Methods of purifying recombinant adeno-associated viruses

Abstract

Provided are methods for isolating recombinant adeno-associated virus (AAV)-based vectors using anion exchange chromatography.

Description

Attorney Docket No.: 99CR-403196-WOMETHODS OF PURIFYING RECOMBINANT ADENO-ASSOCIATED VIRUSES CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U. S. C. 119(e) of United States Provisional Application No. 63 / 737,418, filed on December 20. 2024, which is hereby incorporated by reference in its entirety.BACKGROUND

[0002] Recombinant Adeno-Associated Virus (AAV)-based vectors are currently one of the most widely used products in the development of gene therapies. Their widespread use can be attributed to several factors. Firstly, the wild-type AAV virus is not linked to any diseases, making it a safer option. Additionally, AAVs have the ability to transduce both non-dividing and dividing cells, which enhances their versatility'. Clinical trials have also demonstrated that AAVs provide robust and long-term transgene expression, underscoring their significant potential for use in gene therapy. Moreover, different naturally occurring and recombinant rAAV vector serotypes have the ability to specifically target various tissues, organs, and cells. This targeting capability helps them bypass any pre-existing immunity7to the vector, thereby broadening the therapeutic applications of AAV-based gene therapies.

[0003] However, one of the major challenges faced by research and development teams in the downstream processing of viral vectors is to achieve efficient separation of functional viral particles from impurities and byproducts, including inactive vector forms, such as empty and partially filled viral capsids (product-related impurities), helper virus, and cell membrane vesicles.

[0004] Product-related impurities, like empty' and partially filled capsids, are difficult to remove because they may have similar properties to functional rAAV particles. However, it is important to remove them because their presence in a therapeutic dose can increase the amount of potentially immune-triggering capsid proteins. By reducing these impurities, a dose can be delivered with fewer capsid proteins, lowering the risk of patients experiencing adverse immune reactions.

[0005] Thus, there is a critical need for efficient and effective processes to effectively isolate functional rAAV particles.Attorney Docket No.: 99CR-403196-WOSUMMARY

[0006] Methods provided herein may be useful for AAV anion exchange (AEX) chromatography for purifying recombinant AAV particles, resulting in high yield and high percentage of full capsids.

[0007] Provided in the present disclosure is a method of for purifying recombinant adeno-associated virus (rAAV) particles, comprising: (a) contacting an anion exchange (AEX) chromatography medium with a recombinant AAV (rAAV) loading composition comprising full rAAV particles and empty rAAV particles, wherein the rAAV loading composition further comprises MgCh; (b) applying to the AEX chromatography medium a wash solution; and (c) applying to the AEX chromatography medium an elution solution to provide an eluate, wherein pH and salt concentration of the elution solution is isocratic.

[0008] In some embodiments, full and empty rAAV particles are one or more selected from the group consisting of: AAV2, AAV5, AAV8, and AAV9. In some embodiments, the full and empty rAAV particles are AAV2. In some embodiments, the full rAAV particles comprises one or more polynucleotides. In some embodiments, the full rAAV particles comprise a single vector. In some embodiments, the full rAAV particles include two or more different polynucleotides. In some embodiments, the rAAV loading composition comprises about 4 mM to about 11 mM of MgCh. In some embodiments, the rAAV loading composition comprises about 5 mM to about 9 mM of MgCh. In some embodiments, the rAAV loading composition comprises about 6 mM to about 8 mM of MgCh. In some embodiments, the rAAV loading composition comprises about 6.5 mM to about 10.5 mM of MgCh. In some embodiments, the concentration of MgCh in the rAAV loading composition is substantially proportional to the percentage of full recombinant AAV particles in the eluate. In some embodiments, the rAAV loading composition has pH of about 8 to about 9. In some embodiments, the rAAV loading composition has pH of about 8.6 to about 8.9. In some embodiments, the rAAV loading composition has pH of about 8.7 to about 8.9. In some embodiments, the rAAV loading composition has pH of about 8.7 to about 8.8. In some embodiments, the rAAV loading composition is about 10-fold to 40-fold diluted from a recombinant AAV (rAAV) sample. In some embodiments, the rAAV loading composition is about 10-fold to 30-fold diluted from a recombinant AAV (rAAV) sample. In some embodiments, the rAAV loading composition is about 10-fold to 20-fold diluted from a recombinant AAV (rAAV) sample. In some embodiments, the rAAV loading composition isAttorney Docket No.: 99CR-403196-WOabout 12.5-fold to 17.5-fold diluted from a recombinant AAV (rAAV) sample. In some embodiments, the rAAV loading composition is about 15-fold diluted from a recombinant AAV (rAAV) sample. In some embodiments, the rAAV loading composition has conductivity of about 1.0 mS / cm to about 6.0 mS / cm. In some embodiments, the rAAV loading composition has conductivity7of about 1.0 mS / cm to about 4.5 mS / cm. In some embodiments, the rAAV loading composition has conductivity of about 1.0 mS / cm to about 4.0 mS / cm. In some embodiments, the rAAV loading composition has conductivity of about 1.0 mS / cm to about 2.4 mS / cm. In some embodiments, the rAAV loading composition comprises the rAAV particles in a concentration of about 1.0 x 1012VP / mL to about 5.0 x 1013VP / mL. In some embodiments, the rAAV loading composition comprises the rAAV particles in a concentration of about 2.0 x 1012VP / mL to about 1.6 x 1013VP / mL. In some embodiments, the rAAV loading composition comprises the rAAV particles in a concentration of about 1.6 x 1013VP / mL to about 2.0 x 1013VP / mL. In some embodiments, the rAAV loading composition further comprises one or more selected from the group consisting of Tris, poloxamer and sucrose. In some embodiments, the AEX chromatography medium comprises quaternary amine. In some embodiments, the AEX chromatography medium comprises a copolymer of glycidyl methacrylate and ethylene dimethacrylate. In some embodiments, the AEX chromatography medium comprises a highly cross-linked agarose with dextran surface extender. In some embodiments, the rAAV loading composition is added to the anion exchange (AEX) chromatography medium in an amount of about 2.5x1013VP / mLresin to about 5.0 x 1014VP / mLresin. In some embodiments, the recombinant AAV composition is added to anion exchange (AEX) chromatography medium in an amount of about 1.6xl014VP / mLresin to about 3.5 xlO14VP / mLresin. In some embodiments, the method further comprising applying a chase solution after the step (a). In some embodiments, the chase solution does not comprise MgCh. In some embodiments, the chase solution has pH of about 8.7. In some embodiments, the chase solution is added in an amount of 5 fold of the volume of the AEX chromatography medium. In some embodiments, the wash solution comprises MgCh. In some embodiments, wherein the wash solution comprises about 3 mM to about 10 mM of MgCh. In some embodiments, the wash solution comprises about 4 mM to about 6 mM of MgCh. In some embodiments, the wash solution has conductivity of about 1.4 mS / cm to about 4.0 mS / cm. In some embodiments, the wash solution has conductivity of about 2.5 mS / cm to about 4.0 mS / cm. In some embodiments, the wash solution has conductivity of about 2.7 mS / cm to about 2.9 mS / cm. In some embodiments, the wash solution has pH of about 7.7 to about 9.5. In some embodiments, the wash solution has pH ofAttorney Docket No.: 99CR-403196-WOabout 8.8 to about 9.1. In some embodiments, the elution solution comprise about 3.0 mM to about 5.0 mM of MgCh. In some embodiments, the elution solution comprises about 4.0 mM of MgCh. In some embodiments, the elution solution has pH of about 7.0 to about 8.0. In some embodiments, the elution solution has pH of about 7.5. In some embodiments, pH of the elution solution is lower than pH of the wash solution. In some embodiments, the elution solution does not substantially comprise NaCl. In some embodiments, the method further comprises adding NaCl to the eluate. In some embodiments, aggregation of AAV is decreased by the addition of NaCl to the eluate. In some embodiments, the eluate comprises at least 70% of full capsid recombinant AAV. In some embodiments, the eluate comprises at least 90% of full capsid recombinant AAV. In some embodiments, the eluate comprises at least 95% of full capsid recombinant AAV. In some embodiments, the full capsid recombinant AAV is measured by HPLC assay, mass photometry. AUC (A260), or AUC (A230). In some embodiments, the eluate comprises less than 10 % of empty capsid recombinant AAV. In some embodiments, the eluate comprises less than 5% of empty capsid recombinant AAV. In some embodiments, the eluate provides VG yield of about 50% or greater. In some embodiments, the eluate provides VG yield of about 60% or greater. In some embodiments, the method further comprises purifying the eluate.

[0009] In another aspect, the present disclosure provides a method for isolating recombinant adeno-associated virus (rAAV) particles, comprising: (a) contacting an anion exchange (AEX) chromatography medium wi th a rAAV composition comprising full rAAV particles and empty rAAV particles; (b) applying to the AEX chromatography medium a wash solution comprising MgCh; and (c) applying to the AEX chromatography medium an elution solution to provide an eluate, wherein the pH and salt concentration of the elution solution is isocratic, and wherein pH concentration of the elution solution is lower than pH of the wash solution.

[0010] In some embodiments, the full and empty' rAAV particles are one or more selected from the group consisting of AAV2, AAV5, AAV8, and AAV9. In some embodiments, the full and empty rAAV particles are AAV2. In some embodiments, the full rAAV particles comprises one or more polynucleotides. In some embodiments, the full rAAV particles comprise a single vector. In some embodiments, the full rAAV particles include two or more different polynucleotides. In some embodiments, the rAAV loading composition has pH of about 8 to about 9. In some embodiments, the rAAV loading composition has pH of about 8.6 to about 8.9. In some embodiments, rAAV loading composition has pH of about 8.7 to aboutAttorney Docket No.: 99CR-403196-WO8.9. In some embodiments, the rAAV loading composition has pH of about 8.7 to about 8.8. In some embodiments, the rAAV loading composition is about 10-fold to 40-fold diluted from a recombinant AAV (rAAV) sample. In some embodiments, the rAAV loading composition is about 10-fold to 30-fold diluted from a recombinant AAV (rAAV) sample. In some embodiments, the rAAV loading composition is about 10-fold to 20-fold diluted from a recombinant AAV (rAAV) sample. In some embodiments, the rAAV loading composition is about 12.5-fold to 17.5-fold diluted from a recombinant AAV (rAAV) sample. In some embodiments, the rAAV loading composition is about 15-fold diluted from a recombinant AAV (rAAV) sample. In some embodiments, the rAAV loading composition has conductivity of about 1.0 mS / cm to about 6.0 mS / cm. In some embodiments, the rAAV loading composition has conductivity of about 1.0 mS / cm to about 4.5 mS / cm. In some embodiments, the rAAV loading composition has conductivity of about 1.0 mS / cm to about 4.0 mS / cm. In some embodiments, the rAAV loading composition has conductivity of about 1.0 mS / cm to about 2.4 mS / cm. In some embodiments, the rAAV loading composition comprises the rAAV particles in a concentration of about 1.0 x 1012VP / mL to about 5.0 x 1013VP / mL. In some embodiments, the rAAV loading composition further comprises one or more selected from the group consisting of Tris, poloxamer and sucrose. In some embodiments, wherein the AEX chromatography medium comprises quaternary amine. In some embodiments, the AEX chromatography medium comprises a copolymer of glycidyl methacrylate and ethylene dimethacrylate. In some embodiments, the AEX chromatography medium comprises a highly cross-linked agarose with dextran surface extender. In some embodiments, the rAAV loading composition is added to the anion exchange (AEX) chromatography medium in an amount of about 2.5x1013VP / mLresin to about 5.0 xlO14VP / mLresin. In some embodiments, the recombinant AAV composition is added to anion exchange (AEX) chromatography medium in an amount of about 1,6xl014VP / mLresin to about 3.5 XlO14VP / mLresin. In some embodiments, the method further comprises applying a chase solution after the step (a). In some embodiments, the chase solution has pH of about 8.7. In some embodiments, the chase solution is added in an amount of 5 fold of the volume of the AEX chromatography medium. In some embodiments, the wash solution comprises MgCh. In some embodiments, the wash solution comprises about 3 mM to about 10 mM of MgCh. In some embodiments, the wash solution comprises about 4 mM to about 6 mM of MgCh. In some embodiments, the wash solution has conductivity of about 1.4 mS / cm to about 4.0 mS / cm. In some embodiments, the wash solution has conductivity of about 2.5 mS / cm to about 4.0 mS / cm. In some embodiments, the wash solution has conductivity ofAttorney Docket No.: 99CR-403196-WOabout 2.7 mS / cm to about 2.9 mS / cm. In some embodiments, the wash solution has pH of about 7.7 to about 9.5. In some embodiments, the wash solution has pH of about 8.8 to about 9.1. In some embodiments, the elution solution comprise about 3.0 mM to about 5.0 mM of MgCh. In some embodiments, the elution solution comprises about 4.0 mM of MgCh. In some embodiments, the elution solution has pH of about 7.0 to about 8.0. In some embodiments, the elution solution has pH of about 7.5. In some embodiments, the elution solution does not substantially comprise NaCl. In some embodiments, the method further comprises adding NaCl to the eluate. In some embodiments, aggregation of AAV is decreased by the addition of NaCl to the eluate. In some embodiments, the eluate comprises at least 70% of full capsid recombinant AAV. In some embodiments, the eluate comprises at least 90% of full capsid recombinant AAV. In some embodiments, the eluate comprises at least 95% of full capsid recombinant AAV. In some embodiments, the eluate comprises less than 10 % of empty capsid recombinant AAV. In some embodiments, the eluate comprises less than 5% of empty capsid recombinant AAV. In some embodiments, the eluate provides VG yield of about 50% or greater. In some embodiments, the eluate provides VG yield of about 60% or greater. In some embodiments, the method further comprises purifying the eluate.

[0011] In another aspect, the present disclosure provides a method for isolating recombinant adeno-associated virus (rAAV) particles, comprising: (a) contacting an anion exchange (AEX) chromatography medium wi th a rAAV composition comprising full rAAV particles and empty rAAV particles, wherein the rAAV composition further comprises MgCh; (b) applying to the AEX chromatography medium a wash solution comprising MgCh; and (c) applying to the AEX chromatography medium an elution solution to provide an eluate, wherein the pH and salt concentration of the elution solution is isocratic, wherein pH concentration of the elution solution is lower than pH of the wash solution.

[0012] In another aspect, the present disclosure provides a method for isolating recombinant adeno-associated virus (rAAV) particles, comprising: (a) contacting an anion exchange (AEX) chromatography medium with a rAAV composition comprising full rAAV particles and empty rAAV particles, wherein the rAAV composition has conductivity of about 1.0 to 4.5 mS / cm; (b) applying to the AEX chromatography medium a wash solution; and (c) applying to the AEX chromatography medium an elution solution to provide an eluate, wherein the pH and salt concentration of the elution solution is isocratic.Attorney Docket No.: 99CR-403196-WOBRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIGS. 1A-1B show the plots reflecting the correlation between load MgCh concentration and MP %full capsid or elution UV area.

[0014] FIGS. 2A-2C show the empty capsid % and VG yield model in terms of load MgCh and pH.

[0015] FIGS. 3A-3B show the plots reflecting the correlation between the load conductivity and full capsid % and VG yield %.

[0016] FIGS. 4A-4B show the plots reflecting the correlation between the load conductivity and MP %full capsid or elution UV area.

[0017] FIG. 5 shows the plots reflecting the correlation between the load ratio and full capsid % and VG yield %.

[0018] FIG. 6 shows the MP data between load MgCh concentration and MP %full capsid.

[0019] FIG. 7 show the full capsid % model in terms of load MgCh and pH.

[0020] FIG. 8 shows the contour plot of VG yield % vs. pH and MgCh.

[0021] FIG. 9 shows the contour plot of full capsid % vs. wash pH and MgCh.

[0022] FIG. 10 shows the contour plot of full capsid % vs. wash pH and MgCh.

[0023] FIG. 11A-11C show the DoE modeling.

[0024] FIG. 12A-12C show the VG yield % vs. pH and MgCh (12A), %empty capsid vs. pH and MgCh (12B) and contour plot of VG yield % and %empty capsid vs. pH and MgCh (12C).DETAILED DESCRIPTIONDefinitions

[0025] It is to be noted that the term 'a" or "an" entity’ refers to one or more of that entity; for example, "an antibody,” is understood to represent one or more antibodies. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

[0026] The term “about” or “approximately” means within about 20%, such as within about 10%, within about 5%, or within about 1% or less of a given value or range.Attorney Docket No.: 99CR-403196-WO

[0027] The term "substantially" in "substantially proportional to" refers to a relationship that is largely or essentially proportional, allowing for reasonable deviations from exact equality. This concept introduce flexibility, recognizing that precise proportionality may not always be feasible.

[0028] '‘AAV” is an abbreviation for adeno-associated virus, and may be used to refer to the virus itself or modifications, derivatives, or pseudot pes thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise. The abbreviation "rAAV" refers to recombinant adeno-associated virus. “Recombinant", as applied to an AAV particle means that the AAV particle is the product of one or more procedures that result in an AAV particle construct that is distinct from an AAV particle in nature.

[0029] The term " AAV" includes AAV type 1 (AAV-1), AAV type 2 (AAV -2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV type 9 (AAV-9), AAV-10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV. Rh74, AAV. RHM4-1, AAV.hu37, AAV. Anc80, AAV. Anc80L65, AAV.7m8, 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, or AAV. HSC16, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, nonprimate AAV, ovine AAV, modifications, derivatives, pseudotypes thereof or other rAAV particles, or combinations of two or more thereof. " Primate AAV" refers to AAV that infect primates, "non-primate AAV" refers to AAV that infect non-primate mammals, "bovine AAV" refers to AAV that infect bovine mammals, etc.

[0030] As used herein, the term “full AAV particle", refers to an AAV particle that includes AAV capsid proteins encapsulating a heterologous nucleic acid of interest which is flanked on both sides by AAV ITRs. In some embodiments, the heterologous nucleic acid of interest is operably linked to additional regulatory sequences (e.g., promoter, enhancer, etc ).

[0031] As used herein, the term “empty AAV particle”, refers to an AAV particle that includes AAV capsid proteins but lacks in whole or in part the heterologous nucleic acid of interest flanked on either side by AAV ITRs.Attorney Docket No.: 99CR-403196-WO

[0032] The term "impurity" or "contaminant" refers to any foreign or objectionable molecule, including a biological macromolecule such as DNA, RNA, one or more host cell proteins, endotoxins, lipids and one or more additives which may be present in a sample containing the rAAV particles that are being separated from one or more of the foreign or objectionable molecules using a disclosed method. The term "impurity " or "contaminant" further encompasses product-related impurities, for example, inactive vector forms, empty viral capsids, aggregated viral particles or capsids, mis-folded viral capsids, degraded viral particles. In some embodiments, an impurity includes an empty viral capsid or a viral aggregate. Additionally, such impurity’ may include any reagent which is used in a step which may occur prior to one or more of the disclosed methods. An impurity may be soluble or insoluble in nature. Insoluble impurities include any undesirable or objectionable entity present in a sample containing rAAV particles, where the entity is a suspended particle or a solid. Exemplar}' insoluble impurities include without limitation, aggregated viral particles or capsids, whole cells, cell fragments and cell debris. Soluble impurities include any undesirable or objectionable entity present in a sample containing rAAV particles where the entity is not an insoluble impunty. Exemplary soluble impurities include without limitation, host cell proteins, DNA, RNA, lipids viruses, endotoxins, and cell culture media components.

[0033] The terms "purifying", "purification", "separate", "separating", "separation", "isolate", "isolating", or "isolation", as used herein, refer to increasing the degree of purity of rAAV particles from a sample including the target product and one or more impurities. Typically, the degree of purity of the target product is increased by removing (completely or partially) at least one impurity from the sample. In some embodiments, the degree of purity of the rAAV in a sample is increased by removing (completely or partially) one or more impurities from the sample by using a method described herein.

[0034] The term "recombinant AAV composition" applied to an anion exchange (AEX) chromatography medium refers to a source of rAAV particles that is loaded onto, passed through, or applied to a filter or chromatographic matrix. Recombinant AAV compositions include production culture harvests, and materials isolated from previous chromatographic steps encompassed by the disclosed methods whether the material was present as flow-through from the previous step, bound and eluted in the previous step, present in the void volume of the previous step or present in any fraction obtained during the purification of rAAV particles. Such rAAV composition may include one or more impurities or contaminants. In some embodiments, the rAAV composition containing rAAV particlesAttorney Docket No.: 99CR-403196-WOfurther includes product-related impurities such as empty viral capsids, aggregated viral particles or capsids, mis-folded viral capsids, degraded viral particles, and / or damaged rAAV particles. In some embodiments, the rAAV composition containing rAAV particles further includes host cell contaminants, helper virus contaminants, and / or cell culture contaminants. In some embodiments, the host cell contaminants include host cell DNA, plasmids, or host cell protein. In additional embodiments, the helper virus contaminants include adenovirus particles, adenovirus DNA, adenovirus proteins, baculovirus particles, baculovirus DNA, or baculovirus proteins. In some embodiments, the cell culture contaminants include media components, serum albumin, or other serum proteins. In additional embodiments, the cell culture contaminants include media components.

[0035] The rAAV particles of the disclosure may be of any serot pe, or any combination of serotypes, (e.g., a population of rAAV particles that includes two or more serotypes, e.g., including two or more of rAAV2, rAAV8, and rAAV9 particles). In some embodiments, the rAAV particles are rAAVl, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV 10, or other rAAV particles, or combinations of two or more thereof. In some embodiments, the rAAV particles are AAV2, AAV5, AAV8, and AAV9 particles. In some embodiments, the rAAV particles are AAV2 particles.

[0036] In some embodiments, the rAAV particles have an AAV capsid protein of one or more serotypes selected from the group consisting of AAV1, AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV. Rh74, AAV. RHM4-1, AAV.hu37, AAV. Anc80, AAV. Anc80L65, AAV.7m8, 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, or AAV. HSC16 or a derivative, modification, or pseudotype thereof.

[0037] In some embodiments, rAAV particles include a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. 99%. 99.5%. etc., i.e. up to 100% identical, to e.g., VP1, VP2 and / or VP3 sequence of an AAV capsid serotype selected from AAV1, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV- 10, AAV-11, AAV- 12, AAV- 13, AAV- 14, AAV- 15 and AAV- 16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV. Rh74, AAV. RHM4-1, AAV.hu37, AAV. Anc80, rAAV. Anc80L65, AAV.7m8, AAV. PHP. B, AAV2.5, AAV2tYF,Attorney Docket No.: 99CR-403196-WOAAV3B, 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, or AAV. HSC16.

[0038] In some embodiments, the rAAV particles have an AAV capsid protein of a serotype of AAV2, AAV5, AAV-8, AAV-9, or a derivative, modification, or pseudotype thereof In some embodiments, the rAAV particles have an AAV capsid protein of a serotype of AAV2 or a derivative, modification, or pseudotype thereof.

[0039] In some embodiments, rAAV particles include an AAV capsid disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: United States Patent Nos. 7,282,199; 7.906,111; 8.524,446; 8,999.678;8,628,966; 8,927,514; 8,734,809; US 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9458517; and 9,587,282; US patent application publication nos. 2015 / 0374803; 2015 / 0126588;2017 / 0067908; 2013 / 0224836; 2016 / 0215024; 2017 / 0051257; and International Patent Application Nos. PCT / US2015 / 034799; PCT / EP2015 / 053335. In some embodiments, rAAV particles have a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and / or VP3 sequence of an AAV capsid disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: United States Patent Nos.7.282, 199; 7,906.111; 8,524.446; 8,999.678;8,628,966; 8,927,514; 8,734,809; US 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9458517; and 9,587,282; US patent application publication nos. 2015 / 0374803; 2015 / 0126588;2017 / 0067908; 2013 / 0224836; 2016 / 0215024; 2017 / 0051257; and International Patent Application Nos. PCT / US2015 / 034799; PCT / EP2015 / 053335.

[0040] In another embodiment, rAAV particles include a mosaic capsid. Mosaic AAV particles are composed of a mixture of viral capsid proteins from different serotypes of AAV. In some embodiments, rAAV particles include a mosaic capsid containing capsid proteins of a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV- 12. AAV-13, AAV- 14, AAV- 15 and AAV- 16, AAV.rh8, AAV.rhlO. AAV.rh20, AAV.rh39, AAV. Rh74. AAV. RHM4-1, AAV.hu37, AAV. Anc80, AAV. Anc80L65, AAV.7m8, 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. and AAV. HSC16.Attorney Docket No.: 99CR-403196-WO

[0041] Nucleic acid sequences of AAV based viral vectors and methods of making recombinant AAV and AAV capsids are taught, for example, in United States Patent Nos. 7,282,199; 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; US 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9458517; and 9,587,282; US patent application publication nos. 2015 / 0374803; 2015 / 0126588; 2017 / 0067908; 2013 / 0224836; 2016 / 0215024;2017 / 0051257; International Patent Application Nos. PCT / US2015 / 034799;PCT / EP2015 / 053335; WO 2003 / 052051, WO 2005 / 033321, WO 03 / 042397, WO 2006 / 068888, WO 2006 / 110689, W02009 / 104964, W02010 / 127097, and WO 2015 / 191508, and U. S. Appl. Publ. No. 20150023924.Methods of Isolating rAA V

[0042] In one aspect, the present disclosure provides methods for purifying recombinant adeno-associated viruses (rAAV). Specifically, the methods involve separating rAAV particles that include a nucleic acid payload (i.e., full rAAV particles) and AAV particles that lack in whole or in part the nucleic acid payload (empty rAAV particles, partially full rAAV particles), using anion-exchange chromatography (AEX).

[0043] Methods of purification using an anion exchange chromatography resin disclosed herein can be used in combination with upstream processing, downstream processing, or upstream and downstream processing methods to isolate rAAV particles.

[0044] For example, before purified using the anion-exchange chromatography, an rAAV sample can be prepared and / or provided by any process known in the art. such as, for example, harvest of a cell culture (e.g., cell culture supernatant or cell lysate or both), clarification of the harvested cell culture or cell culture supernatant (e.g., by centrifugation or depth filtration), tangential flow filtration, affinity chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, sterile filtration, or any combination(s) thereof. In some embodiments, the rAAV sample may be prepared and / or provided by an upstream process including at least 2, at least 3, or at least 4 of: harvest of a cell culture or cell culture supernatant, clarification of the harvested cell culture or cell culture supernatant (e.g., by centrifugation or depth filtration), tangential flow filtration, affinity chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, sterile filtration. In some embodiments, upstream processing includes harvest of a cell culture or cell cultureAttorney Docket No.: 99CR-403196-WOsupernatant, clarification of the harvested cell culture or cell culture supernatant (e.g., by depth filtration), sterile filtration, tangential flow filtration, and affinity chromatography.

[0045] In some embodiments, upstream processing for preparing and or providing the rAAV sample includes clarification of a harvested cell culture or cell culture supernatant, sterile filtration, tangential flow filtration, and affinity chromatography. In some embodiments, upstream processing for preparing and / or providing the rAAV sample includes clarification of a harvested cell culture or cell culture supernatant by depth filtration, sterile filtration, tangential flow filtration, and affinity chromatography.

[0046] In some embodiments, clarification of the harvested cell culture or cell culture supernatant includes sterile filtration. In some embodiments, upstream processing does not include centrifugation.

[0047] In some embodiments, the affinity chromatography may be used as an initial step before the anion exchange chromatography (AEX). The affinity chromatography uses specific interactions between the rAAV particles and immobilized ligands, such as antibodies, to selectively capture the virus and remove major contaminants like host cell proteins and DNA. Following this, the rAAV sample that were subjected to the affinity chromatography may be further purified by the AEX by separating molecules based on charge, effectively removing residual impurities such as empty rAAV particles. This sequential approach leverages the specificity of affinity chromatography and the chargebased separation capabilities of AEX, resulting in highly pure and functional rAAV vectors essential for effective and safe gene therapy applications.Anion Exchange Chromatography

[0048] Anion Exchange Chromatography (AEX) is well-suited for separating full and empty AAV particles due to the inherent charge differences between them, as full particles contain genetic material, altering their surface charge. The empty AAV particles are less tightly bound to the anion-exchange medium and can be eluted earlier. The full AAV particles are more strongly bound and thus can be eluted later. As shown in Examples, it has been discovered that use of certain concentration of salts (e.g., MgCh) in the steps of the AEX was critical in resulting in high percentage of full AAV particles and less empty AAV particles, while maintaining commercially viable yields.

[0049] In one aspect, the present disclosure provides methods for purifying rAAV particles from a rAAV sample including full rAAV particles and empty rAAV particles, using anionAttorney Docket No.: 99CR-403196-WOexchange chromatography. In some embodiments, the methods include contacting a recombinant AAV (rAAV) loading composition including full rAAV particles and empty rAAV particles with an anion exchange chromatography medium under conditions that allow binding of the rAAV particles to the chromatography media (i.e., “loading”); applying to the AEX chromatography medium a wash solution (i.e., “washing”); and applying to the AEX chromatography medium an elution solution to provide an eluate (i.e., “elution”), wherein pH and salt concentration of the elution solution is isocratic.

[0050] In some embodiments, the method includes a) contacting an anion exchange (AEX) chromatography medium with a rAAV loading composition including full rAAV particles and empty rAAV particles, wherein the recombinant AAV composition further includes MgCh; (b) applying to the AEX chromatography medium a wash solution; and (c) applying to the AEX chromatography medium an elution solution to provide an eluate, wherein pH and salt concentration of the elution solution is isocratic.

[0051] In some embodiments, the method includes: (a) contacting an anion exchange (AEX) chromatography medium with a rAAV loading composition including full rAAV particles and empty rAAV particles; (b) applying to the AEX chromatography medium a wash solution comprising MgCh; and (c) applying to the AEX chromatography medium an elution solution to provide an eluate, wherein the pH and salt concentration of the elution solution is isocratic, and wherein MgCh concentration of the wash solution is greater than MgCh concentration of the elution solution.

[0052] In some embodiments, the method includes: (a) contacting an anion exchange (AEX) chromatography medium with a rAAV loading composition including full rAAV particles and empty rAAV particles, wherein the rAAV composition further includes MgCh; (b) applying to the AEX chromatography medium a wash solution comprising MgCh; and (c) applying to the AEX chromatography medium an elution solution to provide an eluate, wherein the pH and salt concentration of the elution solution is isocratic, and wherein MgCh concentration of the w ash solution is greater than MgCh concentration of the elution solution.

[0053] In some embodiments, the method includes (a) contacting an anion exchange (AEX) chromatography medium with a rAAV loading composition comprising full rAAV particles and empty rAAV particles, wherein the rAAV composition has conductivity of about 1.0 mS / cm to about 6.0 mS / cm (e.g., about 1.0 mS / cm to about 4.5 mS / cm. about 1.0 mS / cm to about 4.0 mS / cm, or about 1.0 mS / cm to about 2.4 mS / cm); (b) applying to the AEXAttorney Docket No.: 99CR-403196-WOchromatography medium a wash solution; and (c) applying to the AEX chromatography medium an elution solution to provide an eluate, wherein the pH and salt concentration of the elution solution is isocratic.rAAV Samples

[0054] In some embodiments, in addition to the full and empty rAAV particles, the rAAV sample to be purified with the AEX further includes partially filled rAAV particles and / or a viral aggregate. In some embodiments, the full rAAV particles, the empty rAAV particles, or partially filled viral particles includes one or more selected from the group consisting of: AAV1, AAV2. AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, or other rAAV particles, or combinations of two or more thereof. In some embodiments, the full rAAV particles, empty rAAV particles, or partially filled rAAV particles include one or more selected from the group consisting of: AAV2, AAV5, AAV8, and AAV9. In some embodiments, the full rAAV particles, empty rAAV particles, or partially filled rAAV includes AAV2.

[0055] In some embodiments, the full rAAV particles include a single vector, which includes all elements for gene delivery and expression. For example, the single vector may be an ‘All-in-one” vector, which is a comprehensive construct that integrates all necessary genetic elements for efficient gene delivery and expression in a single vector. The “all-in-one” vector may include the therapeutic transgene, a promoter to initiate transcription, enhancer elements to boost expression, a poly adenylation signal for mRNA stabilization, and / or Inverted Terminal Repeats (ITRs) essential for replication and packaging. Additional regulatory sequences or selectable sequences may also be included in the single vector to optimize transgene stability and targeting.

[0056] In some embodiments, the full rAAV particles may include one or more polynucleotides or vectors, each including one or more of the elements for gene delivery and expression, such as the therapeutic transgene, a promoter to initiate transcription, enhancer elements to boost expression, a polyadenylation signal for mRNA stabilization, and / or inverted terminal repeats (ITRs).

[0057] In some embodiments, the full rAAV particles may include two or more different polynucleotides or vector sets, each including one or more of the elements for gene delivery and expression, such as the therapeutic transgene, a promoter to initiate transcription,Attorney Docket No.: 99CR-403196-WOenhancer elements to boost expression, a polyadenylation signal for mRNA stabilization, and / or inverted terminal repeats (ITRs).rAAV Loading Composition

[0058] In some embodiments, before purification using anion exchange chromatography (AEX), the rAAV sample is provided as a composition, such as, an eluate, produced by the affinity chromatography, for purification. The rAAV sample may be added with additional compounds to provide a rAAV loading composition to be contacted with an AEX chromatography medium. In some embodiments, the rAAV sample itself is added to the AEX chromatography medium as the rAAV loading composition, and additional buffer and / or compounds added afterwards. In some embodiments, the additional buffer and / or compounds are added to the AEX chromatography medium and the rAAV sample is added afterwards.

[0059] As the rAAV loading composition includes the rAAV sample, the rAAV loading composition has all properties of the rAAV sample. For example, the rAAV loading composition may include the full AAV particles, the empty AAV particles. The rAAV loading composition may further include the partially filled AAV particles.

[0060] As shown in Examples, it has been discovered that there is positive relationship between the salt (i. e., MgCb) concentration in the rAAV loading composition.

[0061] In some embodiments, one or more salts are added such that the rAAV loading composition includes the one or more salts. In some embodiments, the rAAV loading composition includes the K+, Na+, Mg2+, Ca2+, or Zn2+salt. In some embodiments, the rAAV loading composition includes a Mg2+salt, such as MgCb. In some embodiments, the rAAV loading composition includes salts, such as one or more of K+, Rb+, Cs+, Mg2+, Ca2+, and / or Zn2+salt. In some embodiments, the rAAV loading composition includes a final concentration of the salt at about 0.2 mM to about 50 mM, for example, about 0.2 mM to about 45 mM, about 0.2 mM to about 40 mM, about 0.2 mM to about 35 mM, about 0.2 mM to about 30 mM, about 0.2 mM to about 25 mM, about 0.2 mM to about 20 mM, about 0.5 mM to about 20 mM, about 0.5 mM to about 15 mM, about 0.5 mM to about 10 mM, about 1 mM to about 20 mM, about 1 mM to about 15mM, about 2 to about 12mM, about 4 mM to about 11 mM, about 2 mM to about 10.5mM, about 5 mM to about 10.5mM, about 5 mM to about 9mM, about 6 mM to about 8mM, or about 6.5mM to about 10.5mM.

[0062] In some embodiments, the rAAV loading composition includes a divalent cation salt, such as MgCb. In some embodiments, the rAAV composition includes the Mg2+salt (e.g.,Attorney Docket No.: 99CR-403196-WOMgCh) in the concentration of about 2mM to about 15mM, about 3mM to about 15mM, about 4mM to about 15mM, about 5mM to about 15mM, about 6mM to about 15mM, about 7mM to about 15mM, about 8mM to about 15mM, about 9mM to about 15mM, about lOmM to about 15mM, about 2mM to about 12mM, about 3mM to about 12mM, about 4mM to about 12mM, about 5mM to about 12mM, about 6mM to about 12mM, about 7mM to about 12mM, about 8mM to about 12mM, about 9mM to about 12mM, about lOmM to about 12mM, about 2mM to about 1 ImM, about 3mM to about 1 ImM, about 4mM to about 1 ImM, about 5mM to about 1 ImM, about 6mM to about 1 ImM, about 6.5mM to about 10.5mM, about 7mM to about 11 mM, about 8mM to about 11 mM, about 9mM to about 1 ImM, about lOmM to about llmM, about 2mM to about lOmM, about 3mM to about lOmM. about 4mM to about lOmM, about 5mM to about lOmM, about 6mM to about lOmM, about 7mM to about 1 OmM, about 8mM to about 1 OmM, about 9mM to about 1 OmM, about 2mM to about 9mM, about 3mM to about 9mM, about 4mM to about 9mM, about 5mM to about 9mM, about 6mM to about 9mM, about 7mM to about 9mM, about 8mM to about 9mM, about 2mM to about 8mM, about 3mM to about 8mM, about 4mM to about 8mM, about 5mM to about 8mM, about 6mM to about 8mM, about 7mM to about 8mM, about 2mM to about 7mM, about 3mM to about 7mM, about 4mM to about 7mM, about 5mM to about 7mM, or about 6mM to about 7mM. In some embodiments, the Mg2’ salt is MgCh.

[0063] In some embodiments, the rAAV composition includes the Mg2+salt (e.g., MgCl?) in the concentration of about 6.5mM to about 7.5mM, about 6.7mM to about 7.5mM, about 6.9mM to about 7.5mM, about 6.5mM to about 7.3mM. about 6.7mM to about 7.3mM, about 6.9mM to about 7.3mM, about 6.5mM to about 7. ImM. about 6.7mM to about 7. ImM, about 6.9mM to about 7. ImM, about 6.5mM to about 7. OmM, about 6.7mM to about 7. OmM. about 6.9mM to about 7. OmM. In some embodiments, the Mg2+salt is MgCh.

[0064] In some embodiments, the rAAV loading composition includes the Mg2+salt in the concentration of about 2mM, about 2.5 mM, about 3 mM, about 3.5mM, about 4mM, about 4.5mM, about 5mM, about 5.5mM, about 6mM, about 6.5mM, about 7mM, about 7.5mM. about 8mM, about 8.5mM, about 9mM, about 9.5mM, about lOmM, about 10.5mM, about 1 ImM, about 11.5mM, about 12mM, about 12.5mM, about 13mM, about 13.5mM, about 14mM, about 14.5mM, or about 15mM. In some embodiments, the rAAV loading composition includes the Mg2+salt in the concentration range formed between two values chosen from the preceding sentence.Attorney Docket No.: 99CR-403196-WO

[0065] In some embodiments, the rAAV sample is diluted or otherwise adjusted to provide the rAAV loading composition before being applied to the AEX medium. For example, the rAAV sample may be diluted to provide a desired conductivity of the rAAV loading composition before being added to the AEX medium.

[0066] In certain embodiments, the rAAV sample is diluted, for example with a loading buffer, to about 1 fold to about 100 fold, about 1 fold to about 75 fold, about 1 fold to about 50 fold, about 1 fold to about 30 fold, about 1 fold to about 20 fold, about 1 fold to about 15 fold, about 3 fold to about 50 fold, about 3 fold to about 30 fold, about 3 fold to about 20 fold, about 3 fold to about 15 fold, about 5 fold to about 100 fold, about 5 fold to about 50 fold, about 5 fold to about 30 fold, about 5 fold to about 25 fold, about 5 fold to about 20 fold, about 5 fold to about 15 fold, about 10 fold to about 40 fold, about 10 fold to about 30 fold, about 10 fold to about 25 fold, about 10 fold to about 20 fold, or about 12.5 fold to about 17.5 fold of the original volume to provide the rAAV loading composition. In some embodiments, the r AAV sample is diluted, for example with a loading buffer, to about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, about 11 fold, about 12 fold, about 13 fold, about 14 fold, about 15 fold, about 16 fold, about 17 fold, about 18 fold, about 19 fold, about 20 fold, about 21 fold, about 22 fold, about 23 fold, about 24 fold, about 25 fold, about 30 fold, about 35 fold, or about 40 fold of the original volume to provide the rAAV loading composition.

[0067] In certain embodiments, after dilution, the rAAV loading composition before loading has conductivity of about 0.5 mS / cm to about 10.0 mS / cm, about 0.5 mS / cm to about 9.5 mS / cm, about 0.5 mS / cm to about 9.0 mS / cm, about 0.5 mS / cm to about 8.5 mS / cm, about 0.5 mS / cm to about 8.0 mS / cm, about 0.5 mS / cm to about 7.5 mS / cm, about 0.5 mS / cm to about 7.0 mS / cm, about 0.5 mS / cm to about 6.5 mS / cm, about 0.5 mS / cm to about 6.0 mS / cm, about 0.5 mS / cm to about 5.5 mS / cm, about 0.5 mS / cm to about 5.0 mS / cm, about 0.5 mS / cm to about 4.5 mS / cm, about 0.5 mS / cm to about 4.0 mS / cm, about 0.5 mS / cm to about 3.5 mS / cm, about 0.5 mS / cm to about 3.0 mS / cm, about 0.5 mS / cm to about 2.5 mS / cm, about 0.5 mS / cm to about 2.0 mS / cm, 1.0 mS / cm to about 10.0 mS / cm, 1.0 mS / cm to about 9.0 mS / cm, 1.0 mS / cm to about 8.0 mS / cm, 1.0 mS / cm to about 7.0 mS / cm, 1.0 mS / cm to about 6.0 mS / cm, 1.0 mS / cm to about 5.0 mS / cm, 1.0 mS / cm to about 4.0 mS / cm, 1.0 mS / cm to about 3.0 mS / cm, 1.0 mS / cm to about 2.5 mS / cm, about 1.0 mS / cm to about 2.4 mS / cm, about 1.0 mS / cm to about 2.0 mS / cm. about 2.0 mS / cm to about 10.0 mS / cm, about 2.0 mS / cm to about 2.0 mS / cm, about 2.0 mS / cm to about 9.0 mS / cm, about 2.0 mS / cm toAttorney Docket No.: 99CR-403196-WOabout 8.5 mS / cm, about 2.0 mS / cm to about 8.0 mS / cm, about 2.0 mS / cm to about 7.5 mS / cm, about 2.0 mS / cm to about 7.0 mS / cm. about 2.0 mS / cm to about 6.5 mS / cm, about 2.0 mS / cm to about 6.0 mS / cm, about 2.0 mS / cm to about 5.5 mS / cm, about 2.0 mS / cm to about 5.0 mS / cm, about 2.0 mS / cm to about 4.5 mS / cm, about 2.0 mS / cm to about 4.0 mS / cm, about 2.0 mS / cm to about 3.5 mS / cm, about 2.0 mS / cm to about 3.0 mS / cm, or about 2.0 mS / cm to about 2.5 mS / cm. In some embodiments, the rAAV loading composition has conductivity less than about 6.0 mS / cm, about 5.0 mS / cm, about 4.5 mS / cm, about 4.0 mS / cm, or about 3.0 mS / cm.

[0068] In certain embodiments, after dilution, the rAAV loading composition before loading has conductivity of about 0.1 mS / cm, about 0.5 mS / cm, about 1.0 mS / cm, about 1.5 mS / cm, about 2.0 mS / cm, about 2.4 mS / cm, about 2.5 mS / cm, about 3.0 mS / cm, about 3.5 mS / cm, about 4.0 mS / cm, about 4.5 mS / cm, about 5.5 mS / cm, about 6.0 mS / cm, about 6.5 mS / cm. about 7.0 mS / cm, about 7.5 mS / cm, about 8.0 mS / cm, about 8.5 mS / cm, about 9.0 mS / cm, about 9.5 mS / cm, about 10.0 mS / cm, about 12.0 mS / cm, about 15.0 mS / cm, or about 20.0 mS / cm. In certain embodiments, the rAAV loading composition has conductivity in range formed by any two values chosen from the preceding sentence.

[0069] In certain embodiments, the rAAV loading composition has a pH between about 7.5 and about 10.2. In certain embodiments, the pH value of the rAAV loading composition has a range of about 7.5 to about 10.0, such as about 7.5 to about 9.5, about 7.5 to about 9.0, about 8.0 to about 9.0, about 8.6 to about 8.9, about 8.7 to about 8.9, or about 8.7 to about 8.8. In some embodiments, the pH value of the rAAV loading composition is about 7.5, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5. about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1. about 9.2, about 9.3. about 9.4, about 9.5. about 10.0. or about 10.2.

[0070] In certain embodiments, the rAAV loading composition includes the rAAV particles in concentration of about 1.0 x 1012VP / mL to about 1.0 x 1014VP / mL, about 5.0 x 1012VP / mL to about 1.0 x 1014VP / mL, about 1.0 x 1013VP / mL to about 1.0 x 1014VP / mL, about 1.5 x 1013VP / mL to about 1.0 x IO14VP / mL, about 2.0 x 1013VP / mL to about 1.0 x 1014VP / mL, about 1.0 x 1012VP / mL to about 5.0 x 1013VP / mL, about 5.0 x 1012VP / mL to about 5 0 x 1013VP / mL, about 1.0 x 1013VP / mL to about 5.0 x 1013VP / mL, about 1.5 x 10k’ VP / mL to about 5.0 x 1013VP / mL, about 2.0 x 1013VP / mL to about 5.0 x 1013VP / mL, about 1.0 x 1012VP / mL to about 3.0 x 10L’ VP / mL, about 5.0 x IO12VP / mL to about 3.0 x 101’ VP / mL, about 1.0 x 1013VP / mL to about 3.0 x 1013VP / mL, about 1.5 x 1013VP / mL to about 3.0 x 1013VP / mL, about 2.0 x 1013VP / mL to about 3.0 x 1013VP / mL, about 1.0 x 1012Attorney Docket No.: 99CR-403196-WOVP / mL to about 2.0 x 1013VP / mL, about 5.0 x 1012VP / mL to about 2.0 x 1013VP / mL, about 1.0 x 1013VP / mL to about 2.0 x 1013VP / mL, about 1.5 x 1013VP / mL to about 1.0 x 1013VP / mL, about 1.6 x 1013VP / mL to about 2.0 x 1013VP / mL, or about 2.0 x 1012VP / mL to about 1.6 x 1013VP / mL.

[0071] In some embodiments, the rAAV loading composition further includes any suitable buffer or components known in the art. For example, the rAAV loading composition may include Tris, such as in an amount of about lOmM to about 40mM (e.g., 25mM), poloxamer, such as in an amount of about 0.01%, and / or sucrose, such as in an amount of 1.0%.Anion-Exchange Medium

[0072] The methods of the present disclosure are not limited to any particular column structure or type of separation media. As used herein, the term ‘'separation medium” refers to a physical structure, such as a column (e.g., a monolith) or a membrane, to which a recombinant AAV preparation is applied in order to achieve separation of certain fractions of the preparation. For example, a recombinant AAV preparation may be applied to a column, which column is then washed with one or more solutions in order to separate (and collect separated fractions) empty and full AAV particles from one another.

[0073] In some embodiments, a separation medium is an anion-exchange medium. In some embodiments, a separation medium is a column. In some embodiments, a separation medium is a membrane.

[0074] In some embodiments, the anion exchange chromatography medium is a strong anion exchange media. In some embodiments, the anion exchange chromatography medium is a weak anion exchange media. In some embodiments, the anion exchange chromatography medium includes methacrylates, agarose based materials, cellulose, acrylamides, polystyrene di vinyl benzene or silica based materials.

[0075] In some embodiments, the anion exchange chromatography medium includes a quaternary ammonium ligand. In some embodiments, the anion exchange chromatography media includes a quaternary polyethyleneimine. In some embodiments, the monolith anion exchange chromatography media includes diethyl aminoethanol (DEAE) ligand.

[0076] The anion-exchange resins can be any suitable resins used in the prior art. In some embodiments, anion-exchange resins can include basic functional groups, such as quaternary amine, and support materials. The support materials may include any suitable materialsAttorney Docket No.: 99CR-403196-WOknown in the art, for example glycidyl methacrylate, ethylene dimethacrylate or a copolymer thereof. In some embodiments, the support material includes highly cross-linked agarose matrix with a dextran surface extender.

[0077] Further exemplary anion-exchange resin or media include, without limitation, CIMmultus™ QA (available from BIA Separations, Ajdovscina, Slovenia), CIMmultus™ DEAE (available from BIA Separations, Ajdovscina, Slovenia), Macro Prep™ Q (available from BioRad, Hercules, CA), Macro Prep™ DEAE (available from BioRad, Hercules, CA), UNOsphere™ Q (available from BioRad, Hercules. CA), Nuvia™ Q (available from BioRad, Hercules, CA), POROS™ 50HQ (available from Thermo Fisher Scientific, Waltham, MA), POROS™ 50XQ (available from Thermo Fisher Scientific, Waltham, MA), POROS™ 50D (available from Thermo Fisher Scientific, Waltham, MA), POROS™ 50PI (available from Thermo Fisher Scientific, Waltham, MA), SOURCE™ 30Q (available from GE Healthcare, Uppsala, Sweden), MacroCap™ Q (available from GE Healthcare, Uppsala, Sweden), DEAE Sepharose™ Fast Flow (available from GE Healthcare, Uppsala, Sweden), Q Sepharose™ Fast Flow (available from GE Healthcare, Uppsala, Sweden), Q Sepharose™ High Performance (available from GE Healthcare, Uppsala, Sweden), Capto™ Q (available from GE Healthcare, Uppsala. Sweden), Capto™ Q ImpRes (available from GE Healthcare.Uppsala, Sweden), Capto™ DEAE (available from GE Healthcare, Uppsala, Sweden), Q Sepharose™ (available from GE Healthcare, Uppsala, Sweden), Q Sepharose™ XL (available from GE Healthcare, Uppsala, Sweden), STREAMLINE™ Q XL (available from GE Healthcare, Uppsala. Sweden), STREAMLINE™ DEAE (available from GE Healthcare, Uppsala, Sweden), ANX Sepharose™ 4 Fast Flow (available from GE Healthcare, Uppsala, Sweden), Eshmuno™ Q (available from Millipore Sigma, Billerica, MA), Fractogel™ TMAE (available from Millipore Sigma, Billerica, MA), Fractogel™ DEAE (available from Millipore Sigma, Billerica, MA), Cellufine™ MAX Q (available from JNC Corporation, Tokyo. Japan). Cellufine™ MAX DEAE (available from JNC Corporation, Tokyo, Japan), Q Ceramic HyperD™ F (available from Pall Corporation, Westborough, MA), DEAE Ceramic HyperD™ F (available from Pall Corporation, Westborough, MA), Sartobind™ Q (available from Sartorius Stedim Biotech, Germany), Sartobind™ STIC (available from Sartorius Stedim Biotech, Germany), Mustang™ Q (available from Pall Corporation, Westborough, MA), NatrixFlo™ HD-Q (available from Burlington, Ontario, Canada).

[0078] Anion-exchange resin or media (e.g., columns, membranes, etc.) may be equilibrated using standard buffers (e.g., a sodium phosphate buffer) and according to manufacturer’sAttorney Docket No.: 99CR-403196-WOspecifications prior to loading with the rAAV loading composition. In some embodiments, an anion-exchange medium may be equilibrated to approximately the same conductivity’ and pH conditions as the recombinant AAV composition.

[0079] The loading ratio, i.e. the proportion of viral particles (VPs) or vector particles applied to a specific amount of chromatography resin indicates the proportion of viral particles applied to a given amount of chromatography resin. Optimizing this ratio is essential for maximizing the resin's binding capacity, ensuring high purity and yield of the viral vector, and maintaining cost-effectiveness by reducing material usage. In some embodiments, the loading ratio can be about 2.5xl013VP / mLresin to about 5.0 xlO14VP / mLresin, about 5.0xl013VP / mLresin tO about 5.0 xlO14VP / mLresin, about l. OxlO14VP / mLresin to about 5.0 xlO14VP / mLresin, about 1.6xl014VP / mLresin to about 5.0 xlO14VP / mLresin, about 2.5xl013VP / mLresin tO about 3.5 xlO14VP / mLresin, about 5.0xl013VP / mLresin to about 3.5 xlO14VP / mLresin, about l. xlO14VP / mLresin to about 3.5 xlO14VP / mLresin, or about 1.6xl014VP / mLresin tO about 3.5 xlO14VP / mLresin.Chase Step

[0080] After the initial loading step, it is optionally to have a chase step. Its main purpose is to ensure that any remaining sample, which hasn't fully entered the resin matrix during loading, is effectively pushed into the resin bed. This step involves introducing a chase solution, to move the sample without causing elution. By doing so, it ensures that the target molecules are properly positioned for effective interaction with the stationary phase. This optimization enhances the efficiency of subsequent washing and elution steps by ensuring optimal binding of target molecules and minimizing impurities, ultimately improving the overall effectiveness of the purification process.

[0081] In certain embodiments, the chase solution is the same as or similar to the rAAV loading composition except that the chase solution does not include the rAAV particles.

[0082] In certain embodiments, the chase solution include cations lower than the rAAV loading composition.

[0083] In certain embodiments, the chase solution includes cations, such as one or more of KT, Rb~, Cs+, Mg2+, Ca2+, and / or Zn2+salt. In some embodiments, the chase solution includes a concentration of the cation salt at about 0.2 mM to about 10 mM, for example, about 0.2 mM to about 10 mM, about 0.2 mM about 8 mM, about 0.2 mM to about 6 mM, about 0.2Attorney Docket No.: 99CR-403196-WOmM to about 4 mM, about 1 mM to about 10 mM, about 1 mM to about 8mM, about 1 mM to about 6 mM, about 1 mM to about 4 mM, or about 1 mM to about 3 mM.

[0084] In some circumstances, a low or absence of Mg salt in the chase solution may increase yield of the purified full rAAV particles in the eluate. In certain embodiments, the chase solution does not include Mg2+salt. In some embodiments, the chase solution does not include MgCh. In some embodiments, the chase solution does not include MgCh substantially.

[0085] In certain embodiments, the chase solution has a pH between about 7.5 and about 10.2. In certain embodiments, the pH value of the chase solution has a range of about 7.5 to about 10.0. such as about 7.5 to about 9.5. about 7.5 to about 9.0. about 8.0 to about 9.0, about 8.6 to about 8.9, about 8.7 to about 8.9, or about 8.7 to about 8.8. In some embodiments, the pH value of the chase solution is about 7.5, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1. about 9.2, about 9.3. about 9.4, about 9.5. about 10.0. or about 10.2.

[0086] In certain embodiments, the volume of the chase solution is about 1 fold to about 10 fold of the volume of the AEX chromatography medium, such as about 1 fold to about 7 fold, about 1 fold to about 5 fold, about 2 fold to about 7 fold, about 2 fold to about 5 fold, about 3 fold to about 7 fold, about 3 fold to about 5 fold of the volume of the AEX chromatography medium. In certain embodiments, the volume of the chase solution is about 1 fold, about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, or about 10 fold of the volume of the AEX chromatography medium.Wash Step

[0087] As described herein, after an anion-exchange medium has been loaded with a rAAV loading composition, a “wash” solution is applied to the anion-exchange medium. One or more “wash” fractions may be collected while the “wash” solution is applied to the anion-exchange medium. In some embodiments a single “wash” fraction is collected while the anion-exchange medium is washed with the “wash” solution. In some embodiments, each of the wash fractions corresponds to multiple anion-exchange medium (e.g., column / membrane) volumes. In some embodiments, each wash fraction corresponds to a column / membrane volume. In some embodiments, each wash fraction corresponds to a fraction of a column / membrane volume. It will be appreciated that the number of column / membrane volumes of the wash solutions that are required to separate empty AAV particles from fullAttorney Docket No.: 99CR-403196-WOAAV particles will vary and depend on the nature of the recombinant AAV preparation, the type of chromatography media and the particular "‘wash” solutions used. In some embodiments, at least 1 column / membrane volume, at least 5 column / membrane volumes, at least 10 column / membrane volumes, or at least 15 column / membrane volumes of each of the wash solutions are applied to the anion-exchange medium after it has been loaded with a recombinant AAV preparation.

[0088] In some embodiments, the disclosed methods include washing the anion exchange chromatography medium including the bound rAAV particles with a wash solution to remove any unbound impurities. In some embodiment, the wash solution includes about 0.1 mM to about 10 mM cation, such as about 0.5 mM to about 10 mM, about 1 mM to about 10 mM, about 3 m to about 10 mM, about 4 mM to about 10 mM, about 1 mM to about 8 mM, about 2 mM to about 8 mM, about 3 mM to about 8 mM, about 4 mM to about 8 mM, about 1 mM to about 6 mM, about 2 mM to about 6 mM, or about 4mM to about 6 mM salt. In some embodiments, the salt is based on one or more of K. Rb+. Cs. Mg2+, Ca2+, and / or Zn2+salt. In some embodiments, the salt is Mg2+salt. In some embodiments, the Mg2+salt is MgCh. In some embodiment, the wash solution includes about 0.5 mM to about 10 mM, about 1 m to about 10 mM, about 3 mM to about 10 mM, about 4 mM to about 10 mM. about 1 mM to about 8 mM, about 2 mM to about 8 mM, about 3 mM to about 8 mM, about 4 mM to about 8 mM, about 1 mM to about 6 mM, about 2 mM to about 6 mM, or about 4mM to about 6 mM MgCh.

[0089] In some embodiment, the wash solution includes about the same or similar cation concentration, and pH as the rAAV loading composition.

[0090] In some embodiment, the wash solution includes lower MgCh concentration, compared with the rAAV loading composition.

[0091] In certain embodiments, the wash solution has conductivity of about 0.5 mS / cm to about 10.0 mS / cm. about 0.5 mS / cm to about 9.5 mS / cm, about 0.5 mS / cm to about 9.0 mS / cm, about 0.5 mS / cm to about 8.5 mS / cm, about 0.5 mS / cm to about 8.0 mS / cm, about 0.5 mS / cm to about 7.5 mS / cm, about 0.5 mS / cm to about 7.0 mS / cm, about 0.5 mS / cm to about 6.5 mS / cm, about 0.5 mS / cm to about 6.0 mS / cm, about 0.5 mS / cm to about 5.5 mS / cm, about 0.5 mS / cm to about 5.0 mS / cm, about 0.5 mS / cm to about 4.5 mS / cm, about 0.5 mS / cm to about 4.0 mS / cm, about 0.5 mS / cm to about 3.5 mS / cm, about 0.5 mS / cm to about 3.0 mS / cm, about 0.5 mS / cm to about 2.5 mS / cm, about 0.5 mS / cm to about 2.0Attorney Docket No.: 99CR-403196-WOmS / cm, 1.0 mS / cm to about 10.0 mS / cm, 1.0 mS / cm to about 9.0 mS / cm, 1.0 mS / cm to about 8.0 mS / cm, 1.0 mS / cm to about 7.0 mS / cm, 1.0 mS / cm to about 6.0 mS / cm, 1.0 mS / cm to about 5.0 mS / cm, 1.0 mS / cm to about 4.0 mS / cm, 1.0 mS / cm to about 3.0 mS / cm, 1.0 mS / cm to about 2.5 mS / cm, about 1.0 mS / cm to about 2.4 mS / cm, about 1.0 mS / cm to about 2.0 mS / cm, about 2.0 mS / cm to about 10.0 mS / cm, about 2.0 mS / cm to about 2.0 mS / cm, about 2.0 mS / cm to about 9.0 mS / cm, about 2.0 mS / cm to about 8.5 mS / cm, about 2.0 mS / cm to about 8.0 mS / cm, about 2.0 mS / cm to about 7.5 mS / cm, about 2.0 mS / cm to about 7.0 mS / cm, about 2.0 mS / cm to about 6.5 mS / cm, about 2.0 mS / cm to about 6.0 mS / cm, about 2.0 mS / cm to about 5.5 mS / cm, about 2.0 mS / cm to about 5.0 mS / cm, about 2.0 mS / cm to about 4.5 mS / cm, about 2.0 mS / cm to about 4.0 mS / cm, about 2.0 mS / cm to about 3.5 mS / cm, about 2.0 mS / cm to about 3.0 mS / cm. or about 2.0 mS / cm to about 2.5 mS / cm. In some embodiments, the rAAV loading composition has conductivity less than about 6.0 mS / cm, about 5.0 mS / cm, about 4.5 mS / cm, about 4.0 mS / cm, or about 3.0 mS / cm.

[0092] In certain embodiments, wash solution has a pH between pH between about 7.5 and about 10.2. In certain embodiments, the pH value of the wash solution has a range of about 7.5 to about 10.0, such as about 7.5 to about 9.5, about 7.5 to about 9.0, about 8.0 to about 9.0, about 8.6 to about 8.9. about 8.7 to about 8.9. or about 8.7 to about 8.8. In some embodiments, the pH value of the rAAV loading composition is about 7.5, about 8 0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 10.0, or about 10.2.Isocratic elution solution

[0093] In some embodiments, the disclosed methods include applying to the AEX chromatography medium an elution solution to provide an eluate.

[0094] As described herein, after an anion-exchange medium has been loaded with a recombinant AAV preparation and washed with wash solution, isocratic separation methods of the present disclosure involve applying an elution solution including a fixed concentration of magnesium chloride (MgCh) and optionally sodium chloride (NaCI) to the anion-exchange medium. In some embodiments a series of elution fractions are collected while this elution solution is applied. In some embodiments, the series includes a first elution fraction, and a second elution fraction collected after the first elution fraction. In some embodiments, certain elution fractions from the series are subsequently pooled. In some embodiments, each elution fraction has approximately the same volume and the elution fractions are collected inAttorney Docket No.: 99CR-403196-WOa consecutive and continuous fashion. In some embodiments, each elution fraction corresponds to multiple column / membrane volumes. In some embodiments, each elution fraction corresponds to a column / membrane volume. In some embodiments, each elution fraction corresponds to a fraction of a column / membrane volume. It will be appreciated that the number of column / membrane volumes that are required to separate empty AAV particles from full AAV particles will vary and depend on the nature of the recombinant AAV preparation, the anion-exchange medium and the particular elution solution used. In some embodiments, at least 1 column / membrane volume, at least 5 column / membrane volumes, at least 10 column / membrane volumes, at least 15 column / membrane volumes, at least 20 column / membrane volumes, at least at least 25 column / membrane volumes, at least 30 column / membrane volumes, or more of the elution solution are applied to the column / membrane after it has been loaded with a recombinant AAV preparation.

[0095] In some embodiments, the pH and salt concentration of the elution solution is constant, so it is isocratic. The term "isocratic" as used herein refers to keeping the composition of the solvent constant during a particular step. In certain embodiments, the pH and / or salt concentration remain substantially unchanged during the elution step.

[0096] In some embodiments, the elution solution includes one or more monovalent or divalent cations. In some embodiments, the cation includes one or more of Na, K+, Rb+. Cs‘, Mg2", Ca2+, and / or Zn2+salt.

[0097] In some embodiments, the elution solution includes between about 0.1 mM to about 10 mM salt, such as about 0.5 mM to about 10 mM, about 1 mM to about 10 mM, about 3 mM to about 10 mM, about 4 mM to about 10 mM, about 1 mM to about 8 mM, about 2 mM to about 8 mM, about 3 mM to about 8 mM, about 4 mM to about 8 mM, about 1 m to about 6 mM, about 2 mM to about 6 mM, or about 4mM to about 6 mM salt. In some embodiments, the salt is based on one or more of K.", Rb+, Cs\ Mg2*, Ca2+, and / or Zn21salt. In some embodiments, the salt is Mg2+salt. In some embodiments, the Mg2* salt is MgCh. In some embodiment, the wash solution includes about 0.5 mM to about 10 mM, about 1 mM to about 10 mM, about 3 mM to about 10 mM, about 4 mM to about 10 mM, about 1 mM to about 8 mM. about 2 mM to about 8 mM, about 3 M to about 8 mM, about 4 mM to about 8 mM, about 1 mM to about 6 mM, about 2 mM to about 6 mM, or about 4 mM to about 6 mM MgCh. In some embodiments, the elution solution includes about 2 mM, 3 mM, 4 mM, 5 mM, or 6 mM MgCh.Attomey Docket No.: 99CR-403196-WO

[0098] In some embodiments, the MgCh concentration of the elution solution is lower than the MgCh concentration of the wash solution. In some embodiments, the MgCh concentration of the elution solution is greater than the MgCh concentration of the wash solution. In some embodiments, the MgCh concentration of the elution solution is substantially similar to the MgCh concentration of the wash solution.

[0099] In some embodiments, the elution solution includes between about 0.1 M and about 2 M, such as about 0.1 M to about 1.5 M, about 0.2 M to about 1.5 M, about 0.3 M to about 1.5 M, about 0.4 M to about 1.5 M, about 0.5 M to about 1.5 M, about 0.1 M to about 1.0 M, about 0.2 M to about 0.5 M, about 0.1 M to about 0.3 M monovalent cation salt. In some embodiments, the monovalent cation is Na" salt. Tn some embodiments, the Na" salt is NaCl. In some embodiments, the elution solution does not include NaCl. In some embodiments, the monovalent cation salt is KC1.

[0100] In some embodiments, the methods involve isocratic elution solutions that include particular concentrations of MgCh and optionally NaCl. In some embodiments, a combination of MgCh and NaCl is used in one or more of the elution solutions.

[0101] The separation of full AAV particles and empty AAV particles in recombinant AAV preparations using anion-exchange chromatography can be improved using a single elution solution which includes a fixed concentration of magnesium chloride (MgCh) and optionally sodium chloride (NaCl). In some embodiments, the methods involve an isocratic elution with a solution that includes particular concentrations of magnesium chloride and optionally sodium chloride. In some embodiments, a combination of magnesium chloride and sodium chloride is used in the elution solution.

[0102] In some embodiments, the elution solution does not include NaCl, and NaCl can be added to the elute instead. As described in the Examples, it has been discovered that the addition of NaCl in the elute could effectively prevent aggregated viral particles or capsids, therefore increasing the yield. It is contemplated that NaCl increases the ionic strength of the solution, which screens charges on the surface of the viral particles, reducing electrostatic interactions that can lead to aggregation. This increased salt concentration helps stabilize the viral capsids, preventing them from sticking together or to the column matrix, thereby maintaining their integrity and functionality. Additionally, NaCl can improve the separation efficiency by reducing non-specific interactions with the resin, enhancing the resolution between full capsids, empty ones, and other impurities.Attorney Docket No.: 99CR-403196-WO

[0103] In some embodiments, the method described herein further includes a step of adding NaCl to the eluate to prevent or reduce the aggregation of viral particles or capsids. In some embodiments, the isolated rAAV particles in the eluate in the eluate include less than about 20% of aggregated viral particles or capsids. In some embodiments, the isolated rAAV particles in the eluate include less than about 15% of aggregated viral particles or capsids. In some embodiments, the isolated rAAV particles in the eluate include less than about 10% of aggregated viral particles or capsids. In some embodiments, the isolated rAAV particles in the eluate include less than about 9% of aggregated viral particles or capsids. In some embodiments, the isolated rAAV particles in the eluate include less than about 8% of aggregated viral particles or capsids. In some embodiments, the isolated rAAV particles in the eluate include less than about 7% of aggregated viral particles or capsids. In some embodiments, the isolated rAAV particles in the eluate include less than about 6% of aggregated viral particles or capsids. In some embodiments, the isolated rAAV particles in the eluate include less than about 5% of aggregated viral particles or capsids. In some embodiments, the isolated rAAV particles in the eluate include less than about 4% of aggregated viral particles or capsids. In some embodiments, the isolated rAAV particles in the eluate include less than about 3% of aggregated viral particles or capsids. In some embodiments, the isolated rAAV particles in the eluate include less than about 2% of aggregated viral particles or capsids. In some embodiments, the isolated rAAV particles in the eluate include less than about 1% of aggregated viral particles or capsids.Quantifying Percentage Full A A V Particles

[0104] In some embodiments, the percentage of full AAV particles (vs. empty AAV particles) in a separated product (e.g., an elution fraction from an isocratic separation or a product generated by pooling several elution fractions from an isocratic) can be measured by an HPLC assay, e.g.. as follows. A sample (e.g., elution fraction) is diluted at least 4-fold with 25 mM Bis-Tris Propane, 1 mM MgCh at pH 10 to promote binding to an anion-exchange HPLC column (BIA Separations CIMAC AAV full / empty-0.1 Analytical Column, Pores 1.3 pm). The column is equilibrated with the same dilution buffer as the sample. A small volume of diluted sample (e g., about 50-200 pL) is injected into the equilibration buffer mobile phase, which is flowing through the column. AAV particles in the sample bind to the column. Subsequently, buffer is applied, whereby MgCb concentration of the buffer is held constant (e.g., 1 mM MgCb), and NaCl concentration is gradually increased (e.g., from 0 to 250 mM). Empty AAV particles and full AAV particles partition / elute off of the HPLCAttomey Docket No.: 99CR-403196-WOcolumn at different concentrations of NaCl, which correlates to actual time spent on the column by the AAV particles (“retention time”).

[0105] To decouple any effect of AAV capsid protein and AAV payload A280 / A260 mutual absorbance, native florescence of AAV capsid protein is measured, at an emittance of 350 nm. The AAV payload (nucleic acid payload encapsidated by AAV particle) does not have significant florescence at emittance of 350 nm. As empty and full AAV particles desorb from the HPLC column, they flow past a fluorescence detector, and the measured fluorescence is recorded on a chromatogram. In some embodiments, empty AAV particles elute at lower NaCl concentrations than full AAV particles. Accordingly, in some such embodiments, a first peak is labeled “empty ”; in these embodiments, full particles elute after empty particles, and a next peak is labeled “full.” It is to be understood that in some embodiments, a first peak may be labeled “full” and a second peak “empty”. It will be known by one of skill in the art that distinguishing between empty and full peaks may be facilitated using an AK. TA gradient, which displays A280 / A260 ratios, and informs order of HPLC column desorption (e.g., empty particles, then full or full particles, then empty).

[0106] Once peaks have been identified, they are integrated. The ratio of the integrated peaks (peak area) is representative of the ratio of empty AAV particles to full AAV particles (i.e., providing a “% Full AAV”).

[0107] In some embodiments, a sample (e.g., elution fraction) is enriched in full AAV particles and such a fraction includes greater than 50% full AAV particles, as measured by an HPLC assay described herein. In some embodiments, a sample (e.g., elution fraction) includes greater than about 60%, 70%, 80%, 90%, 95%, 98%, or 99% full AAV particles, as measured by the HPLC assay described herein.

[0108] In some embodiments, the eluate includes at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% of full capsid recombinant AAV, as measured by the HPLC assay described herein.

[0109] In some embodiments, the % of full capsid recombinant AAV can be measured by mass photometry, AUC (A260), or AUC (A230).

[0110] In some embodiments, the eluate described herein includes a percentage of full capsid recombinant AAV at least 1.5-fold (e.g. at least 1.8-fold, at least 2-fold, at least 2.2-fold, at least 2.5-fold, at least 2.8-fold, at least 3-fold, at least 3.2-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 7-fold, at least 8-Attorney Docket No.: 99CR-403196-WOfold, at least 9-fold, at least 10-fold, at least 15-fold, or at least 20-fold) higher than that before the AEX purification step.[OHl] The presence of empty AAV particles in a gene therapy product may increase the overall dose needed to achieve therapeutic efficacy and may also cause an immune response upon administration to a patient (e.g., neutralization of the AAV particles by host development of neutralizing antibodies, or clearance of AAV -transduced cells due to T-cell activation). Therefore, the percentage of empty capsid recombinant AAV in the eluate should be as low as possible.

[0112] In some embodiments, the eluate includes less than about 10%, less than about 9%. less than about 8%, less than about 7%, less than about 6%. less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of empty capsid recombinant AAV.

[0113] In some embodiments, the eluate described herein includes a percentage of empty capsid recombinant AAV at least 0.9-fold (e.g. at least 0.8-fold, at least 0.7-fold, at least 0.6-fold, at least 0.5-fold, at least 0.4-fold, at least 0.3-fold, at least 0.2-fold, at least 0.1-fold, at least 0.05-fold) lower than that before the AEX purification step.Yield

[0114] The provided methods produce isolated rAAV particles in the eluate without significant losses of rAAV. In some embodiments, the yield of rAAV particles in the eluate is at least about 40%. In some embodiments, the yield of rAAV particles in the eluate is at least about 50%. In some embodiments, the yield of rAAV particles in the eluate is at least about 60%. In some embodiments, the yield of rAAV particles in the eluate is at least about 70%. In some embodiments, the yield of rAAV particles in the eluate is at least about 75%. In some embodiments, the yield of rAAV particles in the eluate is at least about 80%. In some embodiments, the yield of rAAV particles in the eluate is at least about 85%. In some embodiments, the yield of rAAV particles in the eluate is at least about 90%. In some embodiments, the yield of rAAV particles in the eluate is at least about 95%. In some embodiments, the yield of rAAV particles in the eluate is at least about 96%. In some embodiments, the yield of rAAV particles in the eluate is at least about 97%. In some embodiments, the yield of rAAV particles in the eluate is at least about 98%. In some embodiments, the yield of rAAV particles in the eluate is at least about 99%. In one embodiments, the yield is viral genome (VG) yield.Attorney Docket No.: 99CR-403196-WO

[0115] The term "viral genome yield" as used herein refers to the yield of viral genomes (VG) obtained after the purification process. Methods to measure viral genome (VG) yield in the eluate are known to the art, such as quantitative PCR (qPCR) or droplet digital PCR (ddPCR) to quantify the number of viral genomes. Analyze the data by calculating the total yield, expressed as the total number of viral genomes or a percentage of the initial load.

[0116] In some embodiments, at least about 10% of the viral capsids in the eluate are intact rAAV particles. In some embodiments, at least about 20% of the viral capsids in the eluate are intact rAAV particles. In some embodiments, at least about 30% of the viral capsids in the eluate are intact rAAV particles. In some embodiments, at least about 40% of the viral capsids in the eluate are intact rAAV particles. In some embodiments, at least about 50% of the viral capsids in the eluate are intact rAAV particles. In some embodiments, at least about 60% of the viral capsids in the eluate are intact rAAV particles. In some embodiments, at least about 70% of the viral capsids in the eluate are intact rAAV particles. In some embodiments, at least about 80% of the viral capsids in the eluate are intact rAAV particles. In some embodiments, at least about 90% of the viral capsids in the eluate are intact rAAV particles. In some embodiments, at least about 95% of the viral capsids in the eluate are intact rAAV particles. In some embodiments, at least about 97% of the viral capsids in the eluate are intact rAAV particles. In some embodiments, at least about 98% of the viral capsids in the eluate are intact rAAV particles. In some embodiments, at least about 99% of the viral capsids in the eluate are intact rAAV particles.Multi-step purification

[0117] In certain embodiments, the methods described herein can further include a second purification step after the AEX described herein. Such purification step can be selected from any types, such as affinity chromatography, cation exchange chromatography, anion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography or mixed-mode chromatography (combines multiple interaction ty pes, such as ionic, hydrophobic, and hydrogen bonding, to achieve high selectivity and efficiency in separating biomolecules). In some embodiments, the second purification step is another anion exchange chromatography. In some embodiments, a different AEX medium is used for the second purification step from the first purification step.Downstream processes

[0118] The eluate from the one or more purification steps including the anion exchange chromatography described herein can be further processed using methods known in the art. InAttorney Docket No.: 99CR-403196-WOsome embodiments, methods of isolating rAAV particles disclosed herein include processing the eluate by a method including downstream processing such as. for example, tangential flow filtration, affinity chromatography, size exclusion chromatography, cation exchange chromatography, hydroxylapatite chromatography, and hydrophobic interaction chromatography. In some embodiments, downstream processing includes at least 2, at least 3, or at least 4 of: tangential flow filtration, affinity' chromatography, anion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, or sterile filtration. In some embodiments, downstream processing includes tangential flow' filtration. In some embodiments, downstream processing includes sterile filtration. In further embodiments, downstream processing includes tangential flow filtration and sterile filtration.Embodiments

[0119] Embodiment 1. A method for purifying recombinant adeno-associated virus (rAAV) particles, comprising: (a) contacting an anion exchange (AEX) chromatography medium with a recombinant AAV (rAAV) loading composition comprising full rAAV particles and empty rAAV particles, wherein the rAAV loading composition further comprises MgCh; (b) applying to the AEX chromatography medium a w ash solution; and (c) applying to the AEX chromatography medium an elution solution to provide an eluate, wherein pH and salt concentration of the elution solution is isocratic.

[0120] Embodiment 2. The method of embodiment 1, wherein the full and empty rAAV particles are one or more selected from the group consisting of: AAV2, AAV5, AAV8, and AAV9.

[0121] Embodiment 3. The method of embodiment 1 or 2, wherein the full and empty rAAV particles are AAV2.

[0122] Embodiment 4. The method of any one of embodiments 1-3, wherein the full rAAV particles comprises one or more polynucleotides.

[0123] Embodiment 5. The method of any one of embodiments 1-4, wherein the full rAAV particles comprise a single vector.

[0124] Embodiment 6. The method of any one of embodiments 1-4, w herein the full rAAV particles include two or more different polynucleotides.

[0125] Embodiment 7. The method of any one of embodiments 1-6, wherein the rAAV loading composition comprises about 4 mM to about 11 mM of MgCh.Attorney Docket No.: 99CR-403196-WO

[0126] Embodiment 8. The method of any one of embodiments 1-7, wherein the rAAV loading composition comprises about 5 mM to about 9 mM of MgCk.

[0127] Embodiment 9. The method of any one of embodiments 1-8, wherein the rAAV loading composition comprises about 6 mM to about 8 mM of MgCk.

[0128] Embodiment 10. The method of any one of embodiments 1-7, wherein the rAAV loading composition comprises about 6.5 mM to about 10.5 mM of MgCh.

[0129] Embodiment 11. The method of any one of embodiments 1-10, wherein the concentration of MgCb in the rAAV loading composition is substantially proportional to the percentage of full recombinant AAV particles in the eluate.

[0130] Embodiment 12. The method of any one of embodiments 1-11, wherein the rAAV loading composition has pH of about 8 to about 9.

[0131] Embodiment 13. The method of any one of embodiments 1-12, wherein the rAAV loading composition has pH of about 8.6 to about 8.9.

[0132] Embodiment 14. The method of any one of embodiments 1-13, wherein the rAAV loading composition has pH of about 8.7 to about 8.9.

[0133] Embodiment 15. The method of any one of embodiments 1-14, wherein the rAAV loading composition has pH of about 8.7 to about 8.8.

[0134] Embodiment 16. The method of any one of embodiments 1-15, wherein the rAAV loading composition is about 10-fold to 40-fold diluted from a recombinant AAV (rAAV) sample.

[0135] Embodiment 17. The method of any one of embodiments 1-16, wherein the rAAV loading composition is about 10-fold to 30-fold diluted from a recombinant AAV (rAAV) sample.

[0136] Embodiment 18. The method of any one of embodiments 1-17, wherein the rAAV loading composition is about 10-fold to 20-fold diluted from a recombinant AAV (rAAV) sample.

[0137] Embodiment 19. The method of any one of embodiments 1-18, wherein the rAAV loading composition is about 12.5-fold to 17.5-fold diluted from a recombinant AAV (rAAV) sample.Attorney Docket No.: 99CR-403196-WO

[0138] Embodiment 20. The method of any one of embodiments 1-19, wherein the rAAV loading composition is about 15-fold diluted from a recombinant AAV (rAAV) sample.

[0139] Embodiment 21. The method of any one of embodiments 1-20, wherein the rAAV loading composition has conductivity of about 1.0 mS / cm to about 6.0 mS / cm.

[0140] Embodiment 22. The method of any one of embodiments 1-21, wherein the rAAV loading composition has conductivity7of about 1.0 mS / cm to about 4.5 mS / cm.

[0141] Embodiment 23. The method of any one of embodiments 1-22, wherein the rAAV loading composition has conductivity of about 1.0 mS / cm to about 4.0 mS / cm.

[0142] Embodiment 24. The method of any one of embodiments 1-23, wherein the rAAV loading composition has conductivity of about 1.0 mS / cm to about 2.4 mS / cm.

[0143] Embodiment 25. The method of any one of embodiments 1-24, wherein the rAAV loading composition comprises the rAAV particles in a concentration of about 1.0 x 1012VP / mL to about 5.0 x 1013VP / mL.

[0144] Embodiment 26. The method of any one of embodiments 1-24, wherein the rAAV loading composition comprises the rAAV particles in a concentration of about 2.0 x 1012VP / mL to about 1.6 x 1013VP / mL.

[0145] Embodiment 27. The method of any one of embodiments 1-24, wherein the rAAV loading composition comprises the rAAV particles in a concentration of about 1.6 x 1013VP / mL to about 2.0 x 1013VP / mL.

[0146] Embodiment 28. The method of any one of embodiments 1-27, wherein the rAAV loading composition further comprises one or more selected from the group consisting of Tris, poloxamer and sucrose.

[0147] Embodiment 29. The method of any one of embodiments 1-28, wherein the AEX chromatography medium comprises quaternary amine.

[0148] Embodiment 30. The method of any one of embodiments 1-29, wherein the AEX chromatography medium comprises a copolymer of glycidyl methacrylate and ethylene dimethacrylate.Attorney Docket No.: 99CR-403196-WO

[0149] Embodiment 31. The method of any one of embodiments 1-29, wherein the AEX chromatography medium comprises a highly cross-linked agarose with dextran surface extender.

[0150] Embodiment 32. The method of any one of embodiments 1-31, wherein the rAAV loading composition is added to the anion exchange (AEX) chromatography medium in an amount of about 2.5xl013VP / mLresin to about 5.0 xlO14VP / mLiesin.

[0151] Embodiment 33. The method of any one of embodiments 1-31, wherein the recombinant AAV composition is added to anion exchange (AEX) chromatography medium in an amount of about 1.6xl014VP / mLresin to about 3.5 xlO14VP / mLresin.

[0152] Embodiment 34. The method of any one of embodiments 1-33, further comprising applying a chase solution after the step (a).

[0153] Embodiment 35. The method of embodiment 34, wherein the chase solution does not comprise MgCh.

[0154] Embodiment 36. The method of embodiment 34 or 35. wherein the chase solution has pH of about 8.7.

[0155] Embodiment 37. The method of any one of embodiments 34-36. wherein the chase solution is added in an amount of 5 fold of the volume of the AEX chromatography medium.

[0156] Embodiment 38. The method of any one of embodiments 1-37, wherein the wash solution comprises MgCh.

[0157] Embodiment 39. The method of any one of embodiments 1-38, wherein the wash solution comprises about 3 mM to about 10 mM of MgCh.

[0158] Embodiment 40. The method of any one of embodiments 1-39, wherein the wash solution comprises about 4 mM to about 6 mM of MgCh.

[0159] Embodiment 41. The method of any one of embodiments 1-40, wherein the wash solution has conductivity of about 1.4 mS / cm to about 4.0 mS / cm.

[0160] Embodiment 42. The method of any one of embodiments 1-41, wherein the wash solution has conductivity of about 2.5 mS / cm to about 4.0 mS / cm.

[0161] Embodiment 43. The method of any one of embodiments 1-42, wherein the wash solution has conductivity of about 2.7 mS / cm to about 2.9 mS / cm.Attorney Docket No.: 99CR-403196-WO

[0162] Embodiment 44. The method of any one of embodiments 1-43, wherein the wash solution has pH of about 7.7 to about 9.5.

[0163] Embodiment 45. The method of any one of embodiments 1-44, wherein the wash solution has pH of about 8.8 to about 9.1.

[0164] Embodiment 46. The method of any one of embodiments 1-45, wherein the elution solution comprise about 3.0 mM to about 5.0 mM of MgCh.

[0165] Embodiment 47. The method of any one of embodiments 1-46, wherein the elution solution comprises about 4.0 mM of MgCh.

[0166] Embodiment 48. The method of any one of embodiments 1-47, wherein the elution solution has pH of about 7.0 to about 8.0.

[0167] Embodiment 49. The method of any one of embodiments 1-48, wherein the elution solution has pH of about 7.5.

[0168] Embodiment 50. The method of any one of embodiments 1-49, wherein pH of the elution solution is lower than pH of the wash solution.

[0169] Embodiment 51. The method of any one of embodiments 1-50, wherein the elution solution does not substantially comprise NaCl.

[0170] Embodiment 52. The method of any one of embodiments 1-51, further comprising adding NaCl to the eluate.

[0171] Embodiment 53. The method of embodiment 52, wherein aggregation of AAV is decreased by the addition of NaCl to the eluate.

[0172] Embodiment 54. The method of any one of embodiments 1-53, wherein the eluate comprises at least 70% of full capsid recombinant AAV.

[0173] Embodiment 55. The method of any one of embodiments 1-54, wherein the eluate comprises at least 90% of full capsid recombinant AAV.

[0174] Embodiment 56. The method of any one of embodiments 1-55, wherein the eluate comprises at least 95% of full capsid recombinant AAV.

[0175] Embodiment 57. The method of any one of embodiments 1-53, wherein the eluate comprises less than 10 % of empty capsid recombinant AAV.Attorney Docket No.: 99CR-403196-WO

[0176] Embodiment 58. The method of any one of embodiments 1-53, wherein the eluate comprises less than 5% of empty capsid recombinant AAV.

[0177] Embodiment 59. The method of any one of embodiments 1-58, wherein the eluate provides VG yield of about 50% or greater.

[0178] Embodiment 60. The method of any one of embodiments 1-59, wherein the eluate provides VG yield of about 60% or greater.

[0179] Embodiment 61. The method of any one of embodiments 1-63, further comprising purifying the eluate.

[0180] Embodiment 62. A method for isolating recombinant adeno-associated virus (rAAV) particles, comprising: (a) contacting an anion exchange (AEX) chromatography medium with a rAAV composition comprising full rAAV particles and empty rAAV particles; (b) applying to the AEX chromatography medium a wash solution comprising MgCh; and (c) applying to the AEX chromatography medium an elution solution to provide an eluate, wherein the pH and salt concentration of the elution solution is isocratic, and wherein pH concentration of the elution solution is lower than pH of the wash solution.

[0181] Embodiment 63. The method of embodiment 62, wherein the full and empty rAAV particles are one or more selected from the group consisting of: AAV2, AAV5, AAV8, and AAV9.

[0182] Embodiment 64. The method of embodiment 62 or 63, wherein the full and empty rAAV particles are AAV2.

[0183] Embodiment 65. The method of any one of embodiments 62-64, wherein the full rAAV particles comprises one or more polynucleotides.

[0184] Embodiment 66. The method of any one of embodiments 62-65. wherein the full rAAV particles comprise a single vector.

[0185] Embodiment 67. The method of any one of embodiments 62-65. wherein the full rAAV particles include two or more different polynucleotides.

[0186] Embodiment 68. The method of any one of embodiments 62-67, wherein the rAAV loading composition has pH of about 8 to about 9.

[0187] Embodiment 69. The method of any one of embodiments 62-68, wherein the rAAV loading composition has pH of about 8.6 to about 8.9.Attorney Docket No.: 99CR-403196-WO

[0188] Embodiment 70. The method of any one of embodiments 62-69, wherein the rAAV loading composition has pH of about 8.7 to about 8.9.

[0189] Embodiment 71. The method of any one of embodiments 62-70, wherein the rAAV loading composition has pH of about 8.7 to about 8.8.

[0190] Embodiment 72. The method of any one of embodiments 62-71. wherein the rAAV loading composition is about 10-fold to 40-fold diluted from a recombinant AAV (rAAV) sample.

[0191] Embodiment 73. The method of any one of embodiments 62-72, wherein the rAAV loading composition is about 10-fold to 30-fold diluted from a recombinant AAV (rAAV) sample.

[0192] Embodiment 74. The method of any one of embodiments 62-73, wherein the rAAV loading composition is about 10-fold to 20-fold diluted from a recombinant AAV (rAAV) sample.

[0193] Embodiment 75. The method of any one of embodiments 62-74. wherein the rAAV loading composition is about 12.5-fold to 17.5-fold diluted from a recombinant AAV (rAAV) sample.

[0194] Embodiment 76. The method of any one of embodiments 62-75, wherein the rAAV loading composition is about 15-fold diluted from a recombinant AAV (rAAV) sample.

[0195] Embodiment 77. The method of any one of embodiments 62-76, wherein the rAAV loading composition has conductivity of about 1.0 mS / cm to about 6.0 mS / cm.

[0196] Embodiment 78. The method of any one of embodiments 62-77, wherein the rAAV loading composition has conductivity of about 1.0 mS / cm to about 4.5 mS / cm.

[0197] Embodiment 79. The method of any one of embodiments 62-78, wherein the rAAV loading composition has conductivity of about 1.0 mS / cm to about 4.0 mS / cm.

[0198] Embodiment 80. The method of any one of embodiments 62-79. wherein the rAAV loading composition has conductivity of about 1.0 mS / cm to about 2.4 mS / cm.

[0199] Embodiment 81. The method of any one of embodiments 62-80. wherein the rAAV loading composition comprises the rAAV particles in a concentration of about 1.0 x 1012VP / mL to about 5.0 x 1013VP / mL.Attorney Docket No.: 99CR-403196-WO

[0200] Embodiment 82. The method of any one of embodiments 62-81, wherein the rAAV loading composition further comprises one or more selected from the group consisting of Tris, poloxamer and sucrose.

[0201] Embodiment 83. The method of any one of embodiments 62-82. wherein the AEX chromatography medium comprises quaternary amine.

[0202] Embodiment 84. The method of any one of embodiments 62-83, wherein the AEX chromatography medium comprises a copolymer of glycidyl methacrylate and ethylene dimethacrylate.

[0203] Embodiment 85. The method of any one of embodiments 62-84, wherein the AEX chromatography medium comprises a highly cross-linked agarose with dextran surface extender.

[0204] Embodiment 86. The method of any one of embodiments 60-85, wherein the rAAV loading composition is added to the anion exchange (AEX) chromatography medium in an amount of about 2.5xl013VP / mLresin to about 5.0 xlO14VP / mLresin.

[0205] Embodiment 87. The method of any one of embodiments 62-86, wherein the recombinant AAV composition is added to anion exchange (AEX) chromatography medium in an amount of about 1.6xl014VP / mLresin to about 3.5 xlO14VP / mLresin.

[0206] Embodiment 88. The method of any one of embodiments 62-87, further comprising applying a chase solution after the step (a).

[0207] Embodiment 89. The method of embodiment 88, wherein the chase solution has pH of about 8.7.

[0208] Embodiment 90. The method of embodiment 88 or 89, wherein the chase solution is added in an amount of 5 fold of the volume of the AEX chromatography medium.

[0209] Embodiment 91. The method of any one of embodiments 62-90, wherein the wash solution comprises MgCh.

[0210] Embodiment 92. The method of any one of embodiments 62-91. wherein the wash solution comprises about 3 mM to about 10 mM of MgCh.

[0211] Embodiment 93. The method of any one of embodiments 62-92. wherein the wash solution comprises about 4 mM to about 6 mM of MgCh.Attorney Docket No.: 99CR-403196-WO

[0212] Embodiment 94. The method of any one of embodiments 62-93, wherein the wash solution has conductivity of about 1.4 mS / cm to about 4.0 mS / cm.

[0213] Embodiment 95. The method of any one of embodiments 62-94, wherein the wash solution has conductivity of about 2.5 mS / cm to about 4.0 mS / cm.

[0214] Embodiment 96. The method of any one of embodiments 62-95. wherein the wash solution has conductivity of about 2.7 mS / cm to about 2.9 mS / cm.

[0215] Embodiment 97. The method of any one of embodiments 62-96. wherein the wash solution has pH of about 7.7 to about 9.5.

[0216] Embodiment 98. The method of any one of embodiments 62-97, wherein the wash solution has pH of about 8.8 to about 9.1.

[0217] Embodiment 99. The method of any one of embodiments 62-98, wherein the elution solution comprise about 3.0 mM to about 5.0 mM of MgCh.

[0218] Embodiment 100. The method of any one of embodiments 62-99, wherein the elution solution comprises about 4.0 mM of MgCh.

[0219] Embodiment 101. The method of any one of embodiments 62-100, wherein the elution solution has pH of about 7.0 to about 8.0.

[0220] Embodiment 102. The method of any one of embodiments 62-101, wherein the elution solution has pH of about 7.5.

[0221] Embodiment 103. The method of any one of embodiments 62-102, wherein the elution solution does not substantially comprise NaCl.

[0222] Embodiment 104. The method of any one of embodiments 62-103, further comprising adding NaCl to the eluate.

[0223] Embodiment 105. The method of embodiment 104, wherein aggregation of AAV is decreased by the addition of NaCl to the eluate.

[0224] Embodiment 106. The method of any one of embodiments 62-105, wherein the eluate comprises at least 70% of full capsid recombinant AAV.

[0225] Embodiment 107. The method of any one of embodiments 62-106, wherein the eluate comprises at least 90% of full capsid recombinant AAV.Attorney Docket No.: 99CR-403196-WO

[0226] Embodiment 108. The method of any one of embodiments 62-107, wherein the eluate comprises at least 95% of full capsid recombinant AAV.

[0227] Embodiment 109. The method of any one of embodiments 62-108, wherein the eluate comprises less than 10 % of empty capsid recombinant AAV.

[0228] Embodiment 110. The method of any one of embodiments 62-109, wherein the eluate comprises less than 5% of empty capsid recombinant AAV.

[0229] Embodiment 111. The method of any one of embodiments 62-110, wherein the eluate provides VG yield of about 50% or greater.

[0230] Embodiment 112. The method of any one of embodiments 62-111, wherein the eluate provides VG yield of about 60% or greater.

[0231] Embodiment 113. The method of any one of embodiments 62-112, further comprising purifying the eluate.

[0232] Embodiment 114. A method for isolating recombinant adeno-associated virus (rAAV) particles, comprising: (a) contacting an anion exchange (AEX) chromatography medium with a rAAV composition comprising full rAAV particles and empty rAAV particles, wherein the rAAV composition further comprises MgCh; (b) applying to the AEX chromatography medium a wash solution comprising MgC; and (c) applying to the AEX chromatography medium an elution solution to provide an eluate, wherein the pH and salt concentration of the elution solution is isocratic, wherein pH concentration of the elution solution is lower than pH of the wash solution.

[0233] Embodiment 115. A method for isolating recombinant adeno-associated virus (rAAV) particles, comprising: (a) contacting an anion exchange (AEX) chromatography medium with a rAAV composition comprising full rAAV particles and empty rAAV particles, wherein the rAAV composition has conductivity of about 1.0 to 4.5 mS / cm; (b) applying to the AEX chromatography medium a wash solution; and (c) applying to the AEX chromatography medium an elution solution to provide an eluate, wherein the pH and salt concentration of the elution solution is isocratic.

[0234] Embodiment 116. The method of embodiments 54-56, wherein the full capsid recombinant AAV is measured by HPLC assay, mass photometry, AUC (A260), or AUC (A230).Attorney Docket No.: 99CR-403196-WOEXAMPLESMethodsPre-Purification Process

[0235] Recombinant AAV (rAAV, AAV2 serotype), using HEK293 cells. After lysis of the rAAV-containing supernatant, the lysed culture medium was harvested. The harvested material was clarified by depth filtration, followed by concentration and buffer exchange using tangential flow filtration. Thereafter, affinity-based purification was performed to selectively isolate AAV.Default Anion Exchange (AEX) Process

[0236] Unless specified in the subsequent paragraphs, the AEX process was according to the following protocol or substantially similar to following protocol. EQ buffer (without MgCh) was added to the sample (affinity eluate) in an amount of 15 times the sample weight (w / w) (this dilution factor may be adjusted to approximately 10-40x based on dilution factor optimization experiments). The EQ buffer may include 25 mM Tris, 0.01% poloxamer, 1% sucrose, being pH 8.7, but the pH of the sample may be adjusted to the desired value by addition of 1 M Tris buffer. A 1 M MgCl₂ buffer was added to adjust the MgCE concentration of the load sample. AEX columns were equilibrated with EQ buffer, and the prepared sample was loaded. The loading, washing, and elution steps were performed sequentially. For Capto Q, the flow rate at each step was set to 150-300 cm / hr, and for CIMmultus® QA, the flow rate was set to 3-5 CV / min. For the wash step, the buffer included 5 mM Tris, 0.01% poloxamer, 1% sucrose, pH 8.9, 3mM MgCh. Elution was performed by isocratic elution using the buffer (25 mM Tris, 0.01% Poloxamer, 1% Sucrose, 4 mM MgCh, pH 7.4). and immediately after elution. 5 M NaCl was added to the AEX eluate to achieve a final NaCl concentration of 180 mM. The resulting AAV composition w ere analyzed using techniques such as analytical ultracentrifugation (AUC) and / or mass photometry.Example 1. Loading Step

[0237] The loading step for increasing % full capsid rAAV particles was explored by adjusting the concentration of MgCh, pH, dilution fold, and conductivity of the loading buffer containing the AAV sample.Attorney Docket No.: 99CR-403196-WOConcentration of loading MgCh

[0238] The concentration of MgCh in the loading buffer was initially set from 0 mM-4 mM (Table 1. “Run 1”) and was expanded to 0 mM-9 mM (Table 2 and FIG. 1A-1B, “Run 2’"). For AEX column, Capto Q was used. The % empty. % partial filled and % full capsid rAAV particles were measured by mass photometry. As can be seen from the results in Tables 1-2, the % full capsid rAAV particles increased as MgCh concentration in the loading buffer increased.Table 1Load VG Mass Photometry AUC (A260) AUC (A230 MgCh Yield % % % % % % % % % cone. (%) Empty Partial Full Empty Partial Full Empty Partial Full O mM 34.0 17.7 17.5 64.9 6.8 14.3 78.3 13.0 17.2 68.8 2 mM 52.2 12.9 18.1 69.1 5.8 9.0 81.0 10.4 10.9 74.5 3 mM 43.2 12.3 16.0 71.8 5.7 8.2 84.1 10.5 7.2 79.84 mM 44.7 11.8 15.1 73.2 5.8 8.4 84.8 11.7 8.9 78.5Table 2Load MgCh Mass Photometry(mM) %Empty %Partial %Full0 17.6 14.1 68.41 16.9 14.2 68.92 16.0 14.4 69.73 12.7 16.7 70.64 9.9 14.6 75.55 9.1 15.2 75.86 9.6 16.2 74.37 4.6 15.4 80.08 5.7 17.9 76.49 7.2 11.7 81.2

[0239] A two factor design of empty capsid % and VG yield model regarding the load MgCh and pH was established as show n in FIG. 2A-2C, and for three conditions (MgCh 6.5 mM and pH 8.8; MgCh 7.0 mM and pH 8.7; and MgCh 7.5 mM and pH 8.6), the actual % empty and VG yield was verified, and the results are shown in Table 3. As shown in Table 3. being consistent with the modeling, loading at these conditions resulted in low % empty and high VG yield.Table 3AUC %Empty VG Yield (%)MgCh(mM) pH Expected Result Verification Expected Result Verification (95% PI Range) Result (95% PI Range) Result 6.5 8.8 4.5 (1.8-7.1) 5.1 46.9 (35.3-58.5) 50.6 7.0 8.7 3.0 (0.2-5.7) 4.3 [SD: 0.51 39.5 (27.3-51.7) 46.7 [SD: 2.81 7.5 8.6 1.8 (-1.0-4.7) 3.3 [SD: 0.9] 30.1 (17.6-42.6) 36.7 [SD: 2.1]Attorney Docket No.: 99CR-403196-WO

[0240] Also for the AEX using CIM QA column, it was confirmed that purity (% Full) tends to increase as load MgCh concentration increases. Table 4 and FIG. 6 shows the data of % Empty, % Partial, % Full at various MgCh concentrations at the loading buffer. Also, a two factor design of full capsid % model regarding the load MgCh and pH using CIM QA column was established as show n in FIG. 7.Table 4Condition Mass PhotometryLoadLoad FlowMgCh Rate Load pH %Empty %Partial %Full Cone. (mL / min)(mM)34.2 6.6 59.2 0 5 9.0[SD: 10.5, n=3] [SD: 1.0, n=3] [SD: 10.1, 11=3] 4 5 9.0 19.1 7.3 73.6|SD: 1.6, n=3| [SD: 1.7, n=3| |SD: 2.8, n=3| 6 5 9.0 20.4 8.9 70.88 5 9.0 17.1 7.0 76.0Dilution Factor

[0241] The dilution factor w as investigated for its contribution to the full capsid % and VP yield process time. Previously, the process time and volume w ere high due to a high dilution (about 40-fold, 80 CV for 3h loading) to achieve AEX load conductivity 1 mS / cm. The correlation between the load conductivity and the full capsid % and VP yield were plotted in FIG. 3A-3B. From the results, the full capsid % and VG yield were kept consistent as the load conductivity' increased, indicating that there is no correlation between the load conductivity and the full capsid % and VG yield. Thus, dilution fold can be low ered, saving time and keeping needed volume low.

[0242] After a screening test (Table 5) and a confirmation test (Table 6), a dilution of 15-fold was used, and the process time and loading volume of the AEX using Capto Q medium was decreased by 30% and 60%, respectively.Table 5Empty (%) Partial (%) Full (%) VG Yield (%) Dilution 25 -fold (N=3) 4.6 [SD: 0.6] 14.5 [SD: 0.6] 81.0 [SD: 1.3] 59.8 [SD: 3.4] Dilution 15-fold (N=3) 3.7 [SD: 0.7] 14.5 [SD: 1.9] 81.9 [SD: 2.1] 59.7 [SD: 5.8]Dilution 10-fold (N=3) 5.2 [SD: 0.6] 16.9 [SD: 2.7] 77.9 [SD: 2.4] 49.6 [SD: 3.9]Attorney Docket No.: 99CR-403196-WOTable 6Empty (%) Partial (%) Full (%) VG Yield Process Loading (%) Time (h) Volume (CV) Previous conditionCapto Q (N=4) 13.3 [SD: 18.3 [SD: 68.6 [SD: 52.2 [SD: 6.8 801.0] 1.6] 2.4] 7.5]Capto Q-CIM 4.9 [SD: 17.1 [SD: 78.1 [SD: 35.1 [SD:QA (N=9) 0.8] _16] _ 2JJ _ 9.0]New conditionDilution 25- 5.5 [SD: 13.9 [SD: 80.7 [SD: 63.5 [SD: 5.7 50 fold (N=3) 0.2] 1.9] 1-7] 3.2]Dilution 15- 5.7 [SD: 13.4 [SD: 81.0 [SD: 59.6 [SD: 4.8 30 fold (N=3) 1.1] 1.7] 2.6] 13.3]Dilution 10- 6.0 [SD: 15.6 [SD: 78.4 [SD: 58.5 [SD: 3.8 20fold (N=3) 0.2] 0.7] 0.7] 4.0]Load Conductivity

[0243] The effect of the conductivity of the loading buffer on the % full capsid rAAV particles was further investigated.

[0244] As can be seen from Table 7 and Table 8 and FIGS. 4A-4B, change in the conductivity in the loading buffer resulted no effect on the % full capsid rAAV particles. Thus, it is contemplated the dilution fold can be lowered to improve the AEX loading stability and reduce the loading volume and loading time.Table 7Load Cond. Mass Photometry(mS / cm) %Empty %Partial %Full0.98 17.6 14.1 68.41.00 16.2 13.5 70.41.06 16.1 11.8 72.11.21 17.7 12.9 69.31.42 16.3 13.6 70.21.60 18.8 13.0 68.31.81 15.7 14.0 70.32.0 19.7 22.9 57.42.2 20.8 13.0 66.22.4 17.0 13.2 69.8Table 8Target Load Dilution Factor Loading Volume Loading Time (min) Conductivity (Predicted) (CV)(mS / cm)1.0 30x 90 1802.4 8x 23 46Attorney Docket No.: 99CR-403196-WOLoading Ratio

[0245] The loading ratio was also tested with respect to the effect to % full and the VG yield. % Empty, % Partial, % Full and VG yield was measured using Capto Q column, at various load ratio, as shown in Tables 9 and 10. and FIG. 5. The VG yield at the loading ratio 1.60E+14 VP / mLresin was 7.2% higher than that at 2.50E+14 VP / mLresin, while the quality (i.e., % full) in the two batches were similar as show n by AUC empty' capsid, as shown in Table 10. Also, load ratios and VG yield for AEX using CIM QA column was measured, and shown in Table 11.Table 9Load Ratio MP (AMD) VG Yield (VP / mLresin) %Empty %Partial %Full (%) 5.00E+14 4.7 14.5 80.9 24.3 2.50E+14 5.2 13.5 81.4 37.1 2.20E+14 3.9 14.3 81.8 38.5 1.90E+14 4.4 13.7 82.0 42.4 1.60E+14 4.2 11.8 84.1 43.9 1.30E+14 4.1 16.3 79.7 49.7 8.00E+13 4.8 18.5 76.7 60.22.50E+13 18.5 17 64.6 35.2Table 10Load MP (AMD) AUC VG Ratio % % % % % % % Yield (VP / Empty Partial Full [SD] Emptv Partial Full Impurity (%)[SD] [SD] [SD] ’ [SD] [SD] [SD] [SD] 2.50E+14 5.6 18.2 76.3 4.3 12.5 78.0 4.8 46.7|0.4| |3.1| |3.1| |0.5| |0.7| |6.4| |7.1| |2.8| 1.60E+14 5.9 15.8 78.4 4.3 13.7 78.2 3.4 53.9_[OA] _ JOT] _ I LJJ _ ro-5] J04] _ ro-3i _U_2J _ [2-3]

[0246] The loading ratio was also tested with respect to the effect to % full and the VG yield. % Empty, % Partial. % Full and VG yield was measured at various load ratio using CIM QA, and the results are shown in Table 11. As shown in Table 11, there was no significant effect on VG yield based on load ratio.Table 11Load Ratio AEX-HPLC VG Yield (VP / mLresin) %Empty %Partial %Full (%) 7.30E+14 1.8 10.4 87.9 84.0 4.00E+14 1.1 17.3 81.6 86.87.00E+13 0.4 6.3 93.3 74.1Attorney Docket No.: 99CR-403196-WOExample 2. Chase Step

[0247] In order to improve the yield, the chase step was also modified. For the first run (“Run 1”), the load sample had an MgCE concentration of 7.5 and a pH of 8.8. The sample was prepared at a dilution factor of x25. This run was conducted under conditions in which the chase was not optimized, and MgCE was included in the chase. For the second run (“Run 2”), The load sample had an MgCE concentration of 7.0 and a pH of 8.9. The sample was prepared at a dilution factor of x 10. This run was conducted under optimized chase conditions, and MgCk was not included in the chase. The TEC AN result and AKTA result were shown in Table 12 and Table 13 and FIG. 8.Table 12Empty (%) Partial (%) Full (%) VG Yield (%) Control (N=8) 15.8 [SD: 1.9] 12.7 [SD: 1.2] 71.6 [SD: 2.5] 39.7 [SD: 12.0] Run 1(N=4) 7.2 [SD: 1.1] 12.3 [SD: 0.9] 80.1 [SD: 2.0] 14.2 [SD: 2.0]Run 2 (N=3) 5.2 [SD: 0.6] 16.9 [SD: 2.7] 77.9 [SD: 2.4] 49.6 [SD: 3.9]Table 13AEX (Capto Q)Empty (%) Partial (%) Full (%) VG Yield (%)[SD] [SD] [SD] [SD] Control 13.3 18.3 [1.6] 68.6 [2.4] 52.2 [7.5] (N=4a, N=9b) [1.0]Run 1(N=3) 4.9 12.0 [0.6] 83.1 [0.7] 14.5 [2.1][0.2]Run 2 (N=3) 6.0 15.6 [0.7] 78.4 [0.7] 58.5 [4.0][0.2]Example 3. Wash Step

[0248] The conditions of the wash step was studied. The previous estimated range was pH 8.5-9.5, MgCb 0.7-7.3 mM. And the new range was pH 7.7-9.9, MgCk 5-19 mM. The results were shown in Table 14. It can be seen that, the harsher wash condition results higher purity of the full capsid rAAV particles (see eluate fraction: from pH 8.9, %full 63.7% (control) to pH 8.1-8.5, %full 69.6%-70.2% (“Run 042”- “Run 044”)). Even in the mild wash condition, the % empty can be selectively lowered (wash fraction at pH 9.5 with 1 ImM MgCh results in the empty capsid of 91% (“Run 024”)).Attorney Docket No.: 99CR-403196-WOTable 14Fraction Run ID pH MgCh Mass Photometry(mM) %Empty %Partial %Full Eluate Control 8.9 5 20.6 15.8 63.7(N = 6) [SD: 2.6] [SD: 1.3] [SD: 2.2] Run 042 8.5 5 17.3 12.7 70.1Run 043 8.3 5 16.0 13.9 70.2Run 044 8.1 5 18.6 11.9 69.6 Wash Control 8.9 5 78.8 14.7 6.5Run 024 9.5 11 91.0 6.3 2.7

[0249] The relationship between the wash conditions (MgCh concentration of pH) and % full capsid is further shown in FIG. 9 and Table 15 based on the DoE range was pH 8.5-9.5, MgCh 0.7-7.3 mM. The conditions of the wash step was studied by AKTA. The results were shown in Table 15. It can be seen that, the harsher wash condition results higher purity of the full capsid rAAV particles (see eluate fraction: from pH 8.9, %full 63.7% (control) to pH 8.1-8.5, %full 69.6%-70.2% (“Run 042"- “Run 044”)). Even in the mild wash condition, the % empty can be selectively lowered (wash fraction at pH 9.5 with 1 ImM MgCh results in the empty capsid of 91% (“Run 024”)).Table 15X-factor Y-responseWash %Full CapsidCondition [MgClJ Wash AEX-HPLCpH Expected AUC(mM) Result Actual Result84.7Condition # 1 4.0 9.1 88.6 78.3(75.0-94.3) [SD: 0.3, n=3] [SD: 1.1, n=2] 96.7Condition #2 5.0 8.9 94.5 83.9(87.1-106.4) [SD: 0.8, n=3] [SD: 3.2, n=2]98.1Condition #3 6.0 8.8 94.7 78.6(88.0-108.2) [SD: 0.5, n=3] [SD: 4.8, n=2]CIMmultus QA (n=6) - 91.9 [SD: 1.8, n=6] 82.2 [SD: 5.2, n=5]

[0250] The relationship between the wash conditions (MgCh concentration and pH) and the percentage of full capsids using CIM QA is further shown in FIG. 10 and Table 16. The DoE range evaluated was pH 8.5-9.5 and MgCh 5.9-14.1 mM. It was observed that the percentage of full capsids increased with increasing MgCh concentration. However, when yield was taken into consideration, an MgCh concentration of 11.0-12.0 mM provided a high percentage of full capsids while maintaining a yield of 60% or higher.Attorney Docket No.: 99CR-403196-WOTable 16X-factor Y-responseWash Wash %Full Capsid VG Yield (%)MgCh(mM) pH (AEX-HPLC) (qPCR)93.2 76.411.0 8.85[SD: 0.8, n=3] 91.9 [SD: 4.7, n=3] 69.3[SD: 1.8. [SD: 10.6, 90.6 62.312.0 8.75 n=6] n=6][SD: 1.6, n=3] [SD: 10.5. n=3]As-was (n=6) 85.9 [SD: 2.6, n=6] 71.5 [SD: 15.4, n=6]Example 4. Elution StepCapto Q Column

[0251] For the process using CaptoQ AEX column, the elution step was modified using 96-well screening method (TEC AN) and the DoE model was conducted. FIG. 11A-11C show the DoE modeling, and Table 17 shows confirmatory % Full data and VG yield (%) in elution conditions (isocratic) corresponding to three data points from the DoE modeling. For “Point 1 ”, Elution conditions was a pH of 7.7 and an MgCE concentration of 7.3 mM. For “Current Condition”, elution conditions was a pH of 7.5 and an MgCE concentration of 4.0 mM. For “Point 2”, elution conditions was a pH of 7.3 and an MgCE concentration of 1.6 mM.Table 17Study Point (N=3) %Full (AUC) VG Yield (%)Point 1 79.8 [SD: 0.7] 53.0 [SD: 2.3]Current condition 82.2 [SD: 1.2] 42.7 [SD: 4.7]Point 2 85.5 [SD: 0.4] 38.8 [SD: 2.1]

[0252] The yield and quality in the 96 well screening method was show n in Table 18 and Table 19. FIGs. 12A-12C show results based on confirming DoE experiments using AKTA, based on the screening tests of the 96 well screening method, showing the possibility' of existence of optimal data point at the right upper side. However, after the DoE experiment, the elution condition containing 4.0 mM MgCb at pH 7.5 (isocratic) was maintained.Table 18MgCE (mM) 1 4 7 10 15 20 pH 8.9 0.007 0.013 0.036 0.059 0.085 0.0918.6 0.005 0.017 0.040 0.050 0.076 0.079 8.3 0.004 0.023 0.053 0.067 0.079 0.079 8 0.007 0.030 0.057 0.067 0.075 0.079 7.7 0.010 0.042 0.062 0.068 0.076 0.077 7.4 0.017 0.055 0.067 0.069 0.075 0.079 7.1 0.023 0.048 0.061 0.064 0.094 0.0726.8 0.036 0.056 0.064 0.065 0.071 0.076Attorney Docket No.: 99CR-403196-WOTable 19MgCh (mM) 1 4 7 10 15 20pH 8.9 -0.643 -0.319 -0.216 -0.216 -0.214 -0.2118.6 -0.743 -0.285 -0.216 -0.233 -0.215 -0.2068.3 -1.203 -0.302 -0.241 -0.245 -0.222 -0.2158 -0.664 -0.268 -0.241 -0.237 -0.220 -0.2127.7 -0.436 -0.270 -0.243 -0.239 -0.223 -0.2177.4 -0.376 -0.249 -0.230 -0.228 -0.221 -0.2137.1 -0.327 -0.288 -0.232 -0.247 -0.244 -0.2146.8 -0.252 -0.226 -0.214 -0.225 -0.206 -0.204 CIM QA Column

[0253] For the AEX process using CIM QA AEX column, purity and yield was confirmed at two elusion conditions (control-salt gradient elution and pH 7.5 step elusion), and the result is shown in Table 19. The salt gradient was formed with a shallow linear KC1 gradient starting at 0 mM of KC1 and reaching 250 mM NaCl over 50CV. (Buffer A: 25 mM Tris, 0.01% poloxamer, 1% sucrose, 10 mM MgCh, pH 9.0; Buffer B 25 mM Tris, 0.5 M KC1, 0.01% pol oxamer, 1% sucrose, 10 mM MgCh; pH 9.0; Buffer B from 0-50%).Table 20X-Factor Y-Response WashCondition Load Ratio Wash MgCh VG Yield %Full Capsid (El 4 VP / mLresin) pH cone. (mM) (%) (AEX-HPLC) Control 4.0 83.88.8 11.5 87.5 [0.6] (salt gradient, pH 9.0) [5.3]pH 7.5 4.0 53.58.8 11.5 90.9 [1.9]step elution _ [^4] _Example 5. Eluate ProcessingAggregates formation in eluate

[0254] The sample aggregation problem was observed for the eluate with high rAAV concentration. It was contemplated that this might be due to the low salt concentration in the elution buffer (25 mM Tris, 0.01% Poloxamer, 1% Sucrose, 4 mM MgCh, pH 7.5). Thus, further processes to the eluate was tested in order to reduce the viral aggregation, such as filtration with 0.2pm filter, or salt spiking by adding 0.2 M NaCl in the eluate (Table 20). In both steps, no detectable viral aggregates were observed. However, the VG concentration after the 0.2pm filter filtration was significantly decreased. Thus, 0.2 M NaCl was added (as spiking) to all the DoE samples for PQ analysis. Elution buffer containing salt can also be used to optimize the elution step.Attorney Docket No.: 99CR-403196-WOTable 21Run Order Sample VG Cone. (VG / mL) Yield (%) AX004 Load 7.26E+10 - Eluate (filtered) 4.92E+09 0.1 AX004_re Load 8.34E+10 - Eluate (no filtered) 2.43E+12 39.3Eluate (w / 0.2 M NaCl) 2.58E+12 41.7VG titer gap in AEX eluate

[0255] There was a 20% gap between the expected and actual VG titer in the AEX eluate. This was perhaps because of the low VG stability after the freeze / thaw and filtration steps, as the visible precipitates in the AEX eluate were removed or adsorbed by 0.22pm filter, and there was no salt in the AEX eluate, potentially causing the compromised (low) AAV stability.

[0256] The VG stability was tested in the AEX eluate (25 mM Tris, 0.01% Poloxamer, 4 mM MgC12, pEI 7.5). The VG titer was significantly decreased after 0.22pm filtration without NaCl in the AEX eluate. Adding NaCl to the elution buffer (Condition 1) results in lower %Full (79.7%) and higher %I4MW (2.5%). By adding 180 mM NaCl in the AEX eluate, no change of %Full, %HMW, VG yield was observed. Thus, to prevent the VG titer decrease, the AEX elution buffer can be changed or NaCl can be added to the AEX eluate. VG yield before & after the 0.22 pm filtration is shown in Table 22.Table 22Condition FT & VG Cone. VG Yield P- %FullFiltration (VG / mL) (%) %HM value (AEX- W HPLC) (SE-HPLC) As-is Elution X 2.90E12 39.4 [SD: 3.7, 0.000 96.0 [SD: 0.9 [SD:buffer n=3] 0.4, n=3] 0.8, n=3] (w / o 0 1.37E11 1.9 [SD: 0.6,NaCl) n=3] 97.4 [SD: 0.0 [SD: No NaCl 0.6, n=2] 0.0, n=2] spikingCondi Elution X 4.52E12 46.0 [SD: 4.3, 0.740 79.3 [SD: 2.5 [SD: tion 1 buffer n=3] 1.2, n=3] 0.1, n=3] (w / 0 4.34E12 44.7 [SD: 4.5, 79.7 [SD: 2.5 [SD: NaCl) n=3] 1.5, n=3] 0.1, n=3] Condi Elution X 2.64E12 38.2 [SD: 2.6, 0.814 96.1 [SD: 0.7 [SD: tion 2 buffer n=2| 0.4, n=2| 1.0, n=2| (w / o 0 2.57E12 37.2 [SD: 9.1,NaCl) n=2] 96.6 [SD: 0.7 [SD: NaCl 0.6, n=2] 0.9, n=2]spikingAttorney Docket No.: 99CR-403196-WO * * 4=

[0257] The present disclosure is not to be limited in scope by the specific embodiments described which are intended as single illustrations of individual aspects of the disclosure, and any compositions or methods which are functionally equivalent are within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present disclosure without departing from the spirit or scope of the disclosure. Thus, it is intended that the present disclosure cover the modifications and variations of this disclosure provided they come within the scope of the appended claims and their equivalents.

[0258] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

Attorney Docket No.: 99CR-403196-WOCLAIMS1. A method for purifying recombinant adeno-associated virus (rAAV) particles, comprising:(a) contacting an anion exchange (AEX) chromatography medium with a recombinant AAV (rAAV) loading composition comprising full rAAV particles and empty rAAV particles, wherein the rAAV loading composition further comprises MgCl2;(b) applying to the AEX chromatography medium a wash solution; and (c) applying to the AEX chromatography medium an elution solution to provide an eluate, wherein pH and salt concentration of the elution solution is isocratic.

2. The method of claim 1, wherein the full and empty rAAV particles are one or more selected from the group consisting of: AAV2, AAV5, AAV8, and AAV9.

3. The method of claim 1 or 2, wherein the full and empty rAAV particles are AAV2.

4. The method of any one of claims 1-3, wherein the full rAAV particles comprises one or more polynucleotides.

5. The method of any one of claims 1-4, wherein the full rAAV particles comprise a single vector.

6. The method of any one of claims 1-4, wherein the full rAAV particles include two or more different polynucleotides.Attorney Docket No.: 99CR-403196-WO7. The method of any one of claims 1-6, wherein the rAAV loading composition comprises about 4 mM to about 13 mM of MgCh.

8. The method of any one of claims 1-7, wherein the rAAV loading composition comprises about 5 mM to about 12 mM of MgCh.

9. The method of any one of claims 1-8, wherein the rAAV loading composition comprises about 9 mM to about 11 mM of MgCh.

10. The method of any one of claims 1-7, wherein the rAAV loading composition comprises about 6.5 mM to about 10.5 mM of MgCh.

11. The method of any one of claims 1-10. wherein the concentration of MgCh in the rAAV loading composition is substantially proportional to the percentage of full recombinant AAV particles in the eluate.

12. The method of any one of claims 1-11, wherein the rAAV loading composition has pH of about 8 to about 9.

13. The method of any one of claims 1-12, wherein the rAAV loading composition has pH of about 8.6 to about 8.9.

14. The method of any one of claims 1-13, wherein the rAAV loading composition has pH of about 8.7 to about 8.9.

15. The method of any one of claims 1-14. wherein the rAAV loading composition has pH of about 8.7 to about 8.8.Attorney Docket No.: 99CR-403196-WO16. The method of any one of claims 1-15, wherein the rAAV loading composition is about 10-fold to 40-fold diluted from a recombinant AAV (rAAV) sample.

17. The method of any one of claims 1-16, wherein the rAAV loading composition is about 10-fold to 30-fold diluted from a recombinant AAV (rAAV) sample.

18. The method of any one of claims 1-17, wherein the rAAV loading composition is about 10-fold to 20-fold diluted from a recombinant AAV (rAAV) sample.

19. The method of any one of claims 1-18. wherein the rAAV loading composition is about 12.5-fold to 17.5-fold diluted from a recombinant AAV (rAAV) sample.

20. The method of any one of claims 1-19. wherein the rAAV loading composition is about 15-fold diluted from a recombinant AAV (rAAV) sample.

21. The method of any one of claims 1-20, wherein the rAAV loading composition has conductivity of about 1.0 mS / cm to about 6.0 mS / cm.

22. The method of any one of claims 1-21, wherein the rAAV loading composition has conductivity of about 1.0 mS / cm to about 4.5 mS / cm.

23. The method of any one of claims 1-22, wherein the rAAV loading composition has conductivity of about 1.0 mS / cm to about 4.0 mS / cm.

24. The method of any one of claims 1-23, wherein the rAAV loading composition has conductivity of about 1.0 mS / cm to about 2.4 mS / cm.Attorney Docket No.: 99CR-403196-WO25. The method of any one of claims 1-24, wherein the rAAV loading composition comprises the rAAV particles in a concentration of about 1.0 x 1012VP / mL to about 5.0 x 1013VP / mL.

26. The method of any one of claims 1-24, wherein the rAAV loading composition comprises the rAAV particles in a concentration of about 2.0 x 1012VP / mL to about 1.6 x 1013VP / mL.

27. The method of any one of claims 1-24, wherein the rAAV loading composition comprises the rAAV particles in a concentration of about 1.6 x 1013VP / mL to about 2.0 x 1013VP / mL.

28. The method of any one of claims 1-27. wherein the rAAV loading composition further comprises one or more selected from the group consisting of Tris, poloxamer and sucrose.

29. The method of any one of claims 1-28, wherein the AEX chromatography medium comprises quaternary amine.

30. The method of any one of claims 1-29, wherein the AEX chromatography medium comprises a copolymer of glycidyl methacrylate and ethylene dimethacrylate.

31. The method of any one of claims 1-29, wherein the AEX chromatography medium comprises a highly cross-linked agarose with dextran surface extender.

32. The method of any one of claims 1-31. wherein the rAAV loading composition is added to the anion exchange (AEX) chromatography medium in an amount of about 2.5xl013VP / mLresin to about 8.0 xlO14VP / mLresin.Attorney Docket No.: 99CR-403196-WO33. The method of any one of claims 1 -31, wherein the recombinant AAV composition is added to anion exchange (AEX) chromatography medium in an amount of about 1.6xl014VP / mLresin to about 3.5 xlO14VP / mLresin, or about 7.0xl013VP / mLresin to about 7.3 xlO14VP / mLresin.

34. The method of any one of claims 1-33, further comprising applying a chase solution after the step (a).

35. The method of claim 34, wherein the chase solution does not comprise MgCh.

36. The method of claim 34 or 35, wherein the chase solution has pH of about 8.7.

37. The method of any one of claims 34-36, wherein the chase solution is added in an amount of 5 fold of the volume of the AEX chromatography medium.

38. The method of any one of claims 1-37, wherein the wash solution comprises MgCh.

39. The method of any one of claims 1-38, wherein the wash solution comprises about 3 mM to about 10 mM of MgCh.

40. The method of any one of claims 1-39, wherein the wash solution comprises about 4 mM to about 6 mM of MgCh.

41. The method of any one of claims 1-40, wherein the wash solution has conductivity of about 1.4 mS / cm to about 4.0 mS / cm.Attorney Docket No.: 99CR-403196-WO42. The method of any one of claims 1 -41, wherein the wash solution has conductivity of about 2.5 mS / cm to about 4.0 mS / cm.

43. The method of any one of claims 1-42, wherein the wash solution has conductivity of about 2.7 mS / cm to about 2.9 mS / cm.

44. The method of any one of claims 1-43, wherein the wash solution has pH of about 7.7 to about 9.5.

45. The method of any one of claims 1-44. wherein the wash solution has pH of about 8.8 to about 9.1.

46. The method of any one of claims 1-45. wherein the elution solution comprise about 3.0 mM to about 5.0 mM of MgCh.

47. The method of any one of claims 1-46, wherein the elution solution comprises about 4.0 rnM of MgCh.

48. The method of any one of claims 1-47, wherein the elution solution has pH of about 7.0 to about 8.0.

49. The method of any one of claims 1-48, wherein the elution solution has pH of about 7.5.

50. The method of any one of claims 1-49, wherein pH of the elution solution is lower than pH of the wash solution.Attorney Docket No.: 99CR-403196-WO51. The method of any one of claims 1 -50, wherein the elution solution does not substantially comprise NaCl.

52. The method of any one of claims 1-51, further comprising adding NaCl to the eluate.

53. The method of claim 52, wherein aggregation of AAV is decreased by the addition of NaCl to the eluate.

54. The method of any one of claims 1-53. wherein the eluate comprises at least 70% of full capsid recombinant AAV.

55. The method of any one of claims 1-54. wherein the eluate comprises at least 90% of full capsid recombinant AAV.

56. The method of any one of claims 1-55, wherein the eluate comprises at least 95% of full capsid recombinant AAV.

57. The method of any one of claims 1-53, wherein the eluate comprises less than 10 % of empty capsid recombinant AAV.

58. The method of any one of claims 1-53, wherein the eluate comprises less than 5% of empty capsid recombinant AAV.

59. The method of any one of claims 1-58, wherein the eluate provides VG yield of about 50% or greater.Attorney Docket No.: 99CR-403196-WO60. The method of any one of claims 1-59, wherein the eluate provides VG yield of about 60% or greater.

61. The method of any one of claims 1-63, further comprising purifying the eluate.

62. A method for isolating recombinant adeno-associated virus (rAAV) particles, comprising:(a) contacting an anion exchange (AEX) chromatography medium with a rAAV composition comprising full rAAV particles and empty rAAV particles;(b) applying to the AEX chromatography medium a wash solution comprising MgCh; and(c) applying to the AEX chromatography medium an elution solution to provide an eluate, wherein the pH and salt concentration of the elution solution is isocratic, and wherein pH concentration of the elution solution is lower than pH of the wash solution.

63. The method of claim 62, wherein the full and empty rAAV particles are one or more selected from the group consisting of: AAV2, AAV5. AAV8. and AAV9.

64. The method of claim 62 or 63, wherein the full and empty rAAV particles are AAV2.

65. The method of any one of claims 62-64, wherein the full rAAV particles comprises one or more polynucleotides.

66. The method of any one of claims 62-65, wherein the full rAAV particles comprise a single vector.Attorney Docket No.: 99CR-403196-WO67. The method of any one of claims 62-65, wherein the full rAAV particles include two or more different polynucleotides.

68. The method of any one of claims 62-67, wherein the rAAV loading composition has pH of about 8 to about 9.

69. The method of any one of claims 62-68, wherein the rAAV loading composition has pH of about 8.6 to about 8.9.

70. The method of any one of claims 62-69, wherein the rAAV loading composition has pH of about 8.7 to about 8.9.

71. The method of any one of claims 62-70, wherein the rAAV loading composition has pH of about 8.7 to about 8.8.

72. The method of any one of claims 62-71, wherein the rAAV loading composition is about 10-fold to 40-fold diluted from a recombinant AAV (rAAV) sample.

73. The method of any one of claims 62-72, wherein the rAAV loading composition is about 10-fold to 30-fold diluted from a recombinant AAV (rAAV) sample.

74. The method of any one of claims 62-73, wherein the rAAV loading composition is about 10-fold to 20-fold diluted from a recombinant AAV (rAAV) sample.

75. The method of any one of claims 62-74, wherein the rAAV loading composition is about 12.5-fold to 17.5-fold diluted from a recombinant AAV (rAAV) sample.Attorney Docket No.: 99CR-403196-WO76. The method of any one of claims 62-75, wherein the rAAV loading composition is about 15-fold diluted from a recombinant AAV (rAAV) sample.

77. The method of any one of claims 62-76, wherein the rAAV loading composition has conductivity of about 1.0 mS / cm to about 6.0 mS / cm.

78. The method of any one of claims 62-77, wherein the rAAV loading composition has conductivity of about 1.0 mS / cm to about 4.5 mS / cm.

79. The method of any one of claims 62-78, wherein the rAAV loading composition has conductivity of about 1.0 mS / cm to about 4.0 mS / cm.

80. The method of any one of claims 62-79, wherein the rAAV loading composition has conductivity of about 1.0 mS / cm to about 2.4 mS / cm.

81. The method of any one of claims 62-80, wherein the rAAV loading composition comprises the rAAV particles in a concentration of about 1.0 x 1012VP / mL to about 5.0 x 1013VP / mL.

82. The method of any one of claims 62-81, wherein the rAAV loading composition further comprises one or more selected from the group consisting of Tris, poloxamer and sucrose.

83. The method of any one of claims 62-82, wherein the AEX chromatography medium comprises quaternary amine.

84. The method of any one of claims 62-83, wherein the AEX chromatography medium comprises a copolymer of glycidyl methacrylate and ethylene dimethacrylate.Attorney Docket No.: 99CR-403196-WO85. The method of any one of claims 62-84, wherein the AEX chromatography medium comprises a highly cross-linked agarose with dextran surface extender.

86. The method of any one of claims 60-85, wherein the rAAV loading composition is added to the anion exchange (AEX) chromatography medium in an amount of about 2.5xl013VP / mLreSm to about 5.0 xlO14VP / mLresin.

87. The method of any one of claims 62-86, wherein the recombinant AAV composition is added to anion exchange (AEX) chromatography medium in an amount of about 1.6xl014VP / mLresin tO about 3.5 XlO14VP / mLresm.

88. The method of any one of claims 62-87, further comprising applying a chase solution after the step (a).

89. The method of claim 88, wherein the chase solution has pH of about 8.7.

90. The method of claim 88 or 89, wherein the chase solution is added in an amount of 5 fold of the volume of the AEX chromatography medium.

91. The method of any one of claims 62-90, wherein the wash solution comprises MgCl2.

92. The method of any one of claims 62-91, wherein the wash solution comprises about 3 mM to about 10 mM of MgCh.

93. The method of any one of claims 62-92, wherein the wash solution comprises about 4 mM to about 6 mM of MgCh.Attorney Docket No.: 99CR-403196-WO94. The method of any one of claims 62-93, wherein the wash solution has conductivity of about 1.4 mS / cm to about 4.0 mS / cm.

95. The method of any one of claims 62-94, wherein the wash solution has conductivity of about 2.5 mS / cm to about 4.0 mS / cm.

96. The method of any one of claims 62-95, wherein the wash solution has conductivity of about 2.7 mS / cm to about 2.9 mS / cm.

97. The method of any one of claims 62-96, wherein the wash solution has pH of about 7.7 to about 9.5.

98. The method of any one of claims 62-97, wherein the wash solution has pH of about 8.8 to about 9.1.

99. The method of any one of claims 62-98, wherein the elution solution comprise about 3.0 mM to about 5.0 mM of MgCh.

100. The method of any one of claims 62-99, wherein the elution solution comprises about 4.0 mM of MgCh.

101. The method of any one of claims 62-100, wherein the elution solution has pH of about 7.0 to about 8.0.

102. The method of any one of claims 62-101, wherein the elution solution has pH of about 7.5.Attorney Docket No.: 99CR-403196-WO103. The method of any one of claims 62-102, wherein the elution solution does not substantially comprise NaCl.

104. The method of any one of claims 62-103, further comprising adding NaCl to the eluate.

105. The method of claim 104, wherein aggregation of AAV is decreased by the addition of NaCl to the eluate.

106. The method of any one of claims 62-105, wherein the eluate comprises at least 70% of full capsid recombinant AAV.

107. The method of any one of claims 62-106, wherein the eluate comprises at least 90% of full capsid recombinant AAV.

108. The method of any one of claims 62-107, wherein the eluate comprises at least 95% of full capsid recombinant AAV.

109. The method of any one of claims 62-108, wherein the eluate comprises less than 10 % of empty capsid recombinant AAV.

110. The method of any one of claims 62-109, wherein the eluate comprises less than 5% of empty capsid recombinant AAV.

111. The method of any one of claims 62-110, wherein the eluate provides VG yield of about 50% or greater.Attorney Docket No.: 99CR-403196-WO112. The method of any one of claims 62-111, wherein the eluate provides VG yield of about 60% or greater.

113. The method of any one of claims 62- 112, further comprising purifying the eluate.

114. A method for isolating recombinant adeno-associated virus (rAAV) particles, comprising:(a) contacting an anion exchange (AEX) chromatography medium with a rAAV composition comprising full rAAV particles and empty rAAV particles, wherein the rAAV composition further comprises MgCfy(b) applying to the AEX chromatography medium a wash solution comprising MgCE; and(c) applying to the AEX chromatography medium an elution solution to provide an eluate, wherein the pH and salt concentration of the elution solution is isocratic, wherein pH concentration of the elution solution is lower than pH of the wash solution.

115. A method for isolating recombinant adeno-associated virus (rAAV) particles, comprising:(a) contacting an anion exchange (AEX) chromatography medium with a rAAV composition comprising full rAAV particles and empty rAAV particles, wherein the rAAV composition has conductivity of about 1.0 to 4.5 mS / cm;(b) applying to the AEX chromatography medium a wash solution; and (c) applying to the AEX chromatography medium an elution solution to provide an eluate, wherein the pH and salt concentration of the elution solution is isocratic.Attorney Docket No.: 99CR-403196-WO116. The method of claims 54-56, wherein the full capsid recombinant AAV is measured by HPLC assay, mass photometry, AUC (A260), or AUC (A230).

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