Method for purifying adeno-associated virus vector

Abstract

The invention relates to a purification method of an adeno-associated virus vector, and belongs to the technical field of adenovirus refining and purification. The method comprises the following steps: firstly, removing free nucleic acid in an adeno-associated virus culture supernatant, then carrying out (1) gel filtration chromatography, (2) bifunctional composite chromatography comprising volume exclusion and adsorption, and (3) anion exchange chromatography, or connecting the chromatography modes in series for use, and finally, carrying out centrifugal concentration on a chromatography harvest liquid to obtain the purified adeno-associated virus vector. When the method is used for purifying the adeno-associated virus, the condition is mild, the possible adverse effect of the traditional affinity chromatography technology on the virus stability is avoided, the steps and flow of the whole purification process are greatly shortened, industrial amplification production is facilitated, and the method is suitable for library screening of the new serotype adeno-associated virus and has a wide application prospect. The method has wide applicability and a good application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of adenovirus fine purification, in particular to a method for purifying an adeno-associated virus vector. Background Art

[0002] Gene therapy is a method that aims to safely and stably introduce foreign genes into the patient's body, achieving therapeutic effects by allowing gene fragments to enter cells to express the desired products. The key to gene therapy is to select a suitable gene vector. Recombinant adeno-associated virus vectors are currently recognized as safe and reliable gene therapy vectors that can mediate long-term and stable expression of foreign genes in the body, and have low immunogenicity, and have broad application prospects. Recombinant adeno-associated virus vectors can be divided into multiple serotypes, each with different tissue preferences. When delivering gene therapy drugs to specific tissues, the vast majority of existing serotypes tend to accumulate to varying degrees in other tissues outside the target cells, affecting the therapeutic effect and may cause toxicity or other adverse reactions in certain tissues. It has become an industry consensus to develop new serotypes with strong tissue preferences, high yields, and low toxicity.

[0003] One of the obstacles limiting the use of recombinant adeno-associated viral vectors is the development of a universal and scalable purification process. During the preparation of AAV, various impurities such as proteins, nucleic acids, cell fragments, etc. are inevitably produced. If these impurities are not removed, they may trigger an immune response, causing host cells to produce antibodies, thereby affecting the infection efficiency and therapeutic effect of the viral vector. Some impurities may be potentially toxic and cause damage to host cells. Purification can significantly reduce the content of these impurities, thereby improving the safety of viral vectors. The purified AAV has a higher purity, which means that more viral particles are intact and functional, which helps to improve the infection efficiency of viral vectors and ensure that exogenous genes can be successfully introduced into target cells.

[0004] The traditional laboratory-scale purification method for recombinant adeno-associated virus vectors is ultracentrifugation. The biggest drawback of this method is that it is difficult to scale up to production scale. Chromatographic technology and membrane separation technology, as representatives of modern purification methods, have been widely used in large-scale production of biopharmaceuticals. Currently, all recombinant adeno-associated virus vector drugs on the market are purified by chromatography, but these reported technologies usually have the following problems: (1) Affinity chromatography technology is used as the capture step of recombinant adeno-associated virus vectors, and the new serotype AAV may have a low affinity level due to changes in the capsid, affecting adsorption, or the affinity level is too high, resulting in drastic elution conditions, affecting the stability of the recombinant adeno-associated virus vector; (2) Affinity chromatography fillers are expensive and need to be reused to reduce costs. In order to maintain sufficient affinity, many recombinant adeno-associated virus vector affinity fillers cannot be cleaned and regenerated using sodium hydroxide, and there is a risk of sample residue and cross contamination; (3) Patent CN107630037A discloses a method for capturing adeno-associated virus vectors using cation exchange chromatography, which results in the pH environment of the mobile phase being acidic. In order to achieve satisfactory separation results, some methods often use a pH value below 4. Low pH environments affect the stability and infection activity of AAV. Different serotypes have different sensitivities to acidic environments. For example, the stability of AAV2 decreases below pH = 5.5, but AAV5 is more sensitive to acidic environments, with an optimal pH of up to 7.5. For a new serotype, the effect of long-term exposure to such an acidic environment on viral stability or infection activity is unknown, and it is difficult to become a good universal purification process; (4) Liquid changes are required between multiple chromatography steps, and the purification process route is relatively long, which puts higher requirements on the stability and contamination control of recombinant adeno-associated virus vectors; (5) New serotypes can be obtained through library screening. In order to maintain a high library capacity, the recombinant adeno-associated virus vector library needs to have a large amount of virus and rich diversity. The use of affinity chromatography will selectively retain vectors with high affinity for the filler, resulting in a large loss of library capacity; if traditional ultracentrifugation is used for purification, due to the large amount of virus in the library, ultracentrifugation is difficult to scale up, resulting in a long purification cycle and low purity. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for purifying an adeno-associated virus vector, which first removes free nucleic acids in the adeno-associated virus culture supernatant, then performs gel filtration chromatography, composite chromatography and anion exchange chromatography, or uses the above three chromatography methods in series, the medium used in the composite chromatography has both size exclusion and adsorption functions, and finally the chromatography harvest liquid is centrifuged and concentrated to obtain the purified adeno-associated virus vector.

[0006] The first object of the present invention is to provide a method for purifying an adeno-associated virus vector, removing free nucleic acids in the adeno-associated virus culture supernatant, filtering to obtain a filtrate, sequentially subjecting the filtrate to gel filtration chromatography, composite chromatography and anion exchange chromatography to obtain a chromatography harvest liquid, centrifuging and concentrating the chromatography harvest liquid to obtain a purified adeno-associated virus vector, wherein the composite chromatography uses a multi-mode composite filler.

[0007] Furthermore, the multi-mode composite filler used in the composite chromatography has dual functions of volume exclusion and adsorption.

[0008] Free nucleotides and most small molecule impurities are removed by filtration, small molecule impurities are removed by gel filtration chromatography, free proteins and oligonucleotides are removed by composite chromatography, and impurities adsorbed on the surface of adeno-associated viruses are removed by anion exchange chromatography.

[0009] Furthermore, the gel filtration chromatography column and the composite chromatography column are connected in series, or the gel filtration chromatography column, the composite chromatography column and the anion exchange chromatography column are connected in series.

[0010] Furthermore, the culture supernatant includes microcarrier cell culture supernatant, adherent cell culture supernatant or suspension cell culture supernatant.

[0011] Furthermore, a universal nuclease is added to the adeno-associated virus culture supernatant for incubation, and the amount of the universal nuclease added is 10-40 U / mL.

[0012] Furthermore, the incubation time is 2-4 hours.

[0013] Furthermore, the filtration uses a hollow fiber membrane column or a flat ultrafiltration membrane.

[0014] Furthermore, the pore size of the hollow fiber membrane column or flat ultrafiltration membrane is 100-300 kD.

[0015] In one embodiment of the present invention, a hollow fiber membrane column is used for filtration, and the pore size is 300 kD.

[0016] In one embodiment of the present invention, the filtrate is concentrated and replaced.

[0017] Furthermore, the medium exclusion size of the gel filtration chromatography is 1-100 kD.

[0018] Furthermore, the filler used in the gel filtration chromatography is dextran gel.

[0019] In one embodiment of the present invention, the filler used in the gel filtration chromatography is G25M.

[0020] In one embodiment of the present invention, the mobile phase used in the gel filtration chromatography includes Tris-HCl buffer, sucrose and a non-ionic surfactant, and the pH value thereof is 8.

[0021] In one embodiment of the present invention, the mobile phase includes 0.03 mol / L Tris-HCl buffer, 1% sucrose and 0.01% Poloxamer 188 surfactant.

[0022] Furthermore, the filler of the composite chromatography is selected from Capto Core 700 or Capto Core 400.

[0023] Capto Core is a multi-mode filler with a unique double-layer structure for composite chromatography. Its outer layer is a 5μm passivation layer, which shields all binding sites and precisely controls the pore size. Biomacromolecules with molecular weights greater than 700kDa or 400kDa cannot enter the filler. Molecules smaller than this exclusion limit enter the pores and bind to the octylamine ligand. During the virus purification process, the virus flows through and impurities are adsorbed to the octylamine group in the pores, showing the dual functions of size separation and adsorption chromatography.

[0024] In one embodiment of the present invention, the filler used in the composite chromatography is Capto core 400.

[0025] In one embodiment of the present invention, the mobile phase used in the composite chromatography includes Tris-HCl buffer, sucrose and a non-ionic surfactant, and the pH value thereof is 8.

[0026] In one embodiment of the present invention, the mobile phase includes 0.03 mol / L Tris-HCl buffer, 1% sucrose and 0.01% Poloxamer 188 surfactant.

[0027] Furthermore, the filler of the anion exchange chromatography is a strong quaternary ammonium anion exchange medium.

[0028] Strong quaternary ammonium anion exchange media achieve strong binding ability for anions by introducing quaternary ammonium groups (Q groups). They have stable ion exchange performance in a wide pH range, usually have high dynamic and static loading capacity, can process a large number of biological samples, and are suitable for screening new serotype adeno-associated virus libraries.

[0029] Furthermore, the strong quaternary ammonium anion exchange medium is selected from Diamond Q Mustang, Capto Q, CaptoQ ImpRess or Diamond Q.

[0030] In one embodiment of the present invention, the filler used in the anion exchange chromatography is Capto Q.

[0031] Furthermore, the pH value of the equilibration buffer and the elution buffer used in the anion exchange chromatography is 7-9.5.

[0032] In one embodiment of the present invention, the equilibration buffer comprises 0.03 mol / L Tris-HCl buffer, 1% sucrose and 0.01% Poloxamer 188 surfactant, and the pH value is 8.

[0033] In one embodiment of the present invention, the elution buffer comprises 0.03 mol / L Tris-HCl buffer, 1 mol / L sodium chloride, 1% sucrose and 0.01% Poloxamer 188 surfactant, and the pH value is 8.

[0034] Beneficial effects of the present invention:

[0035] (1) The purification method provided by the present invention does not require the use of traditional affinity chromatography technology, can maintain a stable capture efficiency between different serotypes, while avoiding adverse effects on virus stability, and also ensuring the long-term stability and reusability of the filler, thereby reducing costs in industrial production applications and avoiding waste;

[0036] (2) The purification method provided by the present invention avoids the influence of low pH environment on the stability and infection activity of AAV, ensuring that adeno-associated virus vectors of different serotypes purified by this method can maintain good virus stability and infection activity.

[0037] (3) The purification method provided by the present invention uses gel filtration chromatography, composite chromatography and anion exchange chromatography in series, thereby reducing the steps of liquid replacement, shortening the purification process route, reducing intermediate steps and processing time, and alleviating the pressure that the long process route may cause on the stability and contamination control of the recombinant adeno-associated virus vector;

[0038] (4) The purification method provided by the present invention can reduce the loss of diversity of the recombinant adeno-associated virus library, ensure that the screened viruses have a wide range of genetic diversity and functional characteristics, and is suitable for screening new serotype adeno-associated virus libraries. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0040] Figure 1 The gel filtration column chromatogram in Example 1 of the present invention;

[0041] Figure 2 The adsorption gel filtration column chromatogram in Example 1 of the present invention;

[0042] Figure 3 This is the anion exchange chromatography column spectrum in Example 1 of the present invention;

[0043] Figure 4 The chromatogram of the gel filtration column and the adsorption gel filtration column in series in Example 2 of the present invention;

[0044] Figure 5 The chromatogram of the three-column series connection of the gel filtration column, the adsorption gel filtration column and the anion exchange column in Example 3 of the present invention;

[0045] Figure 6 The SDS-PAGE electrophoresis results of the samples before and after purification in Examples 1-3 of the present invention are shown, wherein lane M is a marker, lanes 1 and 2 are samples before and after purification in Example 1, lanes 3 and 4 are samples before and after purification in Example 2, and lanes 5 and 6 are samples before and after purification in Example 3;

[0046] Figure 7 This is a photo of SDS-PAGE electrophoresis after chromatography purification in the comparative example of the present invention, lane M is a marker, lanes 1-10 are purified adeno-associated virus vectors of different serotypes, and lane 11 is a sample purified by affinity chromatography. DETAILED DESCRIPTION

[0047] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0048] Example 1

[0049] (1) Filtration and clarification

[0050] Collect 500 mL of adeno-associated virus culture supernatant, add 20 U / mL universal nuclease, incubate at 37°C for 2 hours, and deep filter to clarify. Concentrate the clarified liquid using a 300 kd hollow fiber membrane column, change the liquid 3 times, and concentrate it to 50 mL;

[0051] (2) Gel filtration chromatography

[0052] Use mobile phase A with a volume of 320 mL and a flow rate of 80 cm / h to balance the chromatography column. The filler of the chromatography column is G25M and the column volume is 160 mL until the baselines of conductivity, pH value and UV absorption value are stable;

[0053] 50 mL of the filtered clarified collection solution obtained in step (1) was used for sample loading at a sample loading flow rate of 80 cm / h;

[0054] After loading, the column was flushed with mobile phase A with a volume of 420 mL and a flow rate of 80 cm / h, and samples between 0.35 and 0.75 column volumes were collected to obtain 64 mL of collected liquid. The results are as follows: Figure 1 As shown;

[0055] Wherein, mobile phase A: 0.03 mol / L Tris-HCl, 1% sucrose, 0.01% Poloxamer 188, pH=8.0.

[0056] (3) Adsorption gel filtration chromatography

[0057] The column was equilibrated with mobile phase A of 63 mL and a flow rate of 180 cm / h. The column was filled with Capto core 400 and the column volume was 21 mL until the baselines of conductivity, pH value and UV absorbance were stable;

[0058] 64 mL of the gel filtration chromatography collection solution obtained in step (2) was used for sample loading at a flow rate of 180 cm / h and a retention time of 3 minutes;

[0059] After the loading is completed, 53 mL of mobile phase A is used to flush the column, and the loaded flow-through and flushed samples are collected to obtain 117 mL of collection liquid;

[0060] Regenerate the column using 0.5 M NaOH, 30% IPA;

[0061] Wherein, mobile phase A: 0.03 mol / L Tris-HCl, 1% sucrose, 0.01% Poloxamer 188, pH=8.0.

[0062] (4) Anion exchange chromatography

[0063] Use mobile phase A with a volume of 123 mL and a flow rate of 80 cm / h to equilibrate the column. The column packing is Capto Q and the column volume is 41 mL until the conductivity, pH value and UV absorbance baseline are stable;

[0064] 117 mL of the adsorption gel filtration chromatography collection solution obtained in step (3) was used for sample loading at a flow rate of 80 cm / h;

[0065] Use 123 mL of mobile phase A to flush the column;

[0066] Use 0-30% mobile phase B for linear gradient elution over 3 column volumes and collect in separate tubes;

[0067] 30% mobile phase B was maintained for 2 column volumes, and 100% mobile phase B was used to elute the column for 2 column volumes;

[0068] Use 1mol / L NaOH, 2mol / L NaCl to regenerate the column;

[0069] The titer value of the collected solution was tested, and the fractions containing the adeno-associated virus vector were combined to obtain 50 mL of the collected solution;

[0070] Wherein, mobile phase A: 0.03 mol / L Tris-HCl, 1% Sucrose, 0.01% Poloxamer 188, pH = 8.0;

[0071] Mobile phase B: 0.03 mol / L Tris-HCl, 1 mol / L NaCl, 1% Sucrose, 0.01% Poloxamer 188, pH = 8.0.

[0072] (5) Ultrafiltration concentration

[0073] Under room temperature conditions, the anion exchange chromatography collection liquid obtained in step (4) was concentrated by centrifugation using a 30kd ultrafiltration centrifuge tube with a centrifugal force of 3000g to obtain 1 mL of adeno-associated virus vector solution.

[0074] Example 2

[0075] (1) Filtration and clarification

[0076] Collect 1000 mL of adeno-associated virus culture supernatant, add 20 U / mL universal nuclease, incubate at 37°C for 4 hours, and deep filter to clarify. Concentrate the clarified liquid using a 300 kd hollow fiber membrane column, change the liquid 3 times, and concentrate it to 50 mL;

[0077] (2) Gel filtration chromatography and adsorption gel filtration chromatography in series

[0078] Use a volume of 320 mL, flow rate of 80 cm / h, mobile phase A to equilibrate the gel filtration column, filler is G25M, column volume is 160 mL; use a volume of 63 mL, flow rate of 180 cm / h, mobile phase A to equilibrate the adsorption gel filtration column, filler is Captocore 400, column volume is 21 mL, until the conductivity, pH and UV absorption value baselines are stable;

[0079] Connecting the gel filtration column and the adsorption gel filtration column in series;

[0080] 50 mL of the filtered clarified collection liquid obtained in step (1) was used for sample loading at a sample loading flow rate of 7 mL / min, and the sample with UV absorption was collected to obtain 70 mL of collection liquid;

[0081] The gel filtration column was washed with mobile phase A, and the adsorption gel filtration column was regenerated with 0.5 M NaOH, 30% IPA;

[0082] Wherein, mobile phase A: 0.03 mol / L Tris-HCl, 1% sucrose, 0.01% Poloxamer 188, pH=8.0.

[0083] (3) Anion exchange chromatography

[0084] Use mobile phase A with a volume of 123 mL and a flow rate of 80 cm / h to equilibrate the column. The column packing is Capto Q and the column volume is 41 mL until the conductivity, pH and UV absorbance baselines are stable.

[0085] 70 mL of the collected solution obtained in step (2) was used for sample loading at a flow rate of 80 cm / h;

[0086] Use 123 mL of mobile phase A to flush the column;

[0087] Use 0-30% mobile phase B for linear gradient elution over 3 column volumes and collect in separate tubes;

[0088] 30% mobile phase B was maintained for 2 column volumes, and 100% mobile phase B was used to elute the column for 2 column volumes;

[0089] Use 1mol / L NaOH, 2mol / L NaCl to regenerate the column;

[0090] The titer value of the collected solution was tested, and the fractions containing the adeno-associated virus vector were combined to obtain 50 mL of the collected solution;

[0091] Wherein, mobile phase A: 0.03 mol / L Tris-HCl, 1% Sucrose, 0.01% Poloxamer 188, pH = 8.0;

[0092] Mobile phase B: 0.03 mol / L Tris-HCl, 1 mol / L NaCl, 1% Sucrose, 0.01% Poloxamer 188, pH = 8.0.

[0093] (4) Ultrafiltration concentration

[0094] At room temperature, the anion exchange chromatography collection liquid obtained in step (3) was concentrated by centrifugation using a 30 kd ultrafiltration centrifuge tube with a centrifugal force of 3000 g to obtain 0.7 mL of adeno-associated virus vector solution.

[0095] Example 3

[0096] (1) Filter

[0097] 42 mL of adeno-associated virus culture supernatant was collected, 20 U / mL universal nuclease was added, incubated at 37 °C for 4 h, centrifuged at 3000 g for 30 min at room temperature, and filtered through a 0.45 μm PVDF syringe filter;

[0098] (2) Gel filtration chromatography, adsorption gel filtration chromatography and anion exchange chromatography in series

[0099] A volume of 320 mL, flow rate of 80 cm / h, mobile phase A was used to equilibrate the gel filtration column, the filler was G25M, and the column volume was 160 mL; a volume of 63 mL, flow rate of 180 cm / h, mobile phase A was used to equilibrate the adsorption gel filtration column, the filler was Captocore 400, and the column volume was 21 mL; a volume of 123 mL, flow rate of 80 cm / h, mobile phase A was used to equilibrate the anion exchange column, the filler was Capto Q, and the column volume was 41 mL;

[0100] Connecting a gel filtration column, an adsorption gel filtration column and an anion exchange column in series;

[0101] 42 mL of the filtrate obtained in step (1) was used for sample loading at a flow rate of 7 mL / min;

[0102] After loading, use 41 mL of mobile phase A to wash the three columns separately;

[0103] Use 0-30% mobile phase B to perform linear gradient elution over 3 column volumes of the anion exchange column and collect in separate tubes;

[0104] 30% mobile phase B was maintained for 2 column volumes of the anion exchange column, and then 100% mobile phase B was used to elute the anion exchange column for 2 column volumes;

[0105] The collected solution was tested for titer value, and the fractions containing adeno-associated virus vector were combined to obtain 30 mL of collected solution;

[0106] After collection, the three columns were washed and regenerated respectively. The gel filtration column was washed with mobile phase A, the adsorption gel filtration column was regenerated with 0.5 mol / L NaOH, 30% IPA, and the anion exchange column was regenerated with 1 mol / L NaOH, 2 mol / L NaCl;

[0107] Wherein, mobile phase A: 0.03 mol / L Tris-HCl, 1% sucrose, 0.01% Poloxamer 188, pH = 8.0;

[0108] Mobile phase B: 0.03 mol / L Tris-HCl, 1 mol / L NaCl, 1% sucrose, 0.01% Poloxamer 188, pH=8.0.

[0109] (3) At room temperature, the collected solution obtained in step (2) was concentrated by centrifugation using a 30 kd ultrafiltration centrifuge tube with a centrifugal force of 3000 g to obtain 0.4 mL of adeno-associated virus vector solution.

[0110] The samples before and after purification in the above examples 1-3 were analyzed by SDS-PAGE gel electrophoresis. Figure 6 As shown, it is shown that the purification effect of adeno-associated virus is further improved by performing gel filtration chromatography, composite chromatography and anion exchange chromatography in series.

[0111] Comparative Example

[0112] The method provided in Example 3 was used to purify 10 different serotypes of adeno-associated viruses, and one of the serotypes of adeno-associated viruses was purified by affinity chromatography as a comparison. The purified samples were subjected to SDS-PAGE gel electrophoresis. The electrophoresis results are shown in FIG. Figure 7 As shown, it is shown that the purification effect of the method provided in the embodiment of the present invention on adeno-associated viruses is basically consistent with the affinity chromatography effect, and it is universal for adeno-associated viruses of different serotypes and can be applied to the purification of new serum viruses.

[0113] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A method for purifying an adeno-associated virus vector, characterized in that: Free nucleic acid in the adeno-associated virus culture supernatant is removed, and a filtrate is obtained by filtering. The filtrate is subjected to gel filtration chromatography, composite chromatography and anion exchange chromatography in sequence to obtain a chromatography harvest liquid, and the chromatography harvest liquid is centrifuged and concentrated to obtain a purified adeno-associated virus vector. The composite chromatography uses a multi-mode composite filler.

2. The purification method according to claim 1, characterized in that: The gel filtration chromatography column and the composite chromatography column are connected in series, or the gel filtration chromatography column, the composite chromatography column and the anion exchange chromatography column are connected in series.

3. The purification method according to claim 1, characterized in that: The culture supernatant includes microcarrier cell culture supernatant, adherent cell culture supernatant or suspension cell culture supernatant.

4. The purification method according to claim 1, characterized in that: The adeno-associated virus culture supernatant is added with a universal nuclease for incubation, the amount of the universal nuclease added is 10-40 U / mL, and the incubation time is 2-4 hours.

5. The purification method according to claim 1, characterized in that: The filtration uses a hollow fiber membrane column or a flat ultrafiltration membrane, and the pore size of the hollow fiber membrane column or the flat ultrafiltration membrane is 100-300 kD.

6. The purification method according to claim 1, characterized in that: The medium exclusion size of the gel filtration chromatography is 1-100 kD.

7. The purification method according to claim 1, characterized in that: The filler of the composite chromatography is selected from Capto Core 700 or Capto Core 400.

8. The purification method according to claim 1, characterized in that: The filler of the anion exchange chromatography is a strong quaternary ammonium anion exchange medium.

9. The purification method according to claim 8, characterized in that: The filler of the anion exchange chromatography is Diamond Q Mustang, Capto Q, Capto Q ImpRess or Diamond Q.

10. The purification method according to claim 1, characterized in that: The pH value of the equilibration buffer and the elution buffer used in the anion exchange chromatography is 7-9.5.

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