outlet) and concentrated 2-fold (volume after TFF: 110 mL) (sample g). Next, the buffer of the concentrated sample was replaced with HNM buffer through a TFF (replaced with 100 times the volume of concentrated sample with HNM buffer), and ultrafiltration was performed using an Amicon Ultra-15 filter (fraction size: 100 K, Merck) to concentrate 120-fold (sample h), at which point almost no clogging was observed. Upon examination of the obtained sample h for contaminating proteins, it was found that the amount of contaminating proteins decreased after treatment with sodium deoxycholate. Therefore, we also investigated the effects of other surfactants.

[0037] In addition to the anionic surfactant sodium deoxycholate, we investigated the effects of the nonionic surfactant octyl glucoside and the amphoteric surfactant CHAPS. No surfactant treatment The culture supernatant containing AAV1 was centrifuged at 10,000 × g for 15 minutes. The obtained supernatant was passed through a 0.45 μm bottle-top filter. The supernatant (205 mL) passed through the bottle-top filter was filtered through a TFF (without applying pressure to the primary outlet of the hollow fiber) and concentrated (volume after TFF: 50 mL). Then, the buffer of the concentrated sample was replaced with HNM buffer through a TFF (replaced with 2 L of HNM buffer), and the primary side of the hollow fiber was washed three times (volume after washing: 165 mL) (Sample i).

[0038] Nonionic surfactants The culture supernatant containing AAV1 was centrifuged at 10,000 × g for 15 minutes. The obtained supernatant was passed through a 0.45 μm bottle-top filter. Octyl glucoside, a nonionic surfactant, was added to the supernatant after passing through the bottle-top filter to a final concentration of 0.5%, and the mixture was stirred at 37°C for 30 minutes. The supernatant treated with octyl glucoside (212 mL) was filtered through a TFF (no pressure was applied to the primary side outlet of the hollow fiber) and concentrated (volume after TFF: 50 mL). Next, the buffer of the concentrated sample was replaced with HNM buffer through a TFF (replaced with 2 L of HNM buffer), and the primary side of the hollow fiber was washed three times (volume after washing: 164 mL) (sample j).

[0039] Amphoteric surfactant The culture supernatant containing AAV1 was centrifuged at 10,000 × g for 15 minutes. The obtained supernatant was passed through a 0.45 μm bottle-top filter. CHAPS, an amphoteric surfactant, was added to the supernatant after passing through the bottle-top filter to a final concentration of 0.5%, and the mixture was stirred at 37°C for 30 minutes. The supernatant treated with CHAPS (219 mL) was filtered through a TFF (without applying pressure to the primary side outlet of the hollow fiber) and concentrated (volume after TFF: 46 mL). Next, the buffer of the concentrated sample was replaced with HNM buffer through a TFF (replaced with 2 L of HNM buffer), and the primary side of the hollow fiber was washed three times (volume after washing: 167 mL) (sample k).

[0040] Anionic surfactants The culture supernatant containing AAV1 was centrifuged at 10,000 × g for 15 minutes. The obtained supernatant was passed through a 0.45 μm bottle-top filter. Sodium deoxycholate, an anionic surfactant, was added to the supernatant after passing through the bottle-top filter to a final concentration of 0.5%, and the mixture was stirred at 37°C for 30 minutes. The supernatant treated with sodium deoxycholate (214 mL) was filtered through a TFF (no pressure was applied to the primary side outlet of the hollow fiber) and concentrated (volume after TFF: 45 mL). Next, the buffer of the concentrated sample was replaced with HNM buffer through a TFF (replaced with 2 L of HNM buffer), and the primary side of the hollow fiber was washed three times (volume after washing: 160 mL) (sample l).

[0041] Amphoteric surfactant + Anionic surfactant The culture supernatant containing AAV1 was centrifuged at 10,000 × g for 15 minutes. The obtained supernatant was passed through a 0.45 μm bottle-top filter. To the supernatant passed through the bottle-top filter, the amphoteric surfactant CHAPS was added to a final concentration of 1%, and the anionic surfactant sodium deoxycholate was added to a final concentration of 0.5%, and the mixture was stirred at 37°C for 30 minutes. The supernatant treated with surfactants (238 mL) was filtered through a TFF (no pressure was applied to the hollow fiber primary outlet) and concentrated (volume after TFF: 25 mL). Next, the buffer of the concentrated sample was replaced with HNM buffer through a TFF (replaced with 2 L of HNM buffer), and the primary side of the hollow fiber was washed three times (volume after washing: 172 mL) (sample m).

[0042] The above samples i, j, k, l, and m were subjected to SDS-PAGE to confirm the presence of residual proteins. Lanes 1, 2, 3, 4, and 5 in Figure 2 correspond to samples i, j, k, l, and m, respectively. Before filtering with TFF, treatment of the culture supernatant containing AAV1 with an amphoteric surfactant (CHAPS) and an anionic surfactant (sodium deoxycholate) was found to be highly effective in removing contaminating proteins and highly enriching the capsid proteins VP1, VP2, and VP3 (Figure 2, lane 5). Sample processing and electrophoresis for SDS-PAGE were performed under the conditions described in section 2 above.

[0043] Furthermore, the amount of HCP (host cell protein) in samples i, j, k, l, and m was quantified using the HEK 293 Host Cell Protein ELISA Kit (Cygnus), and the amount of DNA was quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher). The quantification results are shown in Table 1. [Table 1] (In Table 1, "SM" indicates a sample that has not been treated with surfactants and has not been filtered using TFF.) As shown in Table 1, compared to the untreated sample (sample i), treatment with amphoteric surfactants and anionic surfactants (sample m) dramatically reduced both HSP and DNA, with the amount of HSP becoming approximately 1 / 10 and the amount of DNA becoming approximately 1 / 5.

[0044] 3-2. Examination using AAV9 Next, we investigated the effect of surfactants on the purification of AAV9, replacing AAV1. The culture supernatant containing AAV9 was centrifuged at 10,000 × g for 15 minutes. The obtained supernatant was passed through a 0.45 μm bottle-top filter. To the supernatant passed through the bottle-top filter, the amphoteric surfactant CHAPS was added to a final concentration of 1%, and the anionic surfactant sodium deoxycholate was added to a final concentration of 0.5%, and the mixture was stirred at 37°C for 30 minutes. The supernatant treated with surfactants was filtered through TFF and concentrated. Next, the buffer of the concentrated sample was replaced with HNM buffer through TFF, and the primary side of the hollow fiber was washed three times. To determine whether the activity of surfactants is lost when stored as a stock solution, we compared the degree of purification when using surfactants prepared fresh for use (sample m) and when using surfactants prepared from stock solutions (CHAPS; 10%, sodium deoxycholate; 10%, both prepared one week prior) (sample 0) (Figure 3). Lane 1 in Figure 3 shows the results using surfactants prepared fresh for use, and lane 2 shows the results using surfactants prepared from stock solutions. These results indicate that the purification method using both anionic and amphoteric surfactants is effective not only for AAV1 but also for AAV9. Furthermore, it was confirmed that the surfactants retain their activity even when stored as a solution, and that high-purity purification of virus particles is possible even when using surfactants prepared from a stock solution.

[0045] 4. Examination of the infectivity of the purified virus. The infectivity of viruses purified by the present invention was compared with that of viruses purified by conventional methods (CsCl (cesium chloride) ultracentrifugation method, affinity chromatography method). The following samples were obtained: culture supernatant (Sup) of ZsGreen-expressed AAV1; solution (TFF) obtained by treating the culture supernatant with 1% CHAPS and 0.5% sodium deoxycholate at 37°C for 30 minutes (stirred), followed by TFF concentration and HNM buffer exchange; a sample (CsCl) obtained by mixing 1135.6 mg / mL CsCl solution and culture supernatant in a 1:1 ratio and allowing it to stand at 4°C for 2 days as a sample mimicking CsCl ultracentrifugation purification; and affinity chromatography (POROS). TM CaptureSelect TM Samples (affinity) were prepared by purifying AAVX Affinity Resin (Thermo Fisher) and eluting with glycine hydrochloride (pH 2.0). After the above treatment, all samples except the TFF samples were replaced with HNM buffer using Amicon 100K. These samples were then added to HEK293 cells cultured in 12 wells, with 1 × 10⁶ samples added. 9Cells were added to a concentration of vg / well (n=3) and cultured for 2 days. Cells were detached from each well, and the percentage of ZsGreen-positive cells was calculated by FACS.

[0046] As shown in Figure 4, the infectivity of the virus purified by the present invention was found to be approximately the same as that of the virus stored under the same conditions as when using CsCl density gradient centrifugation (CsCl), and was found to be far superior to that of the virus purified by affinity chromatography (affinity).

[0047] 5. Morphological observation of purified virus particles using an electron microscope. After treatment with amphoteric and anionic surfactants, the morphology of AAV1 (sample m) or AAV9 (sample o), purified by filtration with TFF, was observed using an electron microscope. A collodion membrane (400 mesh, Cu) was hydrophilized using an ion bombarder (for 1.7 seconds). 3 μl of sample m or sample o was placed on the hydrophilized collodion membrane and allowed to stand for 1 minute. Moisture from the sample was removed by placing filter paper against the edge of the collodion membrane. 3 μl of DDW (diionized distilled water) passed through a 0.22 μm filter was placed on the collodion membrane and allowed to stand for 10 seconds, after which excess moisture from the sample was removed with filter paper. The DDW treatment and excess moisture removal with filter paper steps were repeated a total of two times. Then, staining solution (3 μl) was placed on the collodion membrane and allowed to stand for 10 seconds, after which excess moisture was removed by placing filter paper against the edge of the collodion membrane. The collodion membrane was allowed to stand for 2 minutes, dried overnight in a desiccator, and observed under a microscope. A microscopic image of purified AAV1 is shown in Figure 5, and a microscopic image of purified AAV9 is shown in Figure 6. Almost no hollow particles were observed in the microscopic images in Figures 5 and 6. Therefore, it is considered that the method according to the present invention can efficiently reduce the inclusion of hollow particles, which were difficult to remove by conventional column chromatography methods. [Industrial applicability]

[0048] This invention provides a method for efficiently and highly purely preparing viruses (particles), such as viral vectors. Therefore, it is expected to be useful in medical fields such as gene therapy.

Claims

1. A method for obtaining a non-enveloped virus, comprising the steps of treating a sample containing a non-enveloped virus with an amphoteric surfactant and an anionic surfactant, and further comprising the step of purifying the non-enveloped virus by performing tangential flow filtration (TFF).

2. The method according to claim 1, wherein the amphoteric surfactant is one or more selected from the group consisting of CHAPS, CHAPSO, NDSB-211, and NDSB-201.

3. The method according to claim 1 or 2, wherein the anionic surfactant is sodium cholate and / or sodium deoxycholate.

4. The method according to any one of claims 1 to 3, wherein the non-enveloped virus is an adeno-associated virus vector.

5. A method for producing a non-enveloped virus, (a) A step of culturing non-enveloped virus-producing cells, (b) A step of preparing a sample containing a non-enveloped virus from the non-enveloped virus-producing cells or cell culture medium, and (c) A step of obtaining a non-enveloped virus from a sample containing a non-enveloped virus prepared in step (b) by the method according to any one of claims 1 to 4. The method, including the method described above.

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