Method for purifying recombinant adeno-associated virus

Abstract

Provided in one or more embodiments of the present description are a method for purifying a recombinant adeno-associated virus, a recombinant adeno-associated virus prepared according to the method, and the use thereof in a gene therapy product. The method comprises: performing a first anion exchange chromatography step on an initial recombinant adeno-associated virus sample to obtain a first eluted sample, the initial recombinant adeno-associated virus sample being obtained by processing a recombinant adeno-associated virus feed solution; and performing a second anion exchange chromatography step on the first eluted sample to obtain a purified recombinant adeno-associated virus, the purified recombinant adeno-associated virus having a total yield of greater than 70% and an empty capsid ratio of less than or equal to 20%.

Description

A method for purifying recombinant adeno-associated virus Technical Field

[0001] This specification relates to the field of purification technology, and more particularly to a method for purifying adeno-associated virus. Background Technology

[0002] Adeno-associated virus (AAV) belongs to the Parvoviridae family. It is a single-stranded DNA virus with good safety, high specificity, high delivery efficiency, and long-term effectiveness. It has been widely used in the field of gene therapy.

[0003] The main production methods for recombinant adeno-associated virus (AAV) are transient plasmid co-transfection, stable cell line production, and baculovirus infection. All three methods produce three types of capsids: full capsids, partial capsids, and empty capsids. Full capsids contain the correct DNA sequence and are the desired product; partial and empty capsids do not contain the target gene and are considered impurities in the production process. Empty capsids account for 50%-95% of the total AAV particles produced by cell production, and their presence is considered harmful: 1) affecting product purity, 2) increasing the immunogenicity of the final product, 3) competing with full capsids for vector binding receptors on infected cells, inhibiting transduction of full capsids, and 4) increasing the overall viral load. The presence of empty capsids seriously affects the safety and efficacy of AAV products; therefore, removing empty capsids and monitoring the empty / full capsid ratio (empty capsid rate) during the production process are critical requirements for any AAV production process.

[0004] Removing empty capsids typically involves two methods: density gradient centrifugation (using cesium chloride or iodixanol) and anion-exchange chromatography (AEX). Density gradient centrifugation, an earlier developed method for purifying AAV, is not suitable for large-scale AAV production. Currently, anion-exchange chromatography (AEX) is more commonly used to separate and remove empty capsids from AAV. Intact capsid AAV and empty capsid AAV have different isoelectric points (pI) due to differences in surface charge; the pI of an empty capsid is around 6.3, while the pI of viral particles containing complete genomic DNA is approximately 5.9. The electrostatic interaction between AAV and the matrix depends on the isoelectric point (pI) of the capsid and the pH of the buffer. Based on this principle, anion-exchange chromatography is used to separate and purify samples, removing impurities (such as empty capsids and partial capsids) and effectively recovering the target vector.

[0005] Current reported methods for purifying AAV using anion exchange chromatography suffer from low yields and poor empty virus removal. Therefore, it is desirable to provide a new method for purifying AAV using anion exchange chromatography that can effectively remove empty viruses and achieve high AAV yields. Summary of the Invention

[0006] This specification provides one or more embodiments of a method for purifying recombinant adeno-associated virus (AAV), comprising: performing a first step of anion exchange chromatography on an initial recombinant AAV sample to obtain a first eluted sample, wherein the initial recombinant AAV sample is obtained by processing a recombinant AAV feed solution; and performing a second step of anion exchange chromatography on the first eluted sample to obtain purified recombinant AAV, wherein the total yield of the purified recombinant AAV is greater than 70% and the empty shell rate is less than or equal to 20%.

[0007] In some embodiments, the elution system B used in the first step of anion exchange chromatography includes at least magnesium sulfate.

[0008] In some embodiments, the elution system B used in the second step of anion exchange chromatography includes at least sodium chloride.

[0009] In some embodiments, the elution system B used in the first step of anion exchange chromatography includes at least sodium chloride.

[0010] In some embodiments, the elution system B used in the second step of anion exchange chromatography includes at least magnesium sulfate.

[0011] In some embodiments, the concentration of magnesium sulfate is in the range of 30-70 mM, and the corresponding elution system B further includes a buffer solution with a concentration range of 5-50 mM and a pH range of 9.0-9.5.

[0012] In some embodiments, the concentration of magnesium sulfate is 40-60 mM, the concentration of the buffer solution is 5-30 mM, and the pH is 9.0-9.4.

[0013] In some embodiments, the concentration of sodium chloride is in the range of 130 mM to 2 M, and the corresponding elution system B further includes a buffer solution with a concentration range of 10 to 40 mM and a pH range of 9.5 to 10.5.

[0014] In some embodiments, the concentration of sodium chloride is 0.5-1.5M, the concentration of the buffer solution is 10-30mM, and the pH is 9.6-10.2.

[0015] In some embodiments, the buffer solution includes Bis-Tris propane, N-methylpiperazine, piperazine, Bis-Tris, triethanolamine, Tris, N-methyldiethanolamine, 1,3-diaminopropane, or ethanolamine.

[0016] In some embodiments, if the first step of the anion exchange chromatography uses an elution system B comprising magnesium sulfate, column equilibration and washing are performed using the elution system B used in the first step of the anion exchange chromatography, wherein the concentration of magnesium sulfate is reduced to 0-10 mM; if the second step of the anion exchange chromatography uses an elution system B comprising sodium chloride, column equilibration and washing are performed using the elution system B used in the second step of the anion exchange chromatography, wherein the concentration of sodium chloride is reduced to 0-100 mM.

[0017] In some embodiments, if the first step of anion exchange chromatography uses an elution system B comprising sodium chloride, column equilibration and washing are performed using the elution system used in the first step of anion exchange chromatography, wherein the concentration of sodium chloride is reduced to 0-100 mM; if the second step of anion exchange chromatography uses an elution system B comprising magnesium sulfate, column equilibration and washing are performed using the elution system B used in the second step of anion exchange chromatography, wherein the concentration of magnesium sulfate is reduced to 0-10 mM.

[0018] In some embodiments, both the first and second steps of anion exchange chromatography employ continuous gradient elution with an elution slope ranging from 0.2% to 0.5% per column volume.

[0019] In some embodiments, the elution slope in the first and second steps of anion exchange chromatography ranges from 0.3 to 0.4% per column volume.

[0020] In some embodiments, the recombinant adeno-associated virus feed solution comprises a feed solution containing recombinant adeno-associated virus particles produced from various cells.

[0021] In some embodiments, the initial recombinant adeno-associated virus (AAV) sample is obtained by further processing the AAV solution by: adjusting the pH of the AAV solution to be consistent with the pH of elution system A used in the first step of anion exchange chromatography, and diluting the AAV solution with elution system A used in the first step of anion exchange chromatography until the conductivity is less than or equal to a first preset value, thereby obtaining the initial recombinant AAV sample, wherein elution system A is a system for removing magnesium sulfate or sodium chloride from elution system B.

[0022] In some embodiments, the first preset value is 2.5 mS / cm (magnesium sulfate system) or 5.0 mS / cm (sodium chloride system).

[0023] In some embodiments, performing a second-step anion exchange chromatography on the first eluted sample to obtain purified recombinant adeno-associated virus includes: adjusting the pH of the first eluted sample to match the pH value of elution system A used in the second-step anion exchange chromatography, and diluting the first eluted sample with elution system A used in the second-step anion exchange chromatography until the conductivity is less than or equal to a second preset value to obtain a treated first eluted sample, wherein elution system A is a system in elution system B in which magnesium sulfate or sodium chloride has been removed; and performing a second-step anion exchange chromatography on the treated first eluted sample to obtain purified recombinant adeno-associated virus.

[0024] In some embodiments, the second preset value is 5.0 mS / cm (sodium chloride system) or 2.5 mS / cm (magnesium sulfate system).

[0025] In some embodiments, the support used in the first and second anion exchange chromatography steps includes anion exchange chromatography column A, which includes Fractogel EMD DEAE, Capto Q, Capto Q ImpRes, and POROS. TM 50HQ, Source 15Q, QA, QAHR, CIMmultus and DEAE (Bia Separations).

[0026] In some embodiments, the support used in the first step of anion exchange chromatography and the second step of anion exchange chromatography includes anion exchange chromatography column A, wherein the anion exchange chromatography column A is... QA or QA HR.

[0027] In some embodiments, the recombinant adeno-associated virus includes, but is not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, AAV-16, AAV.rh8, AAV.rh10, AAV.rh39, AAV.rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AA V.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC 6. AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16.

[0028] In some embodiments, the empty shell ratio is detected by analytical ultracentrifugation.

[0029] This specification provides one or more embodiments of a purified recombinant adeno-associated virus, obtained by the above-described method.

[0030] This specification provides one or more embodiments for the application of the above-described recombinant adeno-associated virus in gene therapy products. Attached Figure Description

[0031] This specification will be further illustrated by way of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not limiting, and in these embodiments, the same reference numerals denote the same structures.

[0032] Figure 1 is a flowchart of a method for purifying recombinant adeno-associated virus according to some embodiments of this specification;

[0033] Figures 2A and 2B show the yield and empty shell rate of recombinant adeno-associated virus after the first and second steps of anion exchange chromatography purification as shown in Example 2 of this specification, respectively.

[0034] Figures 3A and 3B show the yield and empty shell rate of recombinant adeno-associated virus after the first and second steps of anion exchange chromatography purification as shown in Example 3 of this specification, respectively.

[0035] Figures 4A and 4B show the yield and empty shell rate of recombinant adeno-associated virus after anion exchange chromatography purification in the first and second steps as shown in Example 4 of this specification, respectively; and

[0036] Figures 5A and 5B show the yield and empty shell rate of recombinant adeno-associated virus after anion exchange chromatography purification in the first and second steps as shown in Example 5 of this specification, respectively. Detailed Implementation

[0037] To more clearly illustrate the technical solutions of the embodiments in this specification, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are merely some examples or embodiments of this specification. For those skilled in the art, these drawings can be applied to other similar scenarios without creative effort. Unless obvious from the context or otherwise specified, the same reference numerals in the drawings represent the same structures or operations.

[0038] As indicated in this specification and claims, unless the context clearly indicates otherwise, the words "a," "an," "an," and / or "the" do not specifically refer to the singular and may also include the plural. Generally speaking, the terms "comprising" and "including" only indicate the inclusion of expressly identified steps and elements, which do not constitute an exclusive list, and the method or apparatus may also include other steps or elements.

[0039] This specification uses flowcharts to illustrate the steps performed by the method according to embodiments of this specification. It should be understood that the preceding or following operations are not necessarily performed in exact order. Instead, the steps can be processed in reverse order or simultaneously. Furthermore, other operations can be added to the method, or one or more operations can be removed from the method.

[0040] Terminology Definition

[0041] The terms "recombinant adeno-associated virus," "recombinant adeno-associated virus particle," "rAAV," and / or "AAV particle" refer to a virus that has been genetically modified from a wild-type adeno-associated virus. For example, inserting a target gene fragment into a wild-type adeno-associated virus yields a desired product, such as a gene therapy product (e.g., a vaccine product), used to treat certain diseases. The adeno-associated viruses mentioned in Examples 1-6 of this specification are all recombinant adeno-associated viruses.

[0042] The term "empty capsid" or "empty particle" refers to an AAV virion containing an AAV protein coat but lacking all or part of the target nucleotide sequence. Therefore, an empty capsid cannot transfer the target gene to a host cell. It is considered an impurity in the production process and needs to be removed as much as possible.

[0043] The term "anion exchange chromatography" refers to a positively charged chromatographic solid phase. Viral particle capsid proteins carry a net charge, which is determined by the pH of the amino acid groups on their surface. Adenoviruses have an isoelectric point at acidic conditions and carry a negative charge under neutral pH conditions, making them suitable for purifying adeno-associated virus (AAV) particles. The examples in this specification employ two-step anion exchange chromatography to purify recombinant AAV, effectively removing empty particles and some particles, achieving a high AAV yield and a low empty capsid percentage.

[0044] Different elution systems can be used in the two-step anion exchange chromatography examples in this specification. The ion exchange chromatography includes two elution systems: elution system A and elution system B. The difference between elution system A and elution system B is that elution system A removes the compound salts present in elution system B.

[0045] The elution systems in the two-step chromatography can be different. Specifically, elution system B used in the first-step anion exchange chromatography includes at least magnesium sulfate, and elution system B used in the second-step anion exchange chromatography includes at least sodium chloride; or elution system B used in the first-step anion exchange chromatography includes at least sodium chloride, and elution system B used in the second-step anion exchange chromatography includes at least magnesium sulfate. An elution system including magnesium sulfate can be called a magnesium sulfate elution system, and an elution system including sodium chloride can be called a sodium chloride elution system. In some embodiments, the magnesium sulfate or sodium chloride elution system may also include a buffer solution, including but not limited to Bis-Tris propane, N-methylpiperazine, piperazine, Bis-Tris, triethanolamine, Tris, N-methyldiethanolamine, 1,3-diaminopropane, ethanolamine, etc.

[0046] The term "elution slope" refers to the rate of change of the concentration of eluting system B in anion exchange chromatography. It can be calculated by dividing the total change in concentration by the total elution time or gradient length. For example, in a 0-100% elution of system B, if the total change is 100% over a length of 300 column volumes (CV), then the elution slope is 100% / 300CV = 0.33% / CV.

[0047] Methods for purifying adeno-associated virus

[0048] On one hand, some embodiments of this specification provide a method for purifying adeno-associated virus. This method includes the following steps.

[0049] Step 101: Perform the first step of anion exchange chromatography on the initial recombinant adeno-associated virus sample to obtain the first eluted sample.

[0050] The initial recombinant adeno-associated virus (AAV) sample refers to a sample containing AAV suitable for the first step of anion exchange chromatography. In some embodiments, the initial AAV sample is obtained by treating the AAV feed solution to meet the conditions for anion exchange chromatography. In some embodiments, the initial AAV sample obtained after treatment may be a clear liquid. In some embodiments, the initial AAV sample may be obtained through preliminary purification, the purpose of which is to remove large particulate matter and / or impurities from the AAV feed solution, avoid column clogging, and increase sample throughput. Preliminary purification methods include, but are not limited to, membrane filtration, depth filtration, affinity chromatography, and tangential flow filtration. The AAV feed solution includes various cells, such as HEK293 cells, insect cells, or feed solutions containing AAV particles produced by other cell matrices. In some embodiments, the AAV feed solution may be obtained by cell lysis of cultured cells or by any other method that yields the feed solution. In some embodiments, the methods of one or more embodiments of this specification can purify recombinant adeno-associated virus (AAV) solutions with any initial empty shell ratio, especially solutions with a higher empty shell ratio (e.g., 80%, 90%), achieving good separation results. In some embodiments, the recombinant AAV solution may contain one or more recombinant AAVs. Exemplary AAVs include, but are not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, AAV-16, AAV.rh8, AAV.rh10, AAV.rh39, AAV.rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, and AAV.7m8. , AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, A AV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16.

[0051] The initial adeno-associated virus sample was further processed by treating the recombinant adeno-associated virus solution to obtain:

[0052] The pH of the recombinant adeno-associated virus (AAV) solution is adjusted to match the pH of elution system A used in the first step of anion exchange chromatography, and the AAV solution is diluted with elution system A used in the first step of anion exchange chromatography until the conductivity is less than or equal to a first preset value, thereby obtaining the initial AAV sample.

[0053] Elution system A is the same system used in elution system B to remove magnesium sulfate or sodium chloride. In other words, elution system A includes the same buffer solution and pH value as elution system B; the only difference is whether it contains a certain concentration of salt.

[0054] In some embodiments, the elution system B used in the first and second anion exchange chromatography steps can be different systems. Specifically, the elution system B used in the first anion exchange chromatography step includes at least magnesium sulfate, and the elution system B used in the second anion exchange chromatography step includes at least sodium chloride; or the elution system B used in the first anion exchange chromatography step includes at least sodium chloride, and the elution system B used in the second anion exchange chromatography step includes at least magnesium sulfate. An elution system including magnesium sulfate can be referred to as a magnesium sulfate elution system, and an elution system including sodium chloride can be referred to as a sodium chloride elution system.

[0055] In some embodiments, the elution system B used in the first and second steps of anion exchange chromatography can be the same system. For example, the elution system B used in both steps of anion exchange chromatography is either a magnesium sulfate elution system or a sodium chloride elution system.

[0056] The concentration range of the magnesium sulfate is 30-70 mM, 40-60 mM, or 35-65 mM, etc. In some embodiments, the concentration of the magnesium sulfate is 45 mM, 50 mM, 55 mM, or 60 mM. The magnesium sulfate elution system further includes a buffer solution with a concentration range of 5-50 mM, 5-30 mM, 15-45 mM, or 20-50 mM, and a pH range of 9.0-9.5 or 9.0-9.4. In some embodiments, the concentration of the buffer solution is 5 mM, 10 mM, 15 mM, 20 mM, 28 mM, 30 mM, 33 mM, or 35 mM, etc. The pH is 9.0, 9.1, 9.2, 9.3, 9.4, or 9.5, etc.

[0057] The concentration range of the sodium chloride is 130mM-2M, 150mM-1.5M, 180mM-1.2M, or 0.5-1.5M, etc. In some embodiments, the concentration of the sodium chloride is 130mM, 150mM, 160mM, 180mM, 0.5mM, 0.7mM, 0.8mM, 1M, 1.2M, or 1.5M. The sodium chloride elution system further includes a buffer solution with a concentration range of 10-30mM, 10-60mM, 15-65mM, or 20-50mM, and a pH range of 9.5-10.5 or 9.6-10.2. In some embodiments, the concentration of the buffer solution is 15mM, 18mM, 20mM, 25mM, 30mM, 38mM, 40mM, 42mM, or 45mM, etc. The pH is 9.6, 9.7, 9.8, 10, 10.1, 10.2, 10.3, 10.4 or 10.5, etc.

[0058] In some embodiments, the conductivity is less than or equal to a first preset value of 2.5 mS / cm (magnesium sulfate system), for example, 2.5 mS / cm, 2.4 mS / cm, 2.3 mS / cm, 2.2 mS / cm, 2.1 mS / cm, 2.0 mS / cm, 1.9 mS / cm, 1.8 mS / cm, 1.7 mS / cm, 1.6 mS / cm, 1.5 mS / cm, 1.4 mS / cm, 1.3 mS / cm, 1.2 mS / cm, 1.1 mS / cm, 1.0 mS / cm, 0.9 mS / cm, 0.8 mS / cm, 0.7 mS / cm, etc. In some embodiments, the conductivity is less than or equal to a first preset value of 5.0 mS / cm (sodium chloride system), for example, 5.0 mS / cm, 4.9 mS / cm, 4.8 mS / cm, 4.7 mS / cm, 4.6 mS / cm, 4.5 mS / cm, 4.4 mS / cm, 4.3 mS / cm, 4.2 mS / cm, 4.1 mS / cm, 4.0 mS / cm, 3.9 mS / cm, 3.8 mS / cm, 3.7 mS / cm, 3.6 mS / cm, 3.5 mS / cm, 3.4 mS / cm, 3.3 mS / cm, 3.2 mS / cm, 3.1 mS / cm. The sample conductivity values ​​are 3.0 mS / cm, 2.9 mS / cm, 2.8 mS / cm, 2.7 mS / cm, 2.6 mS / cm, 2.5 mS / cm, 2.4 mS / cm, 2.3 mS / cm, 2.2 mS / cm, 2.1 mS / cm, 2.0 mS / cm, 1.9 mS / cm, 1.8 mS / cm, 1.7 mS / cm, 1.6 mS / cm, 1.5 mS / cm, 1.4 mS / cm, 1.3 mS / cm, 1.2 mS / cm, 1.1 mS / cm, 1.0 mS / cm, 0.9 mS / cm, 0.8 mS / cm, and 0.7 mS / cm, etc. The sample is loaded onto the chromatography column when its conductivity is less than or equal to a first preset value. By adjusting the sample conductivity to be less than or equal to the first preset value, the effectiveness and accuracy of the separation process are ensured.

[0059] If the first step of the anion exchange chromatography uses an elution system B containing magnesium sulfate, column equilibration and washing are performed using the elution system B used in the first step of the anion exchange chromatography, wherein the concentration of magnesium sulfate is reduced to 0-10 mM.

[0060] If the first step of the anion exchange chromatography uses an elution system B containing sodium chloride, column equilibration and washing are performed using the elution system B used in the first step of the anion exchange chromatography, wherein the concentration of sodium chloride is reduced to 0-100 mM, preferably 0-50 mM.

[0061] In some embodiments, continuous gradient elution may be used in the first anion exchange chromatography step. Specifically, the elution slope in the first anion exchange chromatography step ranges from 0.2% to 0.5% / CV. In some embodiments, the elution slope in the first anion exchange chromatography step ranges from 0.3% to 0.4% / CV. In some embodiments, the elution slope in the first anion exchange chromatography step is 0.2% / CV, 0.25% / CV, 0.3% / CV, 0.31% / CV, 0.32% / CV, 0.33% / CV, 0.34% / CV, 0.35% / CV, 0.4% / CV, and 0.5% / CV, etc. Specifically, 0-100% elution system B with a length of 300 CV continuous gradient elution, or 0-50% elution system B with a length of 150 CV continuous gradient elution, or 0-25% elution system B with a length of 75 CV continuous gradient elution, or 0-20% elution system B with a length of 60 CV continuous gradient elution, or 0-15% elution system B with a length of 45 CV continuous gradient elution can be used.

[0062] Mixtures containing rAAV can be analyzed using ultraviolet absorbance at approximately 260 nm and 280 nm. Because the capsid nucleic acid content affects A260 and A280 absorbance data, the A260 / A280 ratio in the absorbance data can be used to support the identification of intact AAV particles and empty or partially filled adeno-associated virus (AAV) particles. Typically, intact AAV particles containing nucleic acid within the capsid are detectable at UV260 above 280 nm, while empty and partially filled AAV particles are detectable at UV280 above UV260. Therefore, by collecting UV... 260 ≥UV 280 The viral component peaks yield the first eluted sample. The first eluted sample contains adeno-associated virus particles with empty capsids and partial capsids removed, and contains purified recombinant adeno-associated virus particles.

[0063] Additionally or optionally, the first eluted sample may be further purified, including but not limited to tangential flow chromatography, molecular sieve chromatography, etc.

[0064] In some embodiments, analytical ultracentrifugation (AUC) can be used to detect the empty shell rate of recombinant adeno-associated virus in the one-step purified fraction. The empty shell rate can be detected by methods such as analytical ultracentrifugation (AUC), qPCR, ELISA, optical density method, transmission electron microscopy, etc., with AUC being the preferred method.

[0065] Step 103: Perform a second step of anion exchange chromatography on the first eluted sample to obtain purified adeno-associated virus.

[0066] Similar to the first step of chromatography, the pH of the first eluted sample is adjusted to match the pH of the elution system A used in the second step of anion exchange chromatography, and the first eluted sample is diluted with the elution system A used in the second step of anion exchange chromatography until the conductivity is less than or equal to the second preset value, thus obtaining the processed first eluted sample. In some embodiments, the conductivity is less than or equal to a second preset value of 5.0 mS / cm (sodium chloride system), for example, 5.0 mS / cm, 4.9 mS / cm, 4.8 mS / cm, 4.7 mS / cm, 4.6 mS / cm, 4.5 mS / cm, 4.4 mS / cm, 4.3 mS / cm, 4.2 mS / cm, 4.1 mS / cm, 4.0 mS / cm, 3.9 mS / cm, 3.8 mS / cm, 3.7 mS / cm, 3.6 mS / cm, 3.5 mS / cm, 3.4 mS / cm, 3.3 mS / cm, 3.2 mS / cm, 3.1 mS / cm. m, 3.0mS / cm, 2.9mS / cm, 2.8mS / cm, 2.7mS / cm, 2.6mS / cm, 2.5mS / cm, 2.4mS / cm, 2.3mS / cm, 2.2mS / cm, 2.1mS / cm, 2.0mS / cm, 1.9mS / cm , 1.8mS / cm, 1.7mS / cm, 1.6mS / cm, 1.5mS / cm, 1.4mS / cm, 1.3mS / cm, 1.2mS / cm, 1.1mS / cm, 1.0mS / cm, 0.9mS / cm, 0.8mS / cm, 0.7mS / cm, etc. In some embodiments, the conductivity is less than or equal to a second preset value of 2.5 mS / cm (magnesium sulfate system), for example, 2.5 mS / cm, 2.4 mS / cm, 2.3 mS / cm, 2.2 mS / cm, 2.1 mS / cm, 2.0 mS / cm, 1.9 mS / cm, 1.8 mS / cm, 1.7 mS / cm, 1.6 mS / cm, 1.5 mS / cm, 1.4 mS / cm, 1.3 mS / cm, 1.2 mS / cm, 1.1 mS / cm, 1.0 mS / cm, 0.9 mS / cm, 0.8 mS / cm, 0.7 mS / cm, etc. When the conductivity of the first eluted sample is less than or equal to the second preset value, the sample is loaded onto the chromatography column. By adjusting the conductivity of the sample to be less than or equal to the second preset value, the effectiveness and accuracy of the separation process are ensured.

[0067] In some embodiments, the treated first eluted sample is subjected to a second step of anion exchange chromatography to obtain purified adeno-associated virus.

[0068] If the second step of anion exchange chromatography uses elution system B containing magnesium sulfate, column equilibration and washing are performed using elution system B used in the second step of anion exchange chromatography, wherein the concentration range of magnesium sulfate is reduced to 0-10 mM.

[0069] If the second step of anion exchange chromatography uses an elution system B containing sodium chloride, column equilibration and washing are performed using the elution system used in the second step of anion exchange chromatography, wherein the concentration of sodium chloride is reduced to 0-100 mM, preferably 0-50 mM.

[0070] In some embodiments, continuous gradient elution may be used in the second anion exchange chromatography step. Specifically, the elution slope in the second anion exchange chromatography step ranges from 0.2% to 0.5% / CV. In some embodiments, the elution slope in the second anion exchange chromatography step ranges from 0.3% to 0.4% / CV. In some embodiments, the elution slope in the second anion exchange chromatography step is 0.2% / CV, 0.25% / CV, 0.3% / CV, 0.31% / CV, 0.32% / CV, 0.33% / CV, 0.34% / CV, 0.35% / CV, 0.4% / CV, and 0.5% / CV, etc. Specifically, 0-100% elution system B with a length of 300 CV continuous gradient elution, or 0-50% elution system B with a length of 150 CV continuous gradient elution, or 0-25% elution system B with a length of 75 CV continuous gradient elution, or 0-20% elution system B with a length of 60 CV continuous gradient elution, or 0-15% elution system B with a length of 45 CV continuous gradient elution can be used.

[0071] The treated first eluted sample was subjected to a second step of anion exchange chromatography to collect UV. 260 ≥UV 280 The viral component peaks were analyzed to obtain a second eluted sample. This second eluted sample contained purified adeno-associated virus particles.

[0072] Additionally or optionally, the second eluted sample may be further purified, including but not limited to tangential flow chromatography, molecular sieve chromatography, etc.

[0073] According to some implementation schemes, methods for obtaining purified recombinant adeno-associated virus (rAAV) particles include two anion exchange chromatography steps, and may also include three or even four anion exchange chromatography steps. When the method includes more than two anion exchange chromatography steps, the above steps can be performed on the same type of vector or different vectors.

[0074] It should be noted that suitable examples of monolithic chromatographic supports are known in the art, including but not limited to monolithic columns, such as... QA, QA HR, CIMmultus and DEAE (Bia Separations), and non-monolithic columns, such as Fractogel EMD DEAE, Capto Q, Capto Q ImpRes, POROS TM 50HQ or Source 15Q, the present invention is not limited to these.

[0075] In some embodiments, the support used in the first and second anion exchange chromatography steps includes anion exchange chromatography column A, which is... QA or QA HR.

[0076] The purified recombinant adeno-associated virus exhibits good purification efficiency, for example, a total yield greater than 70% and an empty shell rate less than or equal to 20%. Further, the total yield of the purified recombinant adeno-associated virus is greater than 71%, 72%, 73%, 75%, 80%, etc., and the empty shell rate is less than 15%, 12%, 10%, etc.

[0077] Purified adeno-associated virus

[0078] On the one hand, some embodiments of this specification also provide a recombinant adeno-associated virus obtained by purification according to the above-described method for purifying adeno-associated virus, which can effectively remove empty virus and achieve a high AAV yield.

[0079] Due to its high safety and efficient gene delivery capabilities, AAV is increasingly important and widely used in the biomedical field. In some embodiments, recombinant adeno-associated viruses can be used in gene therapy to create gene therapy products. AAV vectors are used to carry and deliver functional genes to repair or replace defective or inactivated genes in vivo. Because of its low immunogenicity and long-term expression characteristics, AAV has great potential in treating genetic diseases such as muscular dystrophy, cystic fibrosis, retinal diseases, and certain neurodegenerative diseases, as well as in cancer treatment and research and vaccine development. Example 1: Two Steps The effect of QA anion exchange chromatography sequence on AAV8 virus purification

[0080] use AAV8 virus was eluted in two steps using a QA-1 column with magnesium sulfate and sodium chloride elution systems, respectively. QA-1 anion exchange chromatography was used to investigate the effect of the order of magnesium sulfate elution and sodium chloride elution on the purification efficiency of AAV8 virus.

[0081] Chromatography A (magnesium sulfate elution followed by sodium chloride elution): Adjust the pH of the affinity-purified AAV8 solution to 9.4, then dilute the sample with pH 9.4, 30mM Bis-Tris propane buffer until the conductivity (Cond) ≤ 2.5 mS / cm, and load the sample. QA-1 anion exchange chromatography column. Chromatographic conditions: 30 mM Bis-Tris propane, 5 mM MgSO4, pH 9.4 buffer for equilibration and washing. Elution conditions: Pump A: pH 9.4, 30 mM Bis-Tris propane buffer; Pump B: 30 mM Bis-Tris propane, 50 mM MgSO4, pH 9.4 buffer; 0-100% elution system B (Concentration B, Conc B); continuous gradient elution length 300 CV; UV collected. 260 ≥UV 280 The peak of the AAV8 viral component.

[0082] The AAV8 eluted sample collected in the first step of anion exchange chromatography was then adjusted to pH 10.2, and then diluted with pH 10.2, 40 mM Bis-Tris propane until the conductivity was ≤3 mS / cm before loading. QA-1 anion exchange chromatography column. Chromatographic conditions: 40 mM Bis-Tris propane, 10 mM NaCl, pH 10.2 buffer for equilibration and washing. Elution conditions: Pump A: pH 10.2, 40 mM Bis-Tris propane; Pump B: 40 mM Bis-Tris propane, 1 M NaCl, pH 10.2 buffer; 0-100% elution system B; continuous gradient elution length 300 CV; UV collected. 260 ≥UV 280 The AAV8 viral component peaks were observed. Detection showed that the first-step anion exchange chromatography yield was 82%, with a viral empty shell rate of 53.51%; the second-step anion exchange chromatography yield was 99%, for a total yield of 81.18%. The viral empty shell rate was 15.16%.

[0083] Chromatography B (sodium chloride elution followed by magnesium sulfate elution): Adjust the AAV8 feed solution, initially purified by affinity chromatography, to pH 10.2. Then, dilute the sample with pH 10.2 and 40 mM Bis-Tris propane until the conductivity is ≤3.0 mS / cm. Load the sample. QA-1 anion exchange chromatography column. Chromatographic conditions: 40 mM Bis-Tris propane, 10 mM NaCl, pH 10.2 buffer for equilibration and washing. Elution conditions: Pump A: pH 10.2, 40 mM Bis-Tris propane; Pump B: 40 mM Bis-Tris propane, 1 M NaCl, pH 10.2 buffer; 0-100% elution system B; continuous gradient elution length 300 CV; UV collected. 260 ≥UV 280 The peak of the AAV8 viral component.

[0084] Then, the AAV8 elution sample collected in the first step of anion exchange chromatography was adjusted to pH 9.4, and then diluted with pH 9.4, 30mM Bis-Tris propane buffer until the conductivity was ≤2.5mS / cm, and loaded onto the sample. QA-1 anion exchange chromatography column. Chromatographic conditions: 30 mM Bis-Tris propane, 5 mM MgSO4, pH 9.4 buffer for equilibration and washing. Elution conditions: Pump A: pH 9.4, 30 mM Bis-Tris propane buffer; Pump B: 30 mM Bis-Tris propane, 50 mM MgSO4, pH 9.4 buffer; 0-100% elution system B; continuous gradient elution length 300 CV; UV collected. 260 ≥UV 280 The AAV8 viral component peaks were observed. Detection showed that the first-step anion exchange chromatography yield was 86%, with a viral empty shell rate of 56.70%; the second-step anion exchange chromatography yield was 89%, for a total yield of 76.54%. The viral empty shell rate was 17.1%.

[0085] The results of chromatography A and chromatography B are summarized in Table 1 below. Table 1 Summary of anion exchange chromatography results under the magnesium sulfate elution system + sodium chloride elution system and the sodium chloride elution system + magnesium sulfate elution system

[0086] Therefore, it can be seen that by arbitrarily changing the order of the first and second anion exchange chromatography steps, empty-shell AAV viruses can be effectively removed while ensuring AAV yield, achieving the same purification effect. Example 2: Purification of AAV5 virus using two-step anion exchange chromatography

[0087] The AAV5 solution, purified by affinity chromatography, was adjusted to pH 9.4. The sample was then diluted with pH 9.4, 30 mM Bis-Tris propane buffer until the conductivity was ≤2.5 mS / cm, and loaded onto the sample. A QA-40 anion exchange chromatography column. Chromatographic conditions were: equilibration and washing with 10 mM Bis-Tris propane, 5 mM MgSO4, and pH 9.4 buffer. Elution conditions were: Pump A with pH 9.4 and 30 mM Bis-Tris propane buffer; Pump B with 10 mM Bis-Tris propane, 50 mM MgSO4, and pH 9.4 buffer; 0-100% elution system B; continuous gradient elution with a length of 300 CV; and collection of AAV5 virus peaks with UV260 ≥ UV280. The single-step AAV5 virus yield was 73%, and the empty shell percentage of AAV5 virus was 17.54% (AUC method). The chromatogram is shown in Figure 2A, where mAU represents milliabsorbance and ml represents milliliters.

[0088] Adjust the pH of the AAV5 elution sample collected by the first step of anion exchange chromatography to 9.6, then dilute the sample with pH 9.6 and 40mM Bis-Tris propane until the conductivity is ≤3mS / cm, and load the sample. QA-40 anion exchange chromatography column. Chromatographic conditions: 40 mM Bis-Tris propane, 10 mM NaCl, pH 9.6 buffer for equilibration and washing. Elution conditions: Pump A: pH 9.6, 40 mM Bis-Tris propane; Pump B: 40 mM Bis-Tris propane, 1 M NaCl, pH 9.6 buffer; 0-100% elution system B; continuous gradient elution length 300 CV; UV collected. 260 ≥UV 280 The AAV5 viral component peaks were observed, with a single-step AAV5 viral yield of 99%. The chromatographic pattern is shown in Figure 2B.

[0089] After two-step anion exchange chromatography purification, the total yield of AAV5 virus was 72.27%, and the empty shell rate of AAV5 virus was 8.78% (AUC method). Example 3: Two-step anion exchange chromatography purification of AAV8 virus

[0090] The AAV8 virus was purified according to the first step anion exchange chromatography conditions in Example 2. The single-step AAV8 virus yield was 83.07%, and the empty shell rate of the AAV8 virus was 60.68% (AUC method). The chromatographic pattern is shown in Figure 3A.

[0091] Adjust the pH of the AAV8 elution sample collected by the first step of anion exchange chromatography to 10.2, then dilute the sample with pH 10.2 and 40mM Bis-Tris propane until the conductivity is ≤3mS / cm, and load the sample. QA-40 anion exchange chromatography column. Chromatographic conditions: 40 mM Bis-Tris propane, 10 mM NaCl, pH 10.2 buffer for equilibration and washing. Elution conditions: Pump A: pH 10.2, 40 mM Bis-Tris propane; Pump B: 40 mM Bis-Tris propane, 1 M NaCl, pH 10.2 buffer; 0-100% elution system B; continuous gradient elution length 300 CV; UV collected. 260 ≥UV 280 The AAV8 viral component peaks were observed, with a single-step AAV8 viral yield of 87.95%. The chromatographic pattern is shown in Figure 3B.

[0092] After two-step anion exchange chromatography purification, the total yield of AAV8 virus was 73.06%, and the empty shell rate of AAV8 virus was 6.01% (AUC method). Example 4: Two-step anion exchange chromatography purification of AAV-rh74 (AAV.rh74) virus

[0093] AAV-rh74 virus was purified according to the first and second anion exchange chromatography conditions described in Example 2. The first anion exchange chromatography yield of AAV-rh74 virus was 87%, and the empty shell percentage was 23.61% (AUC method). The second anion exchange chromatography yield was 103%. The chromatographic patterns are shown in Figures 4A and 4B, respectively.

[0094] After two-step anion exchange chromatography purification, the total yield of AAV-rh74 virus was 89.61%, and the empty shell rate of AAV-rh74 virus was 5.25% (AUC method). Example 5: Two-step anion exchange chromatography purification of AAV9 virus

[0095] AAV9 virus was purified according to the first and second anion exchange chromatography conditions described in Example 2. The first anion exchange chromatography yield of AAV9 virus was 83%, and the empty shell percentage was 23.61% (AUC method). The second anion exchange chromatography yield of AAV9 virus was 102%. The chromatographic patterns are shown in Figures 5A and 5B, respectively.

[0096] After two-step anion exchange chromatography purification, the total yield of AAV9 virus was 84.39%, and the empty shell rate of AAV9 virus was 7.02% (AUC method). Example 6: Effect of starting solutions with different empty shell rates on anion exchange chromatography.

[0097] AAV8 (92.88%), AAV5 (90.99%), AAV9 (91.95%), and AAV-rh74 (90.13%) were used as starting solutions, respectively. Purification was performed according to the first and second anion exchange chromatography conditions described in Example 2. The results are shown in Table 2 below. Table 2: Comparison of purification effects of anion exchange chromatography on starting solutions with different empty shell ratios.

[0098] After two-step anion exchange chromatography purification, the empty shell percentages of AAV5, AAV8, AAV9, and AAV-rh74 viruses were all <10% (AUC method). This demonstrates that even starting solutions containing over 90% empty shell viruses can achieve good removal of empty shell viruses. Comparative Example 1: Comparison with different adeno-associated virus purification methods.

[0099] US11203740B2 discloses a two-step anion chromatography linear salt gradient purification method for AAV, in which the same buffer system is used for both anion chromatography steps, and only the slope of the linear salt gradient in the two chromatography steps is changed.

[0100] US2019 / 0002841A1, US2019 / 0002842A1, and US2019 / 0002844A1 all describe a method for removing empty-shell viruses using one-step QA anion chromatography with a NaCl salt concentration gradient under high pH 10.2 conditions.

[0101] US2021 / 0370199A1 describes a process involving a certain concentration of Mg. 2+ K + Under specific conditions, a one-step anion chromatography process involving equilibration, sample loading, and washing is performed, followed by purification of AAV using a NaCl salt concentration gradient.

[0102] US2023 / 0399656A1 describes a one-step anion chromatography method using gradient elution with 400-600 mM sodium acetate in a pH 8.5-9.5, 50-150 mM Tris buffer system.

[0103] The methods disclosed in the above literature were compared with the two-step anion exchange chromatography method used in this embodiment, and the empty shell rate was detected using the AUC method. The two-step anion exchange chromatography method used in this embodiment can significantly improve the total yield of purified adeno-associated virus particles and reduce the empty shell rate (both empty shell rates are much lower than those of the methods in these literatures). Table 3 Comparison of the effects of different adeno-associated virus purification methods

[0104] The basic concepts have been described above. Obviously, for those skilled in the art, the detailed disclosure above is merely illustrative and does not constitute a limitation of this specification. Although not explicitly stated herein, those skilled in the art may make various modifications, improvements, and corrections to this specification. Such modifications, improvements, and corrections are suggested in this specification and therefore remain within the spirit and scope of the exemplary embodiments described herein.

[0105] Furthermore, this specification uses specific terms to describe embodiments thereof. For example, "an embodiment," "one embodiment," and / or "some embodiments" refer to a particular feature, structure, or characteristic associated with at least one embodiment of this specification. Therefore, it should be emphasized and noted that references to "an embodiment," "one embodiment," or "an alternative embodiment" in different locations throughout this specification do not necessarily refer to the same embodiment. Moreover, certain features, structures, or characteristics in one or more embodiments of this specification can be appropriately combined.

[0106] In some embodiments, numbers describing the quantity of components and attributes are used. It should be understood that such numbers used in the description of embodiments are modified in some examples with the terms "approximately," "approximately," or "generally." Unless otherwise stated, "approximately," "approximately," or "generally" indicates that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may be changed depending on the characteristics required by individual embodiments. In some embodiments, numerical parameters should take into account specified significant digits and employ a general method of digit reservation. Although the numerical ranges and parameters used to confirm their breadth of range in some embodiments of this specification are approximate values, in specific embodiments, such values ​​are set as precisely as feasible.

[0107] For each patent, patent application, patent application publication, and other material, such as articles, books, specifications, publications, and documents, referenced in this specification, the entire contents of which are incorporated herein by reference. This excludes historical application documents that are inconsistent with or conflict with the content of this specification, as well as documents that limit the broadest scope of the claims in this specification (currently or subsequently appended to this specification). It should be noted that in the event of any inconsistency or conflict between the descriptions, definitions, and / or terminology used in the supplementary materials to this specification and the content of this specification, the descriptions, definitions, and / or terminology used in this specification shall prevail.

[0108] Finally, it should be understood that the embodiments described in this specification are merely illustrative of the principles of the embodiments described herein. Other variations may also fall within the scope of this specification. Therefore, alternative configurations of the embodiments described herein are intended to be illustrative rather than limiting, and should be considered consistent with the teachings of this specification. Accordingly, the embodiments described herein are not limited to those explicitly introduced and described herein.

Claims

1. A method for purifying recombinant adeno-associated virus, comprising: The initial recombinant adeno-associated virus (AAV) sample is subjected to a first-step anion exchange chromatography to obtain a first eluted sample, wherein the initial AAV sample is obtained by processing the recombinant AAV feed solution; and The first eluted sample was subjected to a second step of anion exchange chromatography to obtain purified recombinant adeno-associated virus, wherein the total yield of the purified recombinant adeno-associated virus was greater than 70% and the empty shell rate was less than or equal to 20%.

2. The method as described in claim 1, characterized in that, The elution system B used in the first step of anion exchange chromatography includes at least magnesium sulfate.

3. The method as described in claim 1, characterized in that, The elution system B used in the second step of anion exchange chromatography includes at least sodium chloride.

4. The method as described in claim 1, characterized in that, The elution system B used in the first step of anion exchange chromatography includes at least sodium chloride.

5. The method as described in claim 1, characterized in that, The elution system B used in the second step of anion exchange chromatography includes at least magnesium sulfate.

6. The method according to any one of claims 1-5, characterized in that, The concentration of the magnesium sulfate is in the range of 30-70 mM, and the corresponding elution system B also includes a buffer solution with a concentration range of 5-50 mM and a pH range of 9.0-9.

5.

7. The method as described in claim 6, characterized in that, The concentration of magnesium sulfate is 40-60 mM, and the concentration of the buffer solution is 5-30 mM with a pH of 9.0-9.

4.

8. The method according to any one of claims 1-5, characterized in that, The concentration of sodium chloride is in the range of 130mM-2M, and the corresponding elution system B also includes a buffer solution with a concentration range of 10-40mM and a pH range of 9.5-10.

5.

9. The method as described in claim 8, characterized in that, The concentration of sodium chloride is 0.5-1.5M, and the concentration of the buffer solution is 10-30mM with a pH of 9.6-10.

2.

10. The method according to any one of claims 6-9, characterized in that, The buffer solution includes Bis-Tris propane, N-methylpiperazine, piperazine, Bis-Tris, triethanolamine, Tris, N-methyldiethanolamine, 1,3-diaminopropane, or ethanolamine.

11. The method according to any one of claims 1-5, characterized in that, If the first step of the anion exchange chromatography uses an elution system B containing magnesium sulfate, column equilibration and washing are performed using the elution system B used in the first step of the anion exchange chromatography, wherein the concentration of magnesium sulfate is reduced to 0-10 mM. If the second step of the anion exchange chromatography uses an elution system B containing sodium chloride, column equilibration and washing are performed using the elution system B used in the second step of the anion exchange chromatography, wherein the concentration of sodium chloride is reduced to 0-100 mM.

12. The method according to any one of claims 1-5, characterized in that, If the first step of the anion exchange chromatography uses an elution system B containing sodium chloride, column equilibration and washing are performed using the elution system used in the first step of the anion exchange chromatography, wherein the concentration of sodium chloride is reduced to 0-100 mM. If the second step of the anion exchange chromatography uses an elution system B containing magnesium sulfate, column equilibration and washing are performed using the elution system B used in the second step of the anion exchange chromatography, wherein the concentration of magnesium sulfate is reduced to 0-10 mM.

13. The method according to any one of claims 1-5, characterized in that, Both the first and second steps of anion exchange chromatography employ continuous gradient elution with an elution slope ranging from 0.2% to 0.5% per column volume.

14. The method as described in claim 13, characterized in that, The elution slope in the first and second steps of anion exchange chromatography ranges from 0.3% to 0.4% per column volume.

15. The method according to any one of claims 1-5, characterized in that, The recombinant adeno-associated virus feed solution comprises feed solutions containing recombinant adeno-associated virus particles produced from various cells.

16. The method according to any one of claims 1-5, characterized in that, The initial recombinant adeno-associated virus sample was further processed by treating the recombinant adeno-associated virus solution to obtain the following: The pH of the recombinant adeno-associated virus (AAV) solution is adjusted to match the pH of elution system A used in the first step of anion exchange chromatography. The AAV solution is then diluted with elution system A until the conductivity is less than or equal to a first preset value to obtain the initial recombinant AAV sample. Elution system A is the system in elution system B for removing magnesium sulfate or sodium chloride.

17. The method as described in claim 16, characterized in that, The first preset value is 2.5 mS / cm (magnesium sulfate system) or 5.0 mS / cm (sodium chloride system).

18. The method according to any one of claims 1-5, characterized in that, The first eluted sample was subjected to a second step of anion exchange chromatography to obtain purified recombinant adeno-associated virus, including: The pH of the first eluted sample is adjusted to match the pH of elution system A used in the second step of anion exchange chromatography, and the first eluted sample is diluted with elution system A used in the second step of anion exchange chromatography until the conductivity is less than or equal to a second preset value, thus obtaining the processed first eluted sample. Elution system A is the system used in elution system B to remove magnesium sulfate or sodium chloride. The first eluted sample after treatment was subjected to a second step of anion exchange chromatography to obtain purified recombinant adeno-associated virus.

19. The method as described in claim 18, characterized in that, The second preset value is 5.0 mS / cm (sodium chloride system) or 2.5 mS / cm (magnesium sulfate system).

20. The method according to any one of claims 1-5, characterized in that, The support used in the first and second steps of anion exchange chromatography includes anion exchange column A, which includes Fractogel EMD DEAE, Capto Q, Capto Q ImpRes, and POROS. TM 50HQ, Source 15Q, QA, QA HR, CIMmultus and DEAE (Bia Separations).

21. The method as described in claim 20, characterized in that, The support used in the first and second steps of anion exchange chromatography includes anion exchange column A, which is... QA or QA HR.

22. The method according to any one of claims 1-5, characterized in that, The recombinant adeno-associated viruses include, but are not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15, AAV-16, AAV.rh8, AAV.rh10, AAV.rh39, AAV.rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, and AAV.7m.

8. AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16.

23. The method as described in claim 1, characterized in that, The empty shell ratio was detected using analytical ultracentrifugation technology.

24. A purified recombinant adeno-associated virus, obtained by the method of any one of claims 1-22.

25. The use of the purified recombinant adeno-associated virus as described in claim 24 in gene therapy products.

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