Methods for purifying adeno-associated viruses

Abstract

To provide methods for improving the separability of complete AAV capsid and empty AAV capsid for improved quality of gene therapy products.MEANS FOR SOLVING THE PROBLEM: Provided is a method for isolating complete adeno-associated virus (AAV) capsid, including: a first step of preparing a mixture of the complete AAV capsid and empty AAV capsid to have an initial conductivity in a range of 0.5 mS / cm to 10 mS / cm and subjecting a sample to a first anion exchange chromatography medium; a second step of eluting and separating the complete AAV capsid and the empty AAV capsid by increasing the conductivity of a linear gradient by changing a ratio of an equilibration buffer and a salt-containing buffer; and a third step of preparing a fraction containing the complete AAV capsid recovered in the above step to have an initial conductivity in the range of 0.5 mS / cm to 10 mS / cm and subjecting the fraction to a second anion exchange chromatography medium.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for purifying an adeno-associated virus. [Background technology]

[0002] Adeno-associated virus (AAV) is a relatively small virus with a particle size of about 20 nm that belongs to the Parvoviridae family, and can be used as a gene therapy vector. Although it has a single-stranded DNA genome, empty particles in which the genomic DNA is not packaged can be generated during the virus production process. In large-scale production of AAV for use as a gene therapy vector, the separation and removal of these empty particles is a major challenge.

[0003] The presence of empty particles may increase the number of particles actually delivered when a specific gene copy number is designed to be administered, and may induce an undesired immune response. It is very difficult to separate empty AAV particles from properly packaged complete particles because of their similar properties. The current common method, density gradient ultracentrifugation, has issues such as vector activity, recovery rate, and scalability, and it is difficult to obtain complete separation even with chromatography methods that are highly scalable. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2023-37724 A Summary of the Invention [Problem to be solved by the invention]

[0005] The problem to be solved by the present invention is to provide a method for improving the separation of complete and empty AAV capsids in order to improve the quality of gene therapy products. [Means for solving the problem]

[0006] The present invention has been made to solve the above problems, and provides a method for isolating intact AAV capsids from a mixture of intact and empty AAV capsids, comprising a first step of preparing the mixture to have an initial conductivity within a specific range and subjecting the sample to a first anion exchange chromatography medium, a second step of eluting and separating intact and empty AAV capsids by a linear gradient of increasing conductivity by changing the ratio of an equilibration buffer and a salt-containing buffer, and a third step of collecting fractions containing intact AAV capsids separated in the above step and subjecting them to a second anion exchange chromatography medium with an initial conductivity range similar to that of the first anion exchange chromatography medium. Effect of the Invention

[0007] According to the method using the anion exchange chromatography medium of the present invention, it is possible to further reduce empty AAV capsids that are contaminated with AAV vectors and that could not be completely removed by conventional methods, thereby improving purity. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 is a chromatogram showing the separation of intact and empty AAV capsids by gradient elution using an anion exchange chromatography medium as Example 1, a first anion chromatography run. (This step corresponds to Comparative Example 1.)

[0009] [Diagram 2] FIG. 2 is a chromatogram showing separation of intact and empty AAV capsids by gradient elution using an anion exchange chromatography medium as Example 1;

[0010] [Diagram 3]FIG. 2 is a chromatogram showing the separation of intact and empty AAV capsids by gradient elution using a commercial anion exchange chromatography medium as Example 2, a first anion chromatography run. (This step corresponds to Comparative Example 2.)

[0011] [Figure 4] FIG. 2 is a chromatogram showing separation of intact and empty AAV capsids by gradient elution using a commercially available anion exchange chromatography medium as Example 2; DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention includes the following items. [1] A method for isolating complete adeno-associated virus (AAV) capsids from a mixture of complete and empty AAV capsids, comprising: a first step of preparing the mixture to have an initial conductivity in the range of 0.5 mS / cm to 10 mS / cm and subjecting the sample to a first anion exchange chromatography medium; A second step of eluting and separating the intact and empty AAV capsids by a linear gradient of increasing conductivity by varying the ratio of the equilibration buffer and the salt-containing buffer; and a third step of adjusting the fraction containing intact AAV capsids recovered in the previous step to an initial conductivity in the range of 0.5 mS / cm to 10 mS / cm and subjecting the fraction to a second anion exchange chromatography medium. The method includes:

[0013] [2] The method according to item [1], wherein the first anion exchange chromatography medium and the second anion exchange chromatography medium both contain quaternary ammonium salt functional groups.

[0014] [3] The method according to item [1] or [2], wherein the first anion exchange chromatography medium and the second anion exchange chromatography medium are the same type of anion exchange chromatography medium.

[0015] [4] The method according to paragraph [1] or [2], wherein the first anion exchange chromatography and the second anion exchange chromatography are carried out using an anion exchange chromatography medium packed in a single column.

[0016] [5] The method according to item [1] or [2], wherein the first anion exchange chromatography medium and the second anion exchange chromatography medium are different types of anion exchange chromatography medium.

[0017] The present invention provides chromatographic purification methods for isolating intact AAV capsids from a mixture of intact and empty AAV capsids, which may typically result from AAV production in cell culture, a clarification step (e.g., tangential flow filtration or a clarification filter), and initial purification (e.g., purification by affinity chromatography).

[0018] The serotype of AAV used in the method is not particularly limited, and any AAV is suitable. The serotypes of AAV include 1, 2, 3, 4, 5, 6, 7, 8, 9, and derivatives and modifications thereof. Preferably, AAV serotype 2 (AAV2) can be used.

[0019] The anion exchange chromatography medium includes a positively charged resin or membrane, and any chromatography medium can be used, such as Cellufine™ MAX Qh (manufactured by JNC Corporation), CIMmultus™ QA (manufactured by SARTORIUS), POROS™ HQ (manufactured by Thermo Fisher Scientific), and Capto™ Q (manufactured by Global Life Science Technologies Japan, Inc.).

[0020] The method uses an equilibration buffer and an elution buffer. The pH of the equilibration buffer and the pH of the elution buffer are both suitable to be basic. For example, the pH is 7.0 or more, 7.5 or more, 8.0 or more, 8.5 or more, 9.0 or more, 9.5 or more, or 10 or more. It preferably has a pH between 8.5 and 9.5.

[0021] Suitable equilibration and elution buffers are those that have buffering capacity in the basic range (e.g., pH 7.0 or higher). Examples include 2-amino-2-methyl-1-propanol (AMP), N,N-bis-(2-hydroxyethyl)-glycine (bicine), 1,3-bis[tris(hydroxymethyl)-methylamino]propane (bis-tris-propane), boric acid, 3-(cyclohexylamino)-propanesulfonic acid (CAPS), 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO), sodium carbonate, 2-(N-cyclohexylamino)-ethanesulfonic acid (CHES), glycine, glycylglycine, N-(2-hydroxyethyl)-piperazine-N'-ethanesulfonic acid (HEP ES), N-(2-hydroxyethyl)piperazine-N'-3-propanesulfonic acid (HEPPS), N-(2-hydroxyethyl)-piperazine-N'-2-hydroxypropanesulfonic acid (HEPPSO), 3-{[tris(hydroxymethyl)-methyl]-amino}-propanesulfonic acid (TAPS), 2-aminoethanesulfonic acid, triethanolamine (TEA), 2-[tris(hydroxymethyl)-methylamino]-ethanesulfonic acid (TES), N-[tris(hydroxymethyl)-methyl]-glycine (tricine), tris(hydroxymethyl)-aminomethane (Tris) can be used. Preferably, a Tris buffer can be used.

[0022] The equilibration buffer used in the method has a conductivity of 10 mS / cm or less, preferably 5 mS / cm or less, more preferably 2 mS / cm or less.

[0023] The elution buffer used in the method is any of the equilibration buffers described herein with the addition of an inorganic salt. Preferably, the equilibration buffer and the elution buffer are the same type (e.g., Tris buffer). The salt added to the elution buffer can be any inorganic salt, but preferably sodium chloride. In certain embodiments, the elution buffer has a conductivity of 10 mS / cm or more, for example, 15 mS / cm or more, 20 mS / cm or more, 30 mS / cm or more, 40 mS / cm or more, 50 mS / cm or more, 60 mS / cm or more, 70 mS / cm or more, or 80 mS / cm or more.

[0024] In this method, a mixture of intact AAV capsids and empty AAV capsids is diluted with an equilibration buffer to prepare the mixture to have a predetermined initial conductivity. Although the dilution ratio depends on the initial conductivity of the mixture and the equilibration buffer, the mixture is prepared to have any initial conductivity in the range of 0.5 mS / cm to 10 mS / cm. Preferably, the mixture is prepared to have a conductivity in the range of 0.5 mS / cm to 5.0 mS / cm. More preferably, the mixture is prepared to have a conductivity in the range of 2.0 mS / cm to 5.0 mS / cm. The sample prepared to have a conductivity within the range is subjected to an anion exchange chromatography medium, and intact AAV capsids and empty AAV capsids are adsorbed to the anion exchange chromatography medium. Both samples to be subjected to the first anion exchange chromatography and the second anion exchange chromatography are prepared to have the conductivity range.

[0025] In this method, the conductivity is increased in a linear gradient by gradually changing the mixing ratio of the equilibration buffer and the elution buffer. Intact and empty AAV capsids adsorbed on the anion exchange chromatography medium are eluted and separated by the method. The initial mixing ratio of the equilibration buffer and the elution buffer is arbitrary, for example, within the range of 100:0 to 60:40. The final mixing ratio of the equilibration buffer and the elution buffer is also arbitrary, for example, within the range of 50:50 to 0:100.

[0026] In this method, the absorbance at 280 nm and the absorbance at 260 nm are measured during chromatography to distinguish between complete and empty AAV capsids. If the ratio of absorbance at 260 nm to absorbance at 280 nm (absorbance at 260 nm / absorbance at 280 nm) is 1 or more, it is considered to reflect the DNA concentration present inside the complete AAV capsid, and it can be estimated that the abundance ratio of complete AAV capsids is high. On the other hand, if the absorbance ratio is less than 1, it is considered to indicate a high protein concentration, and it can be estimated that the abundance ratio of empty AAV capsids in which no DNA is present inside the particles is high. Since the ratio of absorbance at 260 nm to absorbance at 280 nm reflects the proportion of complete AAV capsids as described above, it can be evaluated that the higher the ratio of absorbance at 260 nm to absorbance at 280 nm, the more complete AAV capsids are contained, and the more empty AAV capsids are removed. From the elution peak obtained in the first anion exchange chromatography, a fraction having an absorbance ratio at 260 nm to 280 nm of, for example, 1 or more is collected, the conductivity is adjusted by the method described herein, and the fraction is subjected to a second anion exchange chromatography. EXAMPLES

[0027] [Example 1] The AAV2 eluate purified by affinity chromatography was used as a purification sample containing complete and empty AAV capsids. This purification sample was subjected to anion exchange chromatography according to the following procedure. In order to sufficiently reduce the conductivity of the purification sample (for example, to 6 mS / cm), the sample was diluted 3.4 times with 50 mM Tris-HCl, pH 9.0, 2 mM MgCl2, 0.01% (V / V) Pluronic (registered trademark, manufactured by BASF) F-68. There is no limit to the conductivity after dilution as long as it is sufficient to retain complete empty particles, and it can be used even if it is reduced to, for example, 2 mS / cm.

[0028] The complete AAV capsid and empty AAV capsid prepared in this manner were subjected to a medium obtained by attaching dextran modified with a quaternary ammonium salt functional group via an epoxy group to porous particles obtained by the method described in Example 1 of JP 2023-37724 A. The method for producing porous particles is shown below. Cellulose acetate was added to a mixed solvent of benzyl alcohol, 1-hexanol, and polypropylene glycol, and stirred, then heated and stirred to obtain a cellulose acetate solution. In addition, PVA and sodium carboxymethylcellulose were added to pure water saturated with the mixed solvent, heated, stirred, and dissolved to obtain a dispersion medium. Next, this dispersion was left at room temperature and cooled, and when it reached 40°C, the obtained cellulose acetate particles were thoroughly washed with a large amount of water and then methanol. After washing again with water, the obtained cellulose acetate spherical particles were passed through a sieve to obtain porous particles with a volume average particle size of 50 μm. The porous particles thus obtained were added to a solution in which Na2SO4 was dissolved in pure water and stirred. Next, NaOH was added and stirred to react, thereby saponifying the particles. After the reaction, the temperature was raised and stirring was continued, and NaOH solution and NaBH4 were added to dissolve and react. After dissolution, NaOH solution and epichlorohydrin were added, and further reaction was performed after the addition was completed. After cooling, acetic acid was added to neutralize. The reaction mixture was filtered and then washed with pure water to obtain the desired crosslinked porous particles. The chromatography system used was AKTA (trademark, manufactured by Global Life Science Technologies Japan, Inc.) Avant 25, and the protocol is shown in Table 1.

[0029] Table 1 TIFF2025074394000001.tif79161

[0030] In elution 1, the mixing ratio of the equilibration buffer and elution buffer is increased stepwise from 0% (V / V) to 100% (V / V) to elute and separate intact and empty AAV capsids. The purpose of elution 2 is to wash the capsid by increasing the salt concentration to a uniform concentration. In the elution fractions from the first anion chromatography, fractions with an absorbance ratio of 260 nm to 280 nm of 1 or more were pooled (Figure 1). The steps up to this point constitute Comparative Example 1.

[0031] In this Example 1, since the conductivity of the first anion chromatography protocol is adjusted to be higher than the range in which the empty AAV capsid is adsorbed to the anion chromatography medium, most of the empty AAV capsid is eluted in the flow-through fraction. The fraction containing the complete AAV capsid thus obtained from the first anion chromatography was diluted with an equilibration buffer (50 mM Tris-HCl, pH 9.0, 2 mM MgCl2, 0.01% (V / V) Pluronic (registered trademark, BASF) F-68) to sufficiently reduce the conductivity (e.g., 2 mS / cm) and then applied to the second anion chromatography medium, which is the same column (FIG. 2). The results of comparing the ratio of absorbance at 260 nm to 280 nm of the complete AAV capsid portion in the first and second anion chromatography are shown in Table 2.

[0032] Table 2 TIFF2025074394000002.tif2668

[0033] The peak top portion is expected to be the most abundant in complete AAV capsids, and the absorbance ratio is considered to be the highest. The peak area value is the ratio of the integral value of the absorbance of the entire peak, and is considered to be appropriate as an evaluation when pooling complete AAV capsids. Comparing the absorbance ratios of the first anion chromatography (Comparative Example 1) and the second anion chromatography (Example 1), both the peak top and peak area values ​​are improved, and it is considered that the content of complete AAV capsids is improved. In addition, the difference between the peak top and peak area values ​​is reduced, suggesting that the removal of empty AAV capsids ultimately resulted in the obtaining of uniform complete AAV capsids.

[0034] [Example 2] In this Example 2, the applicability of the method of the present invention was verified using a commercially available chromatography medium. The purification sample after the affinity chromatography purification used in Example 1 was diluted with an equilibration buffer (50 mM Tris-HCl, pH 9.0, 2 mM MgCl2, 0.01% (V / V) Pluronic (registered trademark, manufactured by BASF) F-68) and then subjected to CIMmultus (registered trademark) QA. The chromatography system used was AKTA (trademark, manufactured by Global Life Science Technologies Japan Co., Ltd.) Avant25, and the protocol is shown in Table 3. As in Example 1, in the elution fraction of the first anion chromatography (corresponding to Comparative Example 2), the range in which the ratio of absorbance at 260 nm to 280 nm was 1 or more was pooled (FIG. 3).

[0035] Table 3 TIFF2025074394000003.tif83153

[0036] In this Example 2, the conductivity was adjusted so that the empty AAV capsid was adsorbed to the anion chromatography medium, and therefore the UV peaks in the eluted portion were obtained in the order of empty AAV capsid and complete AAV capsid. The complete AAV capsid thus obtained from the first anion chromatography was diluted with an equilibration buffer (50 mM Tris-HCl, pH 9.0, 2 mM MgCl2, 0.01% (V / V) Pluronic (registered trademark, BASF) F-68) to sufficiently lower the conductivity (e.g., 2 mS / cm) before being subjected to the second anion chromatography (FIG. 4). The results of comparing the ratio of absorbance at 260 nm to 280 nm of the complete AAV capsid portion in the first and second anion chromatography are shown in Table 4.

[0037] Table 4 TIFF2025074394000004.tif2669

[0038] As in Example 1, it was confirmed that the absorbance ratio between the first anion chromatography (Comparative Example 2) and the second anion chromatography (Example 2) was improved, and the difference between the peak top and peak area values ​​was reduced. Therefore, the purification method of the present invention is applicable even when a commercially available product is used.

Claims

1. 1. A method for isolating adeno-associated virus (AAV) capsids from a mixture of complete AAV capsids and empty AAV capsids, comprising: a first step of preparing the mixture to have an initial conductivity in the range of 0.5 mS / cm to 10 mS / cm and subjecting the mixture to a first anion exchange chromatography medium; A second step of eluting and separating intact and empty AAV capsids by a linear gradient of increasing conductivity by varying the ratio of the equilibration buffer and the salt-containing buffer; and a third step of adjusting the fraction containing intact AAV capsids recovered in the previous step to an initial conductivity in the range of 0.5 mS / cm to 10 mS / cm and subjecting it to a second anion exchange chromatography medium.

2. 2. The method of claim 1, wherein the first anion exchange chromatography medium and the second anion exchange chromatography medium both comprise quaternary ammonium salt functional groups.

3. 3. The method of claim 1 or 2, wherein the first anion exchange chromatography medium and the second anion exchange chromatography medium are the same type of anion exchange chromatography medium.

4. 3. The method of claim 1 or 2, wherein the first anion exchange chromatography and the second anion exchange chromatography are carried out using an anion exchange chromatography medium packed in a single column.

5. 3. The method of claim 1 or 2, wherein the first anion exchange chromatography medium and the second anion exchange chromatography medium are different types of anion exchange chromatography medium.

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