CN116802308A - Method for purifying AAV vectors by anion exchange chromatography - Google Patents

Abstract

The present disclosure provides methods of purifying recombinant AAV (rAAV) vectors from solution by anion exchange chromatography (AEX) to produce an eluate enriched in complete capsids and depleted in empty capsids.

Description

Method for Purification of AAV Vectors by Anion Exchange Chromatography

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 63/253,215 filed on October 7, 2021, U.S. Provisional Patent Application No. 63/217,194 filed on June 30, 2021, and U.S. Provisional Patent Application No. 63/109,049 filed on November 3, 2020, the entire contents of which are incorporated herein by reference.

Sequence Listing

This application contains a sequence listing, which is submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on October 15, 2021, is named PC072555A_Sequence_Listing_ST25.txt, and is 1,048,576 bytes in size.

Field of the Invention

The present invention relates to the purification of AAV, in particular recombinant AAV (rAAV) vectors, by anion exchange chromatography.

Background of the Invention

Gene therapy using recombinant AAV (rAAV) vectors to deliver therapeutic transgenes has the potential to treat a range of serious diseases that are incurable and in many cases have limited treatment options (Wang et al. (2019) Nature Reviews 18: 358-378). The production of gene therapy vectors is complex and requires specialized methods to purify therapeutic rAAV vectors from host cell impurities and viral capsids that do not contain a complete vector genome encoding a therapeutic transgene. In addition to developing purification methods for producing clinical-grade rAAV vector compositions with high purity and good safety and efficacy, the purification methods must also be scalable to high-capacity rAAV production to meet patient needs.

Ultracentrifugation using cesium chloride gradient sedimentation is a robust method for removing host cell proteins and DNA and isolating viral capsids, which are empty (i.e., containing no vector genome), partially packaged (also known as "intermediate capsids," containing partial vector genomes and/or non-transgenic associated DNA), or fully packaged vectors (also known as "complete capsids," containing complete vector genomes) (Burnham et al. (2015) Hum. Gene Ther. Meth. 26: 228-245). However, cesium chloride gradient purification is laborious, time-consuming, and not suitable for large-scale production. Ultracentrifugation using iodixanol gradients is less labor-intensive, but generally results in lower purity vector production (Hermens et al. Hum. Gene Ther. (1999) 10: 1885-1891). Chromatographic methods including affinity and/or ion exchange chromatography have been shown to be useful for large-scale production of clinical-grade rAAV, including separation of empty viral capsids from complete rAAV vectors.

Empty capsids are produced by host cells that produce recombinant vector genomes and package them in viral capsids. In most mammalian expression systems, empty capsids are overproduced relative to complete vectors, and various systems produce 1-30% complete vectors (Penaud-Budloo et al. Molecular Therapy, Methods & Clinical Dev (2018) 8: 166-180). The production of empty capsids can be attributed to the imbalance of the ratio of plasmids encoding transgenes to plasmids encoding rep/cap genes. The presence of empty capsids in drug products may cause undesirable immune responses and/or compete with recombinant vectors for binding sites on target cells.

Certain anion exchange chromatography methods using acetate buffers and resins such as POROS ^™ 50HQ and Q-Sepharose XL have been used to separate empty capsids from rAAV2 vector libraries by relying on the slightly less anionic nature of empty capsids compared to full vectors (US 7,261,544; Qu et al. (2007) J. Virol. Meth. 140(1): 183-192). A similar approach uses a combination of affinity chromatography and ion exchange chromatography (IEX) and a 10 mM to 300 mM Tris acetate gradient at pH 8 with POROS ^™ 50HQ resin to enrich for full AAV vectors of various serotypes (Nass et al. (2018) Molec. Thera. Meth. & Clin. Dev. 9: 33-46). Other studies have identified buffers and conditions that can be used to chromatographically separate empty capsids from full AAV vectors. For example, Urabe determined that AAV1 material could be diluted with Tris-HCl buffer containing _MgCl2 and glycerol for loading on an anion exchange chromatography (AEX) column, and that a solution containing a counter-chaotropic ion was an effective elution buffer for separating empty AAV1 capsids from whole vectors (Urabe et al. (2006) Molec. Ther. 13(4):823-828). Others have described dilution (e.g., 50-fold) of affinity chromatography eluents and the use of a gentle gradient elution (e.g., 20 mM to 180 mM NaCl) from a monolithic support in an AEX method to separate empty capsids from whole AAV vectors (US 2019-0002841; US 2019-0002842; US 2019-0002843; US 2018-0002844). However, these methods also use high pH (9.8 to 10.2), which may lead to deamidation and/or aggregation of rAAV vectors and may result in a reduction in therapeutic efficacy.

Processes using a combination of methods, including, for example but not in any particular order, tangential flow filtration (TFF) of host cell supernatants, precipitation of capsid material (including rAAV vectors and empty capsids) using ammonium sulfate, AEX chromatography, and size exclusion chromatography have been developed to separate rAAV from empty capsids (Tomono et al. (2018) Molec. Ther. Meth. Clin. Dev. 11: 180-190).

There remains a need for methods of preparing clinical-grade rAAV vectors (e.g., rAAV9) with optimal purity, potency, and consistency. These methods include isolating rAAV containing vector genomes with therapeutic transgenes from empty AAV capsids at a scale necessary to meet clinical needs for treating diseases (e.g., Duchenne muscular dystrophy (DMD), Friedreich ataxia (FA)).

SUMMARY OF THE INVENTION

The present disclosure provides an improved AEX method for purifying rAAV vectors, including but not limited to separating complete rAAV vectors (e.g., rAAV9 vectors) from empty capsids. Such purified complete rAAV vectors are suitable for producing a drug product for administration to human subjects (e.g., subjects with DMD). The present disclosure also provides a novel method for preparing a chromatographic eluent containing rAAV vectors (e.g., from affinity chromatography) for further purification of the chromatographic eluent by AEX. The present disclosure also provides a method for regenerating an AEX stationary phase, which allows the stationary phase to be used for multiple chromatographic runs while maintaining the completeness of the process (e.g., successful purification of rAAV vectors, separation of complete vectors from empty capsids) while reducing production costs.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by Embodiment (E) below.

E1. A method for purifying an rAAV vector by AEX, the method comprising the following steps:

i) loading a solution containing the rAAV vector to be purified onto the stationary phase in the column;

ii) performing a gradient elution of material from the stationary phase in the column, wherein the percentage of a first gradient elution buffer varies inversely proportional to the change in the percentage of a second gradient elution buffer;

iii) starting to collect at least one eluate fraction from said column when the absorbance of the column flow-through reaches an absorbance threshold value during gradient elution.

E2. The method according to E1, wherein loading the solution comprising the rAAV vector onto the column comprises applying 2.5×10 ¹⁵ to 3.0×10 ¹⁷ vector genomes (VG)/L column volume to the column.

E3. The method according to E1 or E2, wherein loading comprises applying 8.0×10 ¹² to 2.0×10 ¹⁸ total VG to the column.

E4. The method according to any one of E1 to E3, wherein loading comprises applying a diluted and optionally filtered solution comprising 2.6×10 ¹² to 6.8×10 ¹³ VG/mL column volume to a column (eg, a 6.4 L column) as measured by qPCR analysis of transgene sequences within the vector genome.

E5. The method according to any one of E1 to E4, wherein loading comprises applying a diluted and optionally filtered solution comprising 5×10 ¹³ to 1.3×10 ¹⁴ VG/mL column volume to a column (eg, a 1.3 L column), as measured by qPCR analysis of ITR sequences within the vector genome.

E6. A method according to any one of E1 to E5, wherein the rAAV vector is produced in a container, and wherein the volume of the container is about 1 L, about 50 L, about 100 L, about 250 L, about 500 L, about 1000 L, about 2000 L or more.

E7. The method according to any one of E1 to E6, wherein the container is a disposable bioreactor (SUB).

E8. The method according to any one of E1 to E7, wherein the solution comprising the rAAV vector is selected from an affinity eluate, a supernatant of a cell lysate, and a post-harvest solution that has been diluted and optionally filtered before loading.

E9. The method according to any one of E1 to E8, wherein the solution comprising the rAAV vector is an affinity eluate, which has been diluted and optionally filtered before loading.

E10. Process according to E1 to E9, wherein the solution has been subjected to at least one further purification or treatment step.

E11. The method according to E10, wherein the at least one further purification or treatment step is selected from the group consisting of cell lysis, flocculation, filtration, chromatography (eg affinity chromatography), dilution, pH adjustment, conductivity adjustment and combinations thereof.

E12. The method according to any one of E1 to E12, wherein the solution comprising the rAAV vector is an affinity eluate obtained by affinity chromatography purification of the rAAV vector produced in a disposable bioreactor (SUB) in a certain volume.

E13. The method according to E12, wherein the SUB has a volume of about 1 L to about 2000 L.

E14. The method according to any one of E1 to E13, wherein the stationary phase is an AEX stationary phase.

E15. The method according to any one of E1 to E14, wherein the stationary phase is positively charged.

E16. A method according to any one of E1 to E15, wherein the stationary phase is polystyrene divinylbenzene particles with covalently bound quaternized polyethyleneimine, and optionally wherein the stationary phase is POROS ^™ 50HQ.

E17. The method according to any one of E1 to E16, further comprising, optionally after loading the solution comprising the rAAV vector onto the stationary phase, applying a chase solution to the AEX stationary phase in the column.

E18. A method according to E17, wherein 1 to 15 column volumes (CV) of a loading chase solution comprising a buffer (e.g., Tris, BIS-Tris propane, diethanolamine, diethylamine, tris(hydroxymethyl)methylglycine (Tricine), triethanolamine and/or n,n-bicine (Bicine)) is applied to the stationary phase in the column.

E19. The method according to E17 or E18, wherein the loading chase solution comprises about 10 mM to 30 mM (eg about 20 mM) Tris, pH 8-10.

E20. The method according to any one of E1 to E19, further comprising flushing the stationary phase in the column before use.

E21. The method according to E20, wherein flushing the stationary phase in the column before use is performed before loading the solution containing the rAAV vector onto the column.

E22. The method according to E20 or E21, wherein flushing the stationary phase in the column before use comprises applying water for injection to the stationary phase.

E23. The method according to any one of E1 to E22, further comprising sterilizing the stationary phase in the column.

E24. The method according to E23, wherein sterilizing the stationary phase in the column is performed before loading the solution containing the rAAV vector onto the column.

E25. The method according to E23 or E24, wherein sanitizing the stationary phase in the column comprises applying a solution comprising NaOH to the stationary phase.

E26. The method according to any one of E23 to E25, wherein sanitizing the stationary phase in the column comprises applying a solution comprising about 0.1 M to about 1.0 M (eg, about 0.5 M) NaOH to the stationary phase.

E27. The method according to any one of E23 to E26, wherein sanitizing the stationary phase in the column comprises applying about 5 CV to about 10 CV, or about 14.4 CV to about 17.6 CV of a solution comprising about 0.1 M to about 1.0 M NaOH to the stationary phase.

E28. The method according to any one of E23 to E27, wherein the stationary phase in the column is sterilized by upward flow.

E29. The method according to any one of E1 to E28, further comprising regenerating the stationary phase in the column.

E30. The method according to E29, wherein regenerating the stationary phase in the column is performed before loading the solution containing the rAAV vector onto the column.

E31. The method according to E29 or E30, wherein regenerating the stationary phase in the column comprises applying a solution comprising a component selected from the group consisting of a salt, a buffer, and combinations thereof to the stationary phase.

E32. A method according to any one of E29 to E31, wherein regenerating the stationary phase in the column comprises applying a solution comprising about 1 M to about 3 M (e.g., about 2 M) NaCl, about 50 mM to about 150 mM (e.g., about 100 mM) Tris, and pH 8 to 10 (e.g., about 9) to the stationary phase.

E33. The method according to any one of E29 to E32, wherein regenerating the stationary phase in the column comprises applying a solution comprising about 50 mM to about 150 mM (eg, about 100 mM) Tris, pH 9 to the stationary phase.

E34. The method according to any one of E29 to E33, wherein regenerating the stationary phase in the column comprises applying 4.5 to 5.5 CV of a solution comprising about 100 mM Tris, pH 9 to the stationary phase.

E35. The method according to any one of E29 to E34, wherein regenerating the stationary phase in the column is performed more than once.

E36. The method according to any one of E1 to E35, further comprising equilibrating the stationary phase in the column.

E37. The method according to E36, wherein equilibration of the stationary phase in the column is performed before or after loading the solution comprising the rAAV vector onto the column.

E38. The method according to E36 or E37, wherein equilibrating the stationary phase in the column comprises applying an equilibration buffer comprising at least one component selected from the group consisting of a buffer, a salt, an amino acid, a detergent, and combinations thereof to the stationary phase.

E39. The method according to E38, wherein the buffer is selected from Tris, BIS-Tris propane, diethanolamine, diethylamine, tris(hydroxymethyl)methylglycine, triethanolamine and/or n,n-bicine and combinations thereof.

E40. The method according to E36 or E39, wherein the salt is selected from sodium chloride, sodium acetate, ammonium acetate, magnesium chloride, sodium sulfate and combinations thereof.

E41. The method according to any one of E38 to E40, wherein the salt is sodium acetate.

E42. The method according to any one of E38 to E41, wherein the amino acid is selected from the group consisting of histidine, arginine, glycine, citrulline and combinations thereof.

E43. The method according to any one of E38 to E42, wherein the amino acid is histidine.

E44. The method according to E38 to E43, wherein the detergent is selected from poloxamer 188 (P188), Triton X-100, polysorbate 80 (PS80), Brij-35, nonylphenoxypolyethoxyethanol (NP-40) and combinations thereof.

E45. The method according to any one of E38 to E44, wherein the detergent is P188.

E46. The method according to any one of E36 to E45, wherein equilibrating the stationary phase in the column comprises applying an equilibration buffer comprising about 50 mM to about 150 mM (e.g., about 100 mM Tris) at a pH of about 8 to 10 (e.g., about 9) to the stationary phase.

E47. A method according to any one of E36 to E46, wherein equilibrating the stationary phase in the column comprises applying to the stationary phase an equilibration buffer comprising about 50 mM to about 150 mM (e.g., about 100 mM Tris), about 250 mM to about 750 mM (e.g., about 500 mM) sodium acetate, about 0.005% to about 0.015% (e.g., 0.01%) P188, and a pH of about 8 to about 10 (e.g., about 8.9).

E48. A method according to any one of E36 to E47, wherein equilibrating the stationary phase in the column comprises applying an equilibration buffer comprising about 100 mM to about 300 mM (e.g., about 200 mM) histidine, about 100 mM to about 300 Mm (e.g., about 200 mM Tris), about 0.1% to about 1.0% (e.g., about 0.5%) P188, and a pH of about 8 to about 10 (e.g., about 8.8) to the stationary phase.

E49. A method according to any one of E36 to E48, wherein equilibration of the stationary phase in the column comprises applying to the stationary phase an equilibration buffer comprising about 50 mM to about 150 mM (e.g., about 100 mM) Tris, 0.005% to about 0.015% (e.g., about 0.01%) P188, and a pH of about 8 to about 10 (e.g., about 8.9).

E50. The method according to any one of E36 to E49, wherein equilibrating the stationary phase in the column comprises applying greater than about 4.5 CV of equilibration buffer to the stationary phase.

E51. The method according to any one of E36 to E50, wherein equilibrating the stationary phase in the column comprises applying 4.5 to 5.5 CV of equilibration buffer to the stationary phase.

E52. The method according to any one of E36 to E51, wherein equilibration of the stationary phase in the column is performed more than once.

E53. A method according to any one of E36 to E52, wherein at least one equilibration buffer is applied to the stationary phase before the solution comprising the rAAV vector is loaded onto the column; and wherein at least one equilibration buffer is applied to the stationary phase after the solution comprising the rAAV vector is loaded onto the column.

E54. A method according to any one of E1 to E53, comprising performing a gradient elution of material from the stationary phase in the column.

E55. A method according to E54, wherein the gradient elution comprises applying 10 to 60CV of at least two different solutions (e.g., gradient elution buffer) or a mixture of the two solutions to the stationary phase, and wherein during the gradient elution, the percentage of the first solution changes inversely proportional to the percentage of the second solution.

E56. The method according to E54 or E55, wherein the at least two different solutions (eg, a first gradient elution buffer, a second gradient elution buffer) comprise components selected from the group consisting of a buffer, a salt, a detergent, and combinations thereof.

E57. The method according to E56, wherein the buffer is selected from Tris, BIS-Tris propane, diethanolamine, diethylamine, tris(hydroxymethyl)methylglycine, triethanolamine and/or n,n-bicine.

E58. The method according to E56 or E57, wherein the salt is selected from sodium chloride, sodium acetate, ammonium acetate, magnesium chloride, sodium sulfate and combinations thereof.

E59. The method according to any one of E56 to E58, wherein the detergent is selected from poloxamer 188 (P188), Triton X-100, polysorbate 80 (PS80), Brij-35, nonylphenoxypolyethoxyethanol (NP-40) and combinations thereof.

E60. The method according to any one of E55 to E59, wherein the at least two different solutions have different pH, salt concentration, conductivity and/or modifier concentration.

E61. A method according to any one of E55 to E60, wherein the first solution (e.g., buffer A) comprises about 50 mM to about 150 mM (e.g., about 100 mM) Tris, about 0.005% to about 0.015% (e.g., about 0.01%) P188, and about pH 8.5 to 9.5 (e.g., about 8.9).

E62. A method according to any one of E55 to E61, wherein the second solution (e.g., buffer B) comprises about 400 mM to about 600 mM (e.g., about 500 mM) sodium acetate, about 50 mM to about 150 mM (e.g., about 100 mM) Tris, about 0.005% to about 0.015% (e.g., about 0.01%) P188, and about pH 8.5 to 9.5 (e.g., about 8.9).

E63. The method according to any one of E55 to E62, wherein performing the gradient elution comprises applying about 10 to 60 CV, about 20 to 40 CV or about 20 to 24 CV of the at least two different solutions to the stationary phase.

E64. A method according to any one of E55 to E63, wherein at the beginning of gradient elution, the percentage of the first solution (e.g., a first gradient elution buffer, buffer A) is 50% to 100%, and at the end of gradient elution, the percentage of the second solution (e.g., a second gradient elution buffer, buffer B) is 50% to 100%, and wherein optionally during gradient elution, 10 to 60CV of the first solution, the second solution, or a mixture of the two are applied to the stationary phase.

E65. A method according to any one of E55 to E64, wherein at the beginning of gradient elution, the percentage of the first solution (e.g., the first gradient elution buffer, buffer A) is 100%, and at the end of gradient elution, the percentage of the second solution (e.g., the second gradient elution buffer, buffer B) is 100%, and wherein optionally during the gradient elution, 10 to 60CV of the first solution, the second solution, or a mixture of the two are applied to the stationary phase.

E66. A method according to any one of E55 to E65, wherein the concentration of a component of the first solution (e.g., a first gradient elution buffer) or the second solution (e.g., a second gradient elution buffer) continuously increases or decreases during the gradient elution; wherein the rate of increase or decrease in the concentration of the component of the first solution or the second solution is equal to the change in the concentration of the component per total CV; and wherein the rate of change in the concentration of the component during the gradient elution is approximately 10 mM/CV to 50 mM/CV.

E67. The method according to any one of E54 to E66, wherein the complete capsid is eluted from the stationary phase in the first elution peak and/or the first part of the second elution peak of said gradient elution.

E68. The method according to any one of E54 to E67, wherein empty capsids are recovered in the AEX column flow-through and/or in the last part of the second elution peak of the gradient elution.

E69. The method according to any one of E1 to E68, comprising implementing a gradient hold.

E70. The method according to E69, wherein the gradient maintenance comprises applying 1 to 10 CV of a gradient maintenance solution to the stationary phase in the column, the gradient maintenance solution comprising a component selected from the group consisting of a salt, a buffer, a detergent, and a combination thereof.

E71. A method according to E69 or E70, wherein implementing gradient maintenance comprises applying 1 to 10CV of a gradient maintenance solution to the AEX stationary phase in the column, the gradient maintenance solution comprising about 5mM to about 1M (e.g., about 500mM) sodium acetate, 50mM to 150mM (e.g., about 100mM) Tris, about 0.005% to about 0.015% (e.g., about 0.01%) P188, and a pH of about 8.5 to 9.5 (e.g., about 8.9).

E72. A method according to any one of E1 to E71, comprising performing step elution (eg isocratic elution) from the stationary phase in the column.

E73. A method according to E72, wherein performing step elution comprises applying at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more step elution solutions to the column stationary phase.

E74. A method according to E72 or E73, wherein the at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more step elution solutions each comprise a component selected from a buffer, a salt, a detergent and combinations thereof.

E75. A method according to any one of E72 to E74, wherein the at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more step elution solutions each contain about 10 mM to about 50 mM (e.g., about 20 mM) Tris and about 5 mM to about 600 mM sodium acetate, at a pH of about 8 to 10 (e.g., about 8.9 to about 9.1).

E76. A method according to any one of E72 to E75, wherein step elution solutions having increasing sodium acetate concentrations are applied sequentially to the column.

E77. The method according to any one of E72 to E76, wherein the final step elution solution comprises about 20 mM Tris, about 500 mM sodium acetate, pH about 8.9 to about 9.1.

E78. The method according to any one of E1 to E77, comprising collecting at least one eluate fraction from the column to recover complete rAAV capsids, optionally during gradient elution.

E79. A method according to E78, wherein the volume of at least one eluate fraction is selected from 1/8CV, 1/4CV, 1/3CV, 1/2CV, 3/4CV, 1CV, 2CV, 3CV, 4CV, 5CV, 6CV, 7CV, 8CV, 9CV, 10CV or more.

E80. The method according to E79, wherein the absorbance of the at least one eluate fraction is measured at 280 nm, and wherein optionally the threshold value for measurement at 280 nm is ≥ 0.5 mAU/mm path length.

E81. A method according to E80, wherein the at least one eluate fraction is collected when the A280 of the eluate is ≥ 0.5 mAU/mm path length.

E82. A method according to E80 or E81, wherein the volume of at least one eluate fraction is equal to 1/8CV to 10CV, for example 1/8CV, 1/4CV, 1/3CV, 1/2CV, 3/4CV, 1CV, 2CV, 3CV, 4CV, 5CV, 6CV, 7CV, 8CV, 9CV, 10CV or more CVs, and wherein optionally the A260/A280 ratio of at least one eluate fraction is ≥1.25.

E83. A method according to any one of E78 to E82, wherein at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more eluate fractions are collected.

E84. The method according to any one of E78 to E83, further comprising adjusting the pH of the at least one eluate fraction collected from the column, optionally during gradient elution.

E85. A method according to E84, wherein adjusting the pH of the at least one eluate fraction comprises i) adding 14.3% to 15% eluate volume by weight of a solution comprising a buffer at a pH of approximately 3.5 to the at least one eluate fraction, or ii) collecting the at least one eluate fraction into a container comprising approximately 0.01CV to 0.1CV (e.g., approximately 0.066CV) of a solution comprising a buffer.

E86. The method according to E85, wherein the buffer is about 200 mM to about 300 mM (eg about 250 mM) sodium citrate.

E87. A method according to E84 to E86, wherein the pH of the at least one eluate fraction collected from the column is adjusted from an initial pH of about 8.5 to about 9.1 to a pH of about 6.8 to about 7.6.

E88. A method according to E84 to E87, wherein the pH of the at least one eluate fraction collected from the column is adjusted from an initial pH of about 8.5 to about 9.1 to a pH of about 7.0 to about 7.4.

E89. The method according to any one of E78 to E88, further comprising measuring the absorbance of at least one eluate fraction collected from the column, optionally during gradient elution.

E90. The method according to E89, wherein the absorbance is measured at 260 nm (A260), 280 nm (A280), or 260 nm and 280 nm, and optionally wherein the A260/A280 ratio is determined.

E91. The method according to E90, wherein the absorbance at 260 nm and 280 nm is measured by size exclusion chromatography (SEC).

E92. A method according to E90 or E91, wherein the A260/A280 ratio of at least one eluate fraction is at least 0.5, at least 0.55, at least 0.6, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.95. at least 1.0, at least 1.05, at least 1.10, at least 1.11, at least 1.12, at least 1.13, at least 1.14, at least 1.15, at least 1.16, at least 1.17, at least 1.18, at least 1.19, at least 1.20, at least 1.21, at least 1.22, at least 1.23, at least 1.24, at least 1.25, at least 1.26, at least 1.27, at least 1.28, at least 1.29, at least 1.30, at least 1.31, at least 1.32, at least 1.33, at least 1.34 4, at least 1.35, at least 1.36, at least 1.37, at least 1.38, at least 1.39, at least 1.40, or from about 0.5 to about 2.0, about 0.5 to about 1.8, about 0.5 to about 1.6, about 0.5 to about 1.4, about 0.5 to about 1.2, about 0.5 to about 1.0, about 0.5 to about 0.8, about 0.6 to about 2.0, about 0.8 to about 1.8, about 0.8 to about 1.6, about 0.8 to about 1.4, about 1.0 to about 1.4, or about 1.0 to about 1.2, optionally as measured by SEC.

E93. The method according to any one of E90 to E92, wherein at least one eluate fraction has an A260/A280 ratio of at least 1.25.

E94. The method according to any one of E78 to E93, further comprising combining at least two eluate fractions collected from the column, optionally during gradient elution, to form a combined eluate comprising the rAAV vector.

E95. The method according to E94, wherein 2 to 50 eluate fractions are combined to form a pooled eluate.

E96. A method according to E94 or E95, wherein the at least two eluate fractions each have an A260/A280 ratio of > 1.25.

E97. The method according to any one of E94 to E96, further comprising measuring the absorbance of the combined eluate, and wherein A260/A280 of the combined eluate is ≥ 1.25 (eg, about 1.28 to 1.35).

E98. The method according to any one of E94 to E97, wherein the combined eluate has a pH of 6.8 to 7.6 (eg 7.0 to 7.4).

E99. A method according to any one of E78 to E98, wherein the complete capsids comprise 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 98%, 20% to 99%, 20% to greater than 99%, 40% to 50%, 40% to 60%, 40% to 70%, 40% to 80% (e.g., 44%, 45%, 50%, 53%) of the total capsids in at least one of the eluate fractions or the combined eluates, and optionally wherein the capsids are measured by analytical ultracentrifugation (AUC).

E100. The method according to any one of E78 to E99, wherein complete capsids comprise about 55% (eg 55% +/- 7%) of the total capsids in the at least one eluate fraction or the combined eluates.

E101. The method according to any one of E78 to E99, wherein complete capsids comprise about 49% (eg 49% +/- 2%) of the total capsids in the at least one eluate fraction or the combined eluates.

E102. The method according to any one of E78 to E99, wherein complete capsids represent 52 +/- 7% of the total capsids in said at least one eluate fraction or the combined eluates.

E103. A method according to any one of E78 to E99, wherein the at least one eluate fraction or the combined eluates contains complete rAAV capsids comprising 48% to 62% of the total capsids, and wherein the at least one eluate fraction or the combined eluates is produced by purifying rAAV vectors produced in a 250L SUB.

E104. A method according to any one of E78 to E99, wherein the at least one eluate fraction or the combined eluates contains complete rAAV capsids that are 47% to 51% of the total capsids, and wherein the at least one eluate fraction or the combined eluates is produced by purifying rAAV vectors produced in 2000LSUB.

E105. A method according to any one of E78 to E99, wherein at least one eluate fraction or the combined eluates contains more than 30% (e.g., 40% to 55%, 45% to 65%, 40% to greater than 99%) of complete capsids of the total capsids, and the solution containing the rAAV vector to be purified contains less than 30% (e.g., 12% to 25%) of complete capsids of the total capsids in the solution.

E106. A method according to any one of E78 to E105, wherein the empty capsids comprise 10% to 99%, 10% to 90%, 10% to 80%, 10% to 70%, 10% to 65%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 65%, 20% to 60%, 20% to 50%, 20% to 40% or 18% to 29% (e.g., ≤29%) of the total capsids in the at least one eluate fraction or the combined eluate, and optionally, wherein the capsids are measured by analytical ultracentrifugation (AUC).

E107. The method according to any one of E78 to E106, wherein in the at least one eluate fraction or the combined eluate, the empty capsids comprise 20% +/- 7% (eg, 21%) of the total capsids.

E108. A method according to any one of E78 to E106, wherein the at least one eluate fraction or the combined eluates contains 11% to 31% empty capsids of the total capsids, and wherein the at least one eluate fraction or the combined eluates is produced by purifying rAAV vectors produced in a 250L SUB.

E109. A method according to any one of E78 to E106, wherein the at least one eluate fraction or the combined eluates contains 18% to 22% empty capsids of the total capsids, and wherein the at least one eluate fraction or the combined eluates is produced by purifying rAAV vectors produced in 2000L SUB.

E110. A method according to any one of E78 to E106, wherein the at least one eluate fraction or the combined eluates contains less than 30% of empty capsids of the total capsids, and the solution comprising the rAAV vector to be purified contains 40% to 90% of empty capsids of the total capsids in the solution.

E111. A method according to any one of E78 to E110, wherein in the at least one eluate fraction or the combined eluate the intermediate capsids comprise 10% to 65%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 65%, 20% to 60%, 20% to 50%, 20% to 40% or 18% to 22% of the total capsids, and optionally wherein the capsids are measured by analytical ultracentrifugation (AUC).

E112. The method according to any one of E78 to E111, wherein the intermediate capsids comprise 28% +/- 5% of the total capsids in the at least one eluate fraction or the combined eluates.

E113. A method according to any one of E78 to E111, wherein the at least one eluate fraction or the combined eluates comprises intermediate capsids of 21% to 27% of the total capsids, and wherein the at least one eluate fraction or the combined eluates is produced by purifying rAAV vectors produced in a 250L SUB.

E114. A method according to any one of E78 to E111, wherein the at least one eluate fraction or the combined eluates comprises intermediate capsids of 28% to 36% of the total capsids, and wherein the at least one eluate fraction or the combined eluates is produced by purifying rAAV vectors produced in 2000L SUB.

E115. A method according to any one of E78 to E111, wherein the at least one eluate fraction or the combined eluate contains 45% to 65% of complete rAAV capsids, 19% to 28% of intermediate capsids and 10% to 37% of empty capsids of the total capsids, and wherein the at least one eluate fraction or the combined eluate is produced by purifying rAAV vectors produced in a 250L SUB.

E116. Method according to E115, wherein in said at least one eluate fraction or the combined eluates, complete capsids comprise 55% +/- 7% of the total capsids.

E117. Method according to E115, wherein in said at least one eluate fraction or the combined eluates, intermediate capsids comprise 24% +/- 3% of the total capsids.

E118. The method according to E115, wherein in said at least one eluate fraction or the combined eluates, empty capsids comprise 21% +/- 10% of the total capsids.

E119. A method according to any one of E78 to E118, wherein the at least one eluate fraction or the combined eluates contains 45% to 52% complete rAAV capsids, 27% to 37% intermediate capsids and/or 18% to 22% empty capsids of the total capsids, and wherein the at least one eluate fraction or the combined eluates is produced by purifying an rAAV vector produced in 2000LSUB.

E120. Method according to E119, wherein in said at least one eluate fraction or the combined eluates, complete capsids comprise 49% +/- 2% of the total capsids.

E121. Method according to E119, wherein in said at least one eluate fraction or the combined eluates, intermediate capsids comprise 32% +/- 4% of the total capsids.

E122. The method according to E119, wherein in said at least one eluate fraction or the combined eluates, empty capsids comprise 20% +/- 2% of the total capsids.

E123. The method of any one of E94 to E122, wherein the combined eluate is enriched for complete capsids and/or depleted for empty capsids compared to the solution loaded on the column.

E124. A method according to any one of E78 to E123, wherein the VG step yield percentage of at least one eluate fraction or the combined eluates is 1% to 10%, 1% to 20%, 1% to 30%, 1% to 40%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 99%, 5% to 95%, 10% to 85%, 15% to 75%, 20% to 65%, 25% to 55%, 30% to 45%, 30% to 80%, 40% to 70% or 100%.

E125. The method according to E124, wherein the VG step yield percentage of the at least one eluate fraction or the combined eluates is from 31% to 66% (eg 47% +/- 11%).

E126. The method according to E124, wherein the VG step yield percentage of at least one eluate fraction or the combined eluates produced in the 250L SUB is 30% to 70% (eg, 37% to 60%).

E127. The method according to E124, wherein the VG step yield percentage of the at least one eluate fraction or the combined eluates produced in the 250L SUB is 45% +/- 8%.

E128. The method according to E124, wherein the VG step yield percentage of at least one eluate fraction or the combined eluates produced in the 2000L SUB is from 25% to 75% (eg, from 31% to 66%).

E129. The method according to E124, wherein the VG step yield percentage of the at least one eluate fraction or the combined eluates produced in the 2000LSUB is 50% +/- 13%.

E130. A method according to any one of E124 to E129, wherein the VG step yield percentage of at least one eluate fraction or combined eluates is greater than the VG step yield percentage of other identical eluate fractions or combined eluates purified by ultracentrifugation and cation exchange chromatography.

E131. The method according to any one of E78 to E130, wherein the A260/A280 (SEC) of the at least one eluate fraction or the combined eluates produced in the 250L SUB is 1.29 +/- 0.03.

E132. The method according to any one of E78 to E130, wherein the A260/A280 (SEC) of the at least one eluate fraction or the combined eluate produced in the 2000L SUB is 1.30 +/- 0.01.

E133. A method according to any one of E78 to E132, wherein the VG column yield percentage of at least one eluate fraction or the combined eluate is 1% to 10%, 1% to 20%, 1% to 30%, 1% to 40%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 99%, 5% to 95%, 10% to 85%, 15% to 75%, 20% to 65%, 25% to 55%, 30% to 45%, 30% to 80%, 40% to 70% or 100%.

E134. The method according to E133, wherein the VG column yield percentage of the at least one eluate fraction or the combined eluates is between 20% and 100% (eg 63% +/- 26%).

E135. The method according to E133, wherein the VG column yield percentage of at least one eluate fraction or the combined eluates produced in the 250 L SUB is from 40% to 100%.

E136. The method according to E133, wherein the VG column yield percentage of at least one eluate fraction or the combined eluates produced in the 2000L SUB is from 10% to 70% (eg, from 20% to 61%).

E137. A method according to any one of E78 to E136, wherein the at least one eluate fraction or the combined eluates is further subjected to a filtration method selected from virus filtration, ultrafiltration/diafiltration (UF/DF), filtration through a 0.2 μm filter and a combination thereof to produce the drug substance.

E138. The method according to E137, wherein the complete capsid comprises 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 98%, 20% to 99%, 20% to greater than 99%, 40% to 50%, 40% to 60%, 40% to 70%, 40% to 80% (e.g., 44%, 45%, 50%, 53%) of the total capsid in the drug substance, and optionally wherein the capsid is measured by analytical ultracentrifugation (AUC).

E139. The method according to E137 or E138, wherein the drug substance comprises complete rAAV capsids of 45% to 65% of the total capsids, and optionally wherein the drug substance is produced by purifying rAAV vectors produced in a 250L SUB.

E140. The method according to any one of E137 to E139, wherein the complete capsid in the drug substance comprises 52+/-7% of the total capsid.

E141. The method according to E137 or E138, wherein the drug substance comprises complete rAAV capsids that are 45% to 52% of the total capsids, and wherein the drug substance is produced by purifying rAAV vectors produced in a 2000L SUB.

E142. A method according to E137 or E138, wherein the drug substance contains more than 30% (e.g., 40% to 55%, 45% to 65%, 40% to greater than 99%) of complete capsids of the total capsids in the drug substance, and the solution containing the rAAV vector to be purified contains less than 30% (e.g., 12% to 25%) of complete capsids of the total capsids in the solution.

E143. The method according to any one of E137 to E142, wherein the empty capsid of the drug substance comprises 10% to 99%, 10% to 90%, 10% to 80%, 10% to 70%, 10% to 65%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 65%, 20% to 60%, 20% to 50%, 20% to 40% or 18% to 29% (e.g., ≤29%) of the total capsid, and optionally wherein the capsid is measured by analytical ultracentrifugation (AUC).

E144. The method according to E143, wherein the drug substance comprises 10% to 37% of empty capsids of the total capsids, and optionally wherein the drug substance is produced by purifying rAAV vectors produced in 250L SUB.

E145. The method according to E143 or E144, wherein the empty capsids comprise 20% +/- 7% of the total capsids in the drug substance.

E146. The method according to E143, wherein the drug substance comprises 18% to 22% of empty capsids of total capsids, and optionally wherein the drug substance is produced by purifying rAAV vectors produced in 2000L SUB.

E147. A method according to any one of E143 to E146, wherein the drug substance contains less than 30% of empty capsids of the total capsids in the drug substance, and the solution containing the rAAV vector to be purified contains 40% to 90% of empty capsids of the total capsids in the solution.

E148. The method according to any one of E137 to E147, wherein the intermediate capsid comprises 10% to 65%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 65%, 20% to 60%, 20% to 50%, 20% to 40% or 18% to 22% of the total capsid in the drug substance, and optionally wherein the capsid is measured by analytical ultracentrifugation (AUC).

E149. The method according to any one of E137 to E148, wherein the drug substance comprises intermediate capsids of 19% to 37% of the total capsids, and optionally wherein the drug substance is produced by purifying rAAV vectors produced in 250L or 2000L SUB.

E150. The method according to E148 or E149, wherein the intermediate capsid comprises 28% +/- 5% of the total capsid in the drug substance.

E151. A method according to any one of E137 to E150, wherein the drug substance comprises 45% to 65% of complete rAAV capsids, 19% to 28% of intermediate capsids and 10% to 37% of empty capsids of the total capsids, wherein the drug substance is produced by purifying rAAV vectors produced in a 250L SUB.

E152. The method according to E151, wherein the complete capsid comprises 55% +/- 7% of the total capsid.

E153. The method according to E151 or E152, wherein the intermediate capsid comprises 24% +/- 3% of the total capsid.

E154. The method according to any one of E151 to E153, wherein the empty capsids comprise 21% +/- 10% of the total capsids.

E155. A method according to any one of E137 to E150, wherein the drug substance comprises 45% to 52% of complete rAAV capsids, 27% to 37% of intermediate capsids and/or 18% to 22% of empty capsids of the total capsids, and wherein the drug substance is produced by purifying rAAV vectors produced in a 2000L SUB.

E156. The method according to E155, wherein the complete capsid comprises 49% +/- 2% of the total capsid.

E157. The method according to E155 or E156, wherein the intermediate capsid comprises 32% +/- 4% of the total capsid.

E158. The method according to any one of E155 to E157, wherein the empty capsids comprise 20% +/- 2% of the total capsids.

E159. The method according to any one of E137 to E158, wherein the drug substance is depleted of host cell proteins (HCP) compared to the amount of HCP in the solution comprising the rAAV vector to be purified.

E160. The method according to any one of E137 to E159, wherein the drug substance comprises an amount of HCP below the lowest level of quantification (LLOQ) measured by ELISA.

E161. The method according to any one of E137 to E160, wherein the drug substance comprises an amount of HCP below the LLOQ as measured by ELISA, wherein the solution comprising the rAAV vector to be purified comprises about 1 to 500 pg HCP/1×10 ⁹ VG, and optionally wherein the solution is produced by affinity chromatography purification of rAAV vector produced in 250 L SUB.

E162. The method according to any one of E137 to E161, wherein the drug substance comprises an amount of HCP below the LLOQ as measured by ELISA, wherein the solution comprising the rAAV vector to be purified comprises about 100 to 1000 pg HCP/1×10 ⁹ VG, and optionally wherein the solution is produced by affinity chromatography purification of rAAV vectors produced in a 2000L SUB.

E163. A method according to any one of E137 to E162, wherein the purity of the drug substance is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100%, optionally measured by reverse phase HPLC.

E164. The method according to E163, wherein the drug substance has a purity of about 98.6+/-0.6%, and optionally wherein the drug substance is produced by purifying a rAAV vector produced in a 250L SUB.

E165. The method according to E164, wherein the drug substance has a purity of about 99.3+/-0.3%, and optionally wherein the drug substance is produced by purifying a rAAV vector produced in a 2000L SUB.

E166. The method according to any one of E137 to E165, wherein the drug substance comprises about 0% to 10% HMMS, optionally measured by SEC.

E167. The method according to E166, wherein the drug substance comprises 2.6+/-0.8% HMMS, and optionally wherein the drug substance is produced by purifying a rAAV vector produced in a 250L SUB.

E168. The method according to E166, wherein the drug substance comprises 2.9+/-0.4% HMMS, and optionally wherein the drug substance is produced by purifying a rAAV vector produced in a 2000L SUB.

E169. The method according to E137 to E168, wherein the drug substance comprises about 7.0 to 25 pg/1×10 ⁹ VG of residual HC-DNA.

E170. The method according to E169, wherein the drug substance comprises about 17.4 +/- 6.7 pg/1 x ¹⁰⁹ VG of HC-DNA, and optionally wherein the drug substance is produced by purifying rAAV vector produced in 250L SUB.

E171. The method according to E169, wherein the drug substance comprises about 9.3 +/- 1.2 pg/1 x ¹⁰⁹ VG of HC-DNA, and optionally wherein the drug substance is produced by purifying rAAV vector produced in 2000L SUB.

E172. The method according to any one of E137 to E171, wherein the drug substance has an A260/A280 of about 1.24 to 1.32, optionally measured by size exclusion chromatography (SEC).

E173. The method according to E172, wherein the drug substance has an A260/A280 of 1.24 to 1.32, optionally measured by SEC, and wherein the drug substance is produced by purifying a rAAV vector produced in a 250L SUB.

E174. The method according to E172, wherein the drug substance has an A260/A280 of 1.28 to 1.31, optionally measured by SEC, and optionally wherein the drug substance is produced by purification of a rAAV vector produced in a 2000L SUB.

E175. The method according to any one of E1 to E174, wherein the column volume is 1.0 mL to 6.6 L.

E176. The method according to any one of E1 to E175, wherein the column volume is about 1.0 mL, about 5.1 mL, about 6.67 mL, about 1.256 L, about 1.3 L, about 6.3 L, about 6.4 L or about 6.6 L.

E177. The method according to any one of E1 to E176, wherein the rAAV vector comprises a capsid protein from an AAV serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAVrh74, AAV12, AAV2i8, NP4, NP22, NP66, AAVDJ, AAVDJ/8, AAVDJ/9, AAVLK03, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, RHM4-1 (SEQ ID NO: 101 of WO 2015/013313), NO:5), RHM15-1, RHM15-2, RHM15-3/RHM15-5, RHM15-4, RHM15-6, AAV hu.26, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9,45, AAV2i8, AAV29G, AAV2,8G9, AVV-LK03, AAV2-TT, AAV 2-TT-S312N, AAV3B-S312N, AAVHSC1, AAVHSC2, AAVHSC3, AAVHSC4, AAVHSC5, AAVHSC6, AAVHSC7, AAVHSC8, AAVHSC9, AAVHSC10, AAVHSC11, AAVHSC12, AAVHSC13, AAVHSC14 and AAVHSC15.

E178. The method according to any one of E1 to E177, wherein a purified rAAV vector is produced.

E179. The method according to E178, wherein the purified rAAV vector is a drug substance.

E180. The method according to any one of E137 to E174 or E179, wherein the drug substance and the pharmaceutically acceptable excipient are combined to form a drug product.

E181. The method according to any one of E178 to E180, wherein the purified rAAV vector, drug substance and/or drug product is suitable for administration to a subject for treating a disease, disorder or condition.

E182. The method according to E181, wherein said disease, disorder or condition is Duchenne muscular dystrophy (DMD).

E183. The method according to any one of E1 to E182, wherein the rAAV vector comprises a vector genome comprising a modified nucleic acid encoding human mini-dystrophin.

E184. The method according to E183, wherein the modified nucleic acid comprises or consists of the nucleic acid sequence of SEQ ID NO:1.

E185. The method according to E183 or E184, wherein the modified nucleic acid encodes human micro-dystrophin comprising or consisting of the amino acid sequence of SEQ ID NO:2.

E186. The method according to any one of E183 to E185, wherein the vector genome comprises a muscle-specific promoter and/or enhancer selected from the group consisting of a synthetic hybrid muscle-specific promoter hCK, a synthetic hybrid muscle-specific promoter hCKplus and a synthetic muscle-specific enhancer and promoter.

E187. The method according to E186, wherein said synthetic hybrid muscle-specific promoter hCK comprises or consists of the nucleic acid sequence of SEQ ID NO:3.

E188. The method according to E186, wherein said synthetic hybrid muscle-specific promoter hCKplus comprises or consists of the nucleic acid sequence of SEQ ID NO:4.

E189. The method according to E186, wherein said synthetic muscle-specific enhancer and promoter comprises or consists of the nucleic acid sequence of SEQ ID NO:5.

E190. The method according to any one of E183 to E189, wherein the vector genome comprises a polyadenylation (polyA) signal sequence.

E191. The method according to E190, wherein the polyA signal sequence comprises or consists of the nucleic acid sequence of SEQ ID NO:6.

E192. The method according to any one of E183 to E191, wherein the vector genome comprises a transcription terminator sequence.

E193. The method according to E192, wherein the transcription terminator sequence comprises or consists of the nucleic acid sequence of SEQ ID NO:9.

E194. The method according to any one of E183 to E193, wherein the vector genome comprises at least one ITR.

E195. According to the method of E194, wherein the at least one ITR comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:14 and combinations thereof, or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:14 and combinations thereof.

E196. The method according to any one of E183 to E195, wherein the vector genome comprises an expression cassette, and wherein the expression cassette comprises or consists of the nucleic acid sequence of SEQ ID NO: 10.

E197. The method according to any one of E1 to E196, wherein the rAAV vector comprises the VP1 polypeptide of AAV9.

E198. The method according to E197, wherein said VP1 polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:11.

E199. The method according to any one of E1 to E198, wherein the rAAV vector comprises an AAV9 capsid protein and a transgene comprising the nucleic acid sequence of SEQ ID NO: 1.

E200. The method according to any one of E1 to E199, further comprising preparing a solution comprising the rAAV vector purified by AEX.

E201. The method according to E200, wherein the solution containing the rAAV vector is selected from an affinity eluate, a supernatant of a cell lysate, and a post-harvest solution, which has been diluted and optionally filtered before loading.

E202. The method according to E200 or E201, wherein the solution containing the rAAV vector is an affinity eluate, which has been diluted and optionally filtered prior to loading.

E203. The method according to any one of E200 to E202, wherein the solution has undergone at least one purification and/or treatment step.

E204. The method according to any one of E200 to E203, wherein the solution comprising the rAAV vector is an eluate obtained from affinity chromatography purification of the rAAV vector.

E205. The method according to any one of E200 to E204, wherein the preparation comprises diluting a solution comprising the rAAV vector.

E206. According to the method of E205, diluting the solution containing the rAAV vector includes diluting the solution by about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 11 times, about 12 times, about 13 times, about 14 times, about 15 times, about 16 times, about 17 times, about 18 times, about 19 times, about 20 times, about 25 times to produce a diluted solution.

E207. The method according to E205 or E206, wherein diluting the solution comprising the rAAV vector comprises diluting the solution by about 15 times to produce a diluted solution.

E208. A method according to any one of E205 to E207, wherein dilution of a solution containing rAAV vectors is performed in-line with an AEX column, and wherein the diluted solution is transported to a Y-type connector via a first tubing system, and wherein the solution containing rAAV vectors is transported to the Y-type connector via a second tubing system.

E209. The method according to E208, wherein the diluted solution is delivered at a flow rate of 1 to 5 mL/min, and wherein the solution comprising the rAAV vector is delivered at a flow rate of 0.1 to 2 mL/min, whereby the solution is diluted approximately 15 times.

E210. The method according to E208 or E209, wherein the dilution solution comprises at least one component selected from a buffer, an amino acid, a detergent, and a combination thereof.

E211. The method according to E210, wherein the buffer is selected from Tris, BIS-Tris propane, diethanolamine, diethylamine, tris(hydroxymethyl)methylglycine, triethanolamine, n,n-bicine, and combinations thereof.

E212. The method according to E210 or E211, wherein the amino acid is selected from the group consisting of histidine, arginine, glycine, citrulline, and combinations thereof.

E213. The method according to any one of E210 to E212, wherein the amino acid is histidine.

E214. A method according to any one of E210 to E213, wherein the detergent is selected from poloxamer 188 (P188), Triton X-100, polysorbate 80 (PS80), Brij-35, nonylphenoxypolyethoxyethanol (NP-40) and combinations thereof.

E215. The method according to any one of E210 to E214, wherein the detergent is P188.

E216. A method according to any one of E208 to E215, wherein the dilution solution comprises about 100 mM to about 300 mM (e.g., about 200 mM) histidine, about 100 mM to about 300 mM (e.g., about 200 mM) Tris, about 0.1% to about 1.0% (e.g., about 0.5%) P188, and a pH of about 8.5 to about 9.5 (e.g., about 8.8).

E217. The method according to any one of E205 to E216, wherein the solution comprising the rAAV vector is diluted before loading the solution comprising the rAAV vector onto the column.

E218. The method according to any one of E205 to E217, wherein after dilution, the pH of the solution comprising the rAAV vector is increased compared to the pH of the solution before dilution.

E219. The method according to any one of E205 to E218, wherein the pH of the solution containing the rAAV vector before dilution is 3.0 to 4.4, and the pH of the solution after dilution is 8.5 to 9.5.

E220. The method according to any one of E205 to E219, wherein the conductivity of the diluted solution comprising the rAAV vector is reduced compared to the conductivity of the solution before dilution.

E221. A method according to any one of E205 to E220, wherein the conductivity of the solution containing the rAAV vector before dilution is 5.0 to 7.0 mS/cm (e.g., 5.5 to 6.5 mS/cm), and the conductivity of the solution after dilution is 1.7 to 3.5 mS/cm.

E222. The method according to any one of E205 to E221, further comprising filtering the diluted solution containing the rAAV vector.

E223. The method according to E222, wherein filtering the diluted solution comprises filtering through a 0.2 μm filter.

E224. A method according to E222 or E223, wherein the filter is connected in series with the column.

E225. The method according to any one of E222 to E224, wherein the solution comprising the rAAV vector is diluted and filtered before loading the solution comprising the rAAV vector onto the column.

E226. The method according to any one of E205 to E225, wherein the diluted and optionally filtered solution comprising rAAV vector has a vector genome percentage (% VG) yield of 60% to 100% compared to the amount of VG present in the solution before dilution and optional filtration.

E227. The method according to any one of E205 to E226, wherein the % VG dilution yield of the diluted solution comprising the rAAV vector is 88% +/- 36%.

E228. A method for preparing a solution containing a rAAV vector purified by AEX, the method comprising the following steps:

i) diluting the first solution 2 to 25 times (eg 15 times) with a diluting solution; and optionally

ii) filtering the solution in step i) through a filter to produce a diluted and optionally filtered solution; wherein the pH of the diluted and optionally filtered solution is increased compared to the pH of the first solution; and wherein the conductivity of the diluted and optionally filtered solution is reduced compared to the conductivity of the first solution.

E229.E228 A method for preparing a solution comprising an rAAV vector purified by AEX, wherein i) the pH of the diluted and optionally filtered solution is 8.5 to 9.5; ii) the conductivity of the diluted and optionally filtered solution is 1.7 to 3.3 mS/cm; and/or iii) the %VG dilution yield of the solution after dilution is 35% to 100%.

E230.E228 or E229. A method for preparing a solution comprising an rAAV vector purified by AEX, wherein the rAAV vector comprises an AAV9 capsid protein.

E231. A method for purifying an rAAV vector by AEX, the method comprising the following steps:

i) loading a solution containing the rAAV vector to be purified onto the AEX stationary phase in the column;

ii) performing a gradient elution of the material from the stationary phase in the column, wherein the percentage of a first gradient elution buffer is varied inversely proportional to the change in the percentage of a second gradient elution buffer;

iii) starting to collect at least one eluate fraction from the column during gradient elution when the absorbance of the column flow-through reaches a threshold absorbance value;

iv) measuring the absorbance of said at least one eluate fraction collected from said column and determining the A260/A280 ratio.

E232. A method for purifying a rAAV vector by AEX according to E231, wherein the method further comprises combining at least two eluate fractions collected from the column to form a combined eluate comprising the rAAV vector.

E233. The method for purifying rAAV vectors by AEX according to E231 or E232, wherein the AEX stationary phase is POROS ^™ 50HQ.

E234. A method for purifying a rAAV vector by AEX according to any one of E231 to E233, wherein the solution is an affinity eluate that has been diluted and filtered before loading on the stationary phase.

E235. The method for purifying a rAAV vector according to any one of E231 to E234, wherein the material eluted from the stationary phase comprises the rAAV vector to be purified.

E236. A method for purifying an rAAV vector by AEX according to any one of E231 to E235, wherein the first gradient elution buffer comprises about 50 mM to about 150 mM (e.g., about 100 mM) Tris, about 0.005% to about 0.015% (e.g., about 0.01%) P188, and a pH of about 8.5 to 9.5 (e.g., about 8.9).

E237. A method for purifying an rAAV vector by AEX according to any one of E231 to E236, wherein the second gradient elution buffer comprises about 400 mM to about 600 mM (e.g., about 500 mM) sodium acetate, about 50 mM to about 150 mM (e.g., about 100 mM) Tris, about 0.005% to about 0.015% (e.g., about 0.01%) P188, and a pH of about 8.5 to 9.5 (e.g., about 8.9).

E238. A method for purifying rAAV vectors by AEX according to any one of E231 to E237, wherein at the beginning of gradient elution, the percentage of the first gradient elution buffer is 100%, and at the end of gradient elution, the percentage of the second gradient elution buffer is 100%.

E239. A method for purifying rAAV vectors by AEX according to any one of E236 to E238, wherein about 15 to about 25CV (e.g., 20CV) of a first gradient elution buffer, a second gradient elution buffer, or a mixture of the two are applied to the stationary phase during gradient elution; wherein, during gradient elution, the concentration of sodium acetate varies between 0 mM and 500 mM, whereby the rate of change of the concentration of sodium acetate during the gradient elution is approximately 25 mM/CV.

E240. A method for purifying rAAV vectors by AEX according to any one of E231 to E239, wherein the complete capsids are eluted from the stationary phase in the first elution peak; wherein the complete capsids are eluted from the stationary phase in the first part of the second elution peak and/or wherein the empty capsids are recovered in the AEX column flow and/or the last part of the second elution peak.

E241. A method for purifying a rAAV vector by AEX according to any one of E231 to E240, wherein the absorbance threshold measured at 280 nm is ≥ 0.5 mAU/mm path length.

E242. The method for purifying a rAAV vector by AEX according to any one of E231 to E241, wherein the volume of the at least one eluate fraction is equal to ≥ 1/3 CV.

E243. The method for purifying a rAAV vector by AEX according to any one of E231 to E242, wherein collecting at least one eluate fraction comprises collecting at least 10 eluate fractions.

E244. A method for purifying a rAAV vector by AEX according to any one of E231 to E242, wherein the pH of the at least one eluate fraction is adjusted to a pH of 6.8 to 7.6 (eg, about pH 7.2).

E245. A method for purifying a rAAV vector by AEX according to any one of E231 to E244, wherein the A260/A280 ratio of the at least one eluate fraction, the at least two eluate fractions and/or the combined eluate is ≥1.25 (e.g., about 1.28 to 1.35).

E246. A method for purifying a rAAV vector by AEX according to any one of E231 to E245, wherein the combined eluate has a % VG column yield of 20% to 100% (e.g. 63+/-26%).

E247. A method for purifying a rAAV vector by AEX according to any one of E231 to E246, wherein the combined eluate has a % VG step yield of 31% to 66% (e.g., 47+/-11%).

E248. A method for purifying a rAAV vector by AEX according to any one of E231 to E247, wherein at least one eluate fraction and/or the combined eluate is enriched for complete capsids and/or depleted for empty capsids compared to the diluted and filtered affinity eluate loaded on the column.

E249. A method for purifying a rAAV vector by AEX according to any one of E231 to E248, wherein a purified rAAV vector is produced.

E250. The method for purifying rAAV vector by AEX according to E231 to E249, further comprising filtering the combined eluate by a method selected from virus filtration, ultrafiltration/diafiltration (UF/DF), filtration through a 0.2 μm filter, and a combination thereof, to produce a drug substance.

E251. A method for purifying rAAV vectors by AEX according to E250, wherein the drug substance comprises complete capsids of 45% to 65% (e.g., 52+/-7%) of the total capsids.

E252. A method for purifying a rAAV vector by AEX according to E250 or E251, wherein the drug substance comprises 19% to 37% (e.g., 28+/-5%) of the intermediate capsids of the total capsids.

E253. A method for purifying rAAV vectors by AEX according to E250 to E252, wherein the drug substance comprises 10% to 37% (e.g., 20+/-7%) empty capsids of the total capsids.

E254. A method for purifying a rAAV vector by AEX according to any one of E231 to E253, wherein the rAAV vector comprises an AAV9 capsid protein.

E255. A method for purifying an rAAV vector by AEX, the method comprising the following steps:

i) loading the affinity eluate containing the rAAV vector to be purified onto an AEX stationary phase (e.g. POROS ^™ 50HQ) in a column, wherein the eluate has been:

a) diluting about 14.4 to 15.5 times (e.g., about 15 times) with a buffer comprising about 200 mM histidine, about 200 mM Tris, about 0.5% P188, pH 8.7 to 9.0, and optionally

b) filtered through a 0.2 μm filter before loading onto the stationary phase;

ii) subjecting the material of the stationary phase in the column to a gradient elution, wherein about 20 CV of a first gradient elution buffer (e.g., 100 mM Tris, 0.01% P188, pH 8.9), a second gradient elution buffer (e.g., 500 mM sodium acetate, 100 mM Tris, 0.01% P188, pH 8.9), or a mixture of the two are applied to the stationary phase; wherein the concentration of sodium acetate varies between 0 mM and 500 mM, such that the rate of change of the concentration of sodium acetate during the gradient elution is about 25 mM/CV;

iii) collecting about 10 eluate fractions from the column during gradient elution when the A280 of the eluate is > 0.5 mAU/mm path length, wherein the volume of at least one eluate fraction is equal to ≥ 1/3 CV;

iv) adjusting the pH of about 10 eluate fractions from the column to 6.8 to 7.6 by adding 14.3% to 15% (by weight of the eluate volume) of a solution containing about 250 mM sodium citrate (pH 3.5);

v) measuring the absorbance of the about 10 eluate fractions collected from the column and determining the A260/A280 ratio; and/or

vi) combining at least two eluate fractions collected from said column to form a combined eluate,

wherein the A260/A280 of each of the at least two eluate fractions is ≥ 1.25;

wherein the combined eluate is enriched for complete capsids and/or depleted for empty capsids compared to the diluted and optionally filtered affinity eluate loaded onto the column; and

Wherein purified rAAV vectors are produced.

E256. A method for purifying a rAAV vector by AEX according to E255, wherein the material eluted from the stationary phase comprises the rAAV vector to be purified.

E257. A method for purifying an rAAV vector by AEX, the method comprising the following steps:

i) flushing before use, comprising applying about 5 CV of water for injection to the AEX stationary phase in the column;

ii) sanitizing, comprising applying about 16 CV of a solution containing about 0.5 M NaOH to the AEX stationary phase in the column, optionally flowing upward;

iii) regeneration, comprising applying about 5 CV of a solution comprising about 2 M NaCl, 100 mM Tris, pH 9 to the AEX stationary phase in the column;

iv) equilibration comprising applying about 5 CV of a solution comprising about 100 mM Tris, pH 9, to the AEX stationary phase in the column;

v) equilibration, comprising applying about 5 CV of equilibration buffer (comprising about 100 mM Tris, 500 mM sodium acetate, 0.01% P188, pH 8.9) to the AEX stationary phase in the column;

vi) equilibration, comprising applying about 5 CV of an equilibration buffer (comprising about 200 mM histidine, 200 mM Tris, 0.5% P188, pH 8.8) to the AEX stationary phase in the column;

vii) loading the affinity eluate containing the rAAV vector to be purified onto the AEX stationary phase of the column, optionally wherein the eluate has been:

a) diluting about 15-fold with a buffer containing about 200 mM histidine, 200 mM Tris, 0.5% P188, pH 8.7 to 9.0, and optionally

b) Filter through a 0.2 μm filter before loading on the stationary phase;

viii) equilibration, comprising applying about 5 CV of equilibration buffer (comprising about 100 mM Tris, 0.01% P188, pH 8.9) to the AEX stationary phase in the column;

ix) performing a gradient elution of material from the stationary phase in the column, wherein about 20 CV of a first gradient elution buffer (e.g., 100 mM Tris, 0.01% P188, pH 8.9), a second gradient elution buffer (e.g., 500 mM sodium acetate, 100 mM Tris, 0.01% P188, pH 8.9), or a mixture of both, is applied to the stationary phase; wherein the concentration of sodium acetate varies from 0 mM to 500 mM, whereby the rate of change of the concentration of sodium acetate during the gradient elution is about 25 mM/CV;

x) collecting about 10 eluate fractions from the column during gradient elution when the A280 of the eluate is ≥ 0.5 mAU/mm path length; and wherein the volume of the about 10 eluate fractions is about ≥ 1/3 CV;

xi) adjusting the pH of the approximately 10 eluate fractions to 6.8 to 7.6 by adding 14.3% to 15% (by weight of the eluate volume) of a solution comprising approximately 250 mM sodium citrate (pH 3.5);

xii) measuring the absorbance of the about 10 eluate fractions collected from the column and determining the A260/A280 ratio; and/or

xiii) combining at least two eluate fractions collected from said column to form a combined eluate,

wherein the A260/A280 of each of the at least two eluate fractions is ≥ 1.25;

wherein the combined eluate is depleted of empty capsids and/or enriched for complete capsids compared to the affinity eluate and/or the diluted and optionally filtered affinity eluate;

wherein at least one of steps i) to ix) is performed at a linear velocity of 270 to 330 cm/hr (e.g., about 300 cm/hr), a flow rate of 1.5 to 2.0 L/min (e.g., about 1.8 L/min) or about 314 mL/min or through a 1.3 L column and/or a residence time of about 3.5 to 4.5 min/CV (e.g., 4 min/CV); and/or wherein a purified rAAV vector is produced.

E258. A method for purifying a rAAV vector by AEX according to E257, wherein the material eluted from the stationary phase comprises the rAAV vector to be purified.

E259. A method for preparing a solution containing a rAAV vector purified by AEX, the method comprising the following steps:

i) diluting the affinity eluate approximately 15-fold with a diluent solution containing approximately 200 mM histidine, 200 mM Tris, 0.5% P188, pH 8.8; and

ii) filtering the affinity eluate in step i) through a 0.2 μm filter to produce a diluted and filtered affinity eluate; wherein the pH of the diluted and filtered affinity eluate is increased (e.g., to about 8.5 to 9.5) compared to the pH of the affinity eluate; and wherein the conductivity of the diluted and filtered affinity eluate is decreased (e.g., to about 1.7 mS/cm to 3.3 mS/cm) compared to the conductivity of the affinity eluate.

E260. A method for preparing a solution comprising a rAAV vector purified by AEX according to E259, wherein the diluted and optionally filtered affinity eluate is loaded onto an AEX stationary phase.

E261. A method for preparing a solution comprising an rAAV vector purified by AEX according to E259 or E260, wherein the affinity eluate is produced by affinity chromatography-based purification of an rAAV vector produced in a container having a volume of 250 L or 2000 L.

E262. A method for preparing a solution comprising rAAV vectors purified by AEX according to any one of E259 to E261, wherein the % VG dilution yield of the affinity eluate after dilution is 88% +/- 36%.

E263. A method for preparing a stationary phase for use in a method for purifying an rAAV vector by AEX, the method comprising the following steps:

i) flushing before use, comprising applying ≥ 4.5 CV (e.g., about 5 CV) of water for injection to the AEX stationary phase in the column;

ii) sanitizing, comprising applying about 14.4 to 17.6 CV (e.g. about 16 CV) of a solution containing about 0.1 M to 1.0 M (e.g. about 0.5 M) NaOH to the AEX stationary phase in the column, optionally by upward flow; and/or

iii) regeneration, comprising applying about 4.5 to 5.5CV (e.g., about 5CV) of a solution containing about 1M to 3M NaCl, 50mM to 150mM Tris, pH 8.5 to 9.5 to the AEX stationary phase in the column; optionally, wherein at least one of steps i) to iii) is performed at a linear velocity of 270 to 330cm/hr (e.g., about 300cm/hr), a flow rate of 1.5 to 2.0L/min (e.g., about 1.8L/min), and/or a residence time of 3.5 to 4.5min/CV (e.g., about 4min/CV).

E264. A method for preparing a stationary phase for use in a method for purifying rAAV vectors by AEX according to E263, further comprising equilibration, comprising applying about 5CV of one or more solutions to the AEX stationary phase in the column, the solutions comprising i) about 100mM Tris, pH 9, ii) about 100mM Tris, 500mM sodium acetate, 0.01% P188, pH 8.9 and iii) about 200mM histidine, 200mM Tris, 0.5% P188, pH 8.8.

E265. A method for preparing a stationary phase for use in a method for purifying rAAV vectors by AEX according to E263 or E264, wherein at least one step is performed before loading the solution comprising the rAAV vector to be purified onto the column.

E266. A purified rAAV vector prepared according to the method of any one of E1 to E227 or E231 to E258.

E267. A purified rAAV vector prepared by a method comprising the following steps:

i) loading a solution containing the rAAV vector to be purified onto the AEX stationary phase in the column;

ii) gradient elution of the material of the stationary phase in the column, wherein about 20CV of a first gradient elution buffer (e.g., 100 mM Tris, 0.01% P188, pH 8.9), a second gradient elution buffer (e.g., 500 mM sodium acetate, 100 mM Tris, 0.01% P188, pH 8.9), or a mixture of the two are applied to the stationary phase; wherein the concentration of salt varies between 0 mM and 500 mM, whereby the rate of change of the concentration of the salt during the gradient elution is about 25 mM/CV;

iii) collecting at least one (e.g., about 10) eluate fractions from the column during a chromatography step (e.g., gradient elution) when the absorbance of the column flow-through reaches an absorbance threshold (e.g., A280>0.5 mAU/mm path length);

iv) measuring the absorbance of said at least one eluate fraction collected from said column and determining the A260/A280 ratio; and/or

v) combining at least two eluate fractions collected from said column to form a combined eluate comprising the rAAV vector to be purified.

E268. A purified rAAV vector prepared according to the method of E267, wherein the material eluted from the stationary phase comprises the rAAV vector to be purified.

E269. A purified rAAV vector prepared according to the method of E267 to E268, wherein the salt is sodium acetate.

E270. A purified rAAV vector prepared according to the method of any one of E267 to E269, wherein the solution is an affinity eluate which has been a) diluted about 14.4 to 15.5 times (e.g., about 15 times) with a buffer comprising about 200 mM histidine, 200 mM Tris, 0.5% P188, pH 8.7 to 9.0, and optionally b) filtered through a 0.2 μm filter before loading on the stationary phase.

E271. A purified rAAV vector prepared according to any one of the methods E267 to E270, the method further comprising adjusting the pH of at least one (e.g., about 10) eluate fractions from the column to 6.8 to 7.6 by adding 14.3% to 15% (eluate volume weight) of a solution containing about 250 mM sodium citrate (pH 3.5).

E272. A purified rAAV vector according to any one of E267 to E271, wherein the A260/A280 of each of the at least two eluate fractions is ≥ 1.25; and wherein the combined eluate is enriched for complete capsids and/or depleted for empty capsids compared to the diluted and optionally filtered affinity eluate loaded on the column; and wherein a purified rAAV vector is produced.

E273. A purified rAAV vector according to any one of E267 to E272, wherein the rAAV vector comprises AAV9 capsid protein.

E274. A purified rAAV vector prepared by a method comprising the following steps:

i) loading a solution containing the rAAV vector to be purified onto the AEX stationary phase in the column;

ii) performing a gradient elution of material from the stationary phase in the column, wherein the percentage of a first gradient elution buffer is varied inversely proportional to the percentage of a second gradient elution buffer;

iii) during the chromatography step, when the absorbance of the column flow-through reaches a threshold absorbance value, starting to collect at least one eluate fraction from said column;

iv) measuring the absorbance of said at least one eluate fraction collected from said column and determining the A260/A280 ratio; and/or

v) combining at least two eluate fractions collected from said column to form a combined eluate comprising the rAAV vector to be purified.

E275. A purified rAAV vector prepared according to the method of E274, wherein the material eluted from the stationary phase contains the rAAV vector to be purified.

E276. A purified rAAV vector prepared according to the method of E274 or E275, wherein the rAAV vector comprises AAV9 capsid protein.

E277. A purified rAAV vector prepared according to any one of the methods of E274 to E276, wherein the method further comprises filtering the combined eluate by a method selected from virus filtration, ultrafiltration/diafiltration (UF/DF), filtration through a 0.2 μm filter, and a combination thereof to produce a drug substance.

E278. A purified rAAV vector prepared according to the method of E277, wherein the drug substance is used to prepare a drug product suitable for administration to a human subject to treat a disease, disorder or condition.

E279. A purified rAAV vector prepared according to the method of E278, wherein the disease, disorder or condition is DMD.

E280. A solution comprising a rAAV vector purified by AEX, the solution being prepared by a method comprising the steps of:

i) diluting the first solution (eg affinity eluate) 2 to 25 times (eg about 15 times) with a buffer comprising 200 mM histidine, 200 mM Tris, 0.5% P188, pH 8.8; and optionally

ii) filtering the first solution in step i) through a 0.2 μm filter to produce a diluted and optionally filtered solution; wherein the pH of the diluted and optionally filtered solution is increased compared to the pH of the first solution; and wherein the conductivity of the diluted and optionally filtered solution is reduced compared to the conductivity of the first solution.

E281. A solution comprising a rAAV vector purified by AEX according to E280, wherein the first solution is an affinity eluate.

E282. A solution comprising a rAAV vector purified by AEX according to E281, wherein the affinity eluate is produced by affinity purification of the rAAV vector produced in a vessel having a volume of 250 L or 2000 L (eg, a disposable bioreactor).

E283. A solution comprising a rAAV vector purified by AEX according to any one of E280 to E282, wherein the pH of the diluted and optionally filtered solution is from 8.5 to 9.5.

E284. A solution comprising a rAAV vector purified by AEX according to any one of E280 to E283, wherein the conductivity of the diluted and optionally filtered solution is 1.7 to 3.3 mS/cm.

E285. A solution comprising a rAAV vector purified by AEX according to any one of E280 to E284, wherein the % VG dilution yield of the diluted and optionally filtered solution is 88% +/- 36%.

E286. A solution comprising a rAAV vector purified by AEX, the solution being prepared by a method comprising the steps of:

i) diluting the first solution (eg affinity eluate) by 2 to 25 times (eg 15 times) with a dilution solution comprising histidine, Tris and P188; and optionally

ii) filtering the diluted first solution of step i) through a 0.2 μm filter to produce a diluted and optionally filtered solution; wherein the pH of the diluted and optionally filtered solution is increased compared to the pH of the first solution; and wherein the conductivity of the diluted and optionally filtered solution is reduced compared to the conductivity of the first solution.

E287. A solution comprising an rAAV vector purified by AEX prepared according to the method of E280, wherein the dilution solution comprises about 100mM to 300mM (e.g., about 200mM) histidine, 100mM to 300mM (about 200mM) Tris, 0.1% to 1.0% (about 0.5%) P188, pH 8.5 to 9.5.

E288. A solution comprising an rAAV vector purified by AEX prepared according to the method of E280 or E281, wherein the pH of the first solution is 3.0 to 4.4 before dilution and optional filtration, and the pH of the first solution is 8.5 to 9.5 or 8.7 to 9.0 (e.g., 8.8, 9.0) after dilution and optional filtration.

E289. A solution comprising an rAAV vector purified by AEX prepared according to any one of the methods of E280 to E282, wherein the conductivity of the first solution before the dilution and optional filtration steps is 5.0 to 7.0 mS/cm (e.g., 5.5 to 6.5 mS/cm), and after the dilution and optional filtration steps, the conductivity of the first solution is 1.7 to 3.5 mS/cm, 1.8 to 2.8 mS/cm or 2.2 to 2.6 mS/cm.

E290. A solution comprising a rAAV vector purified by AEX prepared according to the method of any one of E280 to E283, wherein the % VG dilution yield of the diluted and optionally filtered first solution is 35% to 100%.

E291. A solution comprising an rAAV vector purified by AEX prepared according to the method of any one of E280 to E28, wherein the rAAV vector comprises AAV9 or AAV3B capsid protein, and optionally, wherein the diluted and optionally filtered solution is loaded onto an AEX stationary phase.

E292. A method for regenerating an AEX stationary phase, the method comprising the following steps:

i) post-use sterilization of a stationary phase comprising applying to said stationary phase 14.4 to 17.6 CV (e.g. about 16 CV) of a solution comprising about 0.1 M to 1.0 M (e.g. about 0.5 M) NaOH, optionally flowing upwards;

ii) regenerating the stationary phase comprising applying 4.5 to 5.5 CV (e.g., about 5 CV) of a solution comprising about 1 M to 3 M (e.g., about 2 M) NaCl, 50 mM to 150 mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., about pH 9) to the stationary phase;

iii) equilibrating the stationary phase, comprising applying to the stationary phase 4.5 to 5.5 CV (e.g., about 5 CV) of a solution comprising about 50 mM to 150 mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., about pH 9);

iv) flushing the stationary phase after use, comprising applying ≥ 4.5 CV (e.g. about 5 CV) of water for injection to the stationary phase; and/or

v) applying a stock solution to the stationary phase comprises applying 2.7 to 3.3 CV (e.g. about 3 CV) of a solution comprising about 17.5% ethanol to the stationary phase, optionally wherein at least one of steps i) to v) is performed at a linear velocity of 270 to 330 cm/hr (e.g. about 300 cm/hr), a flow rate through a 6.0 to 6.6 L (e.g. 6.4 L) column of 1.5 to 2.0 L/min (e.g. about 1.8 L/min) or a flow rate through a 1.3 L column of about 314 mL/min and/or a residence time of 3.5 to 4.5 min/CV (e.g. about 4 min/CV).

E293. A method for regenerating an AEX stationary phase according to E292, wherein any one of steps i) to v) follows a chromatographic elution step of a method for purifying a rAAV vector by AEX.

E294. A regenerated AEX stationary phase prepared by a method comprising the steps of:

i) post-use sterilization of a stationary phase comprising applying to said stationary phase 14.4 to 17.6 CV (e.g. about 16 CV) of a solution containing about 0.1 M to 1.0 M (e.g. about 0.5 M) NaOH, optionally by upward flow;

ii) regenerating the stationary phase, comprising applying 4.5 to 5.5 CV (e.g., about 5 CV) of a solution comprising about 1 M to 3 M (e.g., about 2 M) NaCl, about 50 mM to 150 mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., about pH 9) to the stationary phase;

iii) equilibrating the stationary phase, comprising applying to the stationary phase 4.5 to 5.5 CV (e.g., about 5 CV) of a solution comprising about 50 mM to 150 mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., pH 9);

iv) flushing the stationary phase after use, comprising applying ≥ 4.5 (e.g. about 5 CV) of water for injection to the stationary phase; and/or

v) applying a storage solution to the stationary phase, comprising applying 2.7 to 3.3 CV (e.g., about 3 CV) of a solution containing about 17% to 17.5% ethanol to the stationary phase; optionally

Wherein, at least one of steps i) to v) is carried out at a linear velocity of 270 to 330 cm/hr (e.g., about 300 cm/hr), at a flow rate of 1.5 to 2.0 L/min (e.g., about 1.8 L/min) through a 6.0 to 6.6 L (e.g., 6.4 L) column, or about 314 mL/min through a 1.3 L column and/or a residence time of 3.5 to 4.5 min/CV (e.g., about 4 min/CV).

E295. The regenerated AEX stationary phase according to E288, wherein the regenerated AEX stationary phase is used for purifying rAAV vectors.

E296. A pharmaceutical composition comprising a purified rAAV vector prepared according to the method of any one of E1 to E227 or E231 to E258.

E297. A pharmaceutical composition comprising the purified rAAV vector according to E267 to E279.

E298. Use of a rAAV vector purified according to any one of the methods of E1 to E227 or E231 to E258 in the production of a medicament for treating and/or preventing a disease, disorder or condition.

E299. The use according to E298, wherein the disease, disorder or condition is DMD.

E300. The method of any one of E1 to E182, wherein the rAAV vector comprises a vector genome comprising a modified nucleic acid encoding a deleted copper-transporting ATPase2 (ATP7B) protein.

E301. The method according to E300, wherein the modified nucleic acid comprises a nucleic acid sequence encoding a deleted copper-transporting ATPase2 (ATP7B) protein, and the protein comprises or consists of the amino acid sequence of SEQ ID NO:15.

E302. The method according to E300 or E301, wherein the deleted copper transport APTase2 comprises the deletion of heavy metal-related sites HMA 1, HMA 2, HMA 3 and HMA 4.

E303. A method according to any one of E300 to E302, wherein the vector genome further comprises an α1-antitrypsin promoter, a polyadenylation (polyA) signal sequence, a 5'ITR and a 3'ITR.

E304. The method according to E303, wherein the α1-antitrypsin promoter comprises or consists of the nucleic acid sequence of SEQ ID NO:16.

E305. The method according to E303, wherein the polyA signal sequence comprises or consists of the nucleic acid sequence of SEQ ID NO:17.

E306. The method according to E303, wherein the 5'ITR and 3'ITR are AAV2 serotype ITRs.

E307. The method according to any one of E1 to E182, wherein the rAAV vector comprises the VP1 polypeptide of AAV3B.

E308. The method according to E307, wherein the VP1 polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:18.

E309. A method for purifying an rAAV vector by AEX, the method comprising the following steps:

i) loading a solution containing the rAAV vector to be purified onto the AEX stationary phase in the column;

ii) performing a gradient elution of the material from the stationary phase in the column, wherein the percentage of a first gradient elution buffer varies inversely proportional to the change in the percentage of a second gradient elution buffer; wherein at the beginning of the gradient elution, the percentage of the first gradient elution buffer is about 75% to about 100%, and at the end of the gradient elution, the percentage of the second gradient elution buffer is about 60% to about 100%; and wherein during the gradient elution, the percentage of the second elution buffer increases at a rate of about 2% to 5% per CV;

iii) collecting at least one eluate fraction from the column when the gradient elution begins when the percentage of the second gradient elution buffer is about 30% to about 35%,

And wherein the at least one eluate fraction contains the rAAV vector to be purified.

E310. The method for purifying rAAV vector by AEX according to E309, wherein the solution containing the rAAV vector is an affinity eluate which has been diluted about 15 times with a buffer containing histidine, Tris and P188.

E311. A method for purifying rAAV vectors by AEX according to E309 or E310, wherein the first gradient elution buffer comprises 50mM to 150mM (e.g., about 100mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., 8.9) and/or the second gradient elution buffer comprises 400mM to 600mM (e.g., about 500mM) sodium acetate, 50mM to 150mM (e.g., about 100mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., pH8.9).

E312. A method for purifying an rAAV vector by AEX according to any one of E309 to E311, wherein collecting at least one eluate fraction from the column comprises collecting the at least one eluate fraction into a container containing about 0.01CV to 0.1CV (e.g., about 0.066CV) of a solution containing 200mM to 300mM (e.g., about 250mM) sodium citrate, pH 3.0 to 4.0 (e.g., about 3.5).

E313. A method for purifying a rAAV vector by AEX according to any one of E310 to E312, wherein the at least one eluate fraction is enriched for complete capsids and/or depleted for empty capsids compared to the affinity eluate; optionally, wherein the rAAV vector is a rAAV3B vector; and optionally, wherein the AEX stationary phase is POROS ^™ 50HQ.

E314. A method for purifying a rAAV vector by AEX according to any one of E309 to E313, wherein the collection of at least one eluate fraction from the column when performing gradient elution is terminated when the percentage of the second gradient elution buffer is about 50% to about 55%.

E315. A method for purifying an rAAV vector by AEX, the method comprising the following steps:

i) loading a solution containing the rAAV vector to be purified onto the AEX stationary phase in the column;

ii) performing a gradient elution of material from the stationary phase in the column, wherein the percentage of a first gradient elution buffer is varied inversely proportional to the change in the percentage of a second gradient elution buffer; and

iii) starting to collect at least one eluate fraction from the column during gradient elution when the absorbance of the column flow-through reaches an absorbance threshold, and wherein the at least one eluate fraction comprises the rAAV vector to be purified.

E316. A method for purifying a rAAV vector by AEX according to E315, wherein the method further comprises measuring the absorbance of the at least one eluate fraction collected from the column and determining the A260/A280 ratio.

E317. A method for purifying rAAV vectors by AEX according to E315 or E316, wherein a solution containing the rAAV vector to be purified is diluted approximately 2 to 25 times (e.g., 15 times) with a dilution solution containing histidine, Tris and P188, and is optionally filtered before being applied to the stationary phase.

E318. A method for purifying rAAV vectors by AEX according to any one of E315 to E317, wherein the solution is an affinity elution solution.

E319. A method for purifying rAAV vectors by AEX according to E315 to E318, wherein the pH of the diluted and optionally filtered affinity eluate is increased compared to the pH of the solution; and wherein the conductivity of the diluted and optionally filtered affinity eluate is decreased compared to the conductivity of the solution.

E320. A method for purifying rAAV vectors by AEX according to any one of E315 to E319, wherein the first gradient elution buffer comprises about 50 mM to about 150 mM Tris, about 0.005% to about 0.015% P188 and a pH of about 8.5 to 9.5; wherein the second gradient elution buffer comprises about 400 mM to about 600 mM sodium acetate, about 50 mM to about 150 mM Tris, about 0.005% to about 0.015% P188 and a pH of about 8.5 to 9.5; and wherein 10 to 60 column volumes (CV) (e.g., about 20 CV, about 37.5 CV) of the first gradient elution buffer, the second gradient elution buffer, or a mixture of the two are applied to the stationary phase during gradient elution.

E321. A method for purifying an rAAV vector by AEX according to any one of E315 to E320, wherein at the beginning of gradient elution, the percentage of the first gradient elution buffer is 50% to 100%, at the end of gradient elution, the percentage of the second gradient elution buffer is 50% to 100%, and wherein the percentage of the second elution buffer increases at a rate of about 2% to 5% per CV during gradient elution.

E322. A method for purifying rAAV vectors by AEX according to any one of E315 to E321, wherein the sodium acetate concentration of the first gradient elution buffer, the second gradient elution buffer, or a mixture of the two continues to increase during gradient elution; and wherein the concentration of sodium acetate increases at a rate of about 10 mM/CV to 50 mM/CV (e.g., about 10 mM/CV, about 25 mM/CV) during the gradient elution.

E323. A method for purifying rAAV vectors by AEX according to any one of E315 to E322, wherein during gradient elution, the complete capsids are eluted from the stationary phase in the first elution peak and/or the first part of the second elution peak.

E324. A method for purifying a rAAV vector by AEX according to any one of E315 to E323, wherein empty capsids are recovered during gradient elution in the column flow-through, in the first elution peak and/or in the last part of the second elution peak.

E325. A method for purifying a rAAV vector by AEX according to any one of E315 to E324, wherein the absorbance of at least one eluate fraction is measured at 280 nm, and wherein, optionally, the absorbance threshold measured at 280 nm is ≥ 0.5 mAU/mm path length.

E326. A method for purifying an rAAV vector by AEX according to any one of E315 to E325, wherein the volume of the at least one eluate fraction is equal to 1/8CV to 10CV, for example, 1/8CV, 1/4CV, 1/3CV, 1/2CV, 3/4CV, 1CV, 2CV, 3CV, 4CV, 5CV, 6CV, 7CV, 8CV, 9CV, 10CV or more CV, and optionally wherein the A260/A280 ratio of the at least one eluate fraction is ≥1.25.

E327. A method for purifying a rAAV vector by AEX according to any one of E315 to E326, wherein at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more eluate fractions are collected.

E328. A method for purifying a rAAV vector by AEX according to any one of E315 to E327, wherein the method further comprises combining at least two eluate fractions collected from the column, each fraction having an A260/A280 ratio ≥0.98 or ≥1.0, to form a combined eluate comprising the rAAV vector.

E329. A method for purifying rAAV vectors by AEX according to E328, wherein the combined eluate has a %VG column yield of 20% to 100% (e.g., 63+/-26%), a %VG step yield of 31% to 66% (e.g., 47+/-11%) and/or an A260/A280 ratio ≥1.0.

E330. A method for purifying rAAV vectors by AEX according to E328 or E329, wherein the combined eluate is enriched for complete capsids and/or depleted for empty capsids compared to the solution loaded on the column.

E331. A method for purifying a rAAV vector by AEX according to any one of E315 to E330, wherein a purified rAAV vector is produced.

E332. The method for purifying rAAV vector by AEX according to any one of E328 to E331, further comprising filtering the combined eluate by a method selected from virus filtration, ultrafiltration/diafiltration (UF/DF), filtration through a 0.2 μm filter, and a combination thereof, to produce a drug substance.

E333. A method for purifying rAAV vectors by AEX according to E332, wherein the drug substance comprises: i) 45% to 65% (e.g., 52+/-7%) complete capsids of the total capsids; ii) 19% to 37% (e.g., 28+/-5%) intermediate capsids of the total capsids; and/or iii) 10% to 37% (e.g., 20+/-7%) empty capsids of the total capsids.

E334. A method for purifying rAAV vectors by AEX according to any one of E332 to E333, wherein the drug substance is enriched in complete capsids and/or depleted in empty capsids compared to the solution loaded on the column.

E335. A method for purifying a rAAV vector by AEX according to any one of E315 to E334, wherein the rAAV vector comprises an AAV capsid protein from an AAV serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAVrh74, AAV12, AAV2i8, NP4, NP22, NP66, AAVDJ, AAVDJ/8, AAVDJ/9, AAVLK03, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, RHM4-1 (SEQ ID NO: WO 2015/013313) NO:5), RHM15-1, RHM15-2, RHM15-3/RHM15-5, RHM15-4, RHM15-6, AAV hu.26, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9,45, AAV2i8, AAV29G, AAV2,8G9, AVV-LK03, AAV2-TT, AAV 2-TT-S312N, AAV3B-S312N, AAVHSC1, AAVHSC2, AAVHSC3, AAVHSC4, AAVHSC5, AAVHSC6, AAVHSC7, AAVHSC8, AAVHSC9, AAVHSC10, AAVHSC11, AAVHSC12, AAVHSC13, AAVHSC14 and AAVHSC15.

E336. A method for purifying a rAAV vector by AEX according to any one of E315 to E335, wherein the rAAV vector comprises an AAV9 capsid protein and a transgene comprising the nucleic acid of SEQ ID NO: 1.

E337. A method for purifying a rAAV vector by AEX according to any one of E315 to E336, wherein the rAAV vector comprises an AAV3B capsid protein and a transgene comprising a nucleic acid encoding the amino acid sequence of SEQ ID NO: 15.

E338. A method for preparing a solution containing a rAAV vector purified by AEX, the method comprising the following steps:

i) diluting the first solution 2 to 25 times (eg 15 times) with a dilution solution comprising histidine, Tris and P188; and optionally

ii) filtering the first solution in step i) through a filter to produce a diluted and optionally filtered solution;

wherein the pH of the diluted and optionally filtered solution is increased compared to the pH of the first solution; and wherein the conductivity of the diluted and optionally filtered solution is decreased compared to the conductivity of the first solution.

E339. A method for preparing a solution comprising rAAV vectors purified by AEX according to E338, wherein the first solution comprising rAAV vectors is selected from the group consisting of affinity eluate, supernatant of cell lysate and post-harvest solution.

E340. A method for preparing a solution comprising an rAAV vector purified by AEX according to E338 or E339, wherein the diluted solution comprises about 100mM to 300mM (e.g., about 200mM) histidine, 100mM to 300mM (e.g., about 200mM) Tris, 0.1% to 1.0% (e.g., about 0.5%) P188, and a pH of 8.5 to 9.5.

E341. A method for preparing a solution comprising an rAAV vector purified by AEX according to any one of E338 to E340, wherein i) the pH of the diluted and optionally filtered solution is 8.5 to 9.5; ii) the conductivity of the diluted and optionally filtered solution is 1.7 to 3.3 mS/cm; and/or iii) the %VG dilution yield of the diluted solution is 35% to 100%.

E342. A method for preparing a solution comprising an rAAV vector purified by AEX according to any one of E338 to E341, wherein the rAAV vector comprises an AAV9 capsid protein or an AAV3B capsid protein.

E343. A purified rAAV vector prepared by a method comprising the following steps:

i) loading a solution containing the rAAV vector to be purified onto the AEX stationary phase in the column;

ii) subjecting the stationary phase in the column to a gradient elution of material, wherein a first gradient elution buffer, a second gradient elution buffer, or a mixture of both, is applied to the stationary phase and the concentration of the salt is varied from 0 mM to 500 mM, whereby the rate of increase in the concentration of the salt during the gradient elution is about 10 mM/CV to 50 mM/CV (e.g., about 25 mM/CV);

iii) commencing collection of at least one eluate fraction from said column during gradient elution when the absorbance of the column flow-through reaches a threshold absorbance value; and/or

vi) measuring the absorbance of said at least one eluate fraction collected from said column and determining the A260/A280 ratio.

E344. A purified rAAV vector prepared according to the method of E343, wherein the method further comprises combining at least two eluate fractions collected from the column when the A260/A280 ratio is ≥1.0 to form a combined eluate comprising the purified rAAV vector.

E345. A purified rAAV vector prepared according to the method of E343 or E344, wherein the salt is sodium acetate.

E346. A purified rAAV vector prepared according to the method of any one of E343 to E345, wherein the rAAV vector comprises AAV9 capsid protein or AAV3B capsid protein.

E347. A purified rAAV vector prepared according to the method of any one of E343 to E346, wherein the solution comprising the rAAV vector is an affinity eluate, which has been diluted and optionally filtered before being loaded onto the stationary phase.

E348. A purified rAAV vector prepared according to the method of any one of E343 to E347, wherein the material eluted from the stationary phase comprises the rAAV vector.

E349. A purified rAAV vector prepared according to the method of any one of E344 to E348, wherein the method further comprises filtering the combined eluate by a method selected from virus filtration, ultrafiltration/diafiltration (UF/DF), filtration through a 0.2 μm filter, and a combination thereof to produce a drug substance.

E350. A purified rAAV vector prepared according to the method of E349, wherein the drug substance is used to prepare a drug product suitable for administration to a human subject to treat a disease, disorder or condition.

E351. A purified rAAV vector prepared according to the method of E350, wherein the disease, disorder or condition is DMD or Wilson's disease, and optionally wherein the rAAV vector comprises a nucleic acid encoding the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:15.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts the elution phase of an exemplary AEX chromatogram produced on a 1 mL POROS ^™ 50HQ column using four elution salts. The _A260 trace is represented by a dashed line, while the _A280 and conductivity traces are represented by solid lines. The solid bars represent the elution fractions used to form the pool.

FIG. 2 depicts SEC A ₂₆₀ /A ₂₈₀ of exemplary AEX elution fractions produced on a 1 mL POROS ^™ 50HQ column using four elution salts.

FIG3 depicts an exemplary AEX chromatogram produced using 9 wash and elution steps of sodium acetate on a 5.1 mL POROS ^™ 50HQ column. The _A260 trace is represented by a dashed line, while the _A280 and conductivity traces are represented by solid lines. Wash (W), elution (E), strip, and regeneration (Regen.) fractions are represented in accordance with Tables 5 and 6.

FIG4A depicts an exemplary AEX chromatogram generated using sodium acetate step elution, the elution run was eluted at 600 cm/hr, 5.1×10 ¹³ vector genomes/mL resin excitation (VG/mL resin, measured by qPCR of ITR sequences), and a 57 mM sodium acetate wash on a 5.1 mL POROS ^™ 50HQ column. The A ₂₆₀ trace is represented by a dashed line, while the A ₂₈₀ and conductivity traces are represented by solid lines. FIG4B depicts an enlarged view of the chromatogram of the wash, elution, and strip phases of the AEX run.

Figure 5 depicts an exemplary method of in-line mixing of AAV9 affinity eluate with 100 mM Tris, pH 9 to produce an AEX load (also referred to herein as diluted affinity eluate). Fluid was delivered to a Y-connector with a peristaltic pump.

FIG6 depicts pH, conductivity, Z-average, and aggregation (given as + or -) of exemplary AAV9 affinity eluates diluted with 100 mM Tris, pH 9.

Figures 7A and 7B depict exemplary % vector genome (VG) yields of affinity eluates diluted 5, 9, or 25 times with 200 mM histidine, 200 mM Tris, X% (w/v) P188, pH 8.8, where X is 0.01%, 0.05%, 0.2%, and 0.5%, and then filtered. Figure 7A depicts a contour plot of VG percent yield (after dilution and filtration) as a function of conductivity (controlled by the dilution factor) and P188 concentration. Figure 7B depicts a one-way ANOVA analysis of VG percent yield (after dilution and filtration) as a function of P188 concentration or conductivity. The data are also listed in Table 13.

Figure 8A depicts an example chromatogram generated using the optimized AEX method. Figure 8B depicts an enlarged representation of the AEX sodium acetate gradient elution, with fractions numbered 1-14, consistent with Table 15. The _A260 trace is shown as a dashed line, while the _A280 and conductivity traces are shown as solid lines.

Figure 9 depicts exemplary SEC _A260 /A280 values of chromatographic fractions generated on 0%, 20%, 40%, 60%, ₈₀ % and 100% Null affinity pool using the optimized AEX method. Flow-through is abbreviated as F/T.

Figure 10 depicts the elution phase of an exemplary 250L SUB AEX chromatogram of batch 250L-4 run on a 10 cm internal diameter (ID) x 16 cm bed height (BH), 1.3L POROS ^™ 50HQ column. The _A260 trace is represented by a dashed line, while the _A280 and conductivity traces are represented by solid lines.

Figure 11 depicts the elution phase of an exemplary 2000L SUB Scale AEX chromatogram of batch 2000L-4 run on a 20 cm ID x 20.5 cm BH, 6.4L POROS ^™ 50HQ column. The _A260 trace is represented by a dashed line, while the _A280 and conductivity traces are represented by solid lines.

Figure 12 depicts an exemplary chromatogram of an AAV3B vector purified using an optimized AEX method. The _A260 trace is represented by a solid line, while the _A280 trace is represented by a dashed line.

Description of the invention

1. Definition

Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the invention belongs. The terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the invention. As used in the specification of the present invention and the appended claims, the singular forms "a", "an" and "the" shall also include plural forms unless the context clearly indicates otherwise. The following terms have the given meanings:

As used herein, the terms "about" or "approximately" refer to a measurable value, such as the amount of biological activity, the length of a polynucleotide or polypeptide sequence, the content of G and C nucleotides, the codon adaptation index, the number of CpG dinucleotides, dosage, time, temperature, etc., and are meant to encompass variations of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% and even 0.1% in either direction (greater or less than) of the specified amount, unless otherwise indicated, otherwise obvious from the context, or unless the number would exceed 100% of the possible value.

As used herein, the term "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as no combination when interpreted with the alternative ("or").

As used herein, the term "adeno-associated virus" and/or "AAV" refers to a parvovirus having a linear single-stranded DNA genome and variants thereof. The term encompasses all subtypes and naturally occurring and recombinant forms unless otherwise required.

The classical AAV wild-type genome comprises 4681 bases (Berns and Bohenzky (1987) Advances in Virus Research 32: 243-307) and includes terminal repeats (e.g., inverted terminal repeats (ITRs)) at each end, which serve as the origin of DNA replication and viral packaging signals in cis form. The genome includes two large open reading frames, referred to as AAV replication ("AAV rep" or "rep") and capsid ("AAV-cap" or "cap") genes. AAV rep and cap may also be referred to as AAV "packaging genes" herein. These genes encode viral proteins involved in viral genome replication and packaging.

In wild-type AAV, the three capsid genes VP1, VP2 and VP3 overlap each other in a single open reading frame and result in the production of VP1, VPN and VP3 capsid proteins through alternative splicing (Grieger and Samulski (2005) J. Virol. 79 (15): 9933-9944). A single P40 promoter allows all three capsid proteins to be expressed in a ratio of 1:1:10 for VP1, VP2 and VP3, respectively, which complements AAV capsid production. More specifically, VP1 is a full-length protein, and VP2 and VP3 become shorter and shorter due to increasing truncation of the N-terminus. A well-known example is the capsid of AAV9 as described in U.S. Patent No. 7,906,111, in which VP1 comprises amino acid residues 1-736 of SEQ ID NO: 123, VP2 comprises amino acid residues 138-736 of SEQ ID NO: 123, and VP3 comprises amino acid residues 203-736 of SEQ ID NO: 123. As used herein, the term "AAV Cap" or "cap" refers to AAV capsid proteins VP1, VP2 and/or VP3 and variants and analogs thereof. The second open reading frame of the capsid gene encodes an assembly factor, called assembly activation protein (AAP), which is essential for the capsid assembly process (Sonntag et al. (2011) J. Virol. 85(23): 12686-12697).

At least four viral proteins are synthesized by the AAV rep gene: Rep 78, Rep 68, Rep 52 and Rep 40, named according to their apparent molecular weight. As used herein, "AAV rep" or "rep" refers to the AAV replication proteins Rep 78, Rep 68, Rep 52 and/or Rep 40, as well as variants and analogs thereof. As used herein, rep and cap refer to wild-type and recombinant (e.g., modified chimeric, etc.) rep and cap genes and the polypeptides encoded therefrom. In some embodiments, the nucleic acid encoding rep comprises nucleotides from more than one AAV serotype. For example, the nucleic acid encoding the rep protein can comprise nucleotides from the AAV2 serotype and nucleic acids from the AAV3 serotype (Rabinowitz et al. (2002) J. Virology 76 (2): 791-801).

As used herein, the terms "recombinant adeno-associated virus vector", "rAAV" and/or "rAAV vector" refer to an AAV capsid containing a vector genome. The vector genome comprises a polynucleotide sequence that is at least partially not derived from naturally occurring AAV (e.g., a heterologous polynucleotide that is not present in wild-type AAV), and the rep and/or cap genes of the wild-type AAV genome have been removed from the vector genome. When the rep and/or cap genes of AAV are removed (and/or the ITRs of AAV are added or retained), the nucleic acid within AAV is referred to as a "vector genome". Therefore, the term rAAV vector encompasses rAAV virus particles that contain a capsid but do not contain a complete AAV genome; in contrast, recombinant virus particles can contain heterologous nucleic acids, i.e., nucleic acids that are not initially present in the capsid, hereinafter referred to as vector genomes. Therefore, "rAAV vector genome" (or "vector genome") refers to a heterologous polynucleotide sequence (including at least one ITR) that can but need not be contained in an AAV capsid. The rAAV vector genome can be double-stranded (dsAAV), single-stranded (ssAAV) or self-complementary (scAAV). Typically, the vector genome comprises a heterologous nucleic acid (heterologous to the original AAV from which it was derived), typically encoding a therapeutic transgene, a gene editing nucleic acid, or the like.

As used herein, the terms "rAAV vector", "rAAV viral particle" and/or "rAAV vector particle" refer to an AAV capsid composed of at least one AAV capsid protein (but generally all capsid proteins of AAV are present, such as VP1, VP2 and VP3, or variants thereof) and containing a vector genome comprising a heterologous nucleic acid sequence. These terms are to be distinguished from non-recombinant "AAV viral particles" or "AAV viruses", wherein the capsid contains a viral genome encoding the rep and cap genes and the AAV virus is capable of replicating when present in a cell that also contains a helper virus (such as an adenovirus and/or herpes simplex virus) and/or the helper genes required therefrom. Therefore, the production of rAAV vector particles necessarily includes the use of recombinant DNA technology to produce a recombinant vector genome, such as the vector genome contained within a capsid to form a rAAV vector, rAAV viral particle or rAAV vector particle.

The genomic sequences of various serotypes of AAV, as well as the sequences of inverted terminal repeats (ITRs), rep proteins, and capsid subunits are known in the art. These sequences can be found in the literature or in public databases such as GenBank. See, for example, GenBank Accession Numbers NC_002077 (AAV1), AF063497 (AAV1), NC_001401 (AAV2), AF043303 (AAV2), NC_001729 (AAV3), AF028705.1 (AAV3B), NC_001829 (AAV 4), U89790 (AAV4), NC_006152 (AAV5), AF028704 (AAV6), AF513851 (AAV7), AF513852 (AAV8), NC_006261 (AAV8), AY530579 (AAV9), AY631965 (AAV10), AY631966 (AAV11), and DQ813647 (AAV12); the disclosures of which are incorporated herein by reference. See also Srivistava et al. (1983) J. Virology 45:555; Chiorini et al. (1998) J. Virology 71:6823; Chiorini et al. (1999) J. Virology 73:1309; Bantel-Schaal et al. (1999) J. Virology 73:939; Xiao et al. (1999) J. .Virology73:3994; Muramatsu et al. (1996) Virology 221:208; Shade et al. (1986) J.Virol.58:921; Gao et al. (2002) Proc.Nat.Acad.Sci.USA 99:11854; Moris et al. (2004) Virology33:375-383; International Patent Publication WO 00/28061, WO 99/61601, WO 98/11244; WO 2013/063379, WO 2014/194132, WO 2015/121501; and U.S. Patent Nos. 6,156,303 and 7,906,111.

As used herein, the term "associated with" means that when referring to each other, the presence, level and/or form of one is associated with the presence, level and/or form of another. For example, if the presence, level and/or form of a particular entity (e.g., a polypeptide, a genetic feature, a metabolite, a microorganism, etc.) is associated with the incidence and/or susceptibility of a disease, disorder or condition (e.g., in a relevant population), the entity is considered to be associated with the particular disease, disorder, or condition. In some embodiments, if two or more entities interact directly or indirectly, bringing them into physical proximity and/or maintaining physical proximity to each other, the two or more entities are physically "associated" with each other. In some embodiments, two or more entities that are physically associated with each other are covalently linked to each other; in some embodiments, two or more entities that are physically associated with each other are not covalently linked to each other, but are non-covalently associated, such as by hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

As used herein, the term "coding sequence" or "coding nucleic acid" refers to a nucleic acid sequence that encodes a protein or polypeptide, and represents a sequence that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences (operably linked). The boundaries of the coding sequence are usually determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. The coding sequence may include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences.

As used herein, the term "chimeric" refers to a viral capsid or particle having a capsid or particle sequence from a different parvovirus, preferably a different AAV serotype, as described in US Pat. No. 6,491,907 to Rabinowitz et al., the disclosure of which is incorporated herein by reference in its entirety. See also Rabinowitz et al. (2004) J. Virol. 78(9):4421-4432. In some embodiments, the chimeric viral capsid is an AAV2.5 capsid having the sequence of an AAV2 capsid with the following mutations: 263Q mutation to A; 265 insertion T; 705N mutation to A; 708V mutation to A; and 716T mutation to N. The nucleotide sequence encoding this capsid is defined as SEQ ID NO: 15, as described in WO 2006/066066. Other preferred chimeric AAV capsids include, but are not limited to, AAV2i8 described in WO 2010/093784, AAV2G9 and AAV8G9 described in WO 2014/144229, and AAV9.45 (Pulicherla et al. (2011) Molecular Therapy 19(6):1070-1078), AAV-NP4, NP22 and NP66, AAV-LK0 to AAV-LK019, described in WO 2013/029030, RHM4-1 and RHM15_1 to RHM5_6, described in WO 2015/013313, AAVDJ, AAVDJ/8, AAVDJ/9, described in WO 2007/120542.

As used herein, the term "eluent" refers to a fluid that is discharged from a chromatographic stationary phase (e.g., a monolithic column, a membrane, a resin, a medium) (e.g., "eluted from the stationary phase"), which consists of a mobile phase and materials that pass through or are removed from the stationary phase. In some embodiments, the stationary phase includes, for example, a monolithic column, a membrane, a resin, or a medium. The mobile phase can be a solution that has been loaded onto the column and has flowed through the column (i.e., a "flow-through fraction"); an equilibrium solution (e.g., an equilibrium buffer); an isocratic elution solution; a gradient elution solution; a solution for regenerating a stationary phase; a solution for disinfecting a stationary phase; a solution for washing; and combinations thereof.

As used herein, the term "flanking" refers to a sequence that is flanked by other elements on both sides and indicates the presence of one or more flanking elements upstream and/or downstream, i.e., 5' and/or 3', relative to the sequence. The term "flanking" does not mean that the sequence must be continuous. For example, there may be intervening sequences between the nucleic acid encoding the transgene and the flanking elements. A sequence (such as a transgene) that is "flanked" by two other elements (such as ITRs) means that one element is located 5' and the other element is located 3' of the sequence, however, there may be intervening sequences between them.

As used herein, the term "flocculation" refers to the process of aggregating fine particles to form flocs. Fine particles may include proteins, nucleic acids, cell fragments produced by host cell lysis. In some embodiments, flocs formed in the liquid phase may float to the top of the liquid (milky), settle to the bottom of the liquid (precipitation), or be filtered from the liquid phase.

As used herein, the term "fragment" refers to a material or entity having a structure that includes discrete portions of a whole but lacks one or more portions found in the whole. In some embodiments, a fragment consists of discrete portions. In some embodiments, a fragment contains or consists of characteristic structural elements or portions found in the whole. In some embodiments, a polymer fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more monomeric units (e.g., amino acid residues, nucleotides) found in the entire polymer.

When the capsid contains a complete or substantially complete vector genome (including a transgene), the rAAV vector is referred to as "complete", "complete capsid", "complete vector" or "completely packaged vector". During the production of rAAV vectors by host cells, vectors having less packaged nucleic acid than a complete capsid and containing, for example, a partial or truncated vector genome can be produced. These vectors are referred to as "intermediates", "intermediate capsids", "partials" or "partially packaged vectors". When analyzed by analytical ultracentrifugation, an intermediate capsid can also be a capsid with an intermediate sedimentation rate (i.e., a sedimentation rate between a complete capsid and an empty capsid). Host cells can also produce viral capsids that do not contain any detectable nucleic acid material. These capsids are referred to as "empty" or "empty capsids". Complete capsids can be distinguished from empty capsids based on the A260/A280 ratio determined by SEC-HPLC, where the A260/A280 ratio has been pre-calibrated for capsids separated by analytical ultracentrifugation (i.e., complete capsids, intermediate capsids and empty capsids). Other methods known in the art for capsid characterization include CryoTEM, capillary isoelectric focusing, and charge detection mass spectrometry. The calculated isoelectric points of empty and fully capsid AAV9 capsids have been reported to be ˜6.2 and ˜5.8, respectively (Venkatakrishnan et al., J. Virology (2013) 87.9:4974-4984).

As used herein, the term "aborted capsid" refers to a capsid that is intentionally produced and lacks a vector genome. Such an aborted capsid can be produced by transfecting a host cell with a rep/cap and a helper plasmid, rather than a plasmid containing a transgenic cassette sequence (also referred to as a vector plasmid).

As used herein, the term "functional" is a biomolecule that exhibits an identified property and/or activity. A biomolecule can have two functions (ie, bifunctional) or multiple functions (ie, multifunctional).

As used herein, the term "gene" refers to a polynucleotide containing at least one open reading frame that can encode a specific polypeptide or protein after being transcribed and translated. "Gene transfer" or "gene delivery" refers to a method or system for reliably inserting exogenous DNA into a host cell. This method can result in transient expression of non-integrated transferred DNA, expression of extrachromosomal replication and transferred replicons (e.g., exosomes), and/or integration of transferred genetic material into the genomic DNA of the host cell.

As used herein, the term "gradient elution" refers to applying a mixture of at least two different solutions having different pH, conductivity and/or modifier concentrations to a chromatographic stationary phase (including, for example, a monolithic column, a medium, a resin, a membrane), the mixture gradually changing during the elution process. Gradient elution can be linear or nonlinear. In contrast, during isocratic elution, the chromatographic mobile phase composition is constant, while during "step elution", the chromatographic mobile phase composition changes in a stepwise manner. During the gradient elution process, the percentage of the first solution changes continuously in an inversely proportional manner to the percentage of the second solution. For example, at the beginning of the gradient elution, the percentage of gradient elution buffer A (e.g., the first gradient elution buffer) in the mixture is 100%, and the percentage of gradient elution buffer B (e.g., the second gradient elution buffer) in the mixture is 0%, thereby generating a gradient of continuous change of pH, conductivity and/or modifier concentration (increase or decrease according to the embodiment) as the solutions mix and flow through the stationary phase. In some embodiments, the concentration of a salt (e.g., sodium acetate) changes at a constant rate over the volume of a linear gradient. For example, for a 1mL column (i.e., 20CV) with a 20mL linear gradient, running at a constant flow rate of 1mL/min, the salt concentration will change at a rate of 5% per minute. In some embodiments, during the loading of a solution containing the rAAV capsid to be purified onto the AEX stationary phase, the rAAV capsid (e.g., complete capsid, intermediate capsid, empty capsid) is combined with the stationary phase. During the gradient elution, as the percentage of buffer B increases, the concentration of salt (e.g., sodium acetate) increases, the complete rAAV vector is preferentially released (eluted) from the stationary phase, and the empty capsid is preferentially retained on the stationary phase. As the percentage of buffer B increases further, the empty capsid is released in greater amounts. During gradient elution, the elution of complete rAAV vector from the stationary phase can be monitored by measuring the A260 and A280 of the eluate, whereby an increase in the ratio of A260/A280 indicates an increase in the percentage of complete rAAV vector in the eluate, and conversely, a decrease in the ratio of A260/A280 indicates a decrease in the percentage of complete rAAV vector and an increase in the percentage of empty capsids. In some embodiments, the absorbance of at least one eluate fraction is measured using a method such as analytical size exclusion chromatography (SEC) in a high performance liquid chromatography (HPLC) system, online UV tracing, offline UV methods, and the like, and wherein the absorbance is measured at one or more wavelengths (e.g., 260 nm and/or 280 nm).

As used herein, the term "heterologous" refers to a nucleic acid inserted into a vector (e.g., an rAAV vector), which is transferred/delivered to a cell via vector-mediated transfer. Heterologous nucleic acids are generally different from vector (e.g., AAV) nucleic acids, that is, heterologous nucleic acids are non-natural relative to viral (e.g., AAV) nucleic acids. Once transferred or delivered to a cell, the heterologous nucleic acid contained in the vector can be expressed (e.g., transcribed and translated, if appropriate). Alternatively, the heterologous nucleic acid transferred or delivered in the cell contained in the vector does not need to be expressed. Although the term "heterologous" is not always used herein to refer to nucleic acids, even in the absence of the modifier "heterologous", reference to nucleic acids is intended to include heterologous nucleic acids. For example, a heterologous nucleic acid is a nucleic acid encoding a dystrophin polypeptide or a fragment thereof, such as the codon-optimized micro-dystrophin transgene described in WO 2017/221145, incorporated herein by reference, for the treatment of Duchenne muscular dystrophy.

Another exemplary heterologous nucleic acid comprises a wild-type coding sequence of one of the following genes, or a fragment thereof (e.g., truncated, internally deleted), and may or may not be codon-optimized:

As used herein, the term "homologous" or "homology" refers to two or more entities (such as nucleotides or polypeptide sequences) mentioned that share at least partial identity in a given region or portion. For example, when an amino acid position in two peptides is occupied by the same amino acid, the peptides are homologous at that position. It is worth noting that homologous peptides will retain the activity or function associated with the unmodified or reference peptide, and the modified peptides generally have an amino acid sequence that is "substantially homologous" to the amino acid sequence of the unmodified sequence. When referring to a polypeptide, nucleic acid or its fragment, "substantially homologous" or "substantially similar" refers to when appropriate insertions or deletions are made with another polypeptide, nucleic acid (or its complementary chain) or its fragment for optimal comparison, there is at least about 95% to 99% sequence identity. The degree of homology (identity) between two sequences can be determined using a computer program or a mathematical algorithm. This algorithm for calculating sequence homology (or identity) percentages generally considers sequence gaps and mismatches on a comparison region or region. Exemplary programs and algorithms are provided below.

As used herein, the terms "host cell," "host cell line," and "host cell culture" are used interchangeably to refer to cells into which exogenous nucleic acid has been introduced, and include the progeny of such cells. Host cells include "transfectants," "transformants," "transformed cells," and "transduced cells," including primary transfected, transformed, or transduced cells and their derived progeny, regardless of the number of passages. In some embodiments, the host cell is a packaging cell for producing rAAV vectors.

As used herein, the term "host cell DNA" or "HCDNA" refers to residual DNA derived from the host cell culture that produced the rAAV vector, present in the chromatography fraction (e.g., affinity eluate, AEX eluate, wash solution) or chromatography load (e.g., affinity load, AEX load). Host cell DNA can be measured by methods known in the art, such as qPCR, to detect sequences unique to the host cell. Fluorescent dyes (e.g., or Green), absorbance measurement (e.g., at 260 nm or 254 nm), or electrophoresis techniques (e.g., agarose gel electrophoresis or capillary electrophoresis) to estimate the general DNA concentration. The amount of HCDNA present in the eluate can be expressed relative to the amount of vg present in the eluate, for example, ng HCDNA/1×10 ¹⁴ vg or pg HCDNA/1×10 ⁹ vg. The amount of HCDNA present in the eluate can be expressed relative to the amount of vg present in a certain volume of eluate, for example, pg HCDNA/mL eluate.

As used herein, the term "host cell protein" or "HCP" refers to residual proteins derived from the host cell culture that produced the rAAV vector, present in the chromatography fractions (e.g., affinity eluate, AEX eluate, wash solution) or chromatography load (e.g., affinity load, AEX load). Host cell proteins can be measured by methods known in the art, such as ELISA. Host cell proteins can be measured by various electrophoretic staining methods (e.g., silver staining SDS-PAGE, The amount of HCP present in the eluate can be expressed relative to the amount of vg present, for example, ng HCP/1×10 ¹⁴ vg or pg HCP/1×10 ⁹ vg.

As used herein, the term "identity" or "same as" refers to the overall relatedness between polymeric molecules, such as the overall relatedness between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be "substantially identical" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more identical.

For example, the calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed by comparing the two sequences to achieve the best comparison purpose (for example, a gap can be introduced in one or both of the first and second sequences to achieve the best comparison, and different sequences can be ignored for comparison purposes). In certain embodiments, the length of the sequence compared for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of the length of the reference sequence. The nucleotides at the corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position of the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, which need to be introduced in order to achieve the best comparison of the two sequences. The comparison of sequences and the determination of the percent identity between the two sequences can be accomplished using a mathematical algorithm.

To determine identity or homology, sequences can be aligned using methods and computer programs including BLAST, which is available on the World Wide Web at ncbi.nlm.nih.gov/BLAST/. Another alignment algorithm is FASTA, derived from the Genetics Computing Group (GCG) software package from Madison, Wis., USA. Other alignment techniques are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc. Of particular interest are alignment programs that allow for gaps in the sequence. Smith-Waterman is an algorithm that allows for gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). In addition, the GAP program using the Needleman and Wunsch alignment method can be used to align sequences. See J. Mol. Biol. 48:443-453 (1970).

Also of interest is the BestFit program (1981, Advances in Applied Mathematics 2:482-489) using the local homology algorithm of Smith and Waterman to determine sequence identity. Gap creation penalty is typically in the range of 1 to 5, typically in the range of 2 to 4, and in some embodiments 3. Gap extension penalty will typically be in the range of about 0.01 to 0.20, and in some cases 0.10. The program has default parameters determined by the sequence to be compared that is input. Preferably, sequence identity is determined using the default parameters determined by the program. This program is also available from the Genetics Computing Group (GCG) software package, from Madison, WI, USA.

Another program of interest is the FastDB algorithm. FastDB is described in Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1988, Alan R. Liss, Inc. FastDB calculates percent sequence identity based on the following parameters: mismatch penalty: 1.00; gap penalty: 1.00; gap size penalty: 0.33; and joining penalty: 30.0.

As used herein, the term "impurity" refers to any molecule other than the complete rAAV vector to be purified that is also present in the solution containing the rAAV vector to be purified. Impurities include empty capsids, intermediate capsids, biomacromolecules such as DNA, RNA, non-AAV proteins (such as host cell proteins), AAV aggregates, damaged AAV capsids, molecules that are part of the absorbent used for chromatography (which may have leached into the sample during previous purification steps), endotoxins, cell debris, and chemicals from cell culture, including media components, plasmid DNA from transfection, incidental factors, bacteria, and viruses.

As used herein, the terms "inverted terminal repeats", "ITRs", "terminal repeats" and "TRs" refer to palindromic terminal repeat sequences at or near the ends of the AAV viral genome, primarily comprising complementary, symmetrically arranged sequences. These ITRs can fold to form a T-shaped hairpin structure, acting as primers at the start of DNA replication. They are also required for the integration of the viral genome into the host genome, for rescue from the host genome; and for encapsidation of viral nucleic acids into mature viral particles. ITRs are required in cis for vector genome replication and its packaging in viral particles. "5'ITR" refers to the ITR at the 5' end of the AAV genome and/or the 5' end of the recombinant transgene. "3'ITR" refers to the ITR at the 3' end of the AAV genome and/or the 3' end of the recombinant transgene. The length of the wild-type ITR is approximately 145 bp. Modified or recombinant ITRs may include fragments or portions of wild-type AAV ITR sequences. One of ordinary skill in the art will appreciate that during successive rounds of DNA replication, ITR sequences can be interchanged, such that the 5'ITR becomes the 3'ITR, and vice versa. In some embodiments, at least one ITR is present at the 5' and/or 3' end of the recombinant vector genome, such that the vector genome can be packaged in a capsid to produce an rAAV vector (also referred to herein as a "rAAV vector particle" or "rAAV virus particle") comprising the vector genome.

ITR is required for vector genome replication and its packaging in viral particles in cis. "5'ITR" refers to the ITR at the 5' end of the AAV genome and/or the 5' end of the recombinant transgene. "3'ITR" refers to the ITR at the 3' end of the AAV genome and/or the 3' end of the recombinant transgene. The length of the wild-type ITR is about 145bp. The modified or recombinant ITR can include a fragment or part of the wild-type AAV ITR sequence. It will be understood by those of ordinary skill in the art that during consecutive rounds of DNA replication, the ITR sequences can be interchanged, so that the 5'ITR becomes the 3'ITR, and vice versa.

As used herein, the term "isolated" refers to a substance or composition that is: 1) artificially designed, generated, prepared and/or produced, and/or 2) separated from at least one component associated with it when it was originally produced (whether in nature or in an experimental environment). Typically, the isolated composition is substantially free of one or more materials that are normally associated with it in nature, such as one or more proteins, nucleic acids, lipids, carbohydrates and/or cell membranes. The term "isolated" does not exclude artificial combinations, such as recombinant nucleic acids, recombinant vector genomes (e.g., rAAV vector genomes), packaged rAAV vector particles such as encapsidated vector genomes and pharmaceutical preparations (e.g., such as, but not limited to, rAAV vector particles comprising AAV9 capsids). The term "isolated" also does not exclude other physical forms of the composition, such as hybrids/chimeras, multimers/oligomers, modifications (e.g., phosphorylation, glycosylation, lipidation), variants or derivative forms, or artificial forms expressed in host cells.

The separated substance or composition can be separated from about 10%, about 20%, about 30%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% of the other components originally associated with it. In some embodiments, the separated preparation is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% pure. As used herein, if a substance is substantially free of other components, the substance is "pure". In some embodiments, as will be appreciated by those skilled in the art, a substance may still be considered "isolated" or even "pure" after being combined with certain other components, such as one or more carriers or excipients (e.g., buffers, solvents, water, etc.); in such embodiments, the percent isolation or purity of the substance is calculated excluding such carriers or excipients.

As used herein, the term "load chase solution" refers to a solution applied to a column after a load or loading solution (defined below) has been applied. A load chase solution is used to complete the application of the load or loading solution and remove unbound material from the column.

As used herein, the term "load" or "loading solution" refers to any material (e.g., solution) containing a product of interest (e.g., a complete rAAV vector) that is loaded onto a chromatographic stationary phase. In some embodiments, the "loading solution" is exposed to a chromatographic stationary phase. In some embodiments, the loading solution is an affinity eluent. In some embodiments, the loading solution is a diluted and optionally filtered affinity eluent.

As used herein, the term "stationary phase" or "chromatographic stationary phase" refers to any substance that can be used to separate a product from another substance (such as an impurity). In some embodiments, the chromatographic stationary phase is a resin, a medium, a membrane, a membrane adsorbent, or a monolithic column. In some embodiments, the chromatographic stationary phase is a medium that binds to the AAV capsid under certain conditions. In some embodiments, the chromatographic stationary phase is an ion exchange medium (e.g., an anion exchange medium, a cation exchange medium). In some embodiments, the chromatographic stationary phase is POROS ^TM 50HQ.

As used herein, the term "modifier" or "mobile phase modifier" is a component of the mobile phase that modifies the mobile phase to change the chromatography. This change in the chromatography results in, for example, the removal or washing of impurities from the stationary phase, or the elution of a product or material of interest (e.g., a rAAV vector) from the stationary phase. Examples of "modifiers" include salts, detergents, amino acids (e.g., arginine, histidine, citrulline, glycine), organic solvents (e.g., ethanol, ethylene glycol), chaotropic agents (e.g., urea), or displacers (also known as selective eluents).

As used herein, the terms "nucleic acid sequence", "nucleotide sequence" and "polynucleotide" interchangeably refer to any molecule consisting of or comprising monomeric nucleotides linked by phosphodiester bonds. Nucleic acids can be oligonucleotides or polynucleotides. Nucleic acid sequences are presented herein in a direction from 5' to 3'. The nucleic acid sequences (i.e., polynucleotides) disclosed herein can be deoxyribonucleic acid (DNA) molecules or ribonucleic acid (RNA) molecules, and refer to all forms of nucleic acids, such as double-stranded molecules, single-stranded molecules, small hairpin RNA or short hairpin RNA (shRNA), microRNA, small interfering RNA or short interfering RNA (siRNA), trans-splicing RNA, antisense RNA, messenger RNA, transfer RNA, ribosomal RNA. In the case where the polynucleotide is a DNA molecule, the molecule can be a gene, cDNA, antisense molecule or a fragment of any of the aforementioned molecules. Nucleotides are represented herein by single-letter codes: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I) and uracil (U). Nucleotide sequences can be chemically modified or artificial. Nucleotide sequences include peptide nucleic acids (PNA), morpholino nucleic acids and locked nucleic acids (LNA), as well as glycol nucleic acids (GNA) and threonine nucleic acids (TNA). Each of these sequences is distinguished from naturally occurring DNA or RNA by changes in the molecular backbone. In addition, thiophosphate nucleotides can be used. Other deoxynucleotide analogs include methylphosphonates, phosphoramidates, dithiophosphates, N3'-P5'-phosphoramidates and oligoribonucleotide thiophosphates and 2'-O-allyl analogs and 2'-O-methylribonucleotide methylphosphonates, which can be used in the nucleotide sequences of the present disclosure.

As used herein, the term "nucleic acid construct" refers to a non-naturally occurring nucleic acid molecule (e.g., recombinant nucleic acid) produced using recombinant DNA technology. A nucleic acid construct is a single-stranded or double-stranded nucleic acid molecule that has been modified to contain segments of nucleic acid sequences that are combined and arranged in a manner not found in nature. A nucleic acid construct can be a "vector" (e.g., a plasmid, an rAAV vector genome, an expression vector, etc.), i.e., a nucleic acid molecule designed to deliver exogenously produced DNA into a host cell.

As used herein, the term "operably linked" refers to a connection in which a nucleic acid sequence (or polypeptide) element is in a functional relationship. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, it is operably linked. For example, if a promoter or other transcription regulatory sequence (e.g., enhancer) affects the transcription of a coding sequence, it is operably linked to the coding sequence. In some embodiments, operably linked means that the nucleic acid sequence being linked is continuous. In some embodiments, operably linked does not mean that the nucleic acid sequence is continuously linked, but rather that there is an insertion sequence between those nucleic acid sequences being linked.

As used herein, the term "vector genome (VG) dilution yield percentage" or "%VG dilution yield" refers to the amount of VG present in the diluted affinity pool (also referred to herein as the diluted affinity eluate) relative to the amount of VG present in the affinity pool (also referred to herein as the affinity eluate) before dilution. For example, %VG dilution yield = ((amount of VG in the diluted affinity pool) / (amount of VG in the affinity pool)) * 100.

As used herein, the term "VG column yield percentage" or "% VG column yield" refers to the amount of vector genome (VG) present in the combined eluate collected from an AEX column (i.e., the AEX pool) as a percentage of the amount of VG in the diluted affinity eluate alone or the diluted and filtered affinity eluate.

In some embodiments, the affinity eluate containing the rAAV vector to be purified is only diluted and is referred to as a "diluted affinity pool". Optionally, the rAAV vector to be purified is harvested from a 250L or 2000L container (e.g., a disposable bioreactor (SUB)). For example, % VG column yield = ((amount of VG in the AEX pool) / (amount of VG in the diluted affinity pool)) * 100.

In some embodiments, the affinity eluate containing the rAAV vector to be purified has been diluted and filtered and is referred to as the "AEX load". Optionally, the rAAV vector to be purified is harvested from a small-scale (e.g., less than 250 L) container (e.g., a bioreactor). For example, % VG column yield = ((amount of VG in the AEX pool) / (amount of VG in the diluted and filtered affinity pool)) * 100.

As used herein, the term "VG step yield percentage" or "% VG step yield" refers to the amount of VG in the combined eluate collected from the AEX column (i.e., the AEX pool) relative to the amount of VG present in the affinity pool (also referred to herein as the affinity eluate) prior to dilution or filtration. For example, % VG step yield = ((amount of VG in the AEX pool) / (amount of VG in the affinity pool)) * 100.

As used herein, the terms "pharmaceutically acceptable" and "physiologically acceptable" refer to biologically acceptable formulations, either gaseous, liquid or solid, or mixtures thereof, suitable for one or more routes of administration, delivery or contact in vivo.

As used herein, the terms "polypeptide", "protein", "peptide" or "encoded by a nucleic acid sequence" (i.e., encoded by a polynucleotide sequence, encoded by a nucleotide sequence) refer to full-length native sequences, naturally occurring proteins, and functional subsequences, modified forms or sequence variants, as long as the subsequences, modified forms or variants retain a certain degree of functionality of the native full-length protein. In the methods and uses of the present disclosure, such polypeptides, proteins and peptides encoded by nucleic acid sequences can be, but are not required to be, identical to endogenous proteins, which are defective, or whose expression is insufficient, or which are defective in subjects treated with gene therapy.

As used herein, the term "recombinant" refers to a vector, polynucleotide (e.g., recombinant nucleic acid), polypeptide or cell that is the product of various combinations of cloning, restriction or ligation steps (e.g., involving polynucleotides or polypeptides contained therein), and/or the product of other methods of obtaining a construct different from the product found in nature. A recombinant virus or vector (e.g., a rAAV vector) comprises a vector genome comprising a recombinant nucleic acid (e.g., a nucleic acid comprising a transgene and one or more regulatory elements). The term includes replication of the original polynucleotide construct and progeny of the original viral construct, respectively.

As used herein, the term "step-wise elution" refers to the application of a solution with a defined pH, conductivity and/or modifier concentration to a chromatographic stationary phase (including, for example, a monolithic column, a medium, a resin, a film). A series of step-wise elutions (such as conductivity or salt concentration increasing) can be performed to optimize separation. Each step elution solution has a defined composition and does not change during its application. In the step-wise elution process, as a series of solutions (such as load additional solution, pH stabilizing solution, washing buffer, elution buffer) are applied to the stationary phase, the pH, conductivity and/or modifier concentration increase or decrease relative to the previous series of solutions. For example, at the beginning of the step-wise elution series, the concentration of the modifier (such as salt such as sodium acetate) in the first solution is relatively low, for example, 0 to 10mM, for example, about 1mM, about 2mM, about 3mM, about 4mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM. In each subsequent solution in the series, the concentration of the salt increases, whereby over the course of 2 to 20 solutions, the concentration of the salt increases to, for example, 50 mM to 300 mM (e.g., about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 120 mM, about 140 mM, about 160 mM, about 180 mM, and about 200 mM). The salt concentrations in the 2 to 20 (or more) solution series do not necessarily change in equal or proportional increments.

In some embodiments, the step elution includes 2 to 20 solutions, 2 to 10 solutions, 10 to 20 solvents, such as 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more solutions. In some embodiments, during the loading of the solution containing the rAAV capsid onto the AEX stationary phase, the rAAV capsid (e.g., complete capsid, intermediate capsid, empty capsid) is bound to the stationary phase. During the step elution, as the pH, conductivity and/or modifier concentration change, the complete rAAV vector is preferentially released (eluted) from the stationary phase, while the empty capsid is preferentially retained on the stationary phase. As the concentration of the modifier (e.g., salt) increases, the empty capsid is released in large quantities. During step elution, the elution of the complete rAAV vector from the stationary phase can be monitored by measuring the A260 and A280 of the eluate, whereby an increase in the A260/A280 ratio indicates an increase in the percentage of complete rAAV vector in the eluate, whereas a decrease in the A260/A280 ratio indicates a decrease in the percentage of complete rAAV vector and an increase in the percentage of empty capsids. In some embodiments, the absorbance of at least one eluate fraction is measured using a method such as analytical size exclusion chromatography (SEC) in a high performance liquid chromatography (HPLC) system, online UV tracing, offline UV methods, and the like, and wherein the absorbance is measured at one or more wavelengths (e.g., 260 nm and/or 280 nm).

As used herein, the term "subject" refers to an organism, such as a mammal (e.g., a human, a non-human mammal, a non-human primate, a primate, a laboratory animal, a mouse, a rat, a hamster, a gerbil, a cat, a dog). In some embodiments, a human subject is an adult, a teenager, or a child subject. In some embodiments, the subject suffers from a disease, disorder, or condition, such as a disease, disorder, or condition that can be treated as provided herein. In some embodiments, the subject suffers from a disease, disorder, or condition associated with a defective or dysfunctional dystrophin, such as Duchenne muscular dystrophy. In some embodiments, the subject is susceptible to a disease, disorder, or condition. In some embodiments, susceptible subjects tend to and/or show an increased risk of developing a disease, disorder, or condition (compared to the average risk observed in a reference subject or population). In some embodiments, the subject exhibits one or more symptoms of a disease, disorder, or condition. In some embodiments, the subject does not exhibit specific symptoms (e.g., clinical manifestations of the disease) or features of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or features of a disease, disorder, or condition. In some embodiments, the subject is a human patient. In some embodiments, the subject is an individual who is being and/or has been administered a diagnosis and/or treatment (e.g., gene therapy for Duchenne muscular dystrophy). In some embodiments, the subject is a human patient suffering from Duchenne muscular dystrophy.

Diseases, disorders, and conditions that can be treated using the rAAV vectors purified according to the methods described herein include, for example, metabolic diseases or disorders (e.g., Fabry disease, Gaucher disease, phenylketonuria, glycogen storage diseases); urea cycle diseases or disorders (e.g., ornithine transcarbamylase deficiency); lysosomal storage diseases or disorders (e.g., metachromatic leukodystrophy, mucopolysaccharidoses); liver diseases or disorders (e.g., progressive familial intrahepatic cholestasis types 1-3); blood diseases or disorders (hemophilia A, hemophilia B, alpha thalassemia); cancer (e.g., carcinoma, sarcoma, blood cancer); hereditary diseases or disorders (e.g., cystic fibrosis) or infectious diseases (e.g., HIV).

Diseases, disorders and conditions that can be treated using the rAAV vectors purified according to the methods described herein include, for example: 21-hydroxylase deficient congenital adrenal hyperplasia, chondrodysplasia type 1B, achondroplasia, achromatopsia, acid sphingomyelinase deficiency (Niemann-Pick disease type A or B), acute intermittent porphyria, adenosine deaminase 2 deficiency, adenosine deaminase deficiency (e.g., severe combined immunodeficiency, X-linked), adrenoleukodystrophy (e.g., X-linked), age-related macular degeneration (e.g., neovascular, wet), Alagill e syndrome, alkaptonuria, alpha-1 antitrypsin deficiency, alpha-thalassemia, Alport syndrome, Alzheimer's disease, Apert syndrome, arginase deficiency, argininosuccinate lyase (ASL) deficiency, argininosuccinate synthetase (ASS1) deficiency (citrullinemia type 1), aromatic L-amino acid decarboxylase deficiency, autosomal recessive congenital ichthyosis, Becker muscular dystrophy, beta-thalassemia, carbamoyl phosphatase synthetase I deficiency, ceroid lipofuscinosis, Charcot-Marie Tooth neuropathy, choroiditis, chronic granulomatous disease, vitamin P deficiency, Crigler-Najjar syndrome types 1 and 2, critical limb ischemia, cystic fibrosis, cystinosis, Danon disease, diabetic macular retinopathy, dominant short stature, Dravet syndrome, Duchenne muscular dystrophy, dysferlinopathy (eg, Miyoshi myopathy, limb-girdle muscular dystrophy type 2B), dystrophic epidermolysis bullosa, Fabry disease, familial hypercholesterolemia, familial lipoprotein lipase deficiency, Fanconia anemia (eg, Fanconia anemia type A), Friedreich ataxia, frontotemporal dementia, Gaucher disease, glycogen storage disease types 1A and 1B (Von Gierke disease), glycogen storage disease type III, glycogen storage disease type IV, glycogen storage disease type V, glycogen storage disease type VI, glycogen storage disease type XV, GM1 gangliosidosis, gyral atrophy, hemophilia A, hemophilia B, hereditary angioedema types I-III, Huntington's disease, inclusion body myositis, junctional epidermolysis bullosa, Kabuki syndrome, Leber congenital amaurosis, leukocyte adhesion deficiency type 1, Limb-girdle muscular dystrophy, Limb-girdle muscular dystrophy type 2C (gamma-limb-girdle muscular dystrophy), Limb-girdle muscular dystrophy type 2D, Metachromatic leukodystrophy, Mucopolysaccharidosis type I, Mucopolysaccharidosis type II (Hunter syndrome), Mucopolysaccharidosis type IIIA, Mucopolysaccharidosis type IIIB, Mucopolysaccharidosis type IIIC, Mucopolysaccharidosis type IIID, Mucopolysaccharidosis type IVA (Morquio A syndrome), Mucopolysaccharidosis type IVB (Morquio B syndrome), mucopolysaccharidosis type VI (Maroteaux-Lamy), myotonic dystrophy type 1, myotonic dystrophy type 2, N-acetylglutamate synthetase (NAGS) deficiency, Netherton syndrome, neuronal ceroid lipofuscinosis, ornithine transposase deficiency, ornithine carbamoyltransferase deficiency, Parkinson disease, phenylketonuria, Pompe, progressive familial intrahepatic cholestasis types 1-3, progressive myofibrillar myopathy, pyruvate kinase deficiency, retinitis pigmentosa, RPE65-related Leber congenital amaurosis, Sandhoff disease, sickle cell disease, spinal muscular atrophy, Tay-Sachs disease, Wilson disease, Wiskott-Aldrich syndrome, Wiskott-Aldrich syndrome 2, X-linked adrenoleukodystrophy, X-linked chronic granulomatous disease, X-linked myotubular myopathy, X-linked retinitis pigmentosa, X-linked retinoschisis, and X-linked severe combined immunodeficiency.

As used herein, the term "substantially" refers to the qualitative condition of exhibiting the full or nearly full extent or degree of a characteristic or property of interest. It will be appreciated by those of ordinary skill in the art that biological and chemical phenomena are rarely, if ever, completed and/or carried out to a complete or achieved or absolute result. Therefore, the term "substantially" is used herein to represent the potential lack of completeness inherent in many biological and chemical phenomena.

As used herein, the term "therapeutic polypeptide" is a peptide, polypeptide or protein (e.g., enzyme, structural protein, transmembrane protein, transporter) that can alleviate or reduce symptoms caused by protein deficiency or defects in target cells (e.g., isolated cells) or organisms (e.g., subjects). Therapeutic polypeptides or proteins encoded by transgenes are polypeptides or proteins that confer benefits to subjects, such as correcting genetic defects, correcting gene defects associated with expression or function. Similarly, a "therapeutic transgene" is a transgene encoding a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide expressed in a host cell is an enzyme expressed from a transgene (i.e., an exogenous nucleic acid that has been introduced into a host cell). In some embodiments, the therapeutic polypeptide is a dystrophin or a fragment thereof expressed from a therapeutic transgene transduced into a muscle cell (e.g., a skeletal muscle cell).

As used herein, the term "therapeutically effective amount" refers to an amount that produces a desired therapeutic effect after administration. In some embodiments, the term refers to an amount that is sufficient to treat the disease, disorder or condition when applied to a population suffering from or susceptible to a disease, disorder or condition according to a therapeutic dosing regimen. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and/or severity of one or more symptoms of a disease, disorder and/or condition and/or delays its onset. One of ordinary skill in the art will appreciate that the term "therapeutically effective amount" does not actually require successful treatment to be achieved in a particular individual. On the contrary, a therapeutically effective amount can be an amount that provides a specific desired pharmacological response in a large number of subjects when applied to a patient who needs such treatment.

As used herein, the term "transgenic" refers to any heterologous polynucleotide for delivery and/or expression in a host cell, a target cell, or an organism (e.g., a subject). Such a "transgenic" can be delivered to a host cell, a target cell, or an organism using a vector (e.g., an rAAV vector). The transgenic can be operably linked to a control sequence, such as a promoter. It will be appreciated by those skilled in the art that expression control sequences can be selected based on the ability to promote the expression of the transgenic in a host cell, a target cell, or an organism. Typically, the transgenic can be operably linked to an endogenous promoter associated with the transgenic in nature, but more typically, the transgenic is operably linked to a promoter that is not associated with the transgenic in nature. An example of a transgenic is a nucleic acid encoding a therapeutic polypeptide such as a dystrophin polypeptide or a fragment thereof, and the promoter exemplified is a promoter that is not operably linked to a nucleotide encoding a dystrophin in nature. Such a non-endogenous promoter can include a CBh promoter or a muscle-specific promoter and many other promoters known in the art.

Nucleic acids of interest can be introduced into host cells by a variety of techniques known in the art, including transfection and transduction.

"Transfection" is generally known as a technique for introducing exogenous nucleic acid into cells without the use of viral vectors. As used herein, the term "transfection" refers to transferring recombinant nucleic acid (e.g., expression plasmid) into cells (e.g., host cells) without the use of viral vectors. Cells into which recombinant nucleic acid has been introduced are referred to as "transfected cells". Transfected cells can be host cells (e.g., CHO cells, Pro10 cells, HEK293 cells) comprising expression plasmids/vectors for producing recombinant AAV vectors. In some embodiments, transfected cells (e.g., packaging cells) may include a plasmid comprising a transgene (e.g., dystrophin transgene), a plasmid comprising an AAV rep gene and an AAV cap gene, and a plasmid comprising an auxiliary gene. Many transfection techniques are known in the art, including but not limited to electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes combined with nuclear localization signals.

As used herein, the term "transduction" refers to the transfer of a nucleic acid (e.g., a vector genome) to a cell (e.g., a target cell such as a muscle cell) by a viral vector (e.g., a rAAV vector). In some embodiments, gene therapy for Duchenne muscular dystrophy includes transducing a vector genome comprising a modified nucleic acid encoding a dystrophin protein or a fragment thereof into a muscle cell. Cells into which a transgene has been introduced by a virus or viral vector are referred to as "transduced cells." In some embodiments, the transduced cells are isolated cells and the transduction occurs ex vivo. In some embodiments, the transduced cells are cells within an organism (e.g., a subject) and the transduction occurs in vivo. The transduced cells can be target cells of an organism that has been transduced by a recombinant AAV vector so that the target cells of the organism express a polynucleotide (e.g., a transgenic, such as a modified nucleic acid encoding a dystrophin protein, or a fragment thereof).

Cells that can be transduced include cells of any tissue or organ type, or cells of any origin (e.g., mesoderm, ectoderm, or endoderm). Non-limiting examples of cells include cells from the liver (e.g., hepatocytes, sinusoidal endothelial cells), pancreas (e.g., β-islet cells, exocrine cells), lungs, central or peripheral nervous systems such as the brain (e.g., neurons or ependymal cells, oligodendrocytes) or spinal cord, kidneys, eyes (e.g., retinal cells), spleen, skin, thymus, testes, lungs, diaphragm, heart (cardiac), muscle or psoas muscle, or intestinal tract (e.g., endocrine cells), adipose tissue (white, brown, or beige), muscle (e.g., fibroblasts, myocytes), synoviocytes, chondrocytes, osteoclasts, epithelial cells, endothelial cells, salivary gland cells, inner ear nerve cells, or hematopoietic (e.g., blood or lymph) cells. Other examples include stem cells, such as multipotent or pluripotent progenitor cells, that develop or differentiate into liver (e.g., hepatocytes, sinusoidal endothelial cells), pancreas (e.g., β-islet cells, exocrine cells), lung, central or peripheral nervous system, such as brain (e.g., neurons or ependymal cells, oligodendrocytes) or spinal cord, kidney, eye (e.g., retinal cells), spleen, skin, thymus, testis, lung, diaphragm, heart (cardiac), muscle or psoas, or intestine (e.g., endocrine cells), adipose tissue (white, brown or beige), muscle (e.g., fibroblasts, myocytes), synoviocytes, chondrocytes, osteoclasts, epithelial cells, endothelial cells, salivary gland cells, inner ear nerve cells, or hematopoietic (e.g., blood or lymphoid) cells.

In some embodiments, specific regions of a tissue or organ (e.g., muscle) can be transduced by an rAAV vector (e.g., a rAAV vector having a dystrophin or a portion of a dystrophin transgene) administered to the tissue or organ. In some embodiments, muscle cells are transduced with an rAAV comprising a dystrophin transgene. In some embodiments, skeletal muscle cells are transduced with an rAAV comprising a dystrophin transgene. In some embodiments, cardiomyocytes are transduced with an rAAV comprising a dystrophin transgene.

As used herein, the term "vector" refers to a plasmid, virus (e.g., rAAV), cosmid, or other carrier that can be manipulated by inserting or incorporating a nucleic acid (e.g., a recombinant nucleic acid). A vector can be used for various purposes, including, for example, genetic manipulation (e.g., a cloning vector) to introduce/transfer a nucleic acid into a cell to transcribe or translate the inserted nucleic acid in the cell. In some embodiments, the vector nucleic acid sequence contains at least a replication origin for propagation in the cell. In some embodiments, the vector nucleic acid includes a heterologous nucleic acid sequence, an expression control element (e.g., a promoter, an enhancer), a selective marker (e.g., antibiotic resistance), a polyadenosine (polyA) sequence, and/or an ITR. In some embodiments, when delivered to a host cell, the nucleic acid sequence proliferates. In some embodiments, when delivered to a host cell in vitro or in vivo, the cell expresses a polypeptide encoded by a heterologous nucleic acid sequence. In some embodiments, when delivered to a host cell, a nucleic acid sequence or a portion of a nucleic acid sequence is packaged into a capsid. The host cell can be a cell separated or a cell in a host organism. In addition to the nucleic acid sequence encoding a polypeptide or protein (e.g., a transgenic), additional sequences (e.g., regulatory sequences) may be present in the same vector (i.e., in cis with the gene) and may be located flanking the gene. In some embodiments, regulatory sequences may be present on a separate (e.g., second) vector that acts in trans to regulate the expression of the gene. Plasmid vectors may be referred to herein as "expression vectors."

As used herein, the term "vector genome" refers to the nucleic acid packaged/encapsidated in the AAV capsid to form a rAAV vector. Typically, the vector genome includes a heterologous polynucleotide sequence (e.g., a transgene, a regulatory element, etc.) and at least one ITR. In the case of using a recombinant plasmid to construct or manufacture a recombinant vector (e.g., an rAAV vector), the vector genome does not include the entire plasmid, but only includes sequences intended to be delivered by a viral vector. This non-vector genome portion of the recombinant plasmid is called a "plasmid backbone," which is important for plasmid cloning, selection, and amplification (which is a process required for the production and proliferation of recombinant viral vectors), but it itself is not packaged or encapsidated into the rAAV vector. Typically, the flank of the heterologous sequence to be packaged into the capsid is an ITR, whereby when cut from the plasmid backbone, it is packaged into the capsid.

As used herein, the term "viral vector" generally refers to a viral particle that acts as a nucleic acid delivery vehicle, which comprises a vector genome (e.g., a transgene comprising wild-type rep and cap replaced) packaged within a viral particle (i.e., capsid) and includes, for example, lentiviruses and parvoviruses, including AAV serotypes and variants (e.g., rAAV vectors). As described elsewhere herein, recombinant viral vectors do not comprise a viral genome having rep and/or cap genes; instead, these sequences have been removed to provide the ability of the vector genome to carry a transgene of interest.

The present disclosure provides a method for purifying rAAV vectors (e.g., complete rAAV vectors) from host cell harvests. In particular, the present disclosure provides a method for purifying rAAV vectors (e.g., complete rAAV vectors) from other nucleic acids and proteins (including empty capsids) produced by host cells. In addition, the present disclosure provides a method for separating empty capsids from complete rAAV vectors (e.g., rAAV vectors comprising vector genomes). Each of these aspects of the present disclosure is further discussed in the following sections.

2. AAV and rAAV vectors

AAV

As mentioned above, "adeno-associated virus" and/or "AAV" refers to a parvovirus with a linear single-stranded DNA genome and variants thereof. The term encompasses all subtypes and naturally occurring and recombinant forms unless otherwise required. Parvoviruses including AAV can be used as gene therapy vectors because they can penetrate cells and introduce nucleic acids (such as transgenes) into the nucleus. In some embodiments, the introduced nucleic acid (e.g., rAAV vector genome) forms a circular concatemer that persists in the nucleus of the transduced cell as an exosome. In some embodiments, the transgene is inserted into a specific site in the host cell genome, such as a site on human chromosome 19. In contrast to random integration, site-specific integration is thought to result in predictable long-term expression profiles. The insertion site of AAV in the human genome is called AAVS1. Once introduced into a cell, the polypeptide encoded by the nucleic acid can be expressed by the cell. Because AAV is not associated with any pathogenic disease in humans, the nucleic acid delivered by AAV can be used to express therapeutic polypeptides for treating diseases, disorders and/or conditions in human subjects.

There are many AAV serotypes in nature, and at least 15 wild-type serotypes (i.e., AAV1-AAV15) have been identified from humans to date. Naturally occurring serotypes and variant serotypes are distinguished by having a protein capsid that is serologically different from other AAV serotypes. AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3) including AAV serotype 3A (AAV3A) and AAV serotype 3B (AAV3B), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), AAV serotype 12 (AAV12), AAVrh10, AAVrh74 (see WO 2016/210170), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV and ovine AAV, as well as recombinantly produced variants (e.g., capsid variants with insertions, deletions and substitutions, etc.), such as variants known as AAV serotype 2i8 (AAV2i8), NP4, NP22, NP66, AAVDJ, AAVDJ/8, AAVDJ/9, AAVLK03, RHM4-1, etc. AAV variants isolated from human CD34+ cells include AAVHSC1, AAVHSC2, AAVHSC3, AAVHSC4, AAVHSC5, AAVHSC6, AAVHSC7, AAVHSC8, AAVHSC9, AAVHSC10, AAVHSC11, AAVHSC12, AAVHSC13, AAVHSC14, and AAVHSC15 (Smith et al. (2014) Molecular Therapy 22(9):1625-1634).

"Primate AAV" refers to AAV that infects primates, and "non-primate AAV" refers to AAV that infects non-primate mammals. "Bovine AAV" refers to AAV that infects bovine mammals, and so on. Serotype distinctiveness is determined based on the lack of cross-reactivity between antibodies to one AAV and another AAV. Such cross-reactivity differences are generally attributed to differences in capsid protein sequences and antigenic determinants (e.g., differences in VP1, VP2 and/or VP3 sequences due to AAV serotypes). However, some naturally occurring AAVs or artificial AAV mutants (e.g., recombinant AAVs) may not show serological differences from any currently known serotype. These viruses can be considered to be subsets of the corresponding serotypes, or more simply variant AAVs. Therefore, as used herein, the term "serotype" refers to both serologically different viruses, such as AAV, and to viruses that are serologically indistinguishable but may belong to a subset or variant of a serotype, such as AAV.

A comprehensive list and alignment of the amino acid sequences of capsids of known AAV serotypes is provided by Marsic et al. (2014) Molecular Therapy 22(11):1900-1909, particularly in Figure 1 of the Supplement.

The genome sequences of various serotypes of AAV, as well as the sequences of natural terminal repeats (ITRs), rep proteins and capsid subunits are known in the art. These sequences can be found in the literature or in public databases such as GenBank. See, e.g., GenBank Accession Nos. NC_002077 (AAV1), AF063497 (AAV1), NC_001401 (AAV2), AF043303 (AAV2), NC_001729 (AAV3), AF028705.1 (AAV3B), NC_001829 (AAV4), U89790 (AAV4), NC_006152 (AAV5), AF028704 (AAV6), AF513851 (AAV7), AF513852 (AAV8), NC_006261 (AAV8), AY530579 (AAV9), AY631965 (AAV10), AY631966 (AAV11), and DQ813647 (AAV12); the disclosures of which are incorporated herein by reference. See also Srivistava et al. (1983) J. Virology 45:555; Chiorini et al. (1998) J. Virology 71:6823; Chiorini et al. (1999) J. Virology 73:1309; Bantel-Schaal et al. (1999) J. Virology 73:939; Xiao et al. (1999) J .Virology 73:3994; Muramatsu et al. (1996) Virology 221:208; Shade et al. (1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99: 11854; Moris et al. (2004) Virology 33: 375-383; International Patent Publication WO 00/28061, WO 99/61601, WO 98/11244; WO 2013/063379; WO 2014/194132; WO 2015/121501, and U.S. Pat. No. 6,156,303 and U.S. Pat. No. 7,906,111. For illustrative purposes only, wild-type AAV2 comprises a small (20-25 nm) icosahedral viral capsid of AAV, composed of three proteins (VP1, VP2 and VP3; a total of 60 capsid proteins make up the AAV capsid) with overlapping sequences. The proteins VP1 (735 amino acids; Genbank accession number AAC03780), VP2 (598 amino acids, Genbank accession number AAC03778) and VP3 (533 amino acids; Genbank accession number AAC03779) are present in the capsid in a ratio of about 1:1:10. That is, for AAV, VP1 is a full-length protein, and VP2 and VP3 are progressively shortened versions of VP1, with increasing degrees of truncation at the N-terminus relative to VP1. In one embodiment of the methods disclosed herein, the rAAV vector comprises AAV9 VP1 comprising the amino acid sequence of SEQ ID NO:11.

Recombinant AAV (rAAV)

As previously described, "recombinant adeno-associated virus" or "rAAV" is distinguished from wild-type AAV by replacing all or part of the viral genome with non-native sequences. The incorporation of non-native sequences into the virus defines the viral vector as a "recombinant" vector and, therefore, a "rAAV vector." "rAAV vectors may include a heterologous polynucleotide (e.g., a human codon-optimized gene encoding human micro-dystrophin, e.g., SEQ ID NO: 1) encoding a desired protein or polypeptide (e.g., a dystrophin polypeptide or fragment thereof, e.g., SEQ ID NO: 2). The recombinant vector sequence may be encapsidated or packaged into an AAV capsid, referred to as an "rAAV vector," "rAAV vector particle," "rAAV viral particle," or simply "rAAV."

The present disclosure provides a method for purifying rAAV vectors, which contain polynucleotide sequences from non-AAV sources (e.g., polynucleotides heterologous to AAV). The heterologous polynucleotides may be flanked by at least one, sometimes two, AAV terminal repeats (e.g., inverted terminal repeats (ITRs)). The heterologous polynucleotides flanked by ITRs, also referred to herein as "vector genomes," typically encode polypeptides of interest or genes of interest ("GOIs"), such as targets for treatment (e.g., nucleic acids encoding dystrophin or fragments thereof for the treatment of Duchenne muscular dystrophy). Delivery or administration of rAAV vectors to subjects (e.g., patients) provides the subject with encoded proteins and peptides. Therefore, rAAV vectors can be used to transfer/deliver heterologous polynucleotides to express, for example, to treat various diseases, disorders, and conditions.

The rAAV vector genome usually retains 145 base ITRs in cis with heterologous nucleic acid sequences, which replace viral rep and cap genes. Such ITRs are necessary for the production of recombinant AAV vectors; however, modified AAV ITRs and non-AAV terminal repeats including partially or completely synthetic sequences can also achieve this purpose. ITRs form hairpin structures and act, for example, as primers for host cell-mediated complementary DNA chain synthesis after infection. ITRs also play a role in viral packaging, integration, etc. ITRs are the only cis AAV viral elements required for AAV genome replication and packaging into rAAV vectors. The rAAV vector genome optionally includes two ITRs, which are usually located at the 5' and 3' ends of the vector genome, and the vector genome includes heterologous sequences (e.g., transgenes encoding genes of interest or nucleic acid sequences of interest, including but not limited to antisense and siRNA, CRISPR molecules, etc.). 5' and 3' ITRs may both contain the same sequence, or may contain different sequences. AAV ITRs can be from any AAV, including but not limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 or any other AAV. ITRs are sequences that mediate AAV genome replication and packaging.

The rAAV vectors of the present disclosure may comprise ITRs from an AAV serotype (e.g., wild-type AAV2, fragments or variants thereof) that is different from the serotype of the capsid (e.g., AAV9 or others). Such rAAV vectors comprising at least one ITR from one serotype but comprising AAV capsid proteins from a different serotype may be referred to as hybrid viral vectors (see U.S. Pat. No. 7,172,893). AAV ITRs may include the entire wild-type ITR sequence, or a variant, fragment or modification thereof, but will retain functionality.

In some embodiments, the heterologous polypeptide comprises ITRs (e.g., ITRs from AAV2, but may comprise ITRs from any wild-type AAV serotype or variants thereof) located at the left and right ends (i.e., the 5' and 3' ends, respectively) of the vector genome. In some embodiments, the left (e.g., 5') ITR comprises or consists of the nucleic acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8. In some embodiments, the left (e.g., 5') ITR comprises a nucleic acid sequence that is about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or 100% identical to SEQ ID NO: 7 or SEQ ID NO: 8. In some embodiments, the right (e.g., 3') ITR comprises or consists of the nucleic acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8. In some embodiments, the right (e.g., 3') ITR comprises a nucleic acid sequence that is about 80%, about 85%, about 90%, about 95%, about 98%, about 99% or 100% identical to SEQ ID NO:7 or SEQ ID NO:8. Each ITR is in cis with each other or with other elements in the vector genome, but can be separated by a variable length of nucleic acid sequence, such as a recombinant nucleic acid comprising a modified nucleic acid encoding a dystrophin protein or a fragment thereof and a regulatory element. In some embodiments, the ITR is an AAV2 ITR or a variant thereof and is located on the side of the dystrophin transgene. In some embodiments, the rAAV comprises a dystrophin transgene (e.g., comprising a nucleic acid sequence of SEQ ID NO:1) flanked by AAV2 ITRs (e.g., ITRs having a sequence described in SEQ ID NO:7 or SEQ ID NO:8).

In some embodiments, the rAAV vector genome is linear, single-stranded and flanked by AAV ITR. Before the transcription and translation of the heterologous gene, the single-stranded DNA genome of about 4700 nucleotides must be converted into a double-stranded form, which is formed by DNA polymerase (e.g., DNA polymerase in transduced cells) using the free 3'-OH of one of the self-starting ITRs to start the second chain synthesis. In some embodiments, the full-length single-stranded vector genome (i.e., sense and antisense) is annealed to produce a full-length double-stranded vector genome. When multiple rAAV vectors carrying opposite polarity (i.e., sense or antisense) genomes are transduced to the same cell simultaneously, this situation can occur. No matter how it is produced, once a double-stranded vector genome is formed, the cell can transcribe and translate double-stranded DNA and express the heterologous gene.

The efficiency of transgene expression from rAAV vectors can be hampered by the need to convert the single-stranded rAAV genome (ssAAV) to double-stranded DNA prior to expression. This step can be circumvented by using self-complementary AAV genomes (scAAVs), which can package inverted repeat genomes that can fold into double-stranded DNA without the need for DNA synthesis or base pairing between multiple vector genomes (McCarty, (2008) Molec. Therapy 16(10):1648-1656; McCarty et al., (2001) Gene Therapy 8:1248-1254; McCarty et al., (2003) Gene Therapy 10:2112-2118). One limitation of scAAV vectors is that the size of the unique transgene, regulatory elements, and IRTs to be packaged in the capsid is approximately half the size of the ssAAV vector genome (i.e., approximately 4,900 nucleotides, including two ITRs) (i.e., approximately 2,500 nucleotides, of which 2,200 nucleotides can be the transgene and regulatory elements, plus two copies of the approximately 145 nucleotide ITRs).

The scAAV vector genome is prepared by deleting the terminal dissociation site (TRS) from one rAAV ITR of the expression plasmid, thereby preventing replication from that end (see U.S. Pat. No. 8,784,799). AAV replication in the host cell starts from the wild-type ITR of the genome, and continues to replicate through the mutant ITR without terminal dissociation, and then returns to the genome to produce a dimer. The dimer is a self-complementary genome with a mutant ITR in the middle and wild-type ITRs at each end. In some embodiments, the mutant ITR with a missing TRS is located at the 5' end of the vector genome. In some embodiments, the mutant ITR with a missing TRS is located at the 3' end of the vector genome. In some embodiments, the mutant ITR comprises the nucleic acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14.

Without wishing to be bound by theory, although the two halves of the scAAV genome are complementary, substantial base pairing is unlikely to occur within the capsid because many bases are in contact with amino acid residues within the capsid shell and the phosphate backbone is sequestered toward the center (McCarty, Molec. Therapy (2008) 16(10): 1648-1656). It is possible that after uncoating, the two halves of the scAAV genome anneal to form a dsDNA hairpin molecule with a covalently closed ITR at one end and two open-ended ITRs at the other end. The ITRs flank the double-stranded region encoding the transgene and its cis-regulatory elements.

The viral capsid of the rAAV vector can be derived from wild-type AAV or variant AAV, such as AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAVrh74 (see WO2016/210170), AAV12, AAV2i8, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, RHM4-1 (SEQ ID NO: WO 2015/013313), NO:5), RHM15-1, RHM15-2, RHM15-3/RHM15-5, RHM15-4, RHM15-6, AAVhu.26, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9,45, AAV2i8, AAV29G, AAV2,8G9, AVV-LK03, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N, AAV avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, snake AAV, goat AAV, shrimp AAV, sheep AAV and variants thereof (see, e.g., Fields et al., VIROLOGY, volume 2, chapter 69 (4 ^th ed., Lippincott-Raven Publishers). Capsids can be derived from many AAV serotypes, disclosed in U.S. Pat. No. 7,906,111; Gao et al. (2004) J. Virol. 78:6381; Morris et al. (2004) Virol. 33:375; WO 2013/063379; WO 2014/194132; and include authentic AAV (AAV-TT) variants, disclosed in WO 2015/121501, and RHM4-1, RHM15-1 to RHM15-6, and variants thereof, disclosed in WO2015/013313. Capsids may also be derived from AAV variants isolated from human CD34+ cells, including AAVHSC1, AAVHSC2, AAVHSC3, AAVHSC4, AAVHSC5, AAVHSC6, AAVHSC7, AAVHSC8, AAVHSC9, AAVHSC10, AAVHSC11, AAVHSC12, AAVHSC13, AAVHSC14, and AAVHSC15 (Smith et al. (2014) Molecular Therapy 22(9):1625-1634). Those skilled in the art will appreciate that there may be other AAV variants that have not yet been identified that perform the same or similar functions. The full complement of AAV cap proteins includes VP1, VP2, and VP3. An ORF comprising a nucleotide sequence encoding an AAV VP capsid protein may comprise less than a full complement of AAV Cap proteins, or may provide a full complement of AAV cap proteins.

In another embodiment, the present disclosure provides the use of ancestral AAV vectors for therapeutic in vivo gene therapy. Specifically, computer-derived sequences can be synthesized from scratch and their biological activity can be identified. In addition to being assembled into rAAV vectors, the prediction and synthesis of ancestral sequences can also be accomplished using the methods described in WO 2015/054653, the contents of which are incorporated herein by reference. It is noteworthy that rAAV vectors assembled from ancestral viral sequences show reduced susceptibility to pre-existing immunity in the population compared to contemporary viruses or portions thereof.

In some embodiments, rAAV vectors comprising capsid proteins encoded by nucleotide sequences derived from more than one AAV serotype (e.g., wild-type AAV serotype, variant AAV serotype) are referred to as "chimeric vectors" or "chimeric capsids" (see U.S. Pat. No. 6,491,907, the entire disclosure of which is incorporated herein by reference). In some embodiments, the chimeric capsid protein is encoded by nucleic acid sequences derived from 2, 3, 4, 5, 6, 7, 8, 9, 10 or more AAV serotypes. In some embodiments, the recombinant AAV vector comprises a capsid sequence derived from, for example, AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh74, AAVrs10, AAV2i8, or a variant thereof, thereby producing a chimeric capsid protein comprising a combination of amino acids from any of the aforementioned AAV serotypes (see Rabinowitz et al. (2002) J. Virology 76(2):791-801). Alternatively, the chimeric capsid may comprise a mixture of VP1 from one serotype, VP2 from a different serotype, VP3 from another different serotype, and combinations thereof. For example, a chimeric viral capsid may comprise an AAV1 cap protein or subunit and at least one AAV2 cap protein or subunit. Chimeric capsids can, for example, include AAV capsids with one or more B19 cap subunits, for example, AAV cap proteins or subunits can be replaced by B19 cap proteins or subunits. For example, in one embodiment, the VP3 subunit of the AAV capsid can be replaced by the VP2 subunit of B19.

In some embodiments, the chimeric vector has been engineered to exhibit altered tropism or tropism for a specific tissue or cell type. The term "tropism" refers to the preferential entry of a virus into certain cell or tissue types and/or preferential interaction with the cell surface, thereby facilitating entry into certain cell and tissue types. AAV tropism is generally determined by specific interactions between different viral capsid proteins and their cognate cell receptors (Lykken et al. (2018) J. Neurodev. Disord. 10: 16). Preferably, once the virus or viral vector enters the cell, the sequence (e.g., heterologous sequence, e.g., transgene) carried by the vector genome (e.g., rAAV vector genome) is expressed.

"Tropogenicity profile" refers to the pattern of transduction of one or more target cells, tissues, and/or organs. For example, an AAV capsid may have a tropism profile characterized by efficient transduction of muscle cells but only low transduction of, for example, brain cells.

3. Recombinant Nucleic Acids

The method disclosed herein includes purifying the rAAV vector comprising the recombinant nucleic acid including the modified nucleic acid and the plasmid and vector genome comprising the modified nucleic acid. The recombinant nucleic acid, plasmid or vector genome may include a regulatory sequence to regulate (e.g., plasmid) proliferation and/or control the expression of the modified nucleic acid (e.g., transgenic). The recombinant nucleic acid may also be provided as a component of a viral vector (e.g., rAAV vector). Typically, the viral vector includes a vector genome, which includes a recombinant nucleic acid packaged in a capsid.

Modified Nucleic Acids

The modified form or variant form of a gene, nucleic acid or polynucleotide (e.g., transgenic) refers to a nucleic acid that deviates from a reference sequence. The reference sequence can be a naturally occurring wild-type sequence (e.g., a gene), and can include naturally occurring variants (e.g., splice variants, variable reading frames). Those skilled in the art will appreciate that the reference sequence can be found in publicly available databases, such as GenBank (ncbi.nlm.nih.gov/genbank). Compared to the reference sequence, the modified/variant nucleic acid can have substantially the same, greater or less activity, function or expression. Preferably, the modified or variant nucleic acids used interchangeably herein show improved protein expression, for example, compared to the expression level of the protein provided by an endogenous gene (e.g., a wild-type gene, a mutant gene) in other identical cells, the protein encoded by it is expressed in cells at a detectable higher level. In some embodiments, the modified or variant nucleic acids used interchangeably herein show improved protein expression, for example, compared to the expression level of the protein provided by an endogenous gene comprising a mutation in other identical cells, the protein encoded thereby is expressed in cells at a detectable higher level.

Modifications to nucleic acids include one or more modifications of nucleotide substitutions (e.g., substitutions of 1-3, 3-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-100 or more nucleotides), additions (e.g., insertions of 1-3, 3-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-100 or more nucleotides), deletions (e.g., deletions of 1-3, 3-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-100 or more nucleotides, deletions of motifs, domains, fragments, etc.) in the reference sequence. The modified nucleic acid can be about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the reference sequence.

The modified nucleic acid can encode a polypeptide having about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99% or 100% identity to a reference polypeptide. In some embodiments, the modified nucleic acid encodes a polypeptide having 100% identity to a reference polypeptide.

In some embodiments, the modified nucleic acid (e.g., transgenic) encodes a wild-type protein. Such modified nucleic acids can be codon-optimized. "Optimized" or "codon-optimized" interchangeably mentioned herein refers to a coding sequence that has been optimized relative to a wild-type coding sequence or a reference sequence (e.g., a coding sequence of a micro-dystrophin polypeptide, e.g., SEQ ID NO: 2, a coding sequence of a deleted copper transport ATPase2 polypeptide, e.g., SEQ ID NO: 15) to increase the expression of the polypeptide, e.g., by minimizing the use of rare codons, reducing the number of CpG dinucleotides, removing hidden splicing donor or acceptor sites, removing Kozak sequences, removing ribosome entry sites, etc. In some embodiments, the expression level of a protein from a codon-optimized sequence is increased compared to the expression level of a protein from a wild-type gene in other identical cells. In some embodiments, the expression level of a protein from a codon-optimized sequence is not increased (e.g., substantially similar expression) compared to the expression level of a protein from a wild-type gene in other identical cells. In some embodiments, the expression level of a protein from a codon-optimized sequence is increased compared to the expression level of a protein from a mutant gene in an otherwise identical cell.

Examples of modifications include elimination of one or more cis-acting motifs and introduction of one or more Kozak sequences. In some embodiments, one or more cis-acting motifs are eliminated and one or more Kozak sequences are introduced.

Examples of cis-acting motifs that can be eliminated include internal TATA boxes; chi sites; ribosome entry sites; ARE, INS and/or CRS sequence elements; repetitive sequences and/or RNA secondary structures; (cryptic) splice donor and/or acceptor sites, branch points; and restriction sites.

In some embodiments, the modified nucleic acid encodes a modified or variant polypeptide. The modified polypeptide encoded by the modified nucleic acid (e.g., a codon-optimized micro-dystrophin) can retain all or part of the function or activity of the polypeptide encoded by the wild-type or reference sequence. In some embodiments, the modified polypeptide has one or more non-conservative or conservative amino acid changes. In some embodiments, certain domains that have been shown to have limited or ineffective effects in the function of the polypeptide are not present in the modified polypeptide (e.g., certain binding domains) (e.g., WO 2016/097219). Due to the packaging capacity of the rAAV capsid, the modified nucleic acid present in the rAAV vector may contain fewer nucleotides than the wild-type coding sequence or reference sequence (e.g., shortened micro-dystrophin transgene, see WO 2001/83695; B domain deleted human factor VIII transgene, see WO 2017/074526, all of which are incorporated herein by reference), and also includes truncated and codon-optimized shortened transgenes (e.g., codon-optimized micro-dystrophin transgene described in WO 2017/221145; deleted copper-transporting ATPase2 with metal binding site (MBS) 1-4 deletion, see WO 2016/097219 and WO 2016/07218, all of which are incorporated herein by reference). In some embodiments, the polypeptide encoded by the modified nucleic acid has a function or activity that is smaller, the same, or larger than that of the polypeptide encoded by the reference sequence, but is at least a portion thereof.

Relative to a reference sequence and/or a wild-type sequence, a modified nucleic acid can have a modified GC content (e.g., the number of G and C nucleotides present in a nucleic acid sequence), a modified (e.g., increased or decreased) CpG dinucleotide content, and/or a modified (e.g., increased or decreased) codon adaptation index (CAI). See, e.g., WO 2017/07451 (discussing various considerations for codon optimization of nucleic acid sequences of interest well known in the art, including publicly available software for analyzing nucleic acid sequences for optimization). As used herein, modification refers to a decrease or increase in a particular value, amount, or effect.

In some embodiments, the GC content of the modified nucleic acid sequence of the present disclosure is increased relative to the reference sequence and/or wild-type gene or coding sequence. The GC content of the modified nucleic acid is at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 14%, at least 15%, at least 17%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% higher than the GC content of the wild-type coding sequence. In some embodiments, the GC content is expressed as the percentage of G (guanine) and C (cytosine) nucleotides in the sequence.

In some embodiments, the codon adaptation index of the modified nucleic acid sequences of the present disclosure is at least 0.74, at least 0.76, at least 0.77, at least 0.80, at least 0.85, at least 0.86, at least 0.87, at least 0.90, at least 0.95, or at least 0.98.

In some embodiments, the modified nucleic acid sequences of the present disclosure have reduced CpG dinucleotide levels compared to wild-type or reference nucleic acid sequences, i.e., reduced by about 10%, 20%, 30%, 50% or more. In some embodiments, the modified nucleic acid has 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-45 or 45-50 or even fewer dinucleotides than a reference sequence (e.g., a wild-type sequence).

It is known that methylation of CpG dinucleotides plays an important role in the regulation of gene expression in eukaryotes. Specifically, methylation of CpG dinucleotides in eukaryotes essentially silences gene expression by interfering with the transcriptional machinery. Therefore, nucleic acids and vectors with reduced numbers of CpG dinucleotides will provide high and lasting transgene expression levels due to gene silencing caused by methylation of CpG motifs.

The modified nucleic acid sequence may include flanking restriction sites to facilitate subcloning into an expression vector. Many such restriction sites are well known in the art and include, but are not limited to, AvaI, SwaI, ApaL1, and XmaI.

The present disclosure includes modified nucleic acids of SEQ ID NO: 1, which encode functionally active fragments of a dystrophin polypeptide. "Functionally active" or "functional dystrophin polypeptide" means that the fragment provides the same or similar biological function and/or activity as the full-length dystrophin polypeptide. That is, the fragment provides the same function, including but not limited to being a structural protein of the myofilaments of muscle fibers. The biological activity of the functional fragments of dystrophin includes reversing or preventing the neuromuscular phenotype associated with Duchenne muscular dystrophy.

Thus, one embodiment of the present invention relates to a method for purifying a rAAV vector comprising a modified nucleic acid encoding a micro-dystrophin protein, the nucleic acid comprising, consisting essentially of, or consisting of a nucleic acid sequence of SEQ ID NO: 1 or a sequence at least about 90% identical thereto. In some embodiments, the nucleic acid is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the length of the nucleic acid is within the capacity of a viral vector, such as a parvoviral vector, such as an rAAV vector. In some embodiments, the length of the nucleic acid is about 5000, 4900, 4800, 4700, 4600, 4500, 4400, 4300, 4200, 4100 or about 4000 nucleotides or less.

In some embodiments, the nucleic acid encodes a micro-dystrophin protein comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO:2, or a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO:2.

In some embodiments, the nucleic acid encodes a deleted copper-transporting ATPase2 protein comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 15, or a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 15.

In some embodiments, the nucleic acid (e.g., SEQ ID NO: 1) is part of a recombinant nucleic acid for producing a dystrophin protein. The recombinant nucleic acid may further comprise a regulatory element for increasing the expression of the dystrophin protein. In some embodiments, the nucleic acid is part of a recombinant nucleic acid for producing a copper transporting ATPase2 protein. The recombinant nucleic acid may further comprise a regulatory element for increasing the expression of the copper transporting ATPase2.

Adjustment element

The method disclosed herein includes purifying rAAV vectors, and the rAAV vectors include recombinant nucleic acids and various regulatory or control elements, and the recombinant nucleic acids include modified nucleic acids encoding polypeptides (e.g., micro-dystrophin). Generally, regulatory elements are nucleic acid sequences that affect the expression of operably connected polynucleotides. The precise properties of regulatory elements that can be used for gene expression will vary from organism to organism and from cell type to cell type, including, for example, promoters, enhancers, introns, etc., with the purpose of promoting appropriate heterologous polynucleotide transcription and translation. Regulatory control can be affected at levels such as transcription, translation, splicing, information stability, etc. Generally, the regulatory control elements that regulate transcription are juxtaposed near the 5' ends of the polynucleotides transcribed (i.e., upstream). Regulatory control elements can also be located at the 3' ends (i.e., downstream) of the sequence transcribed or in the transcript (e.g., in an intron). Regulatory control elements can be located at a certain distance from the sequence transcribed (e.g., 1 to 100, 100 to 500, 500 to 1000, 1000 to 5000, 5000 to 10,000 or more nucleotides). However, due to the length of the AAV vector genome, regulatory control elements are typically within 1 to 1000 nucleotides of the polynucleotide.

Promoter

As used herein, the term "promoter", such as "eukaryotic promoter", refers to a nucleotide sequence that initiates transcription of a specific gene or one or more coding sequences (such as a micro-dystrophin coding sequence) in a eukaryotic cell (such as a muscle cell). A promoter can work with other regulatory elements or regions to direct the level of transcription of a gene or coding sequence. These regulatory elements include, for example, transcription binding sites, repressor and activator protein binding sites, and other nucleotide sequences known to act directly or indirectly to regulate the amount of transcription from the promoter, including, for example, attenuators, enhancers, and silencers. Promoters are usually located on the same chain, near the transcription start site at the 5' end of the gene or coding sequence to which they are operably connected. The length of a promoter is usually 100-1000 nucleotides. A promoter usually increases gene expression relative to the expression of the same gene in the absence of a promoter.

As used herein, "core promoter" or "minimal promoter" refers to the minimum portion of a promoter sequence required to properly initiate transcription. It may include any of the following sites: a transcription start site, a binding site for RNA polymerase, and a general transcription factor binding site. The promoter may also include a proximal promoter sequence (5' end of the core promoter) containing other major regulatory elements (e.g., enhancers, silencers, boundary elements, insulators) and a distal promoter sequence (3' end of the core promoter). In some embodiments, the core or minimal promoter is an alpha 1-antitrypsin core or minimal promoter, optionally comprising or consisting of a nucleic acid of SEQ ID NO: 16.

Examples of suitable promoters include adenovirus promoters, such as the adenovirus major late promoter; heterologous promoters, such as the cytomegalovirus (CMV) promoter; respiratory syncytial virus promoter; Rous sarcoma virus (RSV) promoter; albumin promoter; inducible promoters, such as the mouse mammary tumor virus (MMTV) promoter; metallothionein promoter; heat shock promoter; alpha-1-antitrypsin promoter; hepatitis B surface antigen promoter; transferrin promoter; apolipoprotein A-1 promoter; chicken β-actin (CBA) promoter; elongation factor 1a (EF1a) promoter; a hybrid form of the CBA promoter (CBh promoter); CAG promoter (cytomegalovirus early enhancer element and promoter, the first exon and first intron of the chicken β-actin gene, and the splice acceptor of the rabbit β-globin gene) (Alexopoulou et al. (2008) BioMed. Central Cell Biol. 9:2); creatine kinase promoter; and human dystrophin gene promoter.

In some embodiments, the promoter is a creatine kinase promoter, such as a promoter comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:4.

In some embodiments of the present disclosure, a eukaryotic promoter sequence (e.g., a creatine kinase promoter) is operably linked to a modified nucleic acid encoding, for example, microdystrophin or a deleted copper-transporting ATPase2. In some embodiments, a promoter comprising a nucleic acid sequence of SEQ ID NO:3 or SEQ ID NO:6 (e.g., a creatine kinase promoter) is operably linked to a modified nucleic acid encoding microdystrophin. In some embodiments, a promoter comprising a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence of SEQ ID NO:3 or SEQ ID NO:4 is operably linked to a nucleic acid comprising a nucleic acid sequence of SEQ ID NO:1. In some embodiments, a promoter comprising a nucleic acid sequence of SEQ ID NO:16 (e.g., an α1-antitrypsin promoter) is operably linked to a modified nucleic acid encoding a deleted copper-transporting ATPase2 having an MBS 1-4 deletion (e.g., SEQ ID NO:15). In some embodiments, a promoter comprising a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO:16 is operably linked to a nucleic acid comprising the amino acid sequence of SEQ ID NO:15.

In some embodiments, a promoter comprising a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NO:3 or SEQ ID NO:4 is operably linked to a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NO:1 and induces expression of a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:1 in muscle cells.

Promoters can be constitutive, tissue-specific or regulated. Constitutive promoters are those that cause operably linked genes to be expressed substantially at all times. In some embodiments, constitutive promoters are active in most eukaryotic tissues under most physiological and developmental conditions.

Regulated promoters are those that can be activated or inactivated. Regulated promoters include inducible promoters, which are normally "off" but can be induced to be "on," and "repressible" promoters, which are normally "on" but can be "off." Many different regulators are known, including temperature, hormones, cytokines, heavy metals, and regulatory proteins. The distinction is not absolute; constitutive promoters can usually be regulated to some degree. In some cases, endogenous pathways can be used to provide regulation of transgene expression, such as using promoters that are naturally downregulated when the pathological condition is ameliorated.

Tissue-specific promoters refer to promoters that are active only in specific types of tissues, cells or organs. Typically, tissue-specific promoters are identified by transcriptional activation elements specific to specific tissues, cells and/or organs. For example, tissue-specific promoters may be more active in one or more specific tissues (e.g., two, three or four) than in other tissues. In some embodiments, the expression of genes regulated by tissue-specific promoters in tissues specific to the promoter is much higher than in other tissues. In some embodiments, the promoter may have almost no or substantially no activity in any tissue other than its specific tissue. The promoter may be a tissue-specific promoter active in hepatocytes, such as a mouse albumin promoter or a transthyretin promoter (TTR). Other examples of tissue-specific promoters include promoters from genes encoding skeletal muscle α-actin, myosin light chain 2A, dystrophin, and muscle creatine kinase, which induce expression in skeletal muscle (Li et al. (1999) Nat. Biotech. 17: 241-245). Liver-specific expression can be induced using promoters from the albumin gene (Miyatake et al. (1997) J. Virol. 71:5124-5132), the hepatitis B virus core promoter (Sandig, et al. (1996) Gene Ther. 3:1002-1009), and alpha-fetoprotein (Arbuthnot et al., (1996) Hum. Gene. Ther. 7:1503-1514).

Enhancer

In another aspect, the modified nucleic acid encoding the therapeutic polypeptide further comprises an enhancer to increase the expression of the therapeutic polypeptide. Typically, the enhancer element is located upstream of the promoter element, but may also be located downstream or internal to another sequence (e.g., transgenic). The enhancer may be located 100 nucleotides, 200 nucleotides, 300 nucleotides, or more upstream or downstream of the modified nucleic acid. The enhancer typically increases the expression of the modified nucleic acid (e.g., encoding the therapeutic polypeptide) beyond the expression of the increase provided by the promoter element alone.

Many enhancers are known in the art, including but not limited to the cytomegalovirus major immediate early enhancer. More specifically, the cytomegalovirus (CMV) MIE promoter comprises three regions: a regulator, a unique region and an enhancer (Isomura and Stinski (2003) J. Virol. 77 (6): 3602-3614). The CMV enhancer region can be combined with another promoter or a portion thereof to form a hybrid promoter, thereby further increasing the expression of the nucleic acid operably connected thereto. For example, the chicken beta-actin (CBA) promoter or a portion thereof can be combined with the CMV promoter/enhancer or a portion thereof to produce a CBA version called a "CBh" promoter, which represents a chicken beta -actin hybrid promoter, as described in Gray et al. (2011, Human Gene Therapy 22: 1143-1153). Like a promoter, an enhancer can be constitutive, tissue-specific or regulated.

In some embodiments of the present disclosure, the regulatory element comprises a hybrid enhancer and a promoter, such as a synthetic hybrid enhancer or promoter derived from a creatine kinase (CK) gene, which is used as a muscle-specific transcriptional regulatory element (hCK) and is operably linked to a modified nucleic acid encoding a micro-dystrophin. In some embodiments, a synthetic hybrid enhancer and promoter comprising a nucleic acid sequence of SEQ ID NO:5 is operably linked to a modified nucleic acid encoding a micro-dystrophin. In some embodiments, a synthetic hybrid enhancer and promoter derived from a creatine kinase (CK) gene is operably linked to a nucleic acid comprising a nucleic acid sequence of SEQ ID NO:1, and the creatine kinase gene comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence of SEQ ID NO:5.

In some embodiments, a synthetic hybrid enhancer and promoter derived from a creatine kinase (CK) gene comprises a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NO:5, which is operably linked to a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NO:1, and induces expression of a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:1 in muscle cells.

Filling sequences, spacer sequences, and padding sequences

As disclosed herein, recombinant nucleic acids for rAAV vectors can include additional nucleic acid elements to adjust the length of the nucleic acid to a near or normal size (e.g., about 4.7 to 4.9 kilobases) of a viral genomic sequence acceptable for AAV packaging into rAAV vectors (Grieger and Samulski (2005) J. Virol. 79 (15): 9933-9944). Such sequences can be interchangeably referred to as filler sequences, spacer sequences, or stuffer sequences. In some embodiments, the stuffer DNA is an untranslated (non-protein coding) segment of the nucleic acid. In some embodiments, the filler or stuffer polynucleotide sequence is a sequence of about 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90-90-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1000, 1000-1500, 1500-2000, 2000-3000, or more nucleotides in length.

AAV vectors generally accept DNA inserts of sizes of about 4kb to about 5.2kb or about 4.1 to 4.9kb to optimally package nucleic acids into AAV capsids. In some embodiments, rAAV vectors include a total length of about 3.0kb to about 3.5kb, about 3.5kb to about 4.0kb, about 4.0kb to about 4.5kb, about 4.5kb to about 5.0kb, or about 5.0kb to about 5.2kb of vector genome. In some embodiments, rAAV vectors include a total length of about 4.5kb of vector genome. In some embodiments, rAAV vectors include self-complementary vector genomes. Although the total length of the self-complementary (sc) vector genome in the rAAV vector is equal to the single-stranded (ss) vector genome (i.e., about 4kb to about 5.2kb), the nucleic acid sequence encoding the sc vector genome (i.e., comprising transgene, regulatory elements, and ITR) must only have half the length of the nucleotide sequence encoding the ss vector genome, so that the sc vector genome is packaged in the capsid.

Introns and Exons

In some embodiments, the recombinant nucleic acid includes, for example, introns, exons and/or a portion thereof. Introns can be used as filling or filling polynucleotide sequences to achieve the appropriate length of the vector genome packaged into the rAAV vector. Compared with the expression in the absence of introns and/or exon elements, introns and/or exon sequences can also enhance the expression of polypeptides (e.g., transgenics) (Kurachi et al. (1995) J. Biol. Chem. 270 (10): 576-5281; WO 2017/074526). In addition, filling/filling polynucleotide sequences (also referred to as "insulators") are well known in the art, including but not limited to those described in WO 2014/14486 and WO 2017/074526.

Intronic elements can be derived from the same gene as the heterologous polynucleotide, or from an entirely different gene or other DNA sequence (e.g., chicken β-actin gene, minute virus of mice (MVM)). In some embodiments, the recombinant nucleic acid includes at least one element selected from introns and exons derived from a non-homologous gene (i.e., not derived from a modified nucleic acid such as a transgene).

Polyadenylation signal sequence (polyA)

Further regulatory elements may include stop codons, termination sequences, and polyadenylation (polyA) signal sequences, such as, but not limited to, the bovine growth hormone polyA signal sequence (BHG-polyA). The polyA signal sequence drives the efficient addition of a polyadenosine "tail" at the 3' end of eukaryotic mRNA, which directs the termination of gene transcription (see, e.g., Goodwin and Rottman J. Biol. Chem. (1992) 267(23): 16330-16334). The polyA signal serves as a signal for endonucleolytic cleavage of the newly formed pre-mRNA at its 3' end and for the addition of an RNA sequence consisting only of adenine bases at this 3' end. The polyA tail is important for nuclear export, translation, and stability of the mRNA. In some embodiments, polyA is an SV40 early polyadenylation signal, an SV40 late polyadenylation signal, an HSV thymidine kinase polyadenylation signal, a protamine gene polyadenylation signal, an adenovirus 5E1b polyadenylation signal, a growth hormone polyadenylation signal, a PBGD polyadenylation signal, or a computer-designed polyadenylation signal.

In some embodiments, and optionally in combination with one or more additional regulatory elements described herein, the polyA signal sequence of the recombinant nucleic acid is a polyA signal capable of directing and effecting endonucleolytic cleavage and polyadenylation of a pre-mRNA resulting from transcription of a modified nucleic acid encoding, for example, micro-dystrophin (e.g., SEQ ID NO: 2) or deleted copper-transporting ATPase 2 (e.g., SEQ ID NO: 15). In some embodiments, the polyA sequence comprises or consists of a nucleic acid sequence of SEQ ID NO: 6 or SEQ ID NO: 17. In some embodiments, the polyA sequence comprises a nucleic acid sequence that is about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or 100% identical to a nucleic acid sequence of SEQ ID NO: 6 or SEQ ID NO: 17. In some embodiments, the recombinant nucleic acid comprises at least one of the following elements: a promoter sequence (e.g., SEQ ID NO: 3, SEQ ID NO: 4), a hybrid enhancer and promoter (e.g., SEQ ID NO: 5), and polyA (SEQ ID NO: 6), and regulates the expression of a heterologous polypeptide optionally encoded by the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the recombinant nucleic acid comprises at least one of the following elements: a promoter sequence (e.g., SEQ ID NO: 16) and polyA (SEQ ID NO/17), and regulates the expression of a heterologous polypeptide comprising the amino acid sequence of SEQ ID NO: 15.

In some embodiments, the rAAV9 vector having tropism for muscle cells contains a vector genome comprising AAV ITRs (e.g., AAV2 ITRs) and a recombinant nucleic acid comprising a modified (i.e., codon-optimized) nucleic acid encoding micro-dystrophin and at least one of the following regulatory elements: a promoter (e.g., human CK promoter), a hybrid enhancer, and a poly A signal sequence.

In some embodiments, a rAAV3B vector tropism for hepatocytes contains a vector genome comprising an AAV ITR (e.g., AAV2 ITR) and a recombinant nucleic acid comprising a modified (i.e., codon-optimized) nucleic acid encoding a deleted copper-transporting ATPase 2 (e.g., the amino acid sequence of SEQ ID NO: 15) and at least one of the following regulatory elements: a promoter (e.g., an α1-antitrypsin promoter, e.g., having a nucleic acid sequence of SEQ ID NO: 16) and a poly A signal sequence (e.g., a nucleic acid of SEQ ID NO: 17).

In some embodiments, the rAAV 9 vector having tropism for muscle cells contains a vector genome comprising AAV ITRs (e.g., SEQ ID NO:7, SEQ ID NO:8) and a recombinant nucleic acid comprising a modified (i.e., codon-optimized) nucleic acid encoding micro-dystrophin (e.g., SEQ ID NO:1), and at least one of the following regulatory elements: a promoter (e.g., SEQ ID NO:3 or SEQ ID NO:4), a hybrid enhancer and promoter (e.g., SEQ ID NO:5), and polyA (e.g., SEQ ID NO:6).

4. Assembly of Viral Vectors

Viral vectors (e.g., rAAV vectors) carrying a transgene (e.g., encoding micro-dystrophin) are assembled from polynucleotides encoding the transgene, appropriate regulatory elements, and elements required to produce viral proteins that mediate cell transduction. Examples of viral vectors include, but are not limited to, adenovirus, retrovirus, lentivirus, herpes virus, and adeno-associated virus (AAV) vectors, particularly rAAV vectors (described above).

The vector genome components of the rAAV vectors produced according to the methods of the present disclosure include at least one transgene, such as a codon-optimized micro-dystrophin transgene and associated expression control sequences for controlling the expression of a modified nucleic acid encoding a dystrophin protein or a fragment thereof.

In an exemplary non-limiting embodiment, the vector genome includes a portion of a parvoviral genome, such as an AAV genome and its associated expression control sequences having a rep and cap deletion and/or replaced by a modified nucleic acid (e.g., a transgene, such as a codon-optimized micro-dystrophin transgene). The modified nucleic acid encoding the dystrophin protein or its fragment is typically inserted adjacent to one or two (i.e., flanked by) AAV ITR or ITR elements sufficient for viral replication (Xiao et al. (1997) J. Virol. 71 (2): 941-948) to replace the nucleic acid encoding the viral rep and cap proteins. Other regulatory sequences suitable for promoting tissue-specific expression of the codon-optimized micro-dystrophin transgene in target cells (e.g., muscle cells) may also be included.

Packaging cells

Those skilled in the art will appreciate that rAAV vectors that contain a transgene and lack viral proteins required for viral replication (e.g., cap and rep) cannot replicate because these proteins are essential for viral replication and packaging. The cap and rep genes can be provided to a cell (e.g., a host cell, such as a packaging cell) as part of a plasmid that is separate from the plasmid that provides the transgene for the vector genome.

"Packaging cells" or "production cells" refer to cells or cell lines that can be transfected with vectors, plasmids or DNA constructs, and trans-provide all the missing functions required for complete replication and packaging of viral vectors. The genes required for rAAV vector assembly include the vector genome (e.g., codon-optimized micro-dystrophin transgene, regulatory elements and ITRs), AAVrep genes, AAV cap genes, and certain auxiliary genes from other viruses such as adenovirus. Those skilled in the art will appreciate that the genes required for AAV production can be introduced into packaging cells in various ways, including, for example, transfection of one or more plasmids. However, in some embodiments, some genes (e.g., rep, cap, auxiliary genes) may already be present in the packaging cells, or integrated into the genome, or carried on exosomes. In some embodiments, the packaging cells express one or more deleted viral functions in a constitutive or inducible manner.

Any suitable packaging cell known in the art can be used to produce packaged viral vectors. Preferred mammalian cells or insect cells. Examples of cells that can be used to produce packaging cells in the practice of the present disclosure include, for example, human cell lines, such as PER.C6, WI38, MRC5, A549, HEK293 (which express functional adenovirus E1 under the control of a constitutive promoter), B-50 or any other HeLa cells, HepG2, Saos-2, HuH7 and HT1080 cell lines. Suitable non-human mammalian cell lines include, for example, VERO, COS-1, COS-7, MDCK, BHK21-F, HKCC or CHO cells.

In some embodiments, the packaging cells can be grown in suspension culture. In some embodiments, the packaging cells can be grown in serum-free medium. For example, HEK293 cells are grown in suspension in serum-free medium. In another embodiment, the packaging cells are HEK293 cells, as described in U.S. Patent No. 9,441,206, and deposited in the American Type Culture Collection (ATCC) with PTA No. 13274. Many rAAV packaging cell lines are known in the art, including but not limited to those disclosed in WO2002/46359.

Cell lines used as packaging cells include insect cell lines. According to the present disclosure, any insect cell that allows AAV replication and can be maintained in culture can be used. Examples include Spodopterafrugiperda, such as Sf9 or Sf21 cell lines, Drosophila spp. cell lines, or mosquito cell lines, such as Aedes albopictus derived cell lines. A preferred cell line is the Spodopterafrugiperda Sf9 cell line. The following references are incorporated herein for teaching methods for expressing heterologous polypeptides using insect cells, methods for introducing nucleic acids into such cells, and methods for maintaining such cells in culture: Methods in Molecular Biology, ed. Richard, Humana Press, NJ (1995); O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual, Oxford Univ. Press (1994); Samulski et al. (1989) J. Virol. 63:3822-3828; Kajigaya et al. (1991) Proc. Nat'l. Acad. Sci. USA 88:4646-4650; Ruffing et al. (1992) J. Virol. 66:6922-6930; Kimbauer et al. (1996) Virol. 219:37-44; Zhao et al. (1997) Proc. Nat'l. Acad. Sci. USA 88:4646-4650; et al. (2000) Virol. 272:382-393; and U.S. Patent No. 6,204,059.

As a further alternative, the viral vectors of the present disclosure can be produced in insect cells using baculovirus vectors to deliver the rep/cap genes and using rAAV templates, such as described by Urabe et al. (2002) Human Gene Therapy 13: 1935-1943. When using baculovirus to produce AAV, in some embodiments, the vector genome is self-complementary. In some embodiments, the host cell is a baculovirus-infected cell (e.g., an insect cell), which optionally contains other nucleic acids encoding baculovirus helper functions to promote the production of viral capsids.

Packaging cells typically include one or more viral vector functions as well as auxiliary functions and packaging functions sufficient to cause viral vector replication and packaging. These different functions can be provided to packaging cells together or separately using genetic constructs such as plasmids or amplicons, and they can be present outside the chromosomes of the cell line or integrated into the chromosomes of the host cell. In some embodiments, packaging cells are transfected with the following plasmids: i) a plasmid comprising a vector genome comprising a transgene and AAV ITR and further comprising at least one of the following regulatory elements: enhancers, promoters, exons, introns and poly A, and ii) a plasmid comprising a rep gene (e.g., AAV2 rep) and a cap gene (e.g., AAV9 or other caps).

In some embodiments, a host cell is provided with one or more packaging or helper functions incorporated therein, for example, a host cell line has one or more vector functions incorporated extrachromosomally or integrated into the chromosomal DNA of the cell.

Accessibility

AAV is a dependent virus that cannot replicate in cells without the co-infection of helper viruses. Auxiliary functions include auxiliary virus elements required for establishing active infection of packaging cells, which are required for initiating viral vector packaging. Helper viruses generally include adenovirus or herpes simplex virus. Adenovirus auxiliary functions generally include adenovirus components adenovirus early region 1A (E1a), E1b, E2a, E4 and virus-related (VA) RNA. Auxiliary functions (e.g., E1a, E1b, E2a, E4 and VA RNA) can be provided to packaging cells by transfecting packaging cells with one or more nucleic acids encoding various auxiliary elements. Alternatively, host cells (e.g., packaging cells) can contain nucleic acids encoding auxiliary proteins. For example, HEK293 cells are produced by transforming human cells with adenovirus 5 DNA, and now express many adenovirus genes, including but not limited to E1 and E3 (see, e.g., Graham et al. (1977) J. Gen. Virol. 36: 59-72). Therefore, these auxiliary functions can be provided by HEK 293 packaging cells without the need to provide them to cells by, for example, plasmids encoding them. In some embodiments, the packaging cells are transfected with at least the following plasmids: i) a plasmid comprising a vector genome comprising a transgene and an AAV ITR and further comprising at least one of the following regulatory elements: an enhancer, a promoter, an exon, an intron, and poly A, and ii) a plasmid comprising a rep gene (e.g., AAV2 rep) and a cap gene (such as AAV9 or other cap), and iii) a plasmid comprising helper functions.

Any method for introducing the nucleotide sequence carrying the auxiliary function into the cell host for replication and packaging can be adopted, including but not limited to electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, carrier molecules (e.g., polyethyleneimine (PEI)) and liposomes in combination with nuclear localization signals. In some embodiments, the auxiliary function is provided by transfection using a viral vector or by infection using a helper virus, and standard methods for producing viral infection can be used.

The vector genome may be any suitable recombinant nucleic acid, such as a DNA or RNA construct, and may be single-stranded, double-stranded or duplexed (ie self-complementary as described in WO 2001/92551).

4. Production of Packaged Viral Vectors

Viral vectors can be prepared by several methods known to those skilled in the art (see, e.g., WO 2013/063379). An exemplary non-limiting method is described in Grieger, et al. (2015) Molecular Therapy 24 (2): 287-297, the contents of which are incorporated herein by reference for all purposes. In brief, efficient transfection of HEK293 cells is used as a starting point, where an adherent HEK293 cell line from a qualified clinical master cell bank is used to grow in a suspension without animal components in a shake flask and a WAVE bioreactor, so that rapid and scalable rAAV production is possible. Using a triple transfection method (e.g., WO 96/40240), when harvested 48 hours after transfection, a HEK293 cell line suspension can produce more than 1×10 ⁵ particles (VG)/cell containing a vector genome, or more than 1×10 ¹⁴ VG/L cell culture. More specifically, triple transfection refers to a method in which packaging cells are transfected with three plasmids: one plasmid encodes the AAV rep and cap (e.g., AAV9 cap) genes, another plasmid encodes various helper functions (e.g., adenovirus or HSV proteins, such as E1a, E1b, E2a, E4, and VA RNA), and a third plasmid encodes a transgene (e.g., dystrophin or a fragment thereof) and various elements that control transgene expression.

The single-stranded vector genome is packaged into the capsid as a positive strand or a negative strand in approximately equal proportions. In some embodiments of the rAAV vector, the vector genome is in positive strand polarity (i.e., the sense or coding sequence of the DNA strand). In some embodiments of the rAAV vector, the vector is in negative strand polarity (i.e., the antisense or template DNA strand). In view of the nucleotide sequence of the positive strand in its 5' to 3' direction, the nucleotide sequence of the 5' to 3' direction of the negative strand as the reverse complementary strand of the positive strand nucleotide sequence can be determined.

To achieve the desired yields, many variables are optimized, such as the selection of a compatible serum-free suspension medium that supports growth and transfection, the choice of transfection reagent, transfection conditions, and cell density.

rAAV vectors can be purified by methods standard in the art, such as column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors are known in the art, including Clark et al. (1999) Human Gene Therapy 10(6):1031-1039; Schenpp and Clark (2002) Methods Mol. Med. 69:427-443; U.S. Pat. No. 6,566,118 and methods described in WO 98/09657.

After the rAAV vector of the present disclosure is produced and purified according to the methods disclosed herein, it can be titered (e.g., the amount of rAAV vector in a sample can be quantified) to prepare a composition for administration to a subject (e.g., a human subject suffering from Duchenne muscular dystrophy). rAAV vector titering can be accomplished using methods known in the art.

In some embodiments, the number of viral particles, including particles containing vector genomes and "empty" capsids that do not contain vector genomes, can be determined by electron microscopy, such as transmission electron microscopy (TEM). This TEM-based method can provide the number of vector particles (or viral particles in the case of wild-type AAV) in a sample. In some embodiments, the amount of particles containing vector genomes (complete capsids) and "empty" capsids that do not contain vector genomes can be determined by charge detection mass spectrometry, analytical ultracentrifugation (AUC), and/or measuring absorbance at 260 nm and 280 nm to determine the A260/A280 ratio.

In some embodiments, the rAAV vector genome can be titered using quantitative PCR (qPCR), using primers for any sequence in the vector genome, such as ITR sequences (e.g., SEQ ID NO: 7 or SEQ ID NO: 8) and/or sequences (or regulatory elements) in the transgene. By performing qPCR in parallel on dilutions of known concentrations of standards (e.g., plasmids containing vector genome sequences), a standard curve can be generated, and the concentration of the rAAV vector can be calculated as the number of vector genomes (VG) per unit volume (e.g., microliters or milliliters). By comparing the number of vector particles measured by, for example, SEC or ELISA with the number of vector genomes in the sample, the percentage of empty capsids can be estimated. Because the vector genome contains a therapeutic transgene, the vg/kg or vg/ml of the vector sample may indicate more than the number of vector particles (some of which may be empty and do not contain a vector genome) the therapeutic amount of the vector that the subject will receive. Once the concentration of the rAAV vector genome in the stock solution is determined, it can be diluted or dialyzed in an appropriate buffer for use in preparing a composition (eg, a drug substance) for administration to a subject (eg, a subject suffering from Duchenne muscular dystrophy).

5. Purification of rAAV vectors by anion exchange chromatography (AEX)

A novel general purification strategy based on ion exchange chromatography can be used to produce highly pure rAAV vector preparations of various AAV serotypes and/or from chimeric capsids (e.g., AAV1, AAV2, AAV3 including AAV3A and AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV12, AAVrh10, AAVrh74, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV, and recombinantly produced variants (e.g., capsid variants with insertions, deletions, substitutions, etc.), such as those referred to as AAV2i8, NP4, NP22, NP66, AAVDJ, AAVDJ/8, AAVDJ/9, AAVLK03, RHM4-1, AAVHSC1, AAVHSC2, AAVHSC 3, AAVHSC4, AAVH SC5, AAVHS C6, AAVHs C7, AAVHFSC8, AAVHsc 9, AAVHSC10, AAVHSC11, AAVHSC12, AAVHSC13, AAVHSC14, and variants of AAVHSC15). In some embodiments, this method can be completed in less than a week, resulting in a high ratio of complete capsids to empty capsids (up to 70% complete capsids), providing up to 70% step yields and purity suitable for clinical use. In some embodiments, this method is universal for chimerization of AAV serotypes and/or capsids. As described herein, scalable production technology can be used to produce GMP clinical and commercial grade rAAV vectors to treat diseases (e.g., DMD, Friedreich's ataxia, Wilson's disease, etc.).

The production of recombinant AAV vectors (rAAV) for gene therapy requires purification of rAAV vectors from the host cells in which they were produced (e.g., host cell debris, including but not limited to host cell DNA, RNA, proteins, lipids, membranes and organelles), as well as removal of capsids that do not contain the complete vector genome (e.g., intermediate capsids and/or empty capsids) and therefore do not contain the therapeutic transgene.

Such purification methods typically include multiple steps, including, for example, host cell lysis, precipitation of cellular proteins and DNA, separation of rAAV vectors from host cell proteins and nucleic acids, and separation of rAAV vectors from empty capsids and intermediate capsids, by column purification, low-speed centrifugation, ultracentrifugation, normal flow filtration, ultrafiltration/diafiltration, or any combination of these methods. Column purification can include, for example, ion exchange chromatography (e.g., anions, cations), affinity chromatography, size exclusion chromatography, multimodal chromatography, and/or hydrophobic interaction chromatography. Centrifugation methods can include, for example, ultracentrifugation or low-speed centrifugation (e.g., for removing solids and clarifying). Filtration methods can include, for example, diafiltration, depth filtration, nominal filtration, and/or absolute filtration.

AEX uses a positively charged stationary phase (e.g., resin) to separate substances (e.g., AAV capsids, DNA, proteins, high molar mass substances, amino acids) based on differences in the charge of the substances, and can separate rAAV capsids from impurities based on differences in charge at moderately acidic to basic pH (e.g., greater than pH 6). AEX can also separate empty capsids from rAAV vectors containing a complete vector genome (i.e., complete capsids) by relying on differences in charge of empty capsids compared to complete capsids.

Without wishing to be bound by theory, the tightness of the binding between the AAV capsid and the AEX chromatography stationary phase is related to the strength of the negative charge of the capsid, including the charge contribution of any nucleic acid within the capsid, solution pH, and solution conductivity (Qu, G. et al., J. Virological. Methods (2007) 140: 183-192). In some embodiments, the AEX chromatography stationary phase is a resin comprising polystyrene divinyl benzene particles modified with covalently bound quaternized polyethyleneimine and optionally with OH groups (e.g., POROS ^™ 50HQ resin). The polystyrene divinyl benzene particles may include 500-10,000 angstroms. hole.

In some embodiments, the AEX chromatography stationary phase is a resin comprising agarose particles with cationic ligands (eg, Capto Q ImpRes, Q Sepharose High Performance). In some embodiments, the AEX chromatography stationary phase is a resin selected from the group consisting of Capto Q, Capto Q XP, Q Sepharose XL, STREAMLINE Q XL, CaptoHiRes Q, RESOURCE Q, SOURCE 15Q, SOURCE 30Q, Q Sepharose HP, Q Sepharose FF, Q Sepharose ^™ BB, POROS ^™ 20HQ, POROS ^™ XQ, TOYOPEARL QAE-550C, TOYOPEARL Q-600C AR, TOYOPEARL GigaCap Q-650S, TOYOPEARL GigaCap Q-650M, TOYOPEARL SuperQ-650S, TOYOPEARL SuperQ-650M, TOYOPEARL SuperQ-650C, TSKgel SuperQ-5PW(20), TSKgelSuperQ-5PW(30), QCeramic HyperD F, Q, EMD TMAE(S), EMD TMAE(M), EMD TMAE Hicap(M), EMD TMAE(S), EMD TMAE(M), EMD TMAE Hicap(M), Nuvia Q, Nuvia HP-Q, UNOsphereQ, Macro-Prep High Q, Macro-Prep 25Q, BioRad 1-X2, WorkBeads ^TM 40Q, WorkBeads ^TM 100Q, Cellufine MAX Qr, Cellufine MAX Qh, Praesto ^TM Q65, Praesto ^TM Q90, Praesto ^TM Jetted Q35, BAKERBOND ^TM POLYQUAT, BAKERBOND ^TM POLYPEI, YMC-BioPro Q30, YMC-BioPro Q75 , YMC-BioPro SmartSep Q10, YMC-BioPro SmartSep Q30, DEAE Sepharose FF, ANX Sepharose 4FF (high sub), POROS ^TM 50PI, POROS ^TM 50D, TOYOPEARL NH ₂ -750F, TOYOPEARLGigaCap DEAE-650M, TOYOPEARL DEAE-650S, TOYOPEARL DEAE-650M, TOYOPEARL DEAE-650C, TSKgel DEAE-5PW(20), TSKgel DEAE-5PW(30), Ceramic HyperD DEAE, HypercelStar AX, EMD DEAE(M), EMD DMAE(M)Resin, Macro-Prep DEAE, WorkBeads ^TM 40DEAE, Cellufine MAX DEAE, DEAE PuraBead HF and WorkBeads ^TM 40TREN.

In some embodiments, the AEX chromatography stationary phase is a monolithic column comprising a porous polymethacrylate with cationic ligands (e.g., CIMmultus ^™ QA). In some embodiments, the AEX chromatography stationary phase is a membrane adsorber comprising polyethersulfone with cationic ligands (e.g., Mustang Q, Mustang E, Q. Sartobind PA).

In some embodiments, the rAAV vector can be purified by AEX from a solution that emerges from an affinity chromatography stationary phase (e.g., "eluted from the stationary phase"), which comprises a mobile phase and materials such as rAAV vectors or capsids that pass through or are removed from the stationary phase. This solution can be referred to as an affinity eluate or "affinity pool."

In some embodiments, rAAV vectors can be purified by AEX from a "supernatant of cell lysate" (also referred to as "clarified lysate"), which, as used herein, refers to a solution collected from a host cell culture after the lysed host cells have been pelleted.

In some embodiments, rAAV vectors can be purified by AEX from a "harvest solution," which, as used herein, refers to a solution resulting from cell lysis that has been subjected to flocculation, depth filtration, and/or nominal filtration.

In some embodiments, the rAAV vector can be purified from a solution that has undergone at least one other purification or processing step (e.g., cell lysis, flocculation, filtration, dilution, pH adjustment, chromatography). In some embodiments, the affinity eluate has been diluted and optionally filtered prior to purification of the rAAV vector, e.g., prior to loading the affinity eluate onto an AEX column.

In some embodiments, rAAV vectors can be purified by AEX from an affinity eluate, which has optionally undergone at least one other purification or processing step (e.g., cell lysis, flocculation, filtration, dilution, pH adjustment, chromatography). In some embodiments, rAAV vectors can be purified by AEX from a cell lysate, which has optionally undergone at least one other purification or processing step (e.g., cell lysis, flocculation, filtration, dilution, pH adjustment, chromatography). In some embodiments, rAAV vectors can be purified by AEX from a post-harvest solution, which has optionally undergone at least one other purification or processing step (e.g., cell lysis, flocculation, filtration, dilution, pH adjustment, chromatography).

As the solution containing the substance to be purified (e.g., rAAV vector) and impurities flows through the AEX stationary phase, substances bound to the positively charged AEX stationary phase (e.g., negatively charged proteins, such as AAV capsids or rAAV vectors, etc.) are retained in the stationary phase. Unbound substances pass through the column and are collected during circulation and/or in subsequent washing steps. The bound substances can be eluted from the stationary phase by adjusting the salt concentration and/or pH in the column. For example, and without wishing to be bound by any particular operating theory, the salt concentration of the elution buffer is gradually increased so that the anions of the salt (e.g., acetate (C ₂ H ₃ O ₂ ^- ), Cl ^- SO ₄ ^-2 ) compete with and displace the substances bound to the resin (i.e., elution). In another embodiment, the pH of the solution in the column can be gradually reduced to reduce the negative charge of the bound substance and release it from the stationary phase (i.e., elution). After release from the stationary phase, the substance can be collected as a column eluent.

Without wishing to be bound by theory, the separation of a mixture of substances such as AAV capsids, or more specifically a mixture of rAAV vectors (i.e., complete capsids), AAV capsids (e.g., empty capsids, intermediate capsids), and host cell proteins will depend on the difference in the overall charge of the substances. The charge composition of the ionizable side groups will determine the overall charge of the protein at a particular pH. At the isoelectric point (pi), the overall charge on the protein is 0 and will not bind to the matrix. If the pH is above the pI, the protein will have a negative charge and will be fixedly bound to the anion exchange column.

The AEX protocol for separating complete rAAV vectors from empty capsids includes multiple steps, such as pre-use flushing of the column medium to replace the storage solution, pre-use disinfection of the column stationary phase, post-use disinfection of the column stationary phase, equilibration of the column stationary phase, loading of a solution containing the rAAV vector (e.g., a diluted affinity eluent) onto the column stationary phase, eluting the substance to be purified from the stationary phase (e.g., by gradient elution, by step elution), applying a gradient hold to the column stationary phase, disinfecting the column stationary phase, regenerating the column stationary phase, and applying the storage solution to the column stationary phase. Those skilled in the art will appreciate that the AEX protocol for purifying rAAV vectors may include all or only some of these steps. Those skilled in the art will also appreciate that the order of these steps may vary, and that certain steps may be performed more than once and not necessarily in sequence.

AEX column preparation

The AEX methods of the present disclosure can be performed at various scales using columns with volumes ranging from 1.0 mL to 20 L. In some embodiments, the AEX methods include using a column with a column volume (CV) of about 1.0 mL, about 5.1 mL, about 49 mL, about 52 mL, about 6.67 mL, about 1.256 L, about 1.3 L, about 6.0 L, about 6.1 L, about 6.2 L, about 6.3 L, about 6.4 L, about 6.5 L, about 6.6 L, about 6.7 L, about 6.8 L, about 6.9 L, or about 7.0 L. In some embodiments, the AEX methods of the present disclosure include using a column with a CV of 1.0 mL to 20 L, such as 1.0 mL to 10 mL, 30 mL to 70 mL, 10 mL to 100 mL, 100 mL to 1000 mL, 1 L to 1.5 L, 1.5 L to 2.0 L, 2.0 L to 5 L, 5 L to 7.5 L, 7.5 L to 10 L, 10 L to 15 L, or 15 L to 20 L. In some embodiments, the AEX methods of the present disclosure include using a column with a CV of 1.0 mL to 10 L, 10 mL to 10 L, 100 mL to 20 L, 100 mL to 10 L, 1 L to 20 L, 1 L to 10 L, 1 L to 5 L, 1 L to 2 L, or 1 L to 1.5 L. In some embodiments, the AEX methods of the present disclosure include using a column with a CV of 6.0 L to 6.6 L (e.g., 6.4 L).

For example, the volume of solution applied to a column in order to equilibrate the stationary phase therein is often expressed in "column volumes" (CV), where one CV equals the volume of the column.

In some embodiments, the AEX chromatography stationary phase (also referred to herein as "resin" or "media") of the present disclosure is polystyrene divinylbenzene particles with covalently bound quaternized polyethyleneimine (eg, POROS ^™ 50HQ resin).

Typically, before the solution to be purified (e.g., affinity chromatography eluate, also referred to herein as "affinity eluate" or "affinity pool") is applied (i.e., loaded) to a column comprising a chromatographic stationary phase, at least one solution is applied to the stationary phase, for example, to wash, disinfect, regenerate and/or equilibrate the stationary phase. In some embodiments, the "affinity eluate" or "affinity pool" has been diluted and optionally filtered before the solution is loaded onto the AEX column.

As disclosed herein, a method for preparing an AEX stationary phase for a method of purifying rAAV (e.g., rAAV9, rAAV3B, etc.) vectors from a solution (e.g., affinity eluent) by AEX includes flushing the AEX stationary phase in the column before use. In some embodiments, the flushing of the AEX stationary phase before use is intended to displace the storage solution (e.g., a solution comprising ethanol) from the stationary phase. In some embodiments, the column is flushed before use before the solution containing the rAAV vector to be purified is loaded onto the column. In some embodiments, the flushing before use includes applying water (e.g., water for injection) to the AEX stationary phase in the column. In some embodiments, the flushing before use includes water flowing upward. During the upward flow of the flushing before use, the flow direction is opposite to the flow direction of the chromatographic separation step (e.g., loading, washing, or elution), so that the solution (e.g., water) flows from the bottom of the column to the top of the column, and during the chromatographic separation step (e.g., loading), the solution flows from the top of the column to the bottom of the column. In some embodiments, the pre-use flushing includes applying 1 to 10 column volumes (CV) (e.g., about 5CV) of water to the AEX stationary phase in the column at a linear velocity of 10 cm/hr to 1000 cm/hr and/or a flow rate of 0.2 L/min to 3.0 L/min. In some embodiments, the pre-use flushing includes applying ≥4.5 CV (e.g., about 5 CV) of water for injection to the AEX stationary phase in the column at a linear velocity of 270 cm/hr to 330 cm/hr (e.g., about 300 cm/hr), a flow rate of 1.5 L/min to 2.0 L/min (e.g., about 1.8 L/min), and/or a residence time (i.e., contact time) of 3.5 min/CV to 4.5 min/CV (e.g., about 4 min/CV).

A method for preparing an AEX stationary phase in a method for purifying rAAV (e.g., rAAV9, rAAV3B, etc.) vectors from a solution (e.g., affinity eluent) by AEX, comprising disinfecting the AEX stationary phase in a column. Disinfection of the AEX stationary phase is used to reduce the bioburden (including but not limited to bacteria) in the column and/or inactivate microorganisms and viruses in the column, and more generally remove contaminants such as proteins, particles, etc. In some embodiments, disinfection is performed before loading a solution containing the rAAV vector to be purified onto the column. In some embodiments, disinfection comprises applying a solution containing NaOH, ethanol, acetic acid, phosphoric acid, guanidine hydrochloride, urea, PAB (phosphoric acid, acetic acid, benzyl alcohol), peracetic acid, etc. to the AEX stationary phase in the column. In some embodiments, disinfection includes applying a solution comprising 0.1M to 1.0M, about 0.1M to about 0.8M, about 0.1M to about 0.6M, about 0.2M to about 0.8M, about 0.2M to about 0.6M, or about 0.4M to about 0.6M (e.g., about 0.5M) NaOH to the AEX stationary phase in the column. In some embodiments, disinfection includes applying a solution comprising about 0.5M NaOH to the AEX stationary phase in the column using an upward flow (i.e., the flow direction is opposite to the direction of the chromatographic separation step, such as loading, washing, or elution). In some embodiments, disinfection includes applying a solution comprising about 0.5M NaOH to the AEX stationary phase in the column using a downward flow (i.e., the flow direction is the same as the direction of the chromatographic separation step, such as loading, washing, or elution). In some embodiments, disinfection includes applying 14.4CV to 17.6CV (e.g., about 16CV) of a solution comprising about 0.5M NaOH to the AEX stationary phase in the column. In some embodiments, sanitization comprises applying 5CV to 10CV (e.g., about 8CV) of a solution comprising about 0.5M NaOH to the AEX stationary phase in the column. In some embodiments, sanitization comprises applying 5CV to 20CV of a solution comprising about 0.5M NaOH to the AEX stationary phase in the column at a linear velocity of 100 cm/hr to 1000 cm/hr and/or a flow rate of 0.2 L/min to 3.0 L/min. In some embodiments, sanitization comprises applying 14.4CV to 17.6CV (e.g., about 16CV) of a solution comprising about 0.5M NaOH to the AEX stationary phase in the column at a linear velocity of 270cm/hr to 330cm/hr (e.g., about 300cm/hr), a flow rate of 1.5L/min to 2.0L/min (e.g., about 1.8L/min), and/or a residence time (i.e., the amount of time per column volume that the solution is in contact with the stationary phase in the column, also referred to herein as contact time) of 3.5min/CV to 4.5min/CV (e.g., about 4min/CV). In some embodiments, sanitization comprises applying 5CV to 10CV (e.g., about 8CV) of a solution comprising about 0.5M NaOH to the AEX stationary phase in the column at a linear velocity of 270cm/hr to 330cm/hr (e.g., about 298cm/hr) and/or a residence time of 1.5min/CV to 2.5min/CV (e.g., about 2min/CV).

A method for preparing an AEX stationary phase in a method for purifying rAAV (e.g., rAAV9, rAAV3B, etc.) vectors from a solution (e.g., an affinity eluent) by AEX, comprising regenerating the AEX stationary phase in the column (also referred to herein as "rinsing"). Those skilled in the art will appreciate that regenerating the ion exchange stationary phase is used to replace the ions accepted during the exchange process with the original ions occupying the exchange sites. In some embodiments, regeneration may also refer to restoring the stationary phase to its original state by, for example, removing impurities using a strong solvent. In some embodiments, regeneration is performed before a solution containing the rAAV vector to be purified is loaded onto the stationary phase. In some embodiments, the stationary phase may be regenerated more than once.

In some embodiments, regeneration comprises applying a solution comprising salt and/or buffer at a pH in the range of 8-10 to the AEX stationary phase in the column. In some embodiments, the salt is selected from sodium chloride (NaCl), sodium acetate (NaAcetate, CH ₃ COONa), ammonium acetate (NH ₄ Acetate), magnesium chloride (MgCl ₂ ) or sodium sulfate (Na ₂ SO ₄ ). In some embodiments, the concentration of salt (e.g., NaCl) in the solution ranges from 1M to 5M, for example, from about 1M to about 4.5M, from about 1M to about 4M, from about 1M to about 3.5M, from about 1M to about 3M, from about 1M to about 2.5M, or from about 1.5M to about 2.5M. In some embodiments, the concentration of salt (e.g., NaCl) in the solution is about 1M, about 2M, about 3M, about 4M, or about 5M. In some embodiments, regeneration comprises applying a solution comprising 1M to 3M (e.g., 2M) NaCl to the stationary phase in the column.

In some embodiments, the buffer is selected from Tris (e.g., a mixture of Tris-Base and Tris-HCl), BIS-Tris propane and/or n,n-bicine. In some embodiments, the concentration range of the buffer (e.g., Tris) in the solution is 10 mM to 500 mM (e.g., about 10 mM to about 450 mM, about 10 mM to about 400 mM, about 10 mM to about 350 mM, about 10 mM to about 300 mM, about 10 mM to about 250 mM, about 10 mM to about 200 mM, about 10 mM to about 150 mM, or about 50 mM to about 150 mM). In some embodiments, the concentration of the buffer (e.g., Tris) in the solution is about 10 mM, about 20 mM, about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 300 mM, about 400 mM, or about 500 mM. In some embodiments, regeneration comprises applying a solution comprising 50 mM to 150 mM (e.g., 100 mM) Tris to the stationary phase in the column.

In some embodiments, regeneration comprises applying a solution having a pH of about 7-11 (e.g., about 7.5-10.5, about 8-10, or about 7, 7.5, 8, 8, 5, 9, 9, 5, 10, 10.5, or 11) to the stationary phase in the column.

In some embodiments, regeneration comprises applying a solution comprising about 1M to 3M (e.g., about 2M) NaCl, 50mM to 150mM (e.g., about 100mM) Tris, pH 8.5 to 9.5 (e.g., about 9) to the AEX stationary phase in the column. In some embodiments, regeneration comprises applying 4.5 to 5.5CV (e.g., about 5CV) of a solution comprising about 2M NaCl, 100mM Tris, pH 9 to the AEX stationary phase in the column. In some embodiments, regeneration comprises applying 1 to 10CV of a solution comprising about 2M NaCl, 100mM Tris, pH 9 to the AEX stationary phase in the column at a linear velocity of 100 to 1000cm/hr and/or a flow rate of 0.2 to 3.0L/min. In some embodiments, regeneration comprises applying 4.5 to 5.5 (e.g., about 5) CV of a solution comprising about 2 M NaCl, 100 mM Tris, pH 9 to the AEX stationary phase in the column at a linear velocity of 270 to 330 cm/hr (e.g., about 298 cm/hr, about 300 cm/hr), a flow rate of 1.5 to 2.0 L/min (e.g., about 1.8 L/min), and/or a residence time (i.e., contact time) of 1.5 to 4.5 min/CV (e.g., about 2 min/CV, about 4 min/CV).

In some embodiments, the present disclosure provides a method for preparing an AEX stationary phase for use in a method for purifying rAAV (e.g., rAAV9, rAAV3B, etc.) vectors by AEX, the method comprising the following steps: i) pre-use flushing, comprising applying ≥4.5CV (e.g., about 5CV) of water for injection to the AEX stationary phase in the column; ii) disinfection, comprising applying about 5CV to 10CV (e.g., about 8CV) or about 14.4 to 17.6CV (e.g., about 16CV) of a solution comprising 0.1M to 1.0M (e.g., about 0.5M NaOH) to the AEX stationary phase in the column, optionally flowing upward; and/or iii) regeneration, comprising applying 4.5 to 5.5CV (e.g., about 5CV) of a solution comprising 1M to 3M (e.g., about 2M) NaCl, 50mM to 150mM (e.g., about 100mM) Tris, pH 5. 8.5 to 9.5 (e.g., about 9) of the solution; wherein at least one of steps i) to iii) is performed at a linear velocity of 270 cm/hr to 330 cm/hr (e.g., about 298 cm/hr, about 300 cm/hr), a flow rate of 1.5 L/min to 2.0 L/min (e.g., about 1.8 L/min), and/or a residence time of 1.5 min/CV to 4.5 min/CV (e.g., about 2 min/CV, about 4 min/CV); optionally, wherein at least one step is performed before loading the solution containing the rAAV vector to be purified onto the column; and optionally, wherein the AEX stationary phase is POROS ^™ 50HQ. One of ordinary skill will appreciate that the above steps may be performed in any order and may be performed more than once.

balance

A method for preparing an AEX stationary phase in a method for purifying rAAV (e.g., rAAV9, rAAV3B, etc.) vectors from a solution (e.g., affinity eluent) by AEX, comprising balancing the AEX stationary phase in a column. In some embodiments, the AEX stationary phase in the equilibration column is used to adjust the pH, conductivity, modifier (e.g., salt, detergent, amino acid, etc.) concentration or other conditions of the mobile phase and the stationary phase so that some substances loaded on the column will bind to the stationary phase, while other substances will flow through the stationary phase with the mobile phase. For example, the conditions in the column can be adjusted by applying a series of equilibration buffers to the column so that the complete rAAV vector is bound to the stationary phase and at least a portion of the empty capsid is not bound. In some embodiments, the AEX stationary phase in the column is equilibrated before a solution containing a substance to be purified (e.g., a rAAV vector) is applied to the column. In some embodiments, the AEX stationary phase in the column is equilibrated by applying an equilibration buffer (e.g., a first equilibration buffer, a second equilibration buffer, a third equilibration buffer, a fourth equilibration buffer, etc.). The equilibration buffer may also be referred to herein as a "washing buffer," "post-disinfection rinse," "rinsing solution," or "regeneration buffer." Referring to the equilibration buffer as the first, second, third, fourth, etc. equilibration buffer does not necessarily imply the order in which the buffers are applied to the column.

In some embodiments, the equilibration buffer (e.g., the first equilibration buffer, the second equilibration buffer, the third equilibration buffer, the fourth equilibration buffer, etc.) comprises at least one component selected from a buffer, a salt, an amino acid, a detergent, and/or a combination thereof. In some embodiments, the buffer is Tris (e.g., a mixture of Tris Base and Tris-HCl), BIS-Tris propane, diethanolamine, diethylamine, tris (hydroxymethyl) methylglycine, and/or n, n-bis (hydroxyethyl) glycine. It will be appreciated by those of ordinary skill in the art that a Tris buffer having a desired pH can be prepared using Tris Base, Tris-HCl, or both. In some embodiments, the salt is sodium chloride (NaCl), sodium acetate (NaAcetate, (CH ₃ COONa)), ammonium acetate (NH ₄ Acetate), magnesium chloride (MgCl ₂ ) or sodium sulfate (Na ₂ SO ₄ ). In some embodiments, the salt is sodium acetate. In some embodiments, the amino acid is histidine, arginine, glycine, or citrulline. In some embodiments, the detergent is poloxamer 188 (P188), Triton X-100, polysorbate 80, Brij-35, or nonylphenoxypolyethoxyethanol (NP-40).

In some embodiments, the equilibration buffer comprises 10mM to 350mM selected from Tris (e.g., a mixture of Tris Base and Tris-HCl), BIS-Tris propane, diethanolamine, diethylamine, tris(hydroxymethyl)methylglycine, triethanolamine, and n,n-bis(hydroxyethyl)glycine. In some embodiments, the equilibration buffer comprises 10mM to 350mM, 10mM to 300mM Tris, 10mM to 250mM Tris, 10mM to 200mM Tris, 10mM to 150mM Tris, 10mM to 100mM Tris or 10mM to 50mM Tris. In some embodiments, the equilibration buffer comprises 30mM to 350mM Tris, 30mM to 300mM Tris, 30mM to 250mM Tris, 30mM to 2000mM Tris, 30mM to 150mM Tris, 30mM to 100mM Tris. In some embodiments, the equilibration buffer comprises 50mM to 300mM Tris, 50mM to 250mM Tris, 50mM to 200mM Tris, 50mM to 150mM Tris. In some embodiments, the equilibration buffer comprises 100mM to 350mM Tris, 100mM to 250mM Tris or 100mM to 150mM Tris. In some embodiments, the equilibration buffer comprises about 10 mM Tris, about 20 mM Tris, about 30 mM Tris, about 40 mM Tris, about 50 mM Tris, about 60 mM Tris, about 70 mM Tris, about 80 mM Tris, about 90 mM Tris, about 100 mM Tris, about 110 mM Tris, about 120 mM Tris, about 130 mM Tris, about 140 mM Tris, about 150 mM Tris, about 160 mM Tris, about 170 mM Tris, about 180 mM Tris, about 190 mM Tris, about 200 mM Tris, about 220 mM Tris, about 240 mM Tris, about 250 mM Tris, about 275 mM Tris, about 300 mM Tris, or about 350 mM Tris. In some embodiments, the equilibration buffer comprises about 20 mM Tris, 100 mM Tris, or 200 mM Tris.

In some embodiments, the equilibration buffer comprises 1 mM to 1 M salt, preferably about 500 mM salt. In some embodiments, the equilibration buffer comprises about 10mM to about 950mM, about 10mM to about 900mM, about 10mM to about 850mM, about 10M to about 800mM, 10mM to about 750mM, about 10mM to about 700mM, about 10mM to about 650mM, about 10mM to about 600mM, about 10mM to about 550mM, about 50mM to about 750mM, about 50mM to about 700mM, about 50mM to about 650mM, about 50mM to about 600mM, about 50mM to about 550mM, about 50mM to about 750mM, about 50mM to about 700mM, about 50mM to about 650mM, about 50mM to about 600mM, about 50mM to about 550mM, about 100mM to about 600mM, about 200mM to about 600mM, about 300mM to about 600mM, or about 400mM to about 600mM salt. In some embodiments, the equilibration buffer comprises about 500mM salt. In some embodiments, the equilibration buffer comprises a salt selected from sodium chloride (NaCl), sodium acetate (NaAcetate, CH ₃ COONa), ammonium acetate (NH ₄ Acetate), magnesium chloride (MgCl ₂ ), or sodium sulfate (Na ₂ SO ₄ ).

In some embodiments, the equilibration buffer comprises 5mM to 1M sodium acetate. In some embodiments, the equilibration buffer comprises about 10mM to about 950mM, about 10mM to about 900mM, about 10mM to about 850mM, about 10M to about 800mM, 10mM to about 750mM, about 10mM to about 700mM, about 10mM to about 650mM, about 10mM to about 600mM, about 10mM to about 550mM, about 50mM to about 750mM, about 50mM to about 700mM, about 50mM to about 650mM, about 50mM to about 600mM, about 50mM to about 550mM, about 50mM to about 750mM, about 50mM to about 700mM, about 50mM to about 650mM, about 50mM to about 600mM, about 50mM to about 550mM, about 100mM to about 600mM, about 200mM to about 600mM, about 300mM to about 600mM, or about 400mM to about 600mM sodium acetate. In some embodiments, the equilibration buffer comprises about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 20mM, about 30mM, about 40mM, about 50mM, about 60mM, about 70mM, about 80mM, about 90mM, about 100mM, about 150mM, about 200mM, about 250mM, about 300mM, about 350mM, about 400mM, about 450mM, about 500mM, about 550mM, or about 600mM sodium acetate. In some embodiments, the equilibration buffer comprises about 500mM sodium acetate.

In some embodiments, the equilibration buffer comprises an amino acid, such as histidine, arginine, glycine or citrulline. In some embodiments, the equilibration buffer comprises about 50 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM or about 300 mM amino acid (e.g., histidine, arginine, glycine or citrulline).

In some embodiments, the equilibration buffer comprises an amino acid, such as histidine or arginine. In some embodiments, the equilibration buffer comprises 100 mM to 300 mM amino acids (e.g., histidine, arginine, glycine, or citrulline). In some embodiments, the equilibration buffer comprises about 10 mM to about 600 mM, about 10 mM to about 550 mM, about 10 mM to about 500 mM, about 10 mM to about 450 mM about 10 mM to about 400 mM, about 10 mM to about 350 mM, about 10 mM to about 300 mM, about 50 mM to about 600 mM, about 50 mM to about 550 mM, about 50 mM to about 500 mM, about 50 mM to about 450 mM, about 50 mM to about 400 mM, about 50 mM to about 350 mM, about 50 mM to about 300 mM, about 100 mM to about 600 mM, about 100 mM to about 500 mM, about 100 mM to about 400 mM, about 100 mM to about 300 mM salt, or about 150 mM to about 250 mM amino acids (e.g., histidine). In some embodiments, the equilibration buffer comprises about 200 mM histidine.

In some embodiments, the equilibration buffer comprises a detergent, such as P188, Triton X-100, polysorbate 80, Brij-35 or NP-40. In some embodiments, the equilibration buffer comprises 0.005% to 1.0% detergent (e.g., P188). In some embodiments, the equilibration buffer comprises 0.005% to 0.015% detergent (e.g., P188). In some embodiments, the equilibration buffer comprises 0.1% to 1.0% detergent (e.g., P188). In some embodiments, the equilibration buffer comprises about 0.005% to about 1.0%, about 0.005% to about 0.5%, about 0.005% to about 0.1%, about 0.005% to about 0.05%, about 0.007% to about 0.07%, about 0.008% to about 0.05%, or about 0.008% to about 0.03% P188. In some embodiments, the equilibration buffer comprises about 0.01% to about 1.5%, about 0.01% to about 1.0%, about 0.01% to about 0.75%, about 0.05% to about 1.5%, about 0.05% to about 1.0%, about 0.05% to about 0.75%, about 0.1% to about 1.5%, about 0.1% to about 1.0%, about 0.1% to about 0.75%, or about 0.25% to about 0.75% P188.

In some embodiments, the equilibration buffer comprises about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, 0.95%, or about 1.0% detergent (e.g., P188). In some embodiments, the equilibration buffer comprises about 0.01% P188. In some embodiments, the equilibration buffer comprises about 0.5% P188.

In some embodiments, the pH of the balancing buffer is between 8 and 10. In some embodiments, the pH of the balancing buffer is between 8.7 and 9.3. In some embodiments, the pH of the balancing buffer is between 8.7 and 9.0. In some embodiments, the pH of the balancing buffer is 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, or about 10.0. In some embodiments, the pH of the balancing buffer is about 8.8. In some embodiments, the pH of the balancing buffer is about 8.9. In some embodiments, the pH of the balancing buffer is about 9.0.

In some embodiments, the equilibration buffer comprises 20 mM Tris, pH 9.0. In some embodiments, the equilibration buffer comprises 100 mM Tris, pH 9.

In some embodiments, the equilibration buffer comprises 20 mM Tris and 500 mM NaCl, pH 9.0 +/- 0.3. In some embodiments, the equilibration buffer comprises 20 mM Tris and 500 mM NH ₄ Acetate, pH 9.0 +/- 0.3. In some embodiments, the equilibration buffer comprises about 20 mM Tris, 500 mM sodium acetate, pH 9.0 +/- 0.3. In some embodiments, the equilibration buffer comprises 20 mM Tris and 500 mM Na ₂ SO ₄ , pH 9.0 +/- 0.3.

In some embodiments, the equilibration buffer comprises 20mM Tris, 7mM salt (e.g., NaCl, sodium acetate, ammonium acetate (NH ₄ Acetate), MgCl ₂ , and Na ₂ SO ₄ ) pH 9.0. In some embodiments, the equilibration buffer comprises 20mM Tris, 7mM sodium acetate, pH 9.0. In some embodiments, the equilibration buffer comprises 20mM Tris, 14mM sodium acetate, pH 9.0. In some embodiments, the equilibration buffer comprises 20mM Tris, 21mM sodium acetate, pH 9.0. In some embodiments, the equilibration buffer comprises 20mM Tris, 42mM sodium acetate, pH 9.0. In some embodiments, the equilibration buffer comprises 20mM Tris, 49mM sodium acetate, pH 9.0. In some embodiments, the equilibration buffer comprises 20mM Tris, 57mM sodium acetate, pH 9.0. In some embodiments, the equilibration buffer comprises 20mM Tris, 67mM sodium acetate, pH 9.0.

In some embodiments, the equilibration buffer comprises 50mM to 150mM (e.g., about 100mM) Tris, pH 8.5 to 9.5 (e.g., about 9). In some embodiments, the equilibration buffer comprises 50mM to 150mM (e.g., about 100mM) Tris, 400mM to 600mM (e.g., about 500mM) sodium acetate, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., about 8.9). In some embodiments, the equilibration buffer comprises 100mM to 300mM histidine (e.g., about 200mM) histidine, 100mM to 300mM (e.g., about 200mM) Tris, 0.1% to 1.0% (e.g., about 0.5%) P188, pH 8.5 to 9.5 (e.g., about 8.8). In some embodiments, the equilibration buffer comprises 50 mM to 150 mM (eg, about 100 mM) Tris, 0.005% to 0.015% (eg, about 0.01%) P188, pH 8.5 to 9.5 (eg, about 8.9).

In some embodiments, the equilibration buffer (e.g., the first equilibration buffer) comprises 100 mM Tris, pH 9. In some embodiments, the equilibration buffer (e.g., the first and second equilibration buffers) comprises 100 mM Tris, 500 mM sodium acetate, 0.01% P188, pH 8.9. In some embodiments, the equilibration buffer (e.g., the second or third equilibration buffer) comprises 200 mM histidine, 200 mM Tris, 0.5% P188, pH 8.8. In some embodiments, the equilibration buffer (e.g., the third and fourth equilibration buffers) comprises 100 mM Tris, 0.01% P188, pH 8.9.

In some embodiments, the above-mentioned equilibration buffer can be the first, second, third and fourth equilibration buffer. In some embodiments, the first, second, third or fourth equilibration buffer is applied to the column stationary phase in sequence. In some embodiments, between applying two equilibration buffers, a solution (e.g., affinity eluent) is applied to the column. For example, the first, second and third equilibration buffers can be applied to the column, then the affinity eluent is applied, and then the fourth equilibration buffer is applied. In another example, the first and second equilibration buffers are applied to the column, then the affinity eluent is applied, and then the third equilibration buffer is applied.

In some embodiments, the amount of equilibration buffer applied to the column is 1CV to 5CV, 4CV to 6CV, 4CV to 10CV, 4CV to 15CV, 4CV to 21CV, 10CV to 21CV, 15CV to 21CV, or 19CV to 21CV. In some embodiments, the amount of equilibration buffer applied to the column is ≥ 4.5CV. In some embodiments, the amount of equilibration buffer applied to the column is 4.5CV to 5.5CV. In some embodiments, the amount of equilibration buffer applied to the column is about 2CV, about 5CV, or about 10CV. In some embodiments, the amount of equilibration buffer applied to the column is about 5CV. In some embodiments, the amount of equilibration buffer applied to the column is about 20CV.

The solution applied to the column, including but not limited to the equilibrium buffer, is set to flow through the stationary phase at a specific rate (e.g., cm/hr, mL/min) so that the solution in the column is in contact with the stationary phase within a specific period of time (referred to herein as "residence time" or "contact time"). In some embodiments, the residence time of the solution in the column is 0.1 min/CV to 10 min/CV, such as 0.1 min/CV to 1 min/CV, 1 min/CV to 2 min/CV, 2 min/CV to 4 min/CV, 4 min/CV to 6 min/CV, 6 min/CV to 8 min/CV, or 8 min/CV to 10 min/CV. In some embodiments, the residence time of the solution in the column is 0.1 min/CV, about 0.5 min/CV, about 1.5 min/CV, about 2 min/CV, about 3 min/CV, about 3.6 min/CV, or about 4 min/CV, about 5 min/CV, about 6 min/CV, about 7 min/CV, about 8 min/CV, about 9 min/CV, or about 10 min/CV. In some embodiments, the residence time of the solution in the column is 1.5 to 4.5 min/CV. In some embodiments, the residence time of the solution in the column is 3.5 to 4.5 min/CV.

In some embodiments, the residence time of the solution in a column of about 5 cm in height, about 0.5 cm in diameter, and about 1.0 mL in volume is about 0.5 min/CV. In some embodiments, the residence time of the solution in a column of about 15 cm in height, about 0.66 cm in diameter, and about 5.1 mL in volume is about 0.5 min/CV, about 1.5 min/CV, or about 4 min/CV. In some embodiments, the residence time of the solution in a column of about 19.5 cm in height, about 0.66 cm in diameter, and about 6.67 mL in volume is about 4 min/CV. In some embodiments, the residence time of the solution in a column of about 10 cm in height, about 2.5 cm in diameter, and about 49 mL in volume is 1.5 min/CV to 2.5 min/CV (e.g., about 2 min/CV). In some embodiments, the residence time of the solution in a column of about 16 cm in height, about 10 cm in diameter, and about 1.256 L to 1.3 L in volume is 3.5 min/CV to 4.5 min/CV (e.g., about 4 min/CV). In some embodiments, the residence time of the solution in a column of about 20.5 cm in height, 20 cm in diameter, and about 6.4 L in volume is about 3.6 min/CV. In some embodiments, the residence time of the solution in an about 6.4 L column is 3.5 min/CV to 4.5 min/CV (e.g., about 4 min/CV). In some embodiments, the residence time of the solution including but not limited to the equilibration buffer in a 6.0 L to 6.6 L (e.g., 6.4 L) column comprising an AEX stationary phase is 3.5 min/CV to 4.5 min/CV (e.g., about 4 min/CV).

Those skilled in the art will appreciate that the linear velocity of a solution through a column (also referred to herein as "linear flow rate" or "velocity") is at least partially related to the volume and/or size of the column and the stationary phase therein. In some embodiments, the linear velocity of a solution including but not limited to an equilibration buffer through a stationary phase in a column is from 100 cm/hr to 1800 cm/hr, e.g., from 100 cm/hr to 200 cm/hr, from 200 cm/hr to 400 cm/hr, from 400 cm/hr to 600 cm/hr, from 600 cm/hr to 800 cm/hr, from 800 cm/hr to 1000 cm/hr, from 1000 cm/hr to 1500 cm/hr, or from 1500 cm/hr to 1800 cm/hr. In some embodiments, the linear velocity of the solution through the stationary phase in the column is about 100 cm/hr, about 240 cm/hr, about 298 cm/hr, about 300 cm/hr, about 600 cm/hr, about 611 cm/hr, or about 1790 cm/hr.

In some embodiments, the linear velocity of the solution through the stationary phase in a column of about 5 cm in height, about 0.5 cm in diameter, and about 1.0 mL in volume is about 611 cm/hr. In some embodiments, the linear velocity of the solution through the stationary phase in a column of about 15 cm in height, about 0.66 cm in diameter, and about 5.1 mL in volume is about 600 cm/hr. In some embodiments, the linear velocity of the solution through the stationary phase in a column of about 15 cm in height, about 0.66 cm in diameter, and about 5.1 mL in volume is about 1790 cm/hr. In some embodiments, the linear velocity of the solution through the stationary phase in a column of about 10 cm in height, about 2.5 cm in diameter, and about 49 mL in volume is about 298 cm/hr. In some embodiments, the linear velocity of the solution through the stationary phase in a column of about 16 cm in height, about 10 cm in diameter, and about 1256 mL in volume is about 240 cm/hr. In some embodiments, the linear velocity of the solution through the stationary phase in a column of about 20.5 cm in height, about 20 cm in diameter, and about 6.4 L in volume is 270 cm/hr to 330 cm/hr (e.g., 300 cm/hr). In some embodiments, the linear velocity of a solution including but not limited to an equilibration buffer through an AEX stationary phase in a 6.0 L to 6.6 L (e.g., 6.4 L) column is about 270 cm/hr to 330 cm/hr (e.g., about 300 cm/hr).

In some embodiments, the flow rate (i.e., volume flow rate) of the solution (including but not limited to the equilibrium buffer) through the stationary phase in the column is 1.0 mL/min to 3.0 L/min, such as 1.0 mL/min to 10 mL/min, 10 mL/min to 100 mL/min, 100 mL/min to 500 mL/min, 500 mL/min to 1000 mL/min, 1 mL/min to 1.5 L/min, 1 mL/min to 2 L/min or 2 mL/min to 3 L/min. In some embodiments, the flow rate of the solution through the stationary phase in the column is about 1 mL/min, about 1.28 mL/min, about 1.67 mL/min, about 314 mL/min, about 1.57 L/min, about 1.8 L/min, about 2 L/min, about 3 L/min.

In some embodiments, the flow rate of the solution through the stationary phase in a column with a height of about 15cm, a diameter of about 0.66, and a volume of about 5.1mL is about 1.28mL/min. In some embodiments, the flow rate of the solution through the stationary phase in a column with a height of about 19.5cm, a diameter of about 0.66, and a volume of about 6.67mL is about 1.67mL/min. In some embodiments, the flow rate of the solution through the stationary phase in a column with a height of about 16cm, a diameter of 10cm, and a volume of about 1256mL is about 314mL/min. In some embodiments, the flow rate of the solution through the stationary phase in a column with a height of about 20.5cm, a diameter of about 20cm, and a volume of about 6.4L is about 1.8L/min. In some embodiments, the flow rate of the solution including but not limited to the equilibrium buffer through the AEX stationary phase in a 6.0L to 6.6L (e.g., 6.4L) column is 1.5mL/min to 2.0L/min (e.g., about 1.8L/min).

A method for preparing an AEX stationary phase for use in a method for purifying an rAAV (e.g., rAAV9, rAAV3B, etc.) vector from a solution (e.g., an affinity eluate) by AEX, comprising balancing the AEX stationary phase in a column. In some embodiments, balancing is performed before loading a solution containing the rAAV vector to be purified onto the column. In some embodiments, balancing is performed after loading a solution containing the rAAV vector to be purified onto the column.

In some embodiments, equilibration comprises applying an equilibration buffer comprising 50 mM to 150 mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 to the AEX stationary phase in the column. In some embodiments, equilibration comprises applying 4.5 CV to 5.5 CV (e.g., about 5 CV) of an equilibration buffer comprising 100 mM Tris, pH 9 to a 6.0 L to 6.6 L (e.g., 6.4 L) column comprising the AEX stationary phase, with a linear velocity of 270 cm/hr to 330 cm/hr (e.g., about 300 cm/hr), a flow rate of 1.5 L/min to 2.0 L/min (e.g., about 1.8 L/min), and/or a residence time of 3.5 min/CV to 4.5 min/CV (e.g., about 4 min/CV).

In some embodiments, equilibration comprises applying an equilibration buffer comprising 400 mM to 600 mM sodium acetate, 50 mM to 150 mM Tris, and 0.005% to 0.015% P188, pH 8.5 to 9.5 to the AEX stationary phase in the column. In some embodiments, equilibration comprises applying an equilibration buffer comprising 100 mM Tris, 500 mM sodium acetate, 0.01% P188, pH 8.9 to the column comprising the AEX stationary phase at a linear velocity of 270 cm/hr to 330 cm/hr (e.g., about 298 cm/hr, about 300 cm/hr), a flow rate of 1.5 L/min to 2.0 L/min (e.g., about 1.8 L/min), and/or a residence time of 1.5 min/CV to 4.5 min/CV (e.g., about 2 min/CV, about 4 min/CV). In some embodiments, the column is 6.0 L to 6.6 L (eg, 6.4 L). In some embodiments, the column is 30 mL to 70 mL (eg, about 49 mL, about 52 mL).

In some embodiments, equilibration comprises applying an equilibration buffer comprising 100 mM to 300 mM histidine, 100 mM to 300 mm Tris, and 0.0% to 1.0% P188, pH 8.5 to 9.5 to the AEX stationary phase in the column. In some embodiments, equilibration comprises applying an equilibration buffer comprising 200 mM histidine, 200 mM Tris, 0.5% P188, pH 8.8 to the column comprising the AEX stationary phase at a linear velocity of 270 cm/hr to 330 cm/hr (e.g., about 298 cm/hr, about 300 cm/hr), a flow rate of 1.5 L/min to 2.0 L/min (1.8 L/min), and/or a residence time of 1.5 min/CV to 4.5 min/CV (e.g., about 2 min/CV, about 4 min/CV), ≥4.5 CV (e.g., about 5 CV) of equilibration buffer comprising 200 mM histidine, 200 mM Tris, 0.5% P188, pH 8.8 to the column comprising the AEX stationary phase. In some embodiments, the column is 6.0 L to 6.6 L (eg, 6.4 L). In some embodiments, the column is 30 mL to 70 mL (eg, about 49 mL, about 52 mL).

In some embodiments, balance includes applying an equilibrium buffer comprising 50mM to 150mM Tris and 0.005% to 0.015% P188, pH 8.5 to 9.5 to the AEX stationary phase in the column. In some embodiments, balance includes applying an equilibrium buffer comprising 100mM Tris, 0.01% P188, pH 8.9 to the column comprising the AEX stationary phase at a linear velocity of 270cm/hr to 330cm/hr (e.g., about 298cm/hr, about 300cm/hr), a flow rate of 1.5L/min to 2.0L/min (1.8L/min), and/or a residence time of 1.5min/CV to 4.5min/CV (e.g., 2min/CV, 4min/CV), 4.5CV to 5.5CV (e.g., about 5CV). In some embodiments, the column is 6.0L to 6.6L (e.g., 6.4L). In some embodiments, the column is 30 mL to 70 mL (eg, about 49 mL, about 52 mL).

In some embodiments, the present invention provides a method for preparing an AEX stationary phase for use in a method for purifying rAAV (e.g., rAAV9, rAAV3B, etc.) vectors by AEX, the method comprising the following steps: i) pre-use flushing, comprising applying ≥4.5CV (e.g., about 5CV) of water for injection to the AEX stationary phase in the column; ii) disinfection, comprising applying about 5CV to 10CV (e.g., about 8CV) or about 14.4 to 17.6CV (e.g., about 16CV) of a solution containing 0.1M to 1.0M (e.g., about 0.5M) NaOH to the AEX stationary phase in the column, optionally flowing upward; iii) regeneration, comprising applying 4.5CV to 5.5CV (e.g., about 5CV) of a solution containing 1M to 3M (e.g., about 2M) NaCl, 50mM to 150mM (e.g., about 100mM) Tris, pH 7. iv) equilibration comprising applying 4.5CV to 5.5CV (e.g., about 5CV) of a solution comprising 50mM to 150mM (e.g., about 100mM) Tris, pH 8.5 to 9.5 (e.g., about 9) to the AEX stationary phase in the column; v) equilibration comprising applying 4.5CV to 5.5CV (e.g., about 5CV) of a solution comprising 50mM to 150mM (e.g., about 100mM) Tris, 400mM to 600mM (e.g., about 500mM) sodium acetate, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., about 8.9) equilibration buffer is applied to the AEX stationary phase in the column; vi) equilibration comprising ≥4.5CV (e.g., about 5CV) of an equilibration buffer comprising 100mM to 300mM (e.g., about 200mM) histidine, 100mM to 300mM (e.g., about 200mM) Tris, 0.1% to 1.0% (e.g., about 0.5%) P188, pH 8.5 to 9.5 (e.g., 8.8) is applied to the AEX stationary phase in the column; and/or vii) equilibration comprising applying 4.5CV to 5.5CV (e.g., about 5CV) of an equilibration buffer comprising 50mM to 150mM (e.g., about 100mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., 8.9) of equilibration buffer is applied to the AEX stationary phase in the column; optionally, wherein at least one of steps i) to vii) is performed at a linear velocity of 270 cm/hr to 330 cm/hr (e.g., about 298 cm/hr, about 300 cm/hr) and/or a residence time of 1.5 min/CV to 4.5 min/CV (e.g., about 2 min/CV, about 4 min/CV); optionally, wherein the AEX stationary phase is POROS ^™ 50HQ; optionally, wherein the rAAV vector is a rAAV9 vector or a rAAV3B vector, and optionally, wherein steps (e.g., loading steps) may be performed between any equilibration steps. In some embodiments, at least one of steps i) to vii) is performed at a flow rate of 1.5 L/min to 2.0 L/min (e.g., about 1.8 L/min) through a 6 L to 6.6 L column (e.g., about 6.4 L), or at a flow rate of about 314 mL/min through a 1.3 L column. One of ordinary skill will appreciate that the order of the above steps may vary.

Dilution and Filtration

The method of purifying rAAV (e.g., rAAV9, rAAV3B, etc.) vectors from a solution (e.g., affinity eluent) by AEX includes preparing the solution by diluting and optionally filtering the solution. The solution containing the rAAV vector to be purified can be an affinity eluent, a supernatant from a cell lysate, and/or a post-harvest solution after at least one purification or processing step. The solution containing the rAAV vector to be purified can be diluted and optionally filtered, and then loaded onto an AEX column to make the solution compatible with the treatment by the AEX column. In some embodiments, diluting and optionally filtering the solution containing the rAAV vector to be purified causes the pH and/or conductivity of the solution to change. In some embodiments, the solution containing the rAAV vector to be purified is an eluate obtained by affinity chromatography purification of the rAAV vector produced in a disposable bioreactor (SUB) of 1L to 2000L (or larger).

The method of preparing a solution comprising a rAAV vector purified by AEX comprises: i) diluting an affinity eluate, and optionally ii) filtering the affinity eluate from step i) to produce a diluted affinity eluate (also referred to herein as a "diluted affinity pool", "load" or "AEX load"). In some embodiments, the pH of the affinity eluate after dilution and optional filtration is increased compared to the pH of the affinity eluate before dilution. In some embodiments, the conductivity of the affinity eluate after dilution and optional filtration is reduced compared to the conductivity of the affinity eluate before dilution. In some embodiments, the diluted and optionally filtered affinity eluate is loaded onto an AEX stationary phase.

In some embodiments, the affinity eluate is produced by affinity purification of rAAV vectors produced in a container (e.g., a disposable bioreactor (SUB)) having a volume of 1 mL to 2000 L or greater than 2000 L. In some embodiments, the affinity eluate is produced by affinity purification of rAAV vectors produced in a container (e.g., a SUB) having a volume of about 1 mL, about 10 mL, about 50 mL, about 100 mL, about 250 mL, about 500 mL, about 750 mL, about 1 L, about 50 L, about 100 L, about 250 L, about 500 L, about 1000 L, about 2000 L or greater. In some embodiments, the affinity eluate is produced by affinity purification of a rAAV vector produced in a container (e.g., SUB) having a volume of 1 mL to 100 mL, 100 mL to 500 mL, 500 mL to 750 mL, 750 mL to 1 L, 1 L to 10 L, 10 L to 50 L, 50 L to 100 L, 100 L to 250 L, 250 L to 500 L, 500 L to 750 L, 750 L to 1000 L, 1000 L to 1500 L, 1500 L to 2000 L, 2000 L to 3500 L, 3500 L to 4000 L, or 4500 L to 5000 L. In some embodiments, the affinity eluate is produced by affinity purification of a rAAV vector produced in a container (e.g., SUB) having a volume of 1 mL to 5000L, 100 mL to 5000L, 100 mL to 4000L, 100 mL to 2000L, 100 mL to 1000L, 1L to 5000L, 1L to 4000L, 1L to 2000L, 1L to 1000L, 500 mL to 5000L, 500 mL to 2000L, or 500 mL to 1000L.

In some embodiments, diluting a solution comprising a rAAV vector to be purified (e.g., an affinity eluate) comprises diluting the solution by about 2 to 25 times, or about 5 to 20 times, or about 10 to 20 times (e.g., about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 11 times, about 12 times, about 13 times, about 14 times, about 15 times, about 20 times, about 25 times) to produce a diluted affinity eluate. In some embodiments, diluting a solution comprising a rAAV vector to be purified (e.g., an affinity eluate) comprises diluting the solution by about 2 times. In some embodiments, diluting a solution comprising a rAAV vector to be purified (e.g., an affinity eluate) comprises diluting the solution by about 15 times.

In some embodiments, dilution of a solution containing the rAAV vector to be purified (e.g., an affinity eluate) is performed "in-line" with the column, wherein the diluent solution (diluent) is delivered to the Y-connector via a first tubing system, and the solution containing the rAAV vector to be purified is delivered to the Y-connector via a second tubing system, and optionally wherein a static mixer is contained within a third tubing system located after the Y-connector.

In some embodiments, dilution of a solution containing the rAAV vector to be purified (e.g., affinity eluate) is performed "in series" and introduced into a holding vessel (e.g., a break tank). For example, the dilution solution (diluent) is delivered to a Y-connector via a first tubing system, and the solution containing the rAAV vector to be purified is delivered to a Y-connector via a second tubing system, wherein the end of the Y-connector is connected to a holding vessel that is optionally connected to a chromatography column (e.g., an AEX column).

In some embodiments, diluting comprises delivering the dilution solution to the Y-connector through a first tubing system at a flow rate of 1 to 5 mL/min (e.g., about 3.5 mL/min), and delivering a solution comprising the rAAV vector to be purified (e.g., affinity eluate) through a second tubing system at a flow rate of 0.1 to 2 mL/min (e.g., about 0.25 mL/min).

In some embodiments, diluting comprises delivering the dilution solution through the first tubing to the Y-connector at a flow rate of about 3.5 mL/min and delivering the affinity eluent through the second tubing at a flow rate of about 0.25 mL/min, thereby diluting the affinity eluent by about 15 times.

In some embodiments, diluting comprises diluting a solution (e.g., affinity eluate) containing the rAAV vector to be purified with a diluent solution comprising a buffer (Tris, BIS-Tris propane, diethanolamine, diethylamine, tris(hydroxymethyl)methylglycine, triethanolamine and/or n,n-bicine). In some embodiments, the solution (e.g., affinity eluate) containing the rAAV vector to be purified is diluted with a diluent solution comprising 10 mM to 500 mM buffer (e.g., Tris). In some embodiments, the dilution solution comprises about 10mM to about 450mM, about 10mM to about 400mM, about 10mM to about 350mM, about 10mM to about 300mM, about 50mM to about 450mM, about 50mM to about 400mM, about 50mM to about 350mM, about 50mM to about 300mM, about 100mM to about 450mM, about 100mM to about 400mM, about 100mM to about 350mM, about 100mM to about 300mM, about 100mM to about 400mM, about 100mM to about 350mM, about 100mM to about 300mM, or about 150mM to about 250mM Tris. In some embodiments, the dilution solution comprises about 200mM Tris.

In some embodiments, the dilution solution comprises an amino acid, such as histidine, arginine, glycine, or citrulline. In some embodiments, the dilution solution comprises about 50 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, about 500 mM, about 550 mM, or about 600 mM of an amino acid (e.g., histidine, arginine, glycine, or citrulline).

In some embodiments, the dilution solution comprises an amino acid, such as histidine or arginine. In some embodiments, the dilution solution comprises 10 mM to 600 mM amino acid (e.g., histidine, arginine, glycine, or citrulline). In some embodiments, the equilibration buffer comprises about 10 mM to about 600 mM, about 10 mM to about 550 mM, about 10 mM to about 500 mM, about 10 mM to about 450 mM about 10 mM to about 400 mM, about 10 mM to about 350 mM, about 10 mM to about 300 mM, about 50 mM to about 600 mM, about 50 mM to about 550 mM, about 50 mM to about 500 mM, about 50 mM to about 450 mM, about 50 mM to about 400 mM, about 50 mM to about 350 mM, about 50 mM to about 300 mM, about 100 mM to about 600 mM, about 100 mM to about 500 mM, about 100 mM to about 400 mM, about 100 mM to about 300 mM, or about 150 mM to about 250 mM amino acids (e.g., histidine). In some embodiments, the dilution solution comprises about 200 mM histidine.

In some embodiments, the dilution solution comprises a detergent, such as P188, Triton X-100, polysorbate 80, Brij-35 or NP-40. In some embodiments, the dilution solution comprises 0.005% to 1.5% detergent (e.g., P188). In some embodiments, the dilution solution comprises 0.1% to 1.0% detergent (e.g., P188). In some embodiments, the dilution solution comprises about 0.01% to about 1.5%, about 0.01% to about 1.0%, about 0.01% to about 0.75%, about 0.05% to about 1.5%, about 0.05% to about 1.0%, about 0.05% to about 0.75%, about 0.1% to about 1.5%, about 0.1% to about 1.0%, about 0.1% to about 0.75%, or about 0.25% to about 0.75% detergent (e.g., P188). In some embodiments, the dilution solution comprises about 0.5% P188.

In some embodiments, the pH of the dilution solution is 8-10. In some embodiments, the pH of the dilution solution is 8.5-9.5. In some embodiments, the pH of the dilution solution is 8.7-9.0. In some embodiments, the pH of the dilution solution is 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, or about 10.0. In some embodiments, the pH of the dilution solution is about 8.8. In some embodiments, the pH of the dilution solution is about 8.9. In some embodiments, the pH of the dilution solution is about 9.0.

In some embodiments, diluting comprises diluting a solution containing the rAAV vector to be purified (e.g., affinity eluate) with a buffer selected from the group consisting of: 20 mM Tris, pH 9; 1 M Tris Base, pH 11; 100 mM Tris, pH 9; 100 mM Tris, 0.01% P188, pH 9; 100 mM Tris, 0.1% P188, pH 9; 100 mM Tris, 1.0% P188, pH 9; 1 M Tris, pH 9; 150 mM acetate, 100 mM glycine, 25 mM MgCl ₂ , pH 4.2; 5 mM arginine, 2 mM MgCl ₂ , 0.1% P188, 100 mM Tris, pH 8.9; 50 mM arginine, 2 mM MgCl ₂ , 0.1% P188, 100 mM Tris, pH 9; 500 mM arginine, 2 mM MgCl ₂ , 0.1% P188, 400 mM Tris, pH 9.1; 200 mM glycine, 5 mM MgCl ₂ , 200 mMTris, pH 8.9; 200 mM histidine, 200 mM Tris, pH 8.9; 200 mM histidine, 200 mM Tris, 5 mM MgCl ₂ , pH8.9; 200 mM histidine, 200 mM Tris, 5 mM MgCl ₂ , 5% glycerol, pH 8.9; 200 mM histidine, 250 mM Tris, 10 mM MgCl ₂ , 25% glycerol, pH 8.9; 200 mM histidine, 200 mM Tris, 5 mM MgCl ₂ , 5% iodixanol pH 8.8; 200 mM Histidine, 200 mM Tris, ₁₀ mM MgCl2, 25% Iodixanol, pH 8.8; 200 mM Histidine, 200 mM Tris, 0.5% Triton X-100, pH 8.9; 200 mM Histidine, 200 mM Tris, 0.5% P188, pH 8.8; and combinations thereof.

In some embodiments, diluting comprises diluting a solution containing the rAAV vector to be purified (e.g., affinity eluate) with a buffer containing about 20 mM Tris, pH 9, about 1 M Tris base, pH 11, or both. In some embodiments, diluting comprises diluting a solution containing the rAAV vector to be purified (e.g., affinity eluate) 7 to 8 times (e.g., about 7.1 times) with a buffer containing about 20 mM Tris, pH 9, about 1 M Tris base, pH 11, or both.

In some embodiments, dilution comprises diluting a solution (e.g., affinity eluate) containing the rAAV vector to be purified with a buffer containing 100 mM to 300 mM (e.g., about 200 mM) histidine, 100 mM to 300 mM (e.g., about 200 mM) Tris, 0.1% to 1.0% (e.g., 0.5%) P188, pH 8.7 to 9.0. In some embodiments, dilution comprises diluting a solution (e.g., affinity eluate) containing the rAAV vector to be purified 10 to 20 times (e.g., about 15 times) with a buffer containing about 200 mM histidine, 200 mM Tris, 0.5% P188, pH 8.7 to 9.0 (e.g., about 8.8). In some embodiments, the dilution comprises diluting the affinity eluate containing the rAAV vector to be purified 14.4 to 15.5 times (e.g., about 15 times) with a buffer containing about 200 mM histidine, 200 mM Tris, 0.5% P188, pH 8.7 to 9.0 (e.g., about pH 8.8) to form a diluted affinity eluate.

In some embodiments, prior to diluting a solution containing rAAV vectors to be purified (eg, affinity eluate), the solution is spiked with 20 mM MgCl ₂ such that the concentration of MgCl ₂ in the diluted solution is about 1.7 mM. In some embodiments, MgCl ₂ stabilizes the rAAV vectors in solution.

In some embodiments, filtering includes filtering a solution (e.g., affinity eluate, diluted affinity eluate) containing the rAAV vector to be purified before loading the solution onto the AEX column. In some embodiments, the filter is pre-wetted with water for injection and/or a diluent solution before filtering. In some embodiments, filtering includes filtering a solution (e.g., affinity eluate, diluted affinity eluate) containing the rAAV vector to be purified through a filter that collects aggregates, such as nucleic acid or protein aggregates or other high molecular weight substances, but allows the AAV capsid to flow through. In some embodiments, the filter is a 0.1 μm to 0.45 μm filter (e.g., a 0.2 μm polyethersulfone (PES) filter or a 0.45 μm PES filter). In some embodiments, filtering includes filtering the diluted affinity eluate containing the rAAV vector to be purified through a 0.2 μm filter, and then loading it onto the AEX column. The filter used to filter the solution containing the rAAV vector to be purified (eg, affinity eluate, diluted affinity eluate) can be separate from the column or can be in series with the column or chromatography instrument (also referred to as a chromatography skid).

In some embodiments, filtering comprises filtering the diluted affinity eluate containing the rAAV vector to be purified through an in-line 0.2 μm filter prior to loading the eluate onto an AEX column.

In some embodiments, the pH of the solution containing the rAAV vector to be purified (e.g., affinity eluate) is between 3.0 and 4.4 before dilution and optional filtration, and the pH of the solution containing the rAAV vector to be purified (e.g., affinity eluate) is between 8.5 and 9.5, 8.7 and 9.0, or ≥ 8.6 (e.g., about pH 8.8, pH 9.0) after dilution and optional filtration.

In some embodiments, before dilution and optional filtration, the conductivity of the solution containing the rAAV vector to be purified (e.g., affinity eluate) is 5.0 mS/cm to 7.0 mS/cm (e.g., about 5.5 mS/cm to 6.5 mS/cm), and after dilution and optional filtration, the conductivity of the solution containing the rAAV vector to be purified (e.g., affinity eluate) is 1.7 mS/cm to 3.5 mS/cm, 1.8 mS/cm to 2.8 mS/cm, 2.2 mS/cm to 2.6 mS/cm, or ≤ 2.5 mS/cm. In some embodiments, after dilution and optional filtration, the conductivity of the affinity eluate is about 1.8 mS/cm to about 2.8 mS/cm. In some embodiments, after dilution and optional filtration, the conductivity of the affinity eluate is about 2.3 +/- 0.5 mS/cm.

As used herein, the term "VG dilution yield percentage" or "%VG dilution yield" refers to the amount of VG present in the diluted affinity pool (also referred to herein as the diluted affinity eluate) as a percentage of the amount of VG present in the affinity pool (also referred to herein as the affinity eluate) before dilution. For example, %VG dilution yield = ((amount of VG in the diluted affinity pool)/(amount of VG in the affinity pool))*100.

In some embodiments, the percentage of VG recovered in the diluted and optionally filtered solution (e.g., affinity eluate) containing the rAAV vector to be purified (% VG dilution yield) is 60% to 100% of the VG present in the solution (e.g., affinity eluate) prior to dilution and optional filtration. In some embodiments, the % VG yield of the diluted and optionally filtered solution (e.g., affinity eluate) containing the rAAV vector to be purified is 60% to 70%, 70% to 80%, 80% to 90%, 90% to 100% of the VG present in the solution (e.g., affinity eluate) prior to dilution and optional filtration. In some embodiments, the % VG yield of a diluted and optionally filtered solution containing the rAAV vector to be purified (e.g., an affinity eluate) is about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or about 100% of the VG present in the solution prior to dilution and optional filtration.

In some embodiments of diluting the affinity eluate according to the methods of the present disclosure, the result is a %VG dilution yield of 88% +/- 36%. In some embodiments of diluting the affinity eluate according to the methods of the present disclosure, the result is a %VG dilution yield of 120% +/- 12%. Dilution of the affinity eluate obtained by affinity chromatography purification of rAAV vectors produced in a 250L SUB results in a %VG dilution yield of 35% to 100% (e.g., 41% to 92%). Dilution of the affinity eluate obtained by affinity chromatography purification of rAAV vectors produced in a 2000L SUB results in a %VG dilution yield of 70% to >100% (e.g., 88% to 154%).

In some embodiments, the Z-mean value (given in nm and determined by dynamic light scattering (DLS)) of a diluted and optionally filtered solution (e.g., affinity eluate) containing the rAAV vector to be purified is measured. The Z-mean value measures the aggregation level of the rAAV capsid present in the solution. In some embodiments, the Z-mean value of the diluted and optionally filtered solution containing the rAAV vector to be purified is about 15nm to 40nm, 15nm to 20nm, 20nm to 30nm, or 30nm to 40nm. In some embodiments, the Z-mean value of the diluted and optionally filtered solution containing the rAAV vector to be purified is about 15nm, about 16nm, about 17nm, about 18nm, about 19nm, about 20nm, 21nm, about 22nm, about 23nm, about 24nm, about 25nm, about 26nm, about 27nm, about 28nm, about 29nm, about 30nm, about 35nm, or about 40nm.

A method for purifying an rAAV (e.g., rAAV9, rAAV3B, etc.) vector from a solution (e.g., an affinity eluate) by AEX, comprising diluting the solution 14 to 16 times (e.g., about 15 times) with a buffer comprising 100 mM to 300 mM (e.g., about 200 mM) histidine, 100 mM to 300 mm (e.g., about 200 mM) Tris, 0.1% to 1.0% (e.g., about 0.5%) P188, and pH 8.7 to 9.0 (e.g., about pH 8.8); and optionally comprising filtration, comprising filtering the diluted solution through a 0.1 μm to 0.45 μm (e.g., about 0.2 μm) filter, and wherein the diluted and optionally filtered solution has a pH of about 8.6 to 9.0 (e.g., about pH 8.9) and a conductivity of 1.8 mS/cm to 2.8 mS/cm.

A method for preparing an affinity eluate comprising an rAAV vector purified by AEX chromatography, as disclosed herein, comprising i) diluting the affinity eluate 2 to 25 times (e.g., about 15 times) with a buffer comprising 200 mM histidine, 200 mM Tris, 0.5% P188, pH 8.8; and ii) optionally filtering the affinity eluate from step i) through a 0.2 μm filter to produce a diluted affinity eluate, wherein the pH of the diluted affinity eluate is increased compared to the pH of the affinity eluate; wherein the conductivity of the diluted affinity eluate is decreased compared to the conductivity of the affinity eluate; optionally, wherein the rAAV vector is an AAV9 vector or an AAV3B vector; and optionally wherein the affinity eluate is produced by affinity purification of an rAAV vector produced in a container (e.g., SUB) having a volume of 250 L or 2000 L.

load

The method of purifying rAAV (e.g., rAAV9, rAAV3B, etc.) vectors by AEX disclosed herein comprises loading a solution containing a substance to be purified (e.g., a rAAV vector) into an AEX stationary phase in a column. The load can be applied to the column by gravity feeding or the load can be pumped to a chromatographic column. In some embodiments, the solution containing the rAAV vector to be purified by AEX is selected from an affinity eluate, a supernatant from a cell lysate, and a post-harvest solution, each of which has undergone at least one other purification or processing step (e.g., cell lysis, flocculation, filtration, dilution, pH adjustment, chromatography). Before loading the solution onto the AEX column, the solution containing the rAAV vector to be purified can be diluted, filtered, and/or pH adjusted to make the solution compatible with processing by an AEX column. In some embodiments, the solution containing the rAAV vector to be purified is an eluate produced by affinity chromatography purification of an rAAV vector produced in a 100 L to 500 L (e.g., about 250 L), 1000 L to 3000 L (e.g., about 2000 L), or larger container (e.g., a disposable bioreactor (SUB)), wherein the eluate has been diluted and filtered.

In some embodiments, loading comprises applying a diluted and optionally filtered solution (e.g., affinity eluate) to an AEX column comprising about 2.0×10 ¹² vector genomes (VG)/mL to 2.0×10 ¹⁵ VG/mL, e.g., 2.0×10 ¹² VG/mL to 2.0×10 ¹³ VG/mL, 2.0×10 ¹³ VG/mL to 2.0×10 ¹⁴ VG/mL, 1.0×10 ¹⁴ VG/mL to 3.0×10 ¹⁴ VG/mL, 2.0×10 ¹⁴ VG/mL to 2.0×10 ¹⁵ VG/mL or more column volumes (also referred to as "column challenge VG/mL resin") as measured by qPCR analysis of sequences within the vector genome. In some embodiments, loading comprises applying a diluted solution (e.g., affinity eluate) containing 6.3×10 ¹³ to 9.4×10 ¹³ VG/mL column volume to an approximately 30 mL to 70 mL AEX column, such as measured by qPCR analysis of transgenic sequences within the vector genome (e.g., where the transgene is an ATP7B transgene). In some embodiments, loading comprises applying a diluted and optionally filtered solution (e.g., affinity eluate) containing 5×10 ¹³ to 1.3×10 ¹⁴ VG/mL column volume to an approximately 1.3 L AEX column, such as measured by qPCR analysis of ITR sequences within the vector genome. In some embodiments, loading comprises applying a diluted and optionally filtered solution (e.g., affinity eluate) containing 2.6×10 ¹² to 6.8×10 ¹³ VG/mL column volume to an approximately 6.4 L AEX column, such as measured by qPCR analysis of transgenic sequences within the vector genome.

In some embodiments, loading comprises applying a diluted and optionally filtered solution (e.g., affinity eluent) containing 2.5×10 ¹⁵ VG/L to 2.5×10 ¹⁶ VG/L, 2.5×10 ¹⁶ VG/L to 2.5×10 ¹⁷ VG/L, 2.5×10 ¹⁵ VG/L to 3.0×10 ¹⁷ VG/L, or more column volumes to an AEX column.

In some embodiments, loading comprises applying a diluted and optionally filtered solution (e.g., affinity ^eluent ) comprising 8.0×10 ¹² total VG to 2.0×10 ¹⁸ total VG, e.g., 8.0×10 ¹² total VG to 8.0×10 ¹³ total VG, 8.0×10 ¹³ to 8.0×10 ¹⁴ total VG, 8.0×10 ¹⁴ total VG to 8.0×10 ¹⁵ total VG, 8.0×10 ¹⁵ total VG to 8.0×10 ¹⁶ total VG, 8.0×10 16 total VG to 8.0×10 ¹⁷ total VG, 8.0×10 ¹⁷ total VG to 2.0×10 ¹⁸ total VG, or more, to the AEX column. In one embodiment, loading comprises applying a diluted and optionally filtered solution (eg, affinity eluate) containing ≤15×10 ¹⁶ VG/L column volume to an AEX column, and optionally wherein VG is measured by quantitative polymerase chain reaction (qPCR) analysis of the transgene.

When a solution containing the rAAV vector to be purified (e.g., affinity eluate) is loaded onto the column, the solution flows through the column stationary phase at a specific rate (e.g., cm/hr, mL/min) and is in contact with the stationary phase for a specific period of time (i.e., residence time).

In some embodiments, the residence time of the solution containing the rAAV vector loaded onto the column is 0.1 min/CV to 5 min/CV, for example 0.1 min/CV to 1.0 min/CV, 1.0 min/CV to 2 min/CV, 2 min/CV to 3 min/CV, 3 min/CV to 4 min/CV, 4 min/CV to 5 min/CV or longer. In some embodiments, the residence time of the solution containing the rAAV vector loaded onto the column is about 0.5 min/CV. In some embodiments, the residence time of the solution containing the rAAV vector loaded onto the column is about 1.5 min/CV. In some embodiments, the residence time of the solution containing the rAAV vector loaded onto the column is about 2.0 min/CV. In some embodiments, the residence time of the solution containing the rAAV vector loaded onto the column is 3.5 min/CV to 4.5 min/CV. In some embodiments, the residence time of the diluted and/or filtered affinity eluate containing the rAAV vector loaded on a 6.0 L to 6.6 L (eg, about 6.4 L) AEX column is 3.0 min/CV to 5.0 min/CV (eg, about 4 min/CV).

In some embodiments, the linear velocity of the solution containing the rAAV vector loaded onto the column is 100 cm/hr to 1800 cm/hr, for example, 100 cm/hr to 200 cm/hr, 200 cm/hr to 400 cm/hr, 400 cm/hr to 600 cm/hr, 600 cm/hr to 800 cm/hr, 800 cm/hr to 1000 cm/hr, 1000 cm/hr to 1500 cm/hr, 1500 cm/hr to 1800 cm/hr. In some embodiments, the linear velocity of the solution containing the rAAV vector loaded onto the column is 270 cm/hr to 330 cm/hr (for example, about 298 cm/hr, about 300 cm/hr). In some embodiments, the linear velocity of the solution containing the rAAV vector loaded onto the column is about 300 cm/hr, about 600 cm/hr, about 611 cm/hr or about 1790 cm/hr. In some embodiments, the diluted and optionally filtered affinity eluate containing the rAAV vector is loaded onto a 6.0 L to 6.6 L (eg, about 6.4 L) AEX column at a linear velocity of 270 cm/hr to 330 cm/hr (eg, about 300 cm/hr).

In some embodiments, the flow rate of the solution containing the rAAV vector loaded onto the column is 1.0mL/min to 3.0L/min, for example, 1.0mL/min to 10mL/min, 10mL/min to 100mL/min, 100mL/min to 500mL/min, 500mL/min to 1000mL/min, 1mL/min to 1.5L/min, 1mL/min to 2L/min, 2mL/min to 3L/min. In some embodiments, the flow rate of the solution containing the rAAV vector loaded onto the column is about 1.28mL/min. In some embodiments, the flow rate of the solution containing the rAAV vector loaded onto the column is about 314mL/min. In some embodiments, the flow rate of the solution containing the rAAV vector through the stationary phase in the column is 1.5L/min to 2.0L/min. In some embodiments, the flow rate of the solution containing the rAAV vector loaded onto the column is about 1.8L/min. In some embodiments, the diluted and/or filtered affinity eluate comprising the rAAV vector is loaded onto a 6.0 L to 6.6 L (eg, about 6.4 L) column at a flow rate of 1.5 L/min to 2.0 L/min (eg, about 1.8 L/min).

In some embodiments, a method for purifying a rAAV vector (e.g., rAAV9, rAAV3B, etc.) from an affinity eluate comprises: i) diluting the affinity eluate with a buffer comprising a detergent (e.g., P188), an amino acid (e.g., histidine), and a buffer (e.g., Tris); ii) optionally filtering the diluted affinity eluate; and iii) loading the diluted and optionally filtered affinity eluate onto a column comprising an AEX stationary phase, wherein the AEX stationary phase has been flushed, sterilized, washed and/or equilibrated prior to loading, and optionally wherein the AEX stationary phase is POROS ^™ 50HQ.

In some embodiments, the method for purifying rAAV vectors (e.g., rAAV9, rAAV3B, etc.) from affinity eluates comprises: i) eluting with a 5% lysine buffer containing about 100 mM to 300 mM (e.g., about 200 mM) histidine, 100 mM to 300 mM (e.g., about 200 mM) Tris, 1.0% to 1.5% (e.g., about 0.5%) P188, pH 7. 8.7 to 9.0 buffer to dilute the affinity eluate 14.4 to 15.5 times (e.g., about 15 times); ii) optionally filtering the diluted affinity eluate through a 0.1 to 0.45 μm (e.g., about 0.2 μm) filter in series; and iii) loading the diluted and filtered affinity eluate onto a column comprising an AEX stationary phase; optionally, wherein at least one step is performed at a linear velocity of 270 cm/hr to 330 cm/hr (e.g., about 298 cm/hr, about 300 cm/hr), a flow rate through the column of 1.5 L/min to 2.0 L/min (e.g., about 1.8 L/min) and/or a residence time of 1.5 min/CV to 4.5 min/CV (e.g., about 2 min/CV, about 4 min/CV), and optionally, wherein the AEX stationary phase is POROS ^™ 50HQ. In some embodiments, the chromatography column is a 6.0 L to 6.6 L (eg, about 6.4 L) column.

In some embodiments, the present invention provides a method for purifying rAAV (e.g., rAAV9, rAAV3B, etc.) vectors by AEX, the method comprising the following steps: i) pre-use flushing, comprising applying ≥4.5CV (e.g., about 5CV) of water for injection to the AEX stationary phase in the column; ii) disinfection, comprising applying 5CV to 10CV (e.g., about 8CV) or 14.4 to 17.6CV (e.g., about 16CV) of a solution comprising 0.1M to 1.0M (e.g., about 0.5M) NaOH to the AEX stationary phase in the column, optionally flowing upward; iii) regeneration, comprising applying 4.5CV to 5.5CV (e.g., about 5CV) of a solution comprising 1M to 3M (e.g., about 2M) NaCl, 50mM to 150mM (e.g., about 100mM) Tris, pH 7. iv) equilibration comprising applying 4.5CV to 5.5CV (e.g., about 5CV) of a solution comprising 50mM to 150mM (e.g., about 100mM) Tris, pH 8.5 to 9.5 (e.g., about 9) to the AEX stationary phase in the column; v) equilibration comprising applying 4.5CV to 5.5CV (e.g., about 5CV) of a solution comprising 50mM to 150mM (e.g., about 100mM) Tris, 400mM to 600mM (e.g., about 500mM) sodium acetate, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.0 (e.g., about 8.9) equilibration buffer is applied to the AEX stationary phase in the column; vi) equilibration, comprising applying ≥ 4.5CV (e.g., about 5CV) of an equilibration buffer comprising 100mM to 300mM (e.g., about 200mM) histidine, 100mM to 300mM (e.g., about 200mM) Tris, 0.1% to 1.0% (e.g., about 0.5%) P188, pH 8.5 to 9.5 (e.g., about 8.8) to the AEX stationary phase in the column; vii) loading the affinity eluate onto the AEX stationary phase in the column, optionally wherein the eluate has been a) treated with 100mM to 300mM (e.g., about 200mM) histidine, 100mM to 300mM (e.g., about 200mM) Tris, 0.1% to 1.0% (e.g., about 0.5%) P188, pH 8.5 to 9.5 (e.g., about 8.8) equilibration buffer is applied to the AEX stationary phase in the column; vii) loading the affinity eluate onto the AEX stationary phase in the column, optionally wherein the eluate has been a) treated with 100mM to 300mM (e.g., about 200mM) histidine a) diluting the stationary phase by about 14.4 to 15.5 times (e.g., about 15 times) a buffer containing 50 mM to 150 mM (e.g., about 100 mM) Tris, 0.1% to 1.0% (e.g., about 0.5%) P188, pH 8.7 to 9.0, and optionally b) filtering through a series of 0.1 μm to 0.45 μm (e.g., about 0.2 μm) filters before application to the stationary phase; and/or viii) equilibration comprising adding 4.5 CV to 5.5 CV (e.g., about 5 CV) of a buffer containing 50 mM to 150 mM (e.g., about 100 mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.7 to 9.0. 8.5 to 9.5 (e.g., about 8.9) of the equilibration buffer is applied to the AEX stationary phase in the column; optionally, at least one of steps i) to viii) is performed at a linear velocity of 270 cm/hr to 330 cm/hr (e.g., about 298 cm/hr, about 300 cm/hr) and/or a residence time of 1.5 min/CV to 4.5 min/CV (e.g., about 2 min/CV, about 4 min/CV); optionally, wherein the rAAV vector is a rAAV9 or rAAV3B vector; and optionally, wherein the AEX stationary phase is POROS ^™ 50HQ. In some embodiments, at least one of steps i) to vii) is passed through a 6L to 6.6L column (e.g., about 6.4L) at a flow rate of 1.5 L/min to 2.0 L/min (e.g., about 1.8 L/min) or through a 1.3L column at a flow rate of about 314 mL/min. One of ordinary skill will appreciate that the order of the above steps may vary.

Loading of additional solution

A method for purifying a rAAV vector (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., an affinity eluent) includes applying a loading additional solution to a column stationary phase after applying a solution comprising a rAAV vector. The loading additional solution is used to complete the application of the load or loading solution and remove unbound material from the column. In some embodiments, the loading additional solution is used to remove unbound material from the column. In some embodiments, the loading additional solution comprises 5mM to 50mM (e.g., about 20mM) Tris, pH 8.5 to 9.5 (e.g., about 9). In some embodiments, 9 to 11CV (e.g., about 10CV) of loading additional solution is applied to the column stationary phase. In some embodiments, the loading additional solution is applied to the column stationary phase at a speed of 200cm/hr to 2000cm/hr (e.g., about 1800cm/hr) and/or a residence time of 0.5min/CV. In some embodiments, 9CV to 11CV (e.g., about 10CV) of a loading chase solution comprising 20 mM Tris, pH 9 is applied to the AEX stationary phase in the column, optionally at a speed of 200 cm/hr to 2000 cm/hr (e.g., about 1800 cm/hr) and/or a residence time of about 0.5 min/CV.

Gradient elution

A method for purifying rAAV vectors (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., an affinity eluent) includes recovering complete capsids, intermediate capsids, and/or empty capsids by gradient elution. Gradient elution can include using at least 2 different solutions (e.g., gradient elution buffer) having different pH, conductivity, and/or modifier concentrations. During the gradient elution process, the percentage of the first solution changes inversely proportional to the percentage change of the second solution, so that when the solutions are mixed and flow through the column stationary phase, a gradient of pH, conductivity, and/or modifier concentration is generated. For example, at the beginning of the gradient elution, the percentage of the first solution (e.g., the first gradient elution buffer, buffer A) is 100%, while the percentage of the second solution (e.g., the second gradient elution, buffer B) is 0%, and at the end of the gradient elution, the percentage of the first solution is 0% and the percentage of the second solution is 100%. In another embodiment, at the beginning of the gradient elution, the percentage of the first solution (e.g., the first gradient elution buffer, buffer A) is 100%, the percentage of the second solution (e.g., the second gradient elution, buffer B) is 0%, and at the end of the gradient elution, the percentage of the first solution is 25%, and the percentage of the second solution is 75%. Those of ordinary skill will understand that the percentage of each solution at the beginning of the gradient and at the end of the gradient can be any percentage between 0% and 100%. For example, in some embodiments, the percentage of the first gradient elution buffer relative to the second gradient elution buffer at the beginning of elution, at the end of elution, or at any point during elution is about 100%/0%, about 99%/1%, about 98%/2%, about 97% 3%, about 96%/4%, about 95%/5%, about 90% 10%, about 80% 20%, about 75%/25%, about 70%/30%, about 60%/40%, about 50%/50%, about 40%/60%, about 30%/70%, about 25%/75%, about 20%/80%, about 10%/90%, about 5%/95%, about 4%/96%, about 3%/97%, about 2%/98%, about 1%/99%, or about 0%/100%.

In some embodiments, the percentage of the first gradient elution buffer relative to the second gradient elution buffer at the beginning of elution, at the end of elution, or at any point during elution is about 100% to 90%/0% to 10%, 90% to 80%/10% to 20%, 80% to 70%/20% to 30%, 70% to 60%/30% to 40%, 60% to 50%/40% to 50%, 50% to 40%/50% to 60%, 40% to 30%/60% to 70%, 30% to 20%/70% to 80%, 20% to 10%/80% to 90%, 10% to 0%/90% to 100%.

In some embodiments, during the application of 10 to 60CV of the solution to the column, the percentage of buffer A (e.g., the first gradient elution buffer) is reduced and the percentage of buffer B (e.g., the second gradient elution buffer) is increased, so that at the end of the gradient elution, the percentage of gradient elution buffer A is 0% and the percentage of gradient elution buffer B is 100%. In some embodiments, during the application of about 20CV of the solution to the column, the percentage of buffer A (e.g., the first gradient elution buffer) is reduced and the percentage of buffer B (e.g., the second gradient elution buffer) is increased, so that the rate of increase of buffer B is 5% buffer B/CV, and the final percentage of buffer B in the solution is 100%. In some embodiments, during the application of about 37.5CV of the solution to the column, the percentage of buffer A (e.g., the first gradient elution buffer) is reduced and the percentage of buffer B (e.g., the second gradient elution buffer) is increased, so that the rate of increase of buffer B is about 2% buffer B/CV, and the final percentage of buffer B in the solution is 75%.

In some embodiments, during the application of 10 to 60 CV of solution to the column, the percentage of buffer A (e.g., the first elution buffer) increases and the percentage of buffer B (e.g., the second elution buffer) decreases, such that at the end of the gradient elution, the percentage of gradient elution buffer A is 100% and the percentage of gradient elution buffer B is 0%. One skilled in the art recognizes that gradient elution can be run with different percentages of buffer (e.g., from 0% to 75% buffer B, corresponding to 100% to 25% buffer A; from 0% to 50% buffer B, corresponding to 100% to 50% buffer A).

In some embodiments, the method of purifying rAAV vectors by AEX of the present disclosure includes gradient elution of material from a stationary phase in a column, wherein the concentration of a component of a first gradient elution buffer or a second gradient elution buffer increases or decreases continuously during the gradient elution. In some embodiments, the material eluted from the stationary phase comprises an rAAV vector to be purified. The rate of increase or decrease of the concentration of a component of the first gradient elution buffer or the second gradient elution buffer may be equivalent to the change in component concentration/total CV. In some embodiments, the rate of increase of sodium acetate concentration during gradient elution is equivalent to the change in sodium acetate concentration/total CV applied to the stationary phase during elution. In some embodiments, the change in component concentration is about the concentration of the component at the beginning of elution compared to the concentration of the component at the end of elution. For example, the concentration of a component (e.g., a salt, such as sodium acetate) at the beginning of gradient elution is 0mM to 100mM, and the concentration of the component at the end of elution is 100mM to 1M. In some embodiments, the concentration of salt (e.g., sodium acetate) is 0 mM at the beginning of gradient elution, and the concentration of salt is 400 mM to 600 mM (e.g., about 500 mM) at the end of gradient elution. In some embodiments, during the application of 2CV to 100CV of elution buffer, the change in component concentration is 2 mM to 1 M from the beginning of the gradient to the end of the gradient elution. In some embodiments, during the application of 10CV to 60CV, 10CV to 50CV, 10CV to 40CV, 10CV to 30CV, or 15CV to 25CV (e.g., 20CV) of elution buffer, from the beginning of the gradient elution to the end of the gradient elution, the concentration of salt changes from about 0 mM to about 500 mM, so when the elution gradient contains 20CV solution, the rate of change of sodium acetate concentration is about 500 mM/20CV or 25 mM/CV. In some embodiments, during elution using 10CV to 60CV, 10CV to 50CV, 10CV to 40CV, 10CV to 30CV, or 15CV to 25CV (e.g., 37.5CV) of elution buffer, the salt concentration changes from about 0 mM to about 375 mM from the start of gradient elution to the end of gradient elution, so when the elution gradient contains 37.5CV of solution, the rate of change of sodium acetate concentration is about 375 mM/37.5CV or 10 mM/CV.

In some embodiments, during gradient elution, the sodium acetate concentration of the first gradient elution buffer, the second gradient elution buffer, or a mixture of the two increases continuously during gradient elution; wherein the rate of increase of sodium acetate is equal to the change in sodium acetate concentration applied to the stationary phase/total CV; wherein the rate of change in concentration of sodium acetate during gradient elution is about 5mM/CV to 15mM/CV, 10mM/CV to 50mM/CV, 10mM/CV to 40mM/CV, 10mM to 30mM/CV, or 20mM/CV to 30mM/CV (e.g., about 10mM/CV, about 25mM/CV).

In some embodiments, the change in component concentration during gradient elution is about 1 mM/CV to 1 M/CV, such as 1 mM/CV to 10 mM/CV, 1 mM/CV to 25 mM/CV, 5 mM/CV to 15 mM/CV, 10 mM/CV to 50 mM/CV, 50 mM/CV to 100 mM/CV, 100 mM/CV to 500 mM/CV, 500 mM/CV to 1 M/CV, 1 mM/CV to 750 mM/CV, 1 mM/CV to 500 mM/CV, 1 mM/CV to 100 mM/CV, 10 mM/CV to 750 mM/CV, or 50 mM/CV to 500 mM/CV.

In some embodiments, during the gradient elution process, the concentration of salt in the gradient solution can change. In some embodiments, during the gradient elution process, the concentration of salt (e.g., sodium chloride, sodium acetate, ammonium acetate, magnesium chloride, sodium sulfate, and combinations thereof) in the gradient solution can increase or decrease. For example, at the beginning of the gradient elution, the salt concentration in the gradient solution can be 0mM to 100mM, and increase to 50mM to 1M during the elution process, such as 50mM to 100mM, 100mM to 150mM, 150mM to 200mM, 200mM to 250mM, 250mM to 300mM, 300mM to 400mM, 400mM to 500mM, 500mM to 600mM. mM, 100mM to 1M, 100mM to 750mM, 100mM to 500mM, 100mM to 400mM, or 100mM to 200mM. In a further example, at the beginning of the gradient elution, the salt concentration in the gradient solution can be 50 mM to 1 M, for example, 50 mM to 100 mM, 100 mM to 150 mM, 150 mM to 200 mM, 200 mM to 250 mM, 250 mM to 300 mM, 300 mM to 400 mM, 400 mM to 500 mM, 500 mM to 600 mM, 600 mM to 700 mM, 700 mM to 800 mM, 800 mM to 900 mM, 900 mM to 1000 mM, 1000 mM to 1500 mM, 1500 mM to 2000 mM, 2000 mM to 2500 mM, 2500 mM to 3000 mM, 3000 mM to 4000 mM, 4000 mM to 5000 mM, 5000 mM to 6000 mM, 6000 mM to 7000 mM, 7000 mM to 8000 mM, 8000 mM to 9000 mM, 8000 mM to 1 ... In some embodiments, the concentration of sodium acetate in the gradient solution is about 0 mM at the beginning of the gradient elution, and the concentration of sodium acetate at the end of the gradient elution is about 500 mM. In some embodiments, the concentration of sodium acetate in the gradient elution solution is about 0 mM at the beginning of the gradient elution, and the concentration of sodium acetate in the gradient elution solution is about 375 mM at the end of the gradient elution.

In some embodiments, the pH of the gradient solution can change during the gradient elution process. In some embodiments, the pH of the gradient solution can increase or can be reduced during the gradient elution process. In some embodiments, when the gradient elution starts, the pH of the gradient solution can be between 7.0 and 11.0 (e.g., 7.0 to 7.5, 7.5 to 8.0, 8.0 to 8.5, 8.5 to 9.0, 9.0 to 9.5, 10.0 to 10.5, 10.5 to 11, 7.5 to 10.5, 8.0 to 10.0, 8.5 to 9.5 or 8.0 to 9.0). In some embodiments, at the end of the gradient elution, the pH of the gradient solution can be between 7.0 and 11.0 (e.g., 7.0 to 7.5, 7.5 to 8.0, 8.0 to 8.5, 8.5 to 9.0, 9.0 to 9.5, 10.0 to 10.5, 10.5 to 11, 7.5 to 10.5, 8.0 to 10.0, 8.5 to 9.5, or 8.0 to 9.0).

In some embodiments, during the process of gradient elution, the conductivity of the gradient solution can change. In some embodiments, during the process of gradient elution, the conductivity of the gradient solution can increase or decrease. In some embodiments, at the beginning of gradient elution, the conductivity of the gradient solution can be between 1.0mS/cm and 2.5mS/cm, for example, 1.2mS/cm and 2.0mS/cm. In some embodiments, at the end of gradient elution, the conductivity of the gradient solution can be between 20mS/cm and 35mS/cm, for example, 27mS/cm and 33mS/cm. In some embodiments, at the beginning of gradient elution, the conductivity of the gradient solution is about 1.6mS/cm, and at the end of gradient elution, the conductivity of the gradient solution is about 30mS/cm.

In some embodiments, during gradient elution, the concentration of the buffer in the gradient solution can be varied. In some embodiments, during gradient elution, the concentration of the buffer in the gradient solution (e.g., Tris (e.g., a mixture of Tris-Base and Tris-HCl), BIS-Tris propane, diethanolamine, diethylamine, tris(hydroxymethyl)methylglycine, triethanolamine and/or n,n-bis(hydroxyethyl)glycine) can be increased or decreased. For example, at the beginning of gradient elution, the concentration of the buffer in the gradient solution can be in the range of 10 mM to 500 mM, such as from 10 mM to 400 mM, from 10 mM to 300 mM, from 10 mM to 200 mM, from 10 mM to 50 mM, from 50 mM to 100 mM, from 50 mM to 150 mM, from 100 mM to 200 mM, from 100 mM to 400 mM, from 200 mM to 300 mM, from 300 mM to 400 mM, from 400 mM to 500 mM or more. At the end of the gradient elution, the concentration of the buffer in the gradient solution can be between 10 mM and 500 mM, for example, from 10 mM to 400 mM, from 10 mM to 300 mM, from 10 mM to 200 mM, from 10 mM to 50 mM, from 50 mM to 100 mM, from 50 mM to 150 mM, from 100 mM to 200 mM, from 100 mM to 400 mM, from 200 mM to 300 mM, from 300 mM to 400 mM, from 400 mM to 500 mM, or more.

In some embodiments, during the gradient elution process, the concentration of the detergent in the gradient solution can be changed. In some embodiments, during the gradient elution process, the concentration of the detergent (e.g., poloxamer 188 (P188), TritonX-100, polysorbate 80 (PS80), Brij-35, nonylphenoxypolyethoxyethanol (NP-40) and combinations thereof) in the gradient solution can be increased or decreased. For example, at the beginning of the gradient elution, the concentration of the detergent (e.g., P188) in the gradient solution can be in the range of 0.005% to 1.0%, for example, from 0.005% to 0.01%, from 0.005% to 0.5%, from 0.01% to 1.0%, from 0.01% to 0.5%, from 0.01% to 0.02%, from 0.02% to 0.03%, from 0.03% to 0.05%, from 0.06% to 0.07%, from 0.07% to 0.08%, from 0.09% to 0.10%, from 0.11% to 0.12%, from 0.13% to 0.14%, from 0.15% to 0.16%, from 0.17% to 0.18%, from 0.18% to 0.19%, from 0.20% to 0.21%, from 0.22% to 0.23%, from 0.24% to 0.25%, from 0.26% to 0.27%, from 0.28% to 0.29%, from 0.30% to 0.31%, from 0.31% to 0.32%, from 0.33% to 0.34%, from 0.35% to 0.36%, from 0.37 .03% to 0.04%, from 0.04% to 0.05%, from 0.05% to 0.06%, from 0.05% to 1.0%, from 0.05% to 0.5%, from 0.07% to 0.08%, from 0.08% to 0.09%, from 0.09% to 0.1%, from 0.1% to 0.5%, from 0.1% to 1.0%, from 0.5% to 1.0%.

In some embodiments, at the end of the gradient elution, the concentration of the detergent (e.g., P188) in the gradient solution can range from 0.005% to 1.0%, such as from 0.005% to 0.01%, from 0.005% to 0.5%, from 0.01% to 1.0%, from 0.01% to 0.5%, from 0.01% to 0.02%, from 0.02% to 0.03%, From 0.03% to 0.04%, from 0.04% to 0.05%, from 0.05% to 0.06%, from 0.05% to 1.0%, from 0.05% to 0.5%, from 0.07% to 0.08%, from 0.08% to 0.09%, from 0.09% to 0.1%, from 0.1% to 0.5%, from 0.1% to 1.0%, from 0.5% to 1.0%.

During the gradient elution process, while one or more aspects of the gradient solution (e.g., salt concentration) may vary, other aspects of the gradient, such as conductivity, pH, buffer concentration, detergent concentration, etc., may remain constant. For example, the pH of the gradient solution may be between 7.0 and 11.0, such as from 7.5 to 10.5, from 8.0 to 10.0, from 8.5 to 9.5, or from 8.0 to 9.0, from 7.0 to 7.5, from 7.5 to 8.0, from 8.0 to 8.5, from 8.5 to 9.0, from 9.0 to 9.5, from 9.5 to 10, from 10.0 to 10.5, or from 10.5 to 11.0, but remains constant throughout the gradient elution process (e.g., pH is about 8.8, about 8.9, about 9). In some embodiments, the pH of the gradient elution solution is about 8.9.

In some embodiments, the concentration range of buffer (e.g., Tris, BIS-Tris propane, n, n-bicine and combinations thereof) in gradient elution is 10mM to 500mM, e.g., from 10mM to 30mM, from 10mM to 50mM, from 50mM to 100mM, from 100mM to 200mM, from 200mM to 300mM, from 300mM to 400mM, from 400mM to 500mM, from 10mM to 400mM, from 10mM to 300mM, about 10mM to 200mM, about 50mM to about 150mM or more, but is constant (e.g., about 20mM, about 100mM) during gradient elution. In some embodiments, the concentration of buffer such as Tris in gradient elution is 50mM to 150mM. In some embodiments, the concentration of the buffer, such as Tris, in the gradient elution is about 100 mM.

In some embodiments, in gradient elution, the concentration of detergents such as poloxamer 188 (P188), Triton X-100, polysorbate 80 (PS80), Brij-35, nonylphenoxypolyethoxyethanol (NP-40), and combinations thereof can range from 0.005% to 0.01%, from 0.005% to 0.5%, from 0.01% to 1.0%, from 0.01% to 0.5%, from 0.01% to 0.02%, from 0.02% to 0.03%, from 0.03% to 0.05%, from 0.01% to 0.06%, from 0.01% to 0.08%, from 0.01% to 0.09%, from 0.02% to 0.10%, from 0.03% to 0.11%, from 0.04% to 0.06%, from 0.05% to 0.08%, from 0.06% to 0.09%, from 0.07% to 0.12%, from 0.08% to 0.13%, from 0.09% to 0.14%, from 0.01% to 0.16%, from 0.0 In some embodiments, the concentration of P188 during gradient elution is 0.05% to 0.1%. In some embodiments, the concentration of P188 during gradient elution is about 0.01%.

During gradient elution, material loaded onto the column elutes from the column at different points during the gradient as conditions within the column change, such as changes in pH, conductivity, salt concentration, and/or modifier concentration.

In some embodiments, during loading of a solution containing capsids to be purified, AAV capsids (e.g., complete capsids, intermediate capsids, empty capsids) are combined with a stationary phase. During gradient elution, as the percentage of the gradient elution buffer increases, the concentration of salt (e.g., sodium acetate) increases, and the complete rAAV vector is preferentially released (eluted) from the stationary phase, and the empty capsid is preferentially retained on the stationary phase. As the percentage of the gradient elution buffer further increases (as well as the salt concentration), the empty capsid is released in greater amounts. The empty capsid can also be recovered in the AEX column flow, i.e., the unbound portion. In some embodiments, complete and/or intermediate capsids are recovered in a portion of the first elution peak and the second elution peak of the AEX column (e.g., the first 2/3 of the second elution peak). By measuring the A260 and A280 of the eluent, the complete rAAV vector eluted from the stationary phase can be monitored during gradient elution, whereby the increase in the A260/A280 ratio indicates an increase in the complete rAAV vector present in the eluent.

In some embodiments, performing a gradient elution comprises applying about 20 CV of a solution to the column, wherein the solution is buffer A, buffer B, or a mixture of buffer A and buffer B, wherein at the beginning of the gradient elution, the solution is 100% buffer A and at the end of the step, the solution is 100% B, thereby creating a gradient between buffer A and buffer B during the elution phase, optionally wherein the rate of increase of buffer B is about 5% buffer B/CV, and optionally when buffer B comprises sodium acetate, the concentration of sodium acetate increases at a rate of 25 mM/CV.

In some embodiments, performing a gradient elution comprises applying about 37.5 CV of a solution to the column, wherein the solution is buffer A, buffer B, or a mixture of buffer A and buffer B, wherein at the beginning of the gradient elution, the solution is 100% buffer A, and at the end of the step, the solution is 75% buffer B and 25% buffer A, thereby creating a gradient between buffer A and buffer B during the elution phase, optionally wherein the rate of increase of buffer B is about 2% buffer B/CV, and optionally when buffer B comprises sodium acetate, the concentration of sodium acetate increases at a rate of 10 mM/CV.

In some embodiments, buffer A (e.g., the first gradient elution buffer) comprises 50mM to 150mM (e.g., about 100mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., about 8.9). In some embodiments, buffer B (e.g., the second gradient elution buffer) comprises about 400mM to 600mM (e.g., about 500mM) sodium acetate, 50mM to 150mM (e.g., about 100mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., about 8.9).

In some embodiments, gradient elution begins with applying 100% buffer A to the column and ends with applying 100% buffer B to the column over 20CV to 24CV (e.g., about 20CV), thereby creating a gradient between buffer A and buffer B during the elution phase, wherein buffer A comprises about 100 mM Tris, 0.01% P188, pH 8.9 and buffer B comprises about 500 mM sodium acetate, 100 mM Tris, 0.01% P188, pH 8.9. In some embodiments, gradient elution begins with applying 100% buffer A to the column and ends with applying 75% buffer B and 25% buffer A to the column over 30CV to 40CV (e.g., about 37.5CV), thereby producing a gradient between buffer A and buffer B during the elution phase, wherein buffer A comprises about 100 mM Tris, 0.01% P188, pH 8.9, and buffer B comprises about 500 mM sodium acetate, 100 mM Tris, 0.01% P188, pH 8.9.

In some embodiments, the gradient elution buffer comprises 5mM to 40mM (e.g., about 20mM) Tris, pH 9.0. In some embodiments, the gradient elution buffer comprises 5mM to 40mM (e.g., about 20mM) Tris, 400mM to 600mM (e.g., about 500mM) salt (e.g., NaCl, sodium acetate, ammonium acetate, and Na ₂ SO ₄ ), pH 9.0. In some embodiments, the gradient elution buffer comprises about 20mM Tris, 500mM sodium acetate, pH 9.0.

In some embodiments, the residence time of the gradient elution buffer (e.g., buffer A, buffer B, or a mixture of buffer A and buffer B) in the AEX column is 0.1 min/CV to 15 min/CV, e.g., 0.1 min/CV to 1 min/CV, 1 min/CV to 2 min/CV, 1.5 min/CV to 2.5 min/CV, 2 min/CV to 4 min/CV, 4 min/CV to 6 min/CV, 6 min/CV to 8 min/CV, or 8 min/CV to 10 min/CV, 10 min/CV to 12 min/CV, 12 min/CV to 15 min/CV. In some embodiments, the residence time of the solution in the column is 0.1 min/CV, about 0.5 min/CV, about 1.5 min/CV, about 2.0 min/CV, about 2.5 min/CV, about 3 min/CV, about 3.6 min/CV, or about 4 min/CV, about 5 min/CV, about 6 min/CV, about 7 min/CV, about 8 min/CV, about 9 min/CV, or about 10 min/CV. In some embodiments, the residence time of the gradient elution buffer (e.g., buffer A, buffer B, or a mixture of buffer A and buffer B) in the column is about 3.6 min/CV or 4 min/CV. In some embodiments, the residence time of the gradient elution buffer (e.g., buffer A, buffer B, or a mixture of buffer A and buffer B) in the column is about 2.0 min/CV.

In some embodiments, the residence time of the gradient elution buffer (e.g., buffer A, buffer B, or a mixture of buffer A and buffer B) in the column is 1.5 to 2.5 min/CV (e.g., about 2 min/CV). In some embodiments, the residence time of the gradient elution buffer (e.g., buffer A, buffer B, or a mixture of buffer A and buffer B) in the column is 3.5 to 4.5 min/CV (e.g., about 4 min/CV). In some embodiments, the residence time of the gradient elution buffer (e.g., buffer A, buffer B, or a mixture of buffer A and buffer B) in the column is about 11 min/CV.

In some embodiments, the linear velocity of the gradient elution buffer (e.g., buffer A, buffer B, or a mixture of buffer A and buffer B) through the stationary phase in the column is 50 to 1800 cm/hr, e.g., 50 cm/hr to 100 cm/hr, 100 cm/hr to 200 cm/hr, 200 cm/hr to 400 cm/hr, 400 cm/hr to 600 cm/hr, 600 cm/hr to 800 cm/hr, 800 cm/hr to 1000 cm/hr, 1000 cm/hr to 1500 cm/hr, or 1500 cm/hr to 1800 cm/hr. In some embodiments, the linear velocity of the gradient elution buffer (e.g., buffer A, buffer B, or a mixture of buffer A and buffer B) through the stationary phase in the column is about 298 cm/hr or about 300 cm/hr. In some embodiments, the linear velocity of the gradient elution buffer (e.g., buffer A, buffer B, or a mixture of buffer A and buffer B) through the stationary phase in the column is about 75 cm/hr, about 204 cm/hr, about 298 cm/hr, about 300 cm/hr, about 597 cm/hr, or about 600 cm/hr. In some embodiments, the linear velocity of the gradient elution buffer (e.g., buffer A, buffer B, or a mixture of buffer A and buffer B) through the AEX stationary phase in a 6.0 L to 6.6 L (e.g., 6.4 L) column is about 270 cm/hr to 330 cm/hr (e.g., about 300 cm/hr).

In some embodiments, the flow rate of the gradient elution buffer (e.g., buffer A, buffer B, or a mixture of buffer A and buffer B) through the stationary phase in the column is about 0.2mL/min to 2.0L/min, such as 0.2mL/min to 1mL/min, 1.0mL/min to 10mL/min, 10mL/min to 100mL/min, 100mL/min to 500mL/min, 500mL/min to 1L/min, 1L/min to 1.5L/min, or 1L/min to 2L/min. In some embodiments, the flow rate of the gradient elution buffer (e.g., buffer A, buffer B, or a mixture of buffer A and buffer B) through the stationary phase in the column is about 0.47mL/min. In some embodiments, the flow rate of the gradient elution buffer (e.g., buffer A, buffer B, or a mixture of buffer A and buffer B) through the stationary phase in the column is about 1.67mL/min. In some embodiments, the flow rate of the gradient elution buffer (e.g., buffer A, buffer B, or a mixture of buffer A and buffer B) through the stationary phase in the column is about 314 mL/min. In some embodiments, the flow rate of the gradient elution buffer (e.g., buffer A, buffer B, or a mixture of buffer A and buffer B) through the stationary phase in the column is about 1.8 L/min. In some embodiments, the flow rate of the gradient elution buffer (e.g., buffer A, buffer B, or a mixture of buffer A and buffer B) through the stationary phase in the column is about 1.5 to 2.0 L/min. In some embodiments, the flow rate of the gradient elution buffer (e.g., buffer A, buffer B, or a mixture of buffer A and buffer B) through the AEX stationary phase in a 1.3L column is about 314 mL/min. In some embodiments, the flow rate of the gradient elution buffer (e.g., buffer A, buffer B, or a mixture of buffer A and buffer B) through the AEX stationary phase in a 6.0L to 6.6L (e.g., 6.4L) column is about 1.8 L/min.

In some embodiments, a method of purifying a rAAV vector (eg, rAAV9, rAAV3B, etc.) from a solution (eg, an affinity eluate) comprises applying a gradient elution buffer to a column comprising a POROS ^™ 50HQ stationary phase. In some embodiments, the method of purifying rAAV vectors (e.g., AAV9, AAV3B, etc.) from an affinity eluate comprises starting with a gradient elution using 100% buffer A (e.g., comprising 50 mM to 150 mM (e.g., about 100 mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., about 8.9)), and applying 75% to 100% buffer B (e.g., 50 mM to 150 mM (e.g., about 100 mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., about 8.9)) to the stationary phase in the column over a period of 15 to 40 CV (e.g., about 20 CV, about 37.5 CV). 8.5 to 9.5 (eg, about 8.9)), wherein the percentage change rate of buffer B during the gradient elution is 2% buffer B/CV to 5% buffer B/CV. In some embodiments, the column is a 6.0 L to 6.6 L (eg, 6.4 L) column.

In some embodiments, the method of purifying rAAV vectors (e.g., AAV9, AAV3B, etc.) from an affinity eluate comprises performing a gradient elution starting with applying 100% of a first buffer (comprising about 100 mM Tris, 0.01% P188, pH 8.9), applying 75% to 100% of a second buffer (comprising 500 mM sodium acetate, 100 mM Tris, 0.01% P188, pH 8.9) to a column comprising an AEX stationary phase over a period of 15 CV to 40 CV (e.g., about 20 CV, about 37.5 CV). 8.9), the linear velocity is 270cm/hr to 330cm/hr (e.g., about 298cm/hr, about 300cm/hr), the flow rate is 1.5L/min to 2.0L/min (e.g., about 1.8L/min), and/or the residence time is 1.5min/CV to 4.5min/CV (e.g., 2min/CV, 4min/CV), thereby generating a gradient between the first buffer and the second buffer during elution, wherein the rate of change of the percentage of buffer B during the gradient elution is 2% buffer B/CV to 5% buffer B/CV. In some embodiments, the column is a 6.0L to 6.6L (e.g., 6.4L) column.

In some embodiments, the present invention provides a method for purifying rAAV (e.g., rAAV9, rAAV3B, etc.) vectors by AEX, the method comprising the following steps: i) pre-use flushing, comprising applying ≥4.5CV (e.g., about 5CV) of water for injection to the AEX stationary phase in the column; ii) disinfection, comprising applying 5CV to 10CV (e.g., about 8CV) or 14.4 to 17.6CV (e.g., about 16CV) of a solution containing 0.1M to 1.0M (e.g., about 0.5M) NaOH to the AEX stationary phase in the column, optionally flowing upward; iii) regeneration, comprising 4.5 to 5.5CV (e.g., about 5CV) of a solution containing 1M to 3M (e.g., about 2M) NaCl, 50mM to 150mM (e.g., about 100)mM Tris, pH 5. iv) equilibration, comprising applying 4.5 to 5.5 CV (e.g., about 5 CV) of a solution comprising 50 mM to 150 mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., about 9) to the AEX stationary phase in the column; v) equilibration, comprising applying 4.5 to 5.5 CV (e.g., about 5 CV) of a solution comprising 50 mM to 150 mM (e.g., about 100 mM) Tris, 400 mM to 600 mM (e.g., about 500 mM) sodium acetate, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., about 8.9) equilibration buffer is applied to the AEX stationary phase in the column; vi) equilibration, comprising applying ≥ 4.5CV (e.g., about 5CV) of an equilibration buffer comprising 100mM to 300mM (e.g., about 200mM) histidine, 100mM to 300mM (e.g., 200mM) Tris, 0.1% to 1.0% (e.g., about 0.5%) P188, pH 8.5 to 9.5 (e.g., about 8.8) to the AEX stationary phase in the column; vii) loading an affinity eluate comprising the rAAV vector to be purified onto the AEX stationary phase in the column, optionally wherein the eluate has been a) treated with an affinity eluate comprising 100 to 300mM (e.g., about 200mM) histidine, 100 to 300mM (e.g., about 200mM) Tris, 0.1% to 1.0% (e.g., about 0.5%) P188, pH 8.7 to 9.0 buffer diluted about 14.4 to 15.5 times (e.g., about 15 times), and optionally b) filtered through a 0.2 μm filter in series before application to the AEX stationary phase; viii) equilibration comprising applying 4.5 to 5.5 CV (e.g., about 5 CV) of an equilibration buffer comprising 50 mM to 150 mM (e.g., about 100 mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., about 8.9) to the AEX stationary phase in the column, and/or ix) performing a gradient elution of material from the stationary phase in the column, applying a 50 mM to 150 mM (e.g., about 100 mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., about 8.9) equilibration buffer to the AEX stationary phase. Starting with 100% first buffer at pH 8.5 to 9.0 (e.g., about 8.9), using a buffer comprising 400 mM to 600 mM (e.g., about 500 mM) sodium acetate, 50 mM to 150 mM (e.g., about 100 mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., pH 8.9) over 20 CV to 24 CV (e.g., about 20 CV). 8.9), optionally wherein at least one of steps i) to ix) is performed at a linear velocity of 270 cm/hr to 330 cm/hr (e.g., about 298 cm/hr, about 300 cm/hr) and/or a residence time of 1.5 min/CV to 4.5 min/CV (e.g., about 2 min/CV, about 4 min/CV); optionally wherein the rAAV vector is a rAAV9 or rAAV3B vector; and optionally wherein the AEX stationary phase is POROS ^TM 50HQ. In some embodiments, at least one of steps i) to ix) is performed at a flow rate of 1.5 L/min to 2.0 L/min (e.g., about 1.8 L/min) through a 6 L to 6.6 L (e.g., about 6.4 L) column or at a flow rate of about 314 mL/min through a 1.3 L column. In some embodiments, the material eluted from the stationary phase during the gradient elution comprises the rAAV vector to be purified. One of ordinary skill will appreciate that the order of the above steps may vary.

Gradient hold

In some embodiments, a method for purifying rAAV vectors (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., affinity eluent) includes applying a gradient holding solution to a column comprising an AEX stationary phase (e.g., POROS ^TM 50HQ) to continue the volume to ensure complete gradient formation, preferably after gradient elution. In some embodiments, the gradient holding solution comprises at least one component selected from salts, buffers, detergents, amino acids, and combinations thereof. In some embodiments, the gradient holding solution comprises a salt selected from sodium chloride, sodium acetate, ammonium acetate, magnesium chloride, sodium sulfate, and combinations thereof. In some embodiments, the gradient holding solution comprises a buffer selected from Tris, BIS-Tris propane, n,n-di(hydroxyethyl)glycine, and combinations thereof. In some embodiments, the gradient holding solution comprises a detergent selected from poloxamer 188 (P188), Triton X-100, polysorbate 80 (PS80), Brij-35, nonylphenoxypolyethoxyethanol (NP-40), and combinations thereof. In some embodiments, the gradient holding solution comprises salt, buffer and detergent. In some embodiments, the gradient holding solution comprises an amino acid selected from histidine, arginine, glycine, citrulline and a combination thereof. In some embodiments, the gradient holding solution comprises sodium acetate, Tris and P188.

In some embodiments, the gradient holding solution comprises 5mM to 1M (e.g., about 500mM) sodium acetate, 1mM to 1M (e.g., about 100mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., about 8.9). In some embodiments, a gradient holding solution of 1CV to 10CV, e.g., 1CV to 3CV, 1CV to 5CV, 4.4CV to 5.5CV, 1CV to 8CV, or 5CV to 10CV is applied to the column stationary phase. In some embodiments, 4.5CV to 5.5CV (e.g., about 5CV) of a gradient holding solution comprising about 500 mM sodium acetate, 100 mM Tris, 0.01% P188, pH 8.9 is applied to an AEX column stationary phase (e.g., POROS™ 50HQ) at a linear velocity of 270 cm/hr to 330 cm/hr (e.g., about 300 cm/hr), a flow rate of 0.4 mL/min to 2.0 L/min (e.g., about 1.8 L/min), and/or a residence time of 3.5 to 11 min/ ^CV .

Step elution

Methods for purifying rAAV vectors (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., an affinity eluent) include step elution (also known as "isocratic elution"). In some embodiments, step elution includes applying at least one step elution solution to a column stationary phase, however, more typically, multiple step elution solutions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) are applied to the column stationary phase.

In some embodiments, the step-by-step elution solution comprises at least one component selected from salt, buffer, detergent, amino acid and combination thereof. In some embodiments, the step-by-step elution solution comprises a salt selected from sodium chloride, sodium acetate, ammonium acetate, magnesium chloride, sodium sulfate and combination thereof. In some embodiments, the step-by-step elution solution comprises a buffer selected from Tris, BIS-Tris propane, n,n-di(hydroxyethyl)glycine and combination thereof. In some embodiments, the step-by-step elution solution comprises an amino acid selected from histidine, arginine, glycine, citrulline and combination thereof. In some embodiments, the step-by-step solution comprises a detergent selected from poloxamer 188 (P188), Triton X-100, polysorbate 80 (PS80), Brij-35, nonylphenoxypolyethoxyethanol (NP-40) and combination thereof. In some embodiments, the step-by-step elution solution includes salt, buffer and detergent. In some embodiments, the step-by-step elution solution comprises sodium acetate and Tris.

In some embodiments, the concentration of the buffer (e.g., Tris) in the step elution solution is about 1 mM to 500 mM, e.g., 1 mM to 10 mM, 10 mM to 50 mM, 50 mM to 100 mM, 100 mM to 200 mM, 200 mM to 300 mM, 300 mM to 400 mM, 400 mM to 500 mM. In some embodiments, the concentration of Tris in the step elution solution is about 20 mM.

In some embodiments, the concentration of salt (e.g., sodium acetate) in the step elution solution is about 5 mM to 600 mM, such as 5 mM to 50 mM. 50 mM to 100 mM, 100 mM to 200 mM, 200 mM to 300 mM, 300 mM to 400 mM, 400 mM to 500 mM, 500 mM to 600 mM. In some embodiments, the concentration of sodium acetate in the step elution solution is about 64 mM, about 75 mM, about 85 mM, about 95 mM, about 100 mM, about 105 mM, about 109 mM, about 110 mM, about 150 mM, about 200 mM, about 300 mM, about 400 mM, about 500 mM or more.

In some embodiments, the step elution solution comprises 10mM to 50mM (e.g., about 20mM) Tris, 5 to 600mM salt, pH 8.9 to 9.1. In some embodiments, the salt is sodium acetate. In some embodiments, at least one step elution solution comprises a buffer selected from the group consisting of 20mM Tris, 64mM sodium acetate, pH 9.0; 20mM Tris, 75mM sodium acetate, pH 9.0; 20mM Tris, 85mM sodium acetate, pH 9.0; 20mM Tris, 95mM sodium acetate, pH 9.0; 20mM Tris, 100mM sodium acetate, pH 9.0; 20mM Tris, 105mM sodium acetate, pH 9.0; 20mM Tris, 109mM sodium acetate, pH 9.0; and 20mMTris, 500mM sodium acetate, pH 9.0.

In some embodiments, at least one step elution solution of 1CV to 20CV, such as 1CV to 3CV, 2CV to 3CV, 1CV to 8CV, 4CV to 11CV, 5CV to 10CV, 10CV to 20CV, or 15CV to 20CV is applied to the column stationary phase. In some embodiments, at least one step elution solution of about 2.5CV, about 5CV, about 10CV, or about 20CV is applied to the column stationary phase.

In some embodiments, the step elution solution is applied to the column stationary phase at a linear velocity of 50 cm/hr to 2000 cm/hr (e.g., about 75 cm/hr, about 150 cm/hr, about 204 cm/hr, about 600 cm/hr, and about 1800 cm/hr). In some embodiments, the residence time of the step elution solution in the column stationary phase is 1 min/CV to 15 min/CV (e.g., about 1.5 min/CV, about 6 min/CV, and about 12 min/CV).

In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more step elution solutions are applied to the stationary phase in the column. In some embodiments, 1 CV to 20 CV of at least one step elution solution (e.g., 2, 3, 4, 5, etc.) comprising 20 mM Tris, 5 to 600 mM salt (e.g., sodium acetate), pH 8.9 to 9.1 (e.g., pH 9.0) is applied to an AEX column (e.g., POROS ^™ 50HQ) at a linear velocity of 50 cm/hr to 2000 cm/hr and a residence time of 1 min/CV to 15 min/CV.

In some embodiments, the step elution solution may also be a strip solution and is preferably applied to the column stationary phase as the last step elution step. The last step elution solution (i.e., strip solution) may be applied to the column stationary phase to cause the substance (e.g., rAAV vector) to be released from the stationary phase. In some embodiments, the last step elution solution may have a high salt concentration (e.g., >450mM). In some embodiments, the last step elution solution may comprise 20mM Tris, 500mM salt (e.g., sodium acetate), pH 8.9 to 9.1.

A method for purifying rAAV vectors (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., an affinity eluate) comprises applying a strip solution to a column stationary phase, preferably after applying at least one step elution solution. In some embodiments, the strip solution comprises 20 mM Tris, 500 mM sodium acetate, pH 8.9 to 9.1.

Fraction collection, neutralization and pooling

A method for purifying an rAAV vector (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., an affinity eluent) by AEX includes collecting at least one eluate fraction from an AEX column to recover and enrich complete capsids, optionally during gradient elution. In some embodiments, complete capsids are collected in a portion of the first elution peak and the second elution peak (e.g., the first 2/3 of the second elution peak). Empty capsids can be recovered in the AEX column flow-through, i.e., the unbound portion. Empty capsids can also be recovered in the elution peak, but are generally at a lower level compared to the recovery of the column flow-through. Intermediate capsids can be recovered together with complete capsids or empty capsids.

During elution (e.g., gradient elution) of an AEX method for purifying rAAV vectors, the eluate from the AEX column can be collected as discrete fractions of specific volumes and/or having specific properties (e.g., absorbance at a specific wavelength). For example, the volume of eluate that can be collected from the AEX column during a chromatography step (e.g., gradient elution) is, for example, 1 mL to 4 L, e.g., 1 mL to 10 mL, 1 mL to 3 L, 1 mL to 2 L, 1 mL to 1 L, 1 mL to 100 mL, 10 mL to 50 mL, 50 mL to 100 mL, 100 mL to 250 mL, 250 mL to 500 mL, 500 mL to 1 L, 1 L to 1.5 L, 1.5 L to 2 L, 2 L to 3 L, 3 L to 4 L, or more (e.g., For example, about 1 mL, 5 mL, 10 mL, 100 mL, 500 mL, 1 L, 2 L, 3 L, 4 L, etc.) or specific CV equivalents such as 1/8 CV to 10 CV, for example, 1/8 CV to 1 CV, 1 CV to 2 CV, 2 CV to 5 CV, 5 CV to 8 CV, 8 CV to 10 CV or more (e.g., 1/8 CV, 1/4 CV, 1/3 CV, 1/2 CV, 1 CV, 2 CV, 3 CV, 4 CV, 5 CV, 6 CV, 7 CV, 8 CV, 9 CV or more). In some embodiments, during the chromatography step, ≥ 1/3 CV volume of eluent can be collected from the AEX column. In some embodiments, during the chromatography step, about 1/2 CV volume of eluent can be collected from the AEX column. In some embodiments, collecting at least one eluate fraction from the AEX column during a chromatography step (e.g., gradient elution) comprises collecting the eluate when the absorbance of the column flow-through (e.g., absorbance at 260 nm and/or 280 nm) reaches an absorbance threshold (e.g., ≥0.5 mAU/mm path length, e.g., 10 mAU/mm path length). In some embodiments, collecting at least a portion of the eluate from the AEX column during a chromatography step (e.g., gradient elution) comprises collecting the eluate when the gradient elution solution comprises a specific percentage of elution buffer, e.g., when the gradient elution solution comprises about 30% to about 35% (e.g., about 32%) to about 50% to about 55% (e.g., about 52%) of a second elution buffer (e.g., buffer B). In some embodiments, the second elution buffer (e.g., buffer B) comprises 500 mM sodium acetate, 100 mM Tris, 0.01% P188, pH 8.9.

In some embodiments, the eluent is collected in multiple fractions (e.g., 5 fractions, 10 fractions, 20 fractions or more) of a specific volume (e.g., 1/3CV, 1/2CV). In some embodiments, the eluent is collected as a single fraction. In some embodiments, when the A280 of the eluent is ≥ 0.5mAU, the eluent is collected in a single fraction, and about 2.3CV is optionally collected.

In some embodiments, collecting at least one eluent fraction from the AEX column comprises measuring the absorbance at 260 nm (A260) and/or the absorbance at 280 nm (A280) of the eluent collected from the column, optionally during a gradient elution. In some embodiments, the measurement of the absorbance of the AEX eluent (e.g., at A260 or A280) is performed in series with collecting at least one eluent fraction. In some embodiments, when the eluent collected from the AEX column during a chromatographic elution (e.g., a gradient elution) has an A280 of 0.5 to 10 mAU/mm path length, at least one eluent fraction is collected. In some embodiments, collecting the eluent from the AEX column comprises collecting at least one eluent fraction of a volume ≥ 1/3CV. In some embodiments, optionally during gradient elution, collecting at least one eluate fraction (e.g., a first eluate fraction) from the AEX column comprises collecting at least one eluate fraction when the A280 of the eluate is ≥0.5 mAU/mm path length, wherein the volume of the at least one eluate fraction is ≥1/3CV.

In some embodiments, 1 to 25 eluent fractions, such as 1 to 5 fractions, 5 to 10 fractions, 10 to 15 fractions, 15 to 20 fractions, or 20 to 25 fractions, are collected from the AEX column, optionally during gradient elution. In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more eluent fractions are collected from the AEX column. In some embodiments, at least 10 eluent fractions are collected from the AEX column, optionally during gradient elution, and the volume of each fraction is ≥ 1/3CV. In some embodiments, optionally during gradient elution, at least 20 eluate fractions are collected from the AEX column, each eluate fraction having a volume of about 1/2 CV.

In some embodiments, a method for purifying an rAAV vector (e.g., rAAV9, rAAV3B, etc.) from an affinity eluate by AEX comprises collecting the first of about 10 eluate fractions from an AEX column, optionally during a gradient elution, when the A280 of the eluate is >0.5 mAU/mm path length, and wherein the volume of each fraction is ≥1/3CV.

In some embodiments, a method for purifying an rAAV vector (e.g., rAAV3B, etc.) from an affinity eluate by AEX includes, optionally during gradient elution, collecting the first of about 20 eluate fractions from an AEX column when the percentage of the second elution buffer (e.g., buffer B) of the gradient elution solution is about 30% to about 35% (e.g., about 32%), and continuing to collect until the percentage of the second elution buffer (e.g., buffer B) is about 50% to 55% (e.g., about 52%) of the gradient elution solution, wherein the volume of each fraction is about 1/2CV.

In some embodiments, the method of purifying rAAV vectors (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., an affinity eluent) by AEX includes, optionally during gradient elution, adjusting the pH of at least one eluate fraction collected from the AEX column. In some embodiments, adjusting the pH value of at least one eluate fraction is referred to as a neutralization step. In some embodiments, before adjusting the pH, the pH of at least one eluate fraction collected from the AEX column is pH 8.5 to 9.1. In some embodiments, the pH of at least one eluate fraction is adjusted to 6.8 to 7.6 (e.g., about pH 7.2). In some embodiments, the pH of at least one eluate fraction is adjusted to 7.5 to 7.7 (e.g., about pH 7.6).

In some embodiments, the pH of at least one eluate fraction collected from an AEX column is adjusted to 6.8 to 7.6 by adding 14% to 16% (eluate volume weight) (e.g., 14.3% to 14.7%, 14.3% to 15%, 15% to 16%) of a solution comprising 50mM to 500mM, e.g., about 50mM to 100mM, 50mM to 400mM, 50mM to 300mM, 50mM to 200mM, 100mM to 200mM, 100mM to 300mM, 200mM to 300mM, 300mM to 400mM, or 400mM to 500mM sodium acetate, pH 3.0 to 4.0 (e.g., about 3.5). In some embodiments, optionally adjusting the pH of at least one eluate fraction collected from the AEX column during gradient elution comprises adjusting the pH to 6.8 to 7.6 (e.g., about pH 7.2) by adding 14% to 16% (e.g., about 15%) of the eluate volume weight of a solution comprising about 250 mM sodium citrate, pH 3.5. In some embodiments, the pH of at least one eluate fraction collected from the AEX column is adjusted by adding a solution comprising about 50 mM citrate, pH 3.6.

In some embodiments, the pH of at least one eluate fraction collected from an AEX column is adjusted to about 7.5 to 7.7 by collecting at least one fraction in a container comprising about 0.01 CV to 0.1 CV (e.g., about 0.066 CV) of a solution comprising 50 mM to 500 mM, e.g., about 50 mM to 100 mM, 50 mM to 400 mM, 50 mM to 300 mM, 50 mM to 200 mM, 100 mM to 200 mM, 100 mM to 300 mM, 200 mM to 300 mM, 300 mM to 400 mM, or 400 mM to 500 mM sodium citrate, pH 3.0 to 4.0 (e.g., about 3.5). In some embodiments, optionally during gradient elution, adjusting the pH of at least one eluate fraction collected from the AEX column comprises adjusting the pH to 7.5 to 7.7 (e.g., pH 7.6) by collecting at least one fraction into a container comprising about 0.01 CV to 0.1 CV (e.g., about 0.066 CV) of a solution comprising about 250 mM sodium citrate (pH 3.5).

In some embodiments, the method of purifying a rAAV vector (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., an affinity eluate) by AEX comprises, optionally during gradient elution, measuring the absorbance of at least one eluate fraction collected from the AEX column. In some embodiments, the absorbance of at least one eluate fraction is measured using analytical size exclusion chromatography (SEC) in a high performance liquid chromatography (HPLC) system, and the absorbance is measured at one or more wavelengths (e.g., 260 nm and/or 280 nm).

In some embodiments, measuring the absorbance of at least one eluate fraction collected from the AEX column comprises measuring the absorbance at 260 nm (A260) and 280 nm (A280), and optionally determining the A260/A280 ratio (when measured by SEC, the measurement may be referred to as SEC A260/A280 or A260/A280 (SEC)). The A260/A280 ratio of at least one eluate fraction collected from the AEX column is at least 0.5 to 2.0, such as at least 0.5 to 0.75, 0.75 to 1.0, 1.0 to 1.25, 1.25 to 1.5, 0.5 to 1.5, 1.5 to 2.0 or more. At least one eluate fraction collected from the AEX column may have an A260/A280 ratio of at least 0.5, at least 0.55, at least 0.6, at least 0.65, at least 0.7, at least 0.75, at least 0.8, at least 0.85, at least 0.9, at least 0.95, at least 1.0, at least 1.10, at least 1.11, at least 1.12, at least 1.13, at least 1.14, at least 1.15, at least 1.16, at least 1.17, at least 1.18, at least 1.19, at least 1.20, at least 1.21, at least 1.22, at least 1.23, at least 1.24, at least 1.25, at least 1.26, at least 1.27, at least 1.28, at least 1.29, at least 1.30, at least 1.31 1.25, at least 1.26, at least 1.27, at least 1.28, at least 1.29, at least 1.30, at least 1.31, at least 1.32, at least 1.33, at least 1.34, at least 1.35, at least 1.36, at least 1.37, at least 1.38, at least 1.39, at least 1.40 or more). In some embodiments, optionally during gradient elution, at least one eluate fraction collected from the AEX column has an A260/A280 ratio of at least 1.25.

In some embodiments, the method of purifying a rAAV vector (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., an affinity eluate) by AEX includes, optionally during gradient elution, measuring the percentage of high molecular weight substances (HMMS) of at least one eluate fraction collected from the AEX column. In some embodiments, the percentage of HMMS is measured by SEC. In some embodiments, the percentage of HMMS of at least one eluate fraction collected during AEX purification of rAAV vectors produced in a 250L SUB ranges from 0% to 10% (e.g., 0% to 3.2%). In some embodiments, the percentage of HMMS of at least one eluate fraction collected during AEX purification of rAAV vectors produced in a 2000L SUB ranges from 0.5% to 15% (e.g., 1.2% to 8.3%).

In some embodiments, the method of purifying a rAAV vector (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., an affinity eluate) by AEX includes optionally determining the purity percentage of at least one eluate fraction collected from the AEX column during gradient elution. In some embodiments, the purity percentage is determined by RP-HPLC. In some embodiments, the purity percentage of at least one eluate fraction collected during AEX purification of rAAV vectors produced in 250LSUB ranges from 95% to 100% (e.g., 99.1% to 99.4%). In some embodiments, the purity percentage of at least one eluate fraction collected during AEX purification of rAAV vectors produced in 2000L SUB ranges from 75% to 100% (e.g., 79.6% to 98.7%).

In some embodiments, the method of purifying a rAAV vector (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., an affinity eluate) by AEX includes, optionally during gradient elution, measuring the amount of host cell DNA (HC-DNA) in at least one eluate fraction collected from the AEX column. In some embodiments, the amount of HC-DNA is measured by qPCR. In some embodiments, the amount of HC-DNA in at least one eluate fraction collected during AEX purification of rAAV vectors produced in a 250L SUB is 0.1 pg/1×10 ⁹ VG to 20 pg/1×10 ⁹ VG (e.g., 1.0 pg/1×10 ⁹ VG to 5.9 pg/1×10 ⁹ VG). In some embodiments, the amount of HC-DNA in at least one eluate fraction collected during AEX purification of rAAV vectors produced in 2000L SUB is 0.1 pg/1×10 ⁹ VG to 50 pg/1×10 ⁹ VG (eg, 2.7 pg/1×10 ⁹ VG to 26.5 pg/1×10 ⁹ VG).

In some embodiments, the method of purifying rAAV vectors (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., affinity eluate) by AEX includes, optionally during gradient elution, measuring the amount of host cell protein (HCP) of at least one eluate fraction collected from the AEX column. In some embodiments, the amount of HCP is measured by ELISA. In some embodiments, the amount of HCP in at least one eluate fraction collected during AEX purification of rAAV vectors produced in 250LSUB ranges from below the level of quantification (LLOQ) to 5.78 pg/1×10 ⁹ VG. In some embodiments, the amount of HCP in at least one eluate fraction collected during AEX purification of rAAV vectors produced in 2000L SUB is LLOQ.

In some embodiments, a method of purifying a rAAV vector (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., an affinity eluate) comprises combining at least two eluate fractions collected from an AEX column (e.g., during a gradient elution) to form a combined eluate (also referred to herein as an "AEX pool"). In some embodiments, at least two fractions of the eluate from the AEX column are combined, and the A260/A280 ratio of each fraction (e.g., as measured by SEC) is at least 0.5 to 2.0, such as at least 0.5 to 0.75, 0.75 to 1.0, 1.0 to 1.25, 1.25 to 1.5, 0.5 to 1.5, 1.5 to 2.0, or more. In some embodiments, at least two eluate fractions from an AEX column are combined to form a combined eluate, and the A260/A280 ratio of each fraction (e.g., as measured by SEC) is at least 0.5, at least 0.55, at least 0.6, at least 0.65, at least 0.7, at least 0.75, at least 0.8, at least 0.85, at least 0.9, at least 0.95, at least 1.0, at least 1.10, at least 1.11, at least 1.12, at least 1.13, at least 1.14, at least 1.15, at least 1.16, at least 1.17, at least 1.18, at least 1.19, at least 1.20, at least 1.21, at least 1.22, at least 1.23, at least 1.24, at least 1.25, at least 1.26, at least 1.27, at least 1.28, at least 1.29, at least 1.30, at least 1.31, In some embodiments, optionally during gradient elution, combining at least two eluate fractions collected from the AEX column comprises combining at least two eluate fractions, each eluate fraction having an A260/A280 ratio of ≥0.98, to form a combined eluate. In some embodiments, optionally during gradient elution, combining at least two eluent fractions collected from the AEX column comprises combining at least two eluent fractions, each having an A260/A280 ratio of ≥1.0, to form a combined eluent. In some embodiments, optionally during gradient elution, combining at least two eluent fractions collected from the AEX column comprises combining at least two eluent fractions, each having an A260/A280 ratio of ≥1.22, to form a combined eluent. In some embodiments, optionally during gradient elution, combining at least two eluent fractions collected from the AEX column comprises combining at least two eluent fractions, each having an A260/A280 ratio of ≥1.24, to form a combined eluent. In some embodiments, optionally during gradient elution, combining at least two eluate fractions collected from the AEX column comprises combining at least two eluate fractions, each eluate fraction having an A260/A280 ratio of ≥ 1.25, to form a combined eluate.

In some embodiments, combining at least two eluent fractions to form a combined eluent comprises, optionally during gradient elution, combining 2 to 7, 2 to 10, 2 to 15, 2 to 20, or 2 to 50 eluent fractions collected from an AEX column. In some embodiments, the A260/A280 ratio of the combined eluent is at least 0.5 to 2.0, such as at least 0.5 to 0.75, 0.75 to 1.0, 1.0 to 1.25, 1.25 to 1.5, 0.5 to 1.5, 1.5 to 2.0 or more. In some embodiments, the A260/A280 ratio of the combined eluate is at least 0.5, at least 0.55, at least 0.6, at least 0.65, at least 0.7, at least 0.75, at least 0.8, at least 0.85, at least 0.9, at least 0.95, at least 1.0, at least 1.10, at least 1.11, at least 1.12, at least 1.13, at least 1.14, at least 1.15, at least 1.16, at least 1.17, at least 1.18, at least 1.19, at least 1.20, at least 1.21, at least 1.22, at least 1.23, at least 1.24, at least 1.25, at least 1.26, at least 1.27, at least 1.28, at least 1.29, at least 1.30, at least 1.31 8, at least 1.19, at least 1.20, at least 1.21, at least 1.22, at least 1.23, at least 1.24, at least 1.25, at least 1.26, at least 1.27, at least 1.28, at least 1.29, at least 1.30, at least 1.31, at least 1.32, at least 1.33, at least 1.34, at least 1.35, at least 1.36, at least 1.37, at least 1.38, at least 1.39, at least 1.40 or more). In some embodiments, the A260/A280 ratio of the combined eluent is > 0.97. In some embodiments, the A260/A280 ratio of the combined eluent is between 0.97 and 1.03. In some embodiments, the A260/A280 ratio of the combined eluent is between 1.0 and 1.05. In some embodiments, the A260/A280 ratio of the combined eluate is 1.20 to 1.40. In some embodiments, the A260/A280 ratio of the combined eluate is ≥ 1.25, such as about 1.28 to 1.35, and is enriched for complete capsids compared to the affinity eluate before purification by AEX or the diluted affinity eluate.

In some embodiments, for example, when only one fraction meets a predetermined criterion (e.g., A280 value or A260/A280 ratio), the combined eluent comprises only one fraction. In some embodiments, the combined eluent comprises only a single fraction, for example, when a single fraction is collected during a gradient elution, starting from a specific point (e.g., when a specific A280 value is measured) and ending at a specific point (e.g., when a specific A280 value is measured), a specific volume of eluent is collected.

In some embodiments, the pH of the combined eluate is about 6.5 to 8, 6.8 to 7.6, about 6.8 to 7.8, 7.0 to 7.6, and about 7.0 to 7.4 or about 7.0 to 7.2. In some embodiments, the pH of the combined eluate is about 6.8 to 7.6.

In some embodiments, a method of purifying a rAAV vector (e.g., rAAV9, rAAV3B, etc.) from an affinity eluate comprises: i) collecting a first fraction of at least one (e.g., about 10) eluate fractions from an AEX column during a chromatography step (e.g., gradient elution) when the eluate has an A280 > 0.5 mAU/mm path length, and wherein the volume of the at least one eluate fraction is equal to 1/8 CV to 2 CV (e.g., about 1/3 CV); ii) adjusting the pH of at least one (e.g., about 10) eluate fractions from the column to 6.8 to 7.6 by adding 14.3% to 15% (by weight of the eluate volume) of a solution comprising about 200 mM to 300 mM (e.g., about 250 mM) sodium citrate, wherein the pH of the sodium citrate is 3. 0 to 4.0 (e.g., 3.5); iii) measuring the absorbance of at least one eluate fraction collected from the column and determining the A260/A280 ratio; and/or iv) combining at least two eluate fractions collected from the column to form a combined eluate, wherein the A260/A280 of each of the at least two eluate fractions is ≥1.25; wherein the A260/A280 of the combined eluate is ≥1.25 (e.g., about 1.28 to 1.35), and optionally wherein the pH of at least one eluate fraction or the combined eluate is 6.8 to 7.6, and wherein the at least one eluate fraction or the combined eluate is enriched for complete capsids and/or depleted for empty capsids compared to the affinity eluate before purification by AEX or the diluted and optionally filtered affinity eluate.

In some embodiments, a method for purifying an rAAV vector (e.g., rAAV9, rAAV3B, etc.) from an affinity eluate comprises: i) during a gradient elution step, when the gradient elution solution comprises about 65% to about 70% (e.g., about 68%) of a first elution buffer (e.g., buffer A) comprising about 100 mM Tris, about 0.01% P188, pH 8.9 and 30% to about 35% (e.g., about 32%) of a first elution buffer (e.g., buffer A) comprising about 500 mM sodium acetate, about 100 mM Tris, about 0.01% P188, pH 8.9; 8.9, collecting the first of at least one (e.g., about 20) eluate fractions from the AEX column, and continuing to collect all eluate fractions until the percentage of the first elution buffer is about 45% to about 50% (e.g., about 48%), and the percentage of the second elution buffer is about 50% to about 55% (e.g., about 52%), wherein the volume of at least one eluate fraction is equivalent to 1/8CV to 2CV (e.g., about 1/2CV); ii) collecting at least one eluate fraction into a column containing 0.01CV to 0.1CV (e.g., about 0.066CV) containing about 200mM to 300mM (e.g., about 250mM) sodium citrate, pH 8.9; 3.0 to 4.0 (e.g., about 3.5) in a container, adjusting the pH of at least one (e.g., about 20) eluate fractions from the column to 7.5 to 7.7; iii) measuring the absorbance of at least one eluate fraction collected from the column and determining the A260/A280 ratio; and/or iv) combining at least two eluate fractions collected from the column to form a combined eluate, wherein the A260/A280 of each of the at least two eluate fractions is ≥0.98; wherein the A260/A280 of the combined eluate is ≥1.0, and wherein the at least one eluate fraction or the combined eluate is enriched for complete capsids and/or depleted for empty capsids compared to the affinity eluate before purification by AEX or the diluted affinity eluate.

In some embodiments, the present invention provides a method for purifying rAAV (e.g., rAAV9, rAAV3B, etc.) vectors by AEX, the method comprising the following steps: i) pre-use flushing, comprising applying ≥4.5CV (e.g., about 5CV) of water for injection to the AEX stationary phase in the column; ii) disinfection, comprising applying 14.4CV to 17.6CV (e.g., about 16CV) of a solution comprising 0.1M to 1.0M (e.g., about 0.5M) NaOH to the AEX stationary phase in the column, optionally flowing upward; iii) regeneration, comprising applying 4.5CV to 5.5CV (e.g., about 5CV) of a solution comprising 1M to 3M (e.g., about 2M) NaCl, 50mM to 150mM (e.g., about 100mM) Tris, pH 8.5 to 9.5 (e.g., about pH 8.5 to 9.5) to the AEX stationary phase in the column; 9) to the AEX stationary phase in the column; iv) equilibration, comprising applying 4.5CV to 5.5CV (e.g., about 5CV) of a solution comprising 50mM to 150mM (e.g., about 100mM) Tris, pH 8.5 to 9.5 (e.g., about 9) to the AEX stationary phase in the column; v) equilibration, comprising applying 4.5CV to 5.5CV (e.g., about 5CV) of an equilibration buffer comprising 50mM to 150mM (e.g., about 100mM) Tris, 400mM to 600mM (e.g., about 500mM) sodium acetate, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., about 9) to the AEX stationary phase in the column. 8.5 to 9.5 (e.g., about 8.9); vi) equilibration, comprising applying ≥ 4.5 CV (e.g., about 5 CV) of an equilibration buffer comprising 100 mM to 300 mM (e.g., about 200 mM) histidine, 100 mM to 300 mM (e.g., about 200 mM) Tris, 0.1% to 1.0% (e.g., about 0.5%) P188, pH 8.5 to 9.5 (e.g., about 8.8); vii) loading an affinity eluate comprising the rAAV vector to be purified onto the AEX stationary phase in the column, optionally wherein the eluate has been a) equilibrated with an affinity eluate comprising 100 mM to 300 mM (e.g., about 200 mM) histidine, 100 mM to 300 mM (e.g., about 200 mM) Tris, 0.1% to 1.0% (e.g., about 0.5%) P188, pH 8.7 to 9.0 buffer diluted about 14.4 to 15.5 times (e.g., about 15 times), and optionally b) filtered through a series of 0.2 μm filters before application to the stationary phase; viii) equilibration, comprising applying 4.5CV to 5.5CV (e.g., about 5CV) of an equilibration buffer to the AEX stationary phase in the column, the buffer comprising 50mM to 150mM (e.g., about 100mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., about 8.9); ix) gradient elution from the stationary phase in the column to the material starting with applying 100% of a first buffer to the stationary phase, the buffer comprising 50mM to 150mM (e.g., about 100mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., 8.9), during the application of 20CV to 24CV (e.g., about 20CV) ending with the application of 100% of a second buffer comprising 400mM to 600mM (e.g., about 500mM) sodium acetate, 50mM to 150mM (e.g., about 100mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., pH 8.9); x) when the A280 of the eluent is ≥ 0.5 mAU/mm of path length, collecting the first fraction of at least one (e.g., about 10) eluent fractions from the column during the gradient elution, and wherein the volume of at least one eluent fraction is equal to 1/8 to 2CV (e.g., about 1/3CV); xi) optionally, by adding 14.3% to 15% (by weight of the eluent volume) of a 200 mM to 300 mM (e.g., about 250 mM) sodium citrate, pH The method comprises the steps of: preparing a solution of at least one eluate fraction collected from the column and adjusting the pH of at least one eluate fraction (e.g., about 3.0 to 4.0 (e.g., about 3.5) to adjust the pH of at least one (e.g., about 10) eluate fractions from the column to 6.8 to 7.6; xii) measuring the absorbance of at least one eluate fraction collected from the column and determining the A260/A280 ratio (e.g., by SEC); and/or xiii) combining at least two eluate fractions collected from the column to form a combined eluate, wherein at least one eluate fraction has an A260/A280 of ≥1.25, wherein the combined eluate has an A260/A280 of ≥1.25 (e.g., about 1.28 to 1.35), and optionally wherein the pH of at least one eluate fraction or the combined eluate is 6.8 to 7.6, and wherein the at least one eluate fraction or the combined eluate has a pH of 6.8 to 7.6 and the eluate is enriched for complete capsids and/or depleted for empty capsids compared to the affinity eluate or the diluted and optionally filtered affinity eluate; optionally wherein at least one of steps i) to ix) is run at a linear velocity of 270 cm/hr to 330 cm/hr (e.g., about 300 cm/hr), at a flow rate of 1.5 L/min to 2.0 L/min (e.g., about 1.8 L/min) through a 6.0 L to 6.6 L (e.g., about 6.4 L) column or at a flow rate of about 314 mL/min through a 1.3 L column, and/or a residence time of 3.5 min/CV to 4.5 min/CV (e.g., about 4 min/CV); optionally wherein the rAAV vector is an AAV9 vector; and optionally wherein the AEX stationary phase is POROS ^™ 50HQ. In some embodiments, the material eluted from the stationary phase during the gradient elution comprises the rAAV vector to be purified.

In some embodiments, the present invention provides a method for purifying rAAV (e.g., rAAV9 or AAV3B, etc.) vectors by AEX, the method comprising the following steps: i) sterilization, comprising applying 5CV to 10CV (e.g., about 8CV) of a solution comprising 0.1M to 1.0M (e.g., about 0.5M) NaOH to the AEX stationary phase in the column; ii) regeneration, comprising adding 4.5CV to 5.5CV (e.g., about 5CV) of a solution comprising 1M to 3M (e.g., about 2M) NaCl, 50mM to 150mM (e.g., about 100mM) Tris, pH 8.5 to 9.5 (e.g., about pH 8.5 to 9.5), to the AEX stationary phase in the column; 9); iii) equilibration, comprising applying 4.5CV to 5.5CV (e.g., about 5CV) of an equilibration buffer to the AEX stationary phase in the column, the buffer comprising 50mM to 150mM (e.g., about 100mM) Tris, 400mM to 600mM (e.g., about 500mM) sodium acetate, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., about 8.9); iv) equilibration, comprising applying ≥4.5CV (e.g., about 5CV) of an equilibration buffer to the AEX stationary phase in the column, the buffer comprising 100mM to 300mM (e.g., about 200mM) histidine, 100mM to 300mM (e.g., about 200)mM Tris, 0.1% to 1.0% (e.g., about 0.5%) P188, pH 8.5 to 9.5 (e.g., about 8.8); v) loading the affinity eluate containing the rAAV vector to be purified onto the AEX stationary phase in the column, optionally wherein the eluate has been diluted about 14.4 to 15.5 times (e.g., about 15 times) with a buffer containing 100 mM to 300 mM (e.g., about 200 mM) histidine, 100 mM to 300 mM (e.g., about 200 mM) Tris, 0.1% to 1.0% (e.g., about 0.5%) P188, pH 8.7 to 9.0; vi) equilibration, comprising applying 4.5 CV to 5.5 CV (e.g., about 5 CV) of an equilibration buffer containing 50 mM to 150 mM (e.g., about 100 mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH vii) gradient elution from the stationary phase in the column to the material starting with applying 100% of a first buffer comprising 50 mM to 150 mM (e.g., about 100 mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., 8.9) to the stationary phase and ending with applying 75% of a second buffer comprising 400 mM to 600 mM (e.g., about 500 mM) sodium acetate, 50 mM to 150 mM (e.g., about 100 mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., 8.9) over 20 to 40 CV (e.g., about 37.5 CV). 8.9); viii) when the percentage of the first buffer is about 65% to about 70% (e.g., about 68%), and when the percentage of the second buffer is about 30% to 35% (e.g., about 32%), collecting the first of at least one (e.g., about 20) eluate fractions from the column during the gradient elution, continuing to collect all eluate fractions until the percentage of the first buffer is about 45% to about 50% (e.g., about 48%) and the percentage of the second buffer is about 50% to 55% (e.g., about 52%), and wherein the volume of at least one eluate fraction is equal to 1/8 to 2CV (e.g., about 1/2CV); ix) optionally, by collecting at least one eluate fraction into a column containing about 0.01CV to 0.1CV (e.g., about 0.066CV) of 200mM to 300mM (e.g., about 250mM) sodium citrate, pH 3.0 to 4.0 (e.g., about 3.5) in a container containing a solution having an A260/A280 ratio of ≥ 0.3.0 to 4.0 (e.g., about 3.5), adjusting the pH of at least one (e.g., about 20) eluate fractions from the column to 7.5 to 7.7; x) measuring the absorbance of at least one eluate fraction collected from the column and determining the A260/A280 ratio (e.g., by SEC); and/or xii) combining at least two eluate fractions collected from the column to form a combined eluate, wherein the A260/A280 of at least one eluate fraction is ≥ 0.98, wherein the A260/A280 of the combined eluate is ≥ 1.0, and and wherein at least one eluate fraction or the combined eluate is enriched for complete capsids and/or depleted for empty capsids compared to the affinity eluate or the filtered affinity eluate, optionally wherein at least one of steps i) to vii) is performed at a linear velocity of 270 cm/hr to 330 cm/hr (e.g., about 298 cm/hr) and/or a residence time of 1.5 min/CV to 4.5 min/CV (e.g., about 2 min/CV); optionally wherein the rAAV vector is a rAAV3B vector; and optionally wherein the AEX stationary phase is POROS ^™ 50HQ. In some embodiments, the material eluted from the stationary phase during the gradient elution comprises the rAAV vector to be purified.

In some embodiments, the present invention provides a method for purifying rAAV (e.g., rAAV9, etc.) vectors by AEX, the method comprising the following steps: i) pre-use flushing, comprising applying ≥4.5CV (e.g., about 5CV) of water for injection to the AEX stationary phase in the column; ii) disinfection, comprising applying 14.4CV to 17.6CV (e.g., about 16CV) of a solution comprising 0.1M to 1.0M (e.g., about 0.5M) NaOH to the AEX stationary phase in the column, optionally flowing upward; iii) regeneration, comprising applying 4.5CV to 5.5CV (e.g., about 5CV) of a solution comprising 1M to 3M (e.g., about 2M) NaCl, 50mM to 150mM (e.g., about 100mM) Tris, pH 8.5 to 9.5 (e.g., about pH 8.5 to 9.5), to the AEX stationary phase in the column. 9); iv) equilibration, comprising applying 4.5CV to 5.5CV (e.g., about 5CV) of a solution comprising 50mM to 150mM (e.g., about 100mM) Tris, pH 8.5 to 9.5 (e.g., about 9) to the AEX stationary phase in the column; v) equilibration, comprising applying 4.5CV to 5.5CV (e.g., about 5CV) of an equilibration buffer comprising 50mM to 150mM (e.g., about 100mM) Tris, 400mM to 600mM (e.g., about 500mM) sodium acetate, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., about 8.9); vi) equilibration, comprising applying ≥4.5CV (e.g., about 5CV) of an equilibration buffer to the AEX stationary phase in the column, the buffer comprising 100mM to 300mM (e.g., about 200mM) histidine, 100mM to 300mM (e.g., about 200mM) Tris, 0.1% to 1.0% (e.g., about 0.5%) P188, pH 8.5 to 9.5 (e.g., about 8.8); vii) loading the affinity eluate containing the rAAV vector to be purified onto the AEX stationary phase in the column, optionally wherein the eluate has been a) diluted about 14.4 to 15.5 times (e.g., about 15 times) with a buffer comprising 100 mM to 300 mM (e.g., about 200 mM) histidine, 100 mM to 300 mM (e.g., about 200 mM) Tris, 0.1% to 1.0% (e.g., about 0.5%) P188, pH 8.7 to 9.0, and optionally b) filtering through a series of 0.2 μm filters prior to application to the stationary phase; viii) equilibration comprising applying 4.5 CV to 5.5 CV (e.g., about 5 CV) of an equilibration buffer comprising 50 mM to 150 mM (e.g., about 100 mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., about 8.9) to the AEX stationary phase in the column; ix) gradient eluting the material from the stationary phase in the column starting with applying 100% of a first buffer comprising 50 mM to 150 mM (e.g., about 100 mM) Tris, 0.005% to 0.15% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., 8.9), during the application of 20CV to 24CV (e.g., about 20CV) of the stationary phase, ending with the application of 100% of a second buffer comprising 400 mM to 600 mM (e.g., about 500 mM) sodium acetate, 50 mM to 150 mM (e.g., about 100 mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., pH 8.9); x) when the A280 of the eluent is ≥ 0.5 mAU/mm path length, collecting eluent fractions from the column during gradient elution, and wherein the volume of the eluent fractions corresponds to 1/8 to 2 CV (e.g., about 1/3 CV); and/or xi) optionally, by adding 14.3% to 15% (by weight of the eluent volume) of a column comprising about 200 mM to 300 mM (e.g., about 250 mM) sodium citrate, pH wherein the eluate fraction is enriched in complete capsids and/or depleted in empty capsids compared to the affinity eluate or the diluted and optionally filtered affinity eluate; optionally, wherein at least one of steps i) to ix) is performed at a linear velocity of 270 cm/hr to 330 cm/hr (e.g., about 300 cm/hr), at a flow rate of 1.5 L/min to 2.0 L/min (e.g., about 1.8 L/min) through a 6.0 L to 6.6 L (e.g., about 6.4 L) column or at a flow rate of about 314 mL/min through a 1.3 L column, and/or at a residence time of 3.5 min/CV to 4.5 min/CV (e.g., about 4 min/CV); optionally wherein the rAAV vector is an AAV9 vector; and optionally wherein the AEX stationary phase is POROS ^™ 50HQ. In some embodiments, the material eluted from the stationary phase during gradient elution includes the rAAV vector to be purified.

In some embodiments, the present invention provides a method for purifying rAAV (e.g., rAAV9, AAV3B, etc.) vectors by AEX, the method comprising the following steps: i) sterilization, comprising applying 5CV to 10CV (e.g., about 8CV) of a solution comprising 0.1M to 1.0M (e.g., about 0.5M) NaOH to the AEX stationary phase in the column; ii) regeneration, comprising adding 4.5CV to 5.5CV (e.g., about 5CV) of a solution comprising 1M to 3M (e.g., about 2M) NaCl, 50mM to 150mM (e.g., about 100mM) Tris, pH 8.5 to 9.5 (e.g., about pH 9) to the AEX stationary phase in the column; iii) equilibration, comprising applying 4.5CV to 5.5CV (e.g., about 5CV) of a solution comprising 50mM to 150mM (e.g., 100mM) Tris, pH 8.5 to 9.5 (e.g., about pH 9) to the AEX stationary phase in the column; iv) equilibration, comprising applying 4.5CV to 5.5CV (e.g., about 5CV) of an equilibration buffer to the AEX stationary phase in the column, wherein the equilibration buffer comprises 50mM to 150mM (e.g., about 100mM) Tris, 400mM to 600mM (e.g., about 500mM) sodium acetate, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 5.0, and 1.5V. 8.5 to 9.5 (e.g., about 8.9); v) equilibration, comprising adding ≥4.5CV (e.g., about 5CV) of an equilibration buffer comprising 100mM to 300mM (e.g., about 200mM) histidine, 100mM to 300mM (e.g., about 200mM) Tris, 0.1% to 1.0% (e.g., about 0.5%) P188, pH 8.5 to 9.5 (e.g., about 8.8) to the AEX stationary phase in the column; vi) loading an affinity eluate comprising the rAAV vector to be purified onto the AEX stationary phase in the column, optionally wherein the eluate has been treated with an affinity eluate comprising 100mM to 300mM (e.g., about 200mM) histidine, 100mM to 300mM (e.g., about 200mM) Tris, 0.1% to 1.0% (e.g., about 0.5%) P188, pH 8.7 to 9.0 buffer diluted about 14.4 to 15.5 times (e.g., about 15 times); vii) equilibration, comprising applying 4.5CV to 5.5CV (e.g., about 5CV) of an equilibration buffer to the AEX stationary phase in the column, the buffer comprising 50mM to 150mM (e.g., about 100mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.4 to 9.5 (e.g., about 8.9); viii) gradient elution from the stationary phase in the column, starting with applying 100% of a first buffer to the stationary phase, the buffer comprising 50mM to 150mM (e.g., about 100mM) Tris, 0.005% to 0.15% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., 8.9), during application of 20CV to 40CV (e.g., about 37.5CV) of the stationary phase to the stationary phase, ending with application of 75% of a second buffer comprising 400 mM to 600 mM (e.g., about 500 mM) sodium acetate, 50 mM to 150 mM (e.g., about 100 mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., pH 8.9); ix) during the gradient elution, when the percentage of the first buffer is about 65% to about 70% (e.g., about 68%) and the percentage of the second buffer is about 30% to about 35% (e.g., about 32%), collecting the eluate fraction from the column, and continuing to collect all the eluate fractions until the percentage of the first buffer is about 45% to about 50% (e.g., about 48%) and the percentage of the second buffer is about 50% to 55% (e.g., about 52%), and wherein the volume of the eluate fraction is equivalent to 1/8 to 2CV (e.g., about 1/2CV); and/or x) optionally, by collecting the eluate fraction into a column containing about 0.01CV to 0.1CV (e.g., about 0.066CV) of 200mM to 300mM (e.g., about 250mM) sodium citrate, pH 3.0 to 4.0 (e.g., about 3.5) solution in a container, the pH of the eluate fraction is adjusted to 7.5 to 7.7; and wherein the eluate fraction is enriched in complete capsids and/or depleted in empty capsids compared to the affinity eluate or the diluted affinity eluate; optionally, wherein at least one of steps i) to viii) is performed at a linear velocity of 270 cm/hr to 330 cm/hr (e.g., about 298 cm/hr) and/or a residence time of 1.5 min/CV to 4.5 min/CV (e.g., about 2 min/CV); optionally, wherein the rAAV vector is an AAV3B vector; and optionally, wherein the AEX stationary phase is POROS ^TM 50HQ. In some embodiments, the material eluted from the stationary phase during the gradient elution includes the rAAV vector to be purified.

Identification of the combined eluate and API

A method for purifying a rAAV vector (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., an affinity eluate) by AEX comprises collecting at least one eluate fraction from an AEX column during an elution step (e.g., a gradient elution) and forming a combined eluate enriched for complete capsids compared to the percentage of complete capsids in the solution. A method for purifying a rAAV vector from a solution by AEX, comprising collecting at least one eluate fraction from an AEX column during an elution step and forming a combined eluate, further comprising filtering by a process selected from viral filtration, ultrafiltration/diafiltration (UF/DF), filtering through a 0.2 μm filter, and combinations thereof, to produce a drug substance. In some embodiments, the quality attributes of the combined eluate, including A260/A280 (e.g., measured by SEC), the percentage of complete capsids, intermediate capsids, and empty capsids, % purity, % HMMS, the amount of HCP, and/or the amount of HC-DNA, are not substantially different from the same quality attributes of the drug substance produced from the combined eluate.

In some embodiments, the percentage of complete capsids in the affinity eluate containing the rAAV vector to be purified is less than 20% of the total capsids. In some embodiments, the combined eluate or bulk drug prepared by the methods disclosed herein is enriched for complete capsids, whereby the complete capsids in the combined eluate or bulk drug contain 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 98%, 20% to 99%, 20% to 99%, 20% to 99%, 40% to 50%, 40% to 60%, 40% to 70%, 40% to 80% (e.g., 44%, 45%, 50%, 53%) of the total capsids, and optionally wherein the capsids are measured by analytical ultracentrifugation (AUC)) (Burnham B. et al. Human Gene Therapy Methods (2015) 26; 228-242). In some embodiments, the combined eluate or bulk drug substance prepared by the methods disclosed herein is enriched for complete capsids, whereby the complete capsids comprise 52+/-7% of the total capsids in the combined eluate or bulk drug substance. In some embodiments, a method of purifying rAAV vectors from an affinity eluate comprises increasing the percentage of complete capsids from less than 30% (e.g., 12% to 25%) of the total capsids in the affinity eluate to greater than 30% (e.g., 40% to 55%, 45% to 65%, 40% to greater than 99%) of the total capsids in the combined AEX eluate or bulk drug substance.

In some embodiments, the combined AEX eluates prepared by the methods disclosed herein are enriched for complete capsids, whereby the complete capsids comprise 22.9+/-2.9% of the total capsids in the combined eluates. In some embodiments, a method of purifying rAAV vectors from an affinity eluate comprises increasing the percentage of complete capsids from less than 20% (e.g., 10% to 19%) of the total capsids in the affinity eluate to 20% or more (e.g., 20% to 30%, 30% to 40%, 40% to 55%, 45% to 65%, 40% to greater than 99%) of the total capsids in the combined AEX eluate. In some embodiments, the method of purifying rAAV vectors from an affinity eluate comprises increasing the percentage of complete capsids from 11.1±2.1 of the total capsids in the affinity eluate to 22.9±2.9% of the total capsids in the combined AEX eluate.

A method for purifying rAAV vectors (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., an affinity eluate) by AEX comprises collecting at least one eluate fraction from an AEX column during an elution step (e.g., a gradient elution) and forming a combined eluate depleted in a percentage of empty capsids compared to a percentage of empty capsids in the solution, and wherein the combined eluate is further subjected to a filtration selected from the group consisting of viral filtration, ultrafiltration/diafiltration (UF/DF), filtration through a 0.2 μm filter, and combinations thereof, to produce a drug substance. In some embodiments, the percentage of empty capsids in the affinity eluate comprising the rAAV vector to be purified is 70% or more of the total capsids. In some embodiments, the combined eluate or bulk drug prepared by the methods disclosed herein is depleted of empty capsids, whereby empty capsids account for 10% to 65%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 65%, 20% to 60%, 20% to 50%, 20% to 40%, or 18% to 29% (e.g., ≤ 29%) of the total capsids in the combined eluate or bulk drug, and optionally wherein the capsids are measured by analytical ultracentrifugation (AUC). In some embodiments, the combined eluate or bulk drug prepared by the methods disclosed herein is depleted of empty capsids, whereby empty capsids account for 20% +/- 7% of the total capsids in the combined eluate or bulk drug. In some embodiments, a method of purifying a rAAV vector from an affinity eluate comprises reducing the percentage of empty capsids to total capsids from 40% to 90% in the affinity eluate to ≤ 30% in the combined AEX eluate or bulk drug substance. In some embodiments, a method of purifying a rAAV vector from an affinity eluate comprises reducing the percentage of empty capsids to total capsids from 79.7 ± 2.5% in the affinity eluate to 67.5 ± 3.8% in the combined AEX eluate or bulk drug substance.

A method of purifying a rAAV vector (e.g., rAAV9, AAV3B, etc.) from a solution (e.g., an affinity eluate) by AEX comprises collecting at least one eluate fraction from an AEX column during an elution step (e.g., a gradient elution) and forming a combined eluate comprising an intermediate capsid, and wherein the combined eluate is further subjected to a filtration selected from viral filtration, ultrafiltration/diafiltration (UF/DF), filtration through a 0.2 μm filter, and a combination thereof, to produce a drug substance. In some embodiments, in the combined eluate or drug substance, the intermediate capsid comprises 10% to 65%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 65%, 20% to 60%, 20% to 50%, 20% to 40%, or 18% to 22% of the total capsid, and optionally wherein the capsid is measured by analytical ultracentrifugation (AUC). In some embodiments, the intermediate capsids comprise 28% +/- 5% of the total capsids in the combined eluate or drug substance. In some embodiments, the intermediate capsids comprise 9.6% +/- 1.4% of the total capsids in the combined eluate or drug substance.

A method for purifying rAAV vectors (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., an affinity eluate) by AEX includes collecting at least one eluate fraction from an AEX column during an elution step (e.g., a gradient elution) and forming a combined eluate or bulk drug substance that is enriched in complete capsids and depleted in empty capsids compared to the percentage of complete capsids and empty capsids in a solution containing the rAAV vector to be purified. In addition to complete capsids and empty capsids, in certain non-limiting exemplary embodiments, capsids containing partial vector genomes (also referred to as truncated or fragmented vector genomes) and/or non-transgene-associated DNA (i.e., intermediate capsids) can constitute the balance of capsid species in the combined eluate (e.g., combined AEX eluate) or bulk drug substance.

In some embodiments, the method of purifying rAAV vectors from an affinity eluate by AEX produces a pooled eluate or drug substance comprising about 53% complete rAAV capsids, about 23% intermediate capsids, and about 24% empty capsids.

In some embodiments, the method of purifying rAAV vectors from an affinity eluate by AEX produces a pooled eluate or drug substance comprising about 44% complete rAAV capsids, about 27% intermediate capsids, and about 29% empty capsids.

In some embodiments, methods for purifying rAAV vectors from an affinity eluate by AEX produce a pooled eluate or drug substance that contains about 20% to >99% complete rAAV capsids, about 5% to 65% intermediate capsids, and 10% to 65% empty capsids.

In some embodiments, the method of purifying rAAV vectors from an affinity eluate by AEX produces a pooled eluate or drug substance comprising about 45% to 65% complete rAAV capsids, 19% to 28% intermediate capsids, and 10% to 37% empty capsids. In some embodiments, the affinity eluate is produced by affinity chromatography purification of rAAV vectors produced in a vessel having a volume of 100 L to 500 L (e.g., about 250 L), optionally wherein the vessel is a SUB.

In some embodiments, the pooled eluate or drug substance prepared by the methods disclosed herein is enriched for complete capsids, whereby complete capsids comprise 55% +/- 7% of the total capsids in the pooled eluate or drug substance. In some embodiments, the rAAV vector present in the pooled eluate or drug substance is produced in a vessel having a volume of 100 L to 500 L (e.g., about 250 L), optionally wherein the vessel is a SUB.

In some embodiments, the combined eluate or drug substance prepared by the methods disclosed herein comprises 24% +/- 3% intermediate capsid. In some embodiments, the rAAV vector present in the combined eluate or drug substance is produced in a container having a volume of 100L to 500L (e.g., about 250L), optionally wherein the container is a SUB.

In some embodiments, the pooled eluate or drug substance prepared by the methods disclosed herein is depleted of empty capsids, whereby empty capsids comprise 21% +/- 10% of the total capsids in the pooled eluate or drug substance. In some embodiments, the rAAV vector present in the pooled eluate or drug substance is produced in a vessel having a volume of 100 L to 500 L (e.g., about 250 L), optionally wherein the vessel is a SUB.

In some embodiments, the combined eluate or drug substance prepared by the methods disclosed herein comprises 45% to 52% complete rAAV, 27 to 37% intermediate capsids, and 18% to 22% empty capsids. In some embodiments, the affinity eluate is produced by affinity chromatography purification of rAAV vectors produced in a vessel of 1000L to 3000L (e.g., about 2000L), optionally wherein the vessel is a SUB.

In some embodiments, the combined eluate or drug substance prepared by the methods disclosed herein is enriched for complete capsids, whereby complete capsids account for 49% +/- 2% of the total capsids in the combined eluate or drug substance. In some embodiments, the rAAV vectors present in the combined eluate or drug substance are produced in a vessel of 1000L to 3000L (e.g., about 2000L), optionally wherein the vessel is a SUB.

In some embodiments, the combined eluate or drug substance prepared by the methods disclosed herein comprises intermediate capsids that account for 32% +/- 4% of the total capsids in the combined eluate or drug substance. In some embodiments, the rAAV vectors present in the combined eluate or drug substance are produced in a vessel of 1000L to 3000L (e.g., about 2000L), optionally wherein the vessel is a SUB.

In some embodiments, the pooled eluate or drug substance prepared by the methods disclosed herein is depleted of empty capsids, whereby empty capsids comprise 20% +/- 2% of the total capsids in the pooled eluate or drug substance. In some embodiments, the rAAV vectors present in the pooled eluate or drug substance are produced in a vessel of 1000 L to 3000 L (e.g., about 2000 L), optionally wherein the vessel is a SUB.

A method for purifying a rAAV vector (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., an affinity eluate) by AEX comprises collecting at least one eluate fraction from an AEX column during an elution step (e.g., a gradient elution) and forming a combined eluate comprising the rAAV vector and optionally forming a bulk drug substance, which can be quantified by performing a quantitative polymerase chain reaction (qPCR) analysis of the vector genome (VG or vg). The qPCR analysis can measure the copies of the ITR sequences, the copies of the transgene sequences, and/or the copies of any other nucleotide sequences present in the complete vector genome.

The amount of VG present in the combined eluate from the AEX column can be expressed as a VG column yield percentage, which refers to the percentage of the amount of VG present in the combined eluate collected from the AEX column (i.e., AEX pool) to the amount of VG present in the sample to be purified, such as an affinity eluate, which in some embodiments has only been diluted, or diluted and filtered and applied to the AEX column.

The method of purifying rAAV vectors according to the methods disclosed herein results in a % VG column yield of 63% +/- 26%. The method of purifying rAAV vectors according to the methods disclosed herein results in a % VG column yield of 1%-10%, 1%-20%, 1%-30%, 1%-40%, 1%-50%, 1%-60%, 1%-70%, 1%-80%, 1%-90%, 1%-99%, 5%-95%, 10%-85%, 15%-75%, 20%-65%, 25%-55%, 30%-45%, 30%-80%, 35%-65%, 40%-70% or 100%.

In some embodiments, purification of rAAV vectors produced in 250L SUB by the methods disclosed herein results in a % VG column yield of 40%-100%. In some embodiments, purification of rAAV vectors produced in 2000LSUB by the methods disclosed herein results in a % VG column yield of 10%-70% (e.g., 20%-61%).

The amount of VG present in the combined eluate from the AEX column can be expressed as % VG step yield, which refers to the amount of VG present in the combined eluate collected from the AEX column (i.e., AEX pool) as a percentage of the amount of VG present in the affinity eluate prior to dilution or filtration.

The method of purifying rAAV vectors according to the methods disclosed herein results in a % VG step yield of 47% +/- 11%. The method of purifying rAAV vectors according to the methods disclosed herein results in a % VG step yield of 1%-10%, 1%-20%, 1%-30%, 1%-40%, 1%-50%, 1%-60%, 1%-70%, 1%-80%, 1%-90%, 1%-99%, 5%-95%, 10%-85%, 15%-75%, 20%-65%, 25%-55%, 30%-45%, 30%-80%, 35%-65%, 40%-70% or 100%.

In some embodiments, purification of rAAV vectors produced in 250L SUB by the methods disclosed herein results in a % VG step yield of 30%-70% (e.g., 37%-60%). In some embodiments, purification of rAAV vectors produced in 250L SUB by the methods disclosed herein results in a % VG step yield of 45% +/- 8%.

In some embodiments, purification of rAAV vectors produced in 2000L SUB by the methods disclosed herein results in a % VG step yield of 50% +/- 13%. In some embodiments, purification of rAAV vectors produced in 2000L SUB by the methods disclosed herein results in a % VG step yield of 25% to 75% (e.g., 31% to 66%).

A method of purifying a rAAV vector (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., an affinity eluate) by AEX includes collecting at least one eluate fraction from an AEX column during an elution step (e.g., a gradient elution) and forming a combined eluate or bulk drug having a reduced amount of host cell protein (HCP) compared to the amount of HCP in the solution. In some embodiments, the amount of HCP reduced in the combined eluate, at least one eluate fraction, or bulk drug is below the level of quantification (LLOQ) as measured by ELISA. In some embodiments, the amount of HCP reduced in the combined eluate, at least one eluate fraction, or bulk drug is 10 ng to 2000 ng/1×10 ⁹ VG, 50 ng to 200 ng/1×10 ⁹ VG, 100 ng to 1000 ng/1×109 VG, or 200 to 2000 ng/1×10 ⁹ VG.

In some embodiments, a method of purifying a rAAV vector (e.g., rAAV9, rAAV3b, etc.) from an affinity eluate by AEX comprises reducing the amount of HCP in the affinity eluate from 1-500 pg/1×10 ⁹ VG (e.g., about 50 pg/1×10 ⁹ ) to a LLOQ amount in a pooled eluate, at least one eluate fraction, or a bulk drug substance, wherein the rAAV vector is produced in a 250 LSUB.

In some embodiments, a method of purifying a rAAV vector (e.g., rAAV9, rAAV3B, etc.) from an affinity eluate by AEX comprises reducing the amount of HCP in the affinity eluate from 100-500 pg/1×10 ⁹ VG (e.g., about 330 pg/1×10 ⁹ ) to a LLOQ amount in a pooled eluate, at least one eluate fraction, or a bulk drug substance, wherein the rAAV vector is produced in a 2000L SUB.

A method for purifying a rAAV vector (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., an affinity eluate) by AEX comprises collecting at least one eluate fraction from an AEX column during an elution step (e.g., a gradient elution) and forming a combined eluate or drug substance comprising the rAAV vector, wherein the purity of the rAAV vector is at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100%, as measured by, for example, analytical reverse phase HPLC, capillary gel electrophoresis.

In some embodiments, the rAAV vector produced in a 250L SUB is purified by the methods disclosed herein to obtain a rAAV vector preparation (e.g., a drug substance) with a purity of 98.6% +/- 0.6%. In some embodiments, the rAAV vector produced in a 1000L to 3000L (e.g., about 2000L) container (e.g., SUB) is purified by the methods disclosed herein to obtain a rAAV vector preparation (e.g., a drug substance) with a purity of 99.3% +/- 0.3%.

The method of purifying rAAV vectors (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., affinity eluent) by AEX includes collecting at least one eluate fraction from the AEX column during the elution step (e.g., gradient elution) and forming a combined eluate or bulk drug, and the percentage of HMMS is 0% to 10%. In some embodiments, the percentage of HMMS is measured by size exclusion chromatography (SEC). In some embodiments, the rAAV vector produced in a 100L to 300L (e.g., about 250L) container (e.g., SUB) is purified by the method disclosed herein to obtain an rAAV vector preparation containing 2.6% +/- 0.8% of HMMS, measured by SEC. In some embodiments, the rAAV vector produced in 2000L of Sub is purified by the method disclosed herein to obtain an rAAV vector preparation containing 2.9% +/- 0.4% HMMS.

The method of purifying rAAV vectors (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., affinity eluate) by AEX includes collecting at least one eluate fraction from the AEX column during an elution step (e.g., gradient elution) and forming a combined eluate or bulk drug having about 7.0 to 25 pg residual HC-DNA/1×10 ⁹ VG. In some embodiments, the amount of HC-DNA is measured by qPCR. In some embodiments, the rAAV vector produced in 250 L of SUB is purified by the method disclosed herein to obtain an rAAV vector preparation (e.g., combined eluate, bulk drug) containing 17.4+/-6.7 pg HC-DNA/1×10 ⁹ VG. In some embodiments, the rAAV vector produced in 2000 L of Sub is purified by the method disclosed herein to obtain an rAAV vector preparation (e.g., combined eluate, bulk drug) containing 9.3+/-1.2 pg HC-DNA/1×10 ⁹ VG.

A method for purifying an rAAV vector (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., an affinity eluate) by AEX comprises collecting at least one eluate fraction from an AEX column during an elution step (e.g., a gradient elution) and forming a combined eluate or bulk drug substance having an A260/A280 of about 1.24 to 1.32. In some embodiments, A260/A280 is measured by size exclusion chromatography (SEC). In some embodiments, rAAV vectors produced in a 250 L SUB are purified by the methods disclosed herein to obtain an rAAV vector preparation (e.g., a combined eluate, bulk drug substance) having an A260/A280 of 1.24 to 1.32, as measured by SEC. In some embodiments, purification of rAAV vectors produced in 2000 L of Sub by the methods disclosed herein yields a rAAV vector preparation (e.g., pooled eluates, drug substance) having an A260/A280 of 1.28 to 1.31 as measured by SEC.

A method for purifying a rAAV vector (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., an affinity eluate) by AEX comprises collecting at least one eluate fraction from an AEX column during an elution step (e.g., a gradient elution) and forming a combined eluate, wherein the combined eluate is subjected to a filtration method selected from viral filtration, ultrafiltration/diafiltration (UF/DF), filtration through a 0.2 μm filter, and combinations thereof, to produce a drug substance suitable for producing a therapeutic drug product. In some embodiments, the drug substance is suitable for administration to a human subject to treat a disease, disorder, or condition (e.g., Duchenne muscular dystrophy). In some embodiments, the rAAV vector is an AAV9 vector.

AEX stationary phase regeneration

After elution (e.g., gradient elution) and collection of at least one eluate fraction containing complete rAAV capsids from the AEX column, additional steps may be performed to prepare the column stationary phase for further rAAV purification runs. These steps may include, for example, disinfection, equilibration, regeneration, flushing and/or storage. One skilled in the art will appreciate that one or more steps may be performed in a different order and frequency.

The method for regenerating the AEX stationary phase in the column for further rAAV purification operation includes post-use disinfection of the stationary phase. In some embodiments, the post-use disinfection of the stationary phase is after the elution step (e.g., gradient elution). In some embodiments, disinfection includes applying a solution comprising about 0.1M to 1M, about 0.2M to 0.8M, about 0.3 to about 0.7M, or about 0.4M to about 0.6MNaOH to the AEX stationary phase in the column. In some embodiments, disinfection includes applying a solution comprising about 0.5M NaOH to the AEX stationary phase in the column. In some embodiments, post-use disinfection includes applying a solution containing about 0.5M NaOH to the AEX stationary phase in the column and flowing upward. In some embodiments, post-use disinfection includes applying 14.4 to 17.6CV (e.g., about 16CV) of a solution comprising 0.5M NaOH to the AEX stationary phase in the column. In some embodiments, post-use disinfection comprises applying 2 to 20 CV, 5 to 15 CV, 7 to 13 CV (e.g., about 5, about 7.5, about 10, about 16 CV, etc.) of a solution comprising about 0.5 M NaOH to the AEX stationary phase in the column at a linear velocity of 50 to 2000 cm/hr, a flow rate of 0.2 to 3.0 L/min, and/or a residence time of 2 to 15 min/CV. In some embodiments, post-use disinfection comprises applying 14.4 to 17.6 CV (e.g., about 16 CV) of the solution to the AEX stationary phase in the column at a linear velocity of 270 to 330 cm/hr (e.g., about 300 cm/hr), through a 6.0 to 6.6 L (e.g., about 6.4 L) column at a flow rate of 1.5 to 2.0 L/min (e.g., about 1.8 L/min) or through a 1.3 L column at a flow rate of about 314 mL/min and/or a residence time of 3.5 to 4.5 min/CV (e.g., about 4 min/CV).

A method for regenerating a column stationary phase for further rAAV purification comprises regenerating the stationary phase (in some embodiments, this step may be referred to as "equilibrium"). In some embodiments, the column stationary phase is regenerated after an elution step (e.g., a gradient elution). In some embodiments, regeneration comprises applying a solution comprising a salt (e.g., NaCl, sodium acetate, ammonium acetate (NH ₄ Acetate), MgCl ₂ , and Na ₂ SO ₄ ) and a buffer (e.g., Tris, BIS-Tris propane, diethanolamine, diethylamine, tris(hydroxymethyl)methylglycine, triethanolamine, and/or n,n-bicine) to the stationary phase in the column. In some embodiments, regeneration comprises applying a solution comprising about 0.1 M to 5 M (e.g., 0.1 M to 4 M, 0.1 M to 3.5 M, 0.1 M to 3 M, 0.1 M to 2.5 M, 0.5 M to 4 M, 0.5 M to 3.5 M, 0.5 M to 3.0 M, 0.5 M to 2.5 M, 1 M to 4 M, 1 M to 3.5 M, 1 M to 3 M, 1 M to 2.5 M, or about 1.5 M to 2.5 M) salt to the stationary phase. In some embodiments, regeneration comprises applying a solution comprising about 1 mM to 500 mM (e.g., 1 mM to 450 mM, 1 mM to 400, 1 mM to 350 mM, 1 mM to 300 mM, 1 mM to 250 mM, 1 mM to 200 mM, 50 mM to 450 mM, 50 mM to 400 mM, 50 mM to 350 mM, 50 mM to 300 mM, 50 mM to 250 mM, 50 mM to 200 mM, or 50 mM to 150 mM) of buffer to the stationary phase.

In some embodiments, regeneration comprises applying a solution having a pH between about 7.0 and 11.0 (eg, 7.5 to 10.5, 8.0 to 10.0, 8.5 to 9.5, or 8.0 to 9.0) to the stationary phase.

In some embodiments, regeneration comprises applying a solution comprising about 2M NaCl, 100mM Tris, pH 9 to the AEX stationary phase in the column. In some embodiments, regeneration comprises applying a solution comprising 2M NaCl, 25mM Tris, pH 9 to the AEX stationary phase in the column. In some embodiments, regeneration comprises applying 2 to 15CV (e.g., about 5CV, about 10CV) of a solution (e.g., a regeneration solution) to the AEX stationary phase in the column. In some embodiments, regeneration comprises applying 4.5 to 5.5CV (e.g., about 5CV) of a solution comprising 2M NaCl, 100mM Tris, pH 9 to the AEX stationary phase in the column. In some embodiments, regeneration comprises applying 2 to 15 CV (e.g., about 5 CV, about 10 CV) of a solution comprising 2 M NaCl, 100 mM Tris, pH 9 to the AEX stationary phase in the column at a linear velocity of 100 to 2000 cm/hr, a flow rate of 0.2 to 3.0 L/min, and/or a residence time of 2 to 15 min/CV. In some embodiments, regeneration comprises applying 4.5 to 5.5 CV (e.g., about 5 CV) of a solution comprising 2 M NaCl, 100 mM Tris, pH 9 to the AEX stationary phase in the column at a linear velocity of 270 to 330 cm/hr (e.g., about 300 cm/hr), a flow rate of 1.5 to 2.0 L/min (e.g., about 1.8 L/min) through a 6.0 to 6.6 L (e.g., about 6.4 L) column or at about 314 mL/min through a 1.3 L column and/or a residence time of 3.5 to 4.5 min/CV (e.g., about 4 min/CV).

The method of regenerating the column stationary phase for further rAAV purification runs includes equilibration of the stationary phase (in some embodiments, this step may be referred to as a "regeneration step"). In some embodiments, the equilibration of the stationary phase in the column is after an elution step (e.g., a gradient elution). In some embodiments, the equilibration of the medium in the column includes applying a solution comprising about 100 mM Tris, pH 9 to the AEX stationary phase in the column. In some embodiments, the equilibration of the column includes applying a solution comprising 20 mM Tris, pH 9 to the AEX stationary phase in the column. In some embodiments, the equilibration of the column includes applying 2 to 15 CV (e.g., about 5 CV, 10 CV) of a solution (e.g., an equilibration solution) to the AEX medium in the column. In some embodiments, the equilibration of the column includes applying 4.5 to 5.5 CV (e.g., about 5 CV) of a solution comprising 100 mM Tris, pH 9 to the AEX stationary phase in the column. In some embodiments, equilibration of the column comprises applying 2 to 15 CV (e.g., about 5 CV, about 10 CV) of a solution comprising 100 mM Tris, pH 9 to the AEX stationary phase in the column at a linear velocity of 100 to 2000 cm/hr, a flow rate of 0.2 to 3.0 L/min, and/or a residence time of 2 min/CV to 15 min/CV. In some embodiments, equilibration of the column comprises applying 4.5 to 5.5 CV (e.g., about 5 CV) of a solution comprising 100 mM Tris, pH 9 to the AEX stationary phase in the column at a linear velocity of 270 to 330 cm/hr (e.g., about 300 cm/hr), a flow rate of 1.5 to 2.0 L/min (e.g., about 1.8 L/min) through a 6.0 to 6.6 L (e.g., about 6.4 L) column or 314 mL/min through a 1.3 L column and/or a residence time of 3.5 to 4.5 min/CV (e.g., about 4 min/CV).

The method for regenerating the column stationary phase for further rAAV purification operation includes post-use flushing (i.e., flushing) of the stationary phase. In some embodiments, the post-use flushing of the column is after the elution step (e.g., gradient elution). In some embodiments, the post-use flushing includes applying water for injection (e.g., purified water) to the AEX stationary phase in the column. In some embodiments, the post-use flushing includes applying ≥4.5CV (e.g., about 5CV) of water for injection to the AEX stationary phase in the column. In some embodiments, the post-use flushing includes applying 2 to 15CV (e.g., about 5CV, about 10CV) of a solution containing water for injection to the AEX stationary phase in the column at a linear velocity of 100 to 2000cm/hr, a flow rate of 0.2 to 3.0L/min, and/or a residence time of 2min/CV to 15min/CV. In some embodiments, the post-use flush of the column comprises applying 4.5 to 5.5 CV (e.g., about 5 CV) of a solution comprising water for injection to the AEX stationary phase in the column at a linear velocity of 270 to 330 cm/hr (e.g., about 300 cm/hr), at a flow rate of 1.5 to 2.0 L/min (e.g., about 1.8 L/min) through a 6.0 to 6.6 L (e.g., about 6.4 L) column or at about 314 mL/min through a 1.3 L column and/or a residence time of 3.5 to 4.5 min/CV (e.g., about 4 min/CV).

A method for regenerating a column stationary phase for further rAAV purification runs includes applying a storage solution to the stationary phase. In some embodiments, the storage solution is applied to the column after an elution step (e.g., a gradient elution). In some embodiments, a storage solution comprising 16% to 20% ethanol (e.g., about 17.5%) is applied to the AEX stationary phase in the column. In some embodiments, 2 to 11CV (e.g., about 3CV, about 10CV) of storage buffer is applied to the AEX stationary phase in the column. In some embodiments, 2.7 to 3.3CV (e.g., about 3CV) of a storage solution comprising 17.5% ethanol is applied to the AEX stationary phase in the column. In some embodiments, 2 to 11CV (e.g., about 3CV) of a storage solution comprising 17.5% ethanol is applied to the AEX stationary phase in the column at a linear velocity of 100 to 2000cm/hr, a flow rate of 0.2 to 3.0L/min, and/or a residence time of 2min/CV to 15min/CV. In some embodiments, applying the storage solution to the column comprises applying 2.7 to 3.3 CV (e.g., about 3 CV) of a solution comprising 17.5% ethanol at a linear velocity of 270 to 330 cm/hr (e.g., about 300 cm/hr), a flow rate of 1.5 to 2.0 L/min (e.g., about 1.8 L/min) through a 6.0 to 6.6 L (e.g., 6.4 L) column, or at about 314 mL/min through a 1.3 L column and/or a residence time of 3.5 to 4.5 min/CV (e.g., about 4 min/CV) to the AEX stationary phase in the column.

A method for regenerating a column stationary phase for further rAAV purification, the method comprising the following steps: i) post-use disinfection, comprising applying 14.4 to 17.6 CV (e.g., about 16 CV) of a solution containing about 0.5 M NaOH to the stationary phase; ii) regeneration, comprising applying 4.5 to 5.5 CV (e.g., about 5 CV) of a solution containing about 2 M NaCl, 100 mM Tris, pH 9 to the stationary phase; iii) equilibration, comprising applying 4.5 to 5.5 CV (e.g., about 5 CV) of a solution containing about 100 mM Tris, pH 9 to the stationary phase; 9 solution is applied to the stationary phase; iv) a post-use rinse comprises applying 4.5 to 5.5CV (e.g., about 5CV) of water for injection to the stationary phase; and/or v) applying a storage solution to the stationary phase comprises applying 2.7 to 3.3CV (e.g., about 3CV) of a storage solution comprising about 17.5% ethanol to the column; wherein at least one of steps i) to v) is performed at a linear velocity of 270 to 330 cm/hr (e.g., about 300 cm/hr), at a flow rate of 1.5 to 2.0 L/min (e.g., about 1.8 L/min) through a 6.0 to 6.6 L (e.g., 6.4 L) column or about 314 mL/min through a 1.3 L column, and/or a residence time of 3.5 to 4.5 min/CV (e.g., about 4 min/CV), wherein the stationary phase is an AEX stationary phase, optionally a POROS ^™ 50HQ stationary phase.

The method of regenerating the AEX stationary phase for further rAAV purification runs comprises applying an ethanol rinse to the stationary phase prior to the first step of the method for purifying the rAAV vector (i.e., prior to disinfection, prior to equilibration, etc.). In some embodiments, the ethanol rinse comprises about 20 mM Tris, pH 9. In some embodiments, applying the ethanol rinse solution to the column stationary phase comprises applying 8 to 12 CV (e.g., about 10 CV) of a solution comprising about 20 mM Tris, pH 9 to the AEX stationary phase. In some embodiments, applying the ethanol rinse solution to the AEX stationary phase comprises applying 8 to 12 CV (e.g., about 10 CV) of a solution comprising about 20 mM Tris, pH 9 to the AEX stationary phase at a speed of 100 to 1000 cm/hr (e.g., about 600 cm/hr) and/or at a residence time of 1 to 10 min/CV (e.g., about 1.5 min/CV).

Equivalent

The foregoing written description is considered sufficient to enable those skilled in the art to practice the present disclosure. The foregoing description and examples detail certain exemplary embodiments of the present disclosure. However, it will be understood that no matter how detailed the foregoing appears in text, the present disclosure can still be implemented in a variety of ways, and the present disclosure should be interpreted according to the appended claims and any equivalents thereof.

All references cited herein, including patents, patent applications, articles, textbooks, etc., and the references cited therein, are fully incorporated herein by reference.

Example Implementation

The present invention is further described in detail by reference to the following experimental examples. These examples are only used to illustrate the present invention and are not restrictive unless otherwise stated. Therefore, the present invention should not be construed as being limited to the following examples, but should be construed as covering any and all variations that become apparent from the teachings provided herein.

Example

Example 1: Screening of elution salts by AEX chromatography to enrich for complete AAV9 vector

Preparation of AEX loads for elution salt screening

HEK 293 cells were grown in suspension culture and transfected with three plasmids to produce AAV9 vectors according to standard methods known in the art (Grieger et al. (2016) Molecular Therapy 24(2):287-297). HEK 293 cells were harvested, lysed, flocculated, and the resulting lysate was filtered. AAV9 vectors were purified from the clarified lysate by affinity chromatography. The affinity column was balanced, the clarified lysate was loaded, washed, and the purified AAV9 vector was eluted. The pH of the AAV9 vector affinity pool (also referred to as affinity eluate) was 4.4 and the conductivity was 5.3 mS/cm. The affinity pool was diluted 7.6-fold with 20 mM Tris (pH 9), adjusted to pH 9 with 1 M Tris base (pH 11), and filtered through a 0.2 μM filter. The resulting solution had a pH of 9 and a conductivity of 1.9 mS/cm and was loaded onto an AEX column for elution salt screening studies.

Screening of eluted salts by AEX chromatography on a 1 mL POROS ^™ 50HQ column

The ability of four elution salts to distinguish AAV9 empty capsids (i.e., AAV capsids without a recombinant vector genome) and complete capsids (i.e., AAV capsids containing a recombinant vector genome) during AEX chromatography was investigated. A POROS ^™ 50HQ column (0.5 cm inner diameter, 5 cm column bed height, 1 mL column volume) was equilibrated, loaded, and washed in accordance with Table 1. A 50CV gradient was generated from 0-50% buffer B, followed by a 10CV step elution with 100% buffer B. Four buffer Bs were used, consisting of 20 mM Tris, 500 mM salt, pH 9. The salt was one of NaCl, sodium acetate, ammonium acetate, or Na ₂ SO ₄ .

Table 1: Screening of AEX Chromatography with Gradient Elution Salts on a 1 mL POROS ^™ 50HQ Column

* Elution salt: NaCl, sodium acetate, ammonium acetate, Na ₂ SO ₄

^# Vector genome (VG) was measured by qPCR analysis of ITR sequences

The effect of elution salt on the shape of the elution peak and the _A260 / _A280 ratio is shown in the chromatogram of Figure 1. The _A260 / _A280 ratio provides an estimate of the percentage of AAV capsids containing the recombinant vector genome (% complete), with a higher ratio indicating a higher percentage complete (Sommer et al. Molecular Therapy (2003) 7(1): 122-128). Elution by NaCl resulted in a main peak with high _A260 / _A280 (indicating complete vector) and a tightly connected shoulder with low _A260 / _A280 (indicating empty capsids). Elution by sodium acetate and ammonium acetate produced a main peak with high _A260 / _A280 , as well as two resolved peaks with low _A260 / _A280 . These results indicate that sodium acetate and ammonium acetate separate empty capsids from AAV9 vectors better than NaCl. In contrast, Na ₂ SO ₄ eluted AAV9 material as a sharp peak at a medium A ₂₆₀ /A ₂₈₀ , implying that there was little separation of AAV9 vectors from empty capsids.

1 mL eluate fractions were collected throughout the gradient elution and neutralized with 0.15 mL of 50 mM citrate at pH 3.6. The neutralized fractions were analyzed by HPLC-SEC A ₂₆₀ / A _280. During the HPLC-SEC method, absorbance was monitored at 214, 260, and 280 nm. The _{A 260} / A ₂₈₀ ratio provides an estimate of the percentage of complete capsids of AAV (Sommer et al. Molecular Therapy (2003) 7 (1): 122-128). The characteristic SEC elution peaks of AAV9 at 260 and 280 nm were integrated, and the ratio of these two values was reported as SEC _{A 260} / A ₂₈₀ , as shown in Figure 2. The maximum SEC A ₂₆₀ / A ₂₈₀ of the elution fraction produced by the sodium acetate gradient was 1.27, which is higher than the similar maximum values of NaCl (1.23), ammonium acetate (1.22), and Na ₂ SO ₄ (1.15). In addition, sodium acetate-based elution produced 7 consecutive eluate fractions with SEC _A260 / _A280 ≥1.19, which was higher than similar results for NaCl (3 fractions), ammonium acetate (4 fractions), _{and Na2SO4} (0 fraction). Fractions with SEC _A260 / _A280 ≥1.19 were pooled, ITRs were determined by qPCR to determine vector genome (VG) yield, and analyzed by analytical ultracentrifugation (AUC) to determine empty capsids, complete capsids, and intermediate capsids (AAV capsids that package less nucleic acid than complete capsids and contain, for example, partially fragmented or truncated vector genomes and/or non-transgene-associated DNA).

Elution salt screening studies showed that sodium acetate was superior to NaCl, ammonium acetate, and Na ₂ SO ₄ in terms of VG yield and complete percentage of AAV9 vector recovered, as judged by SEC A ₂₆₀ /A ₂₈₀ and AUC (Table 2). The SEC A ₂₆₀ /A ₂₈₀ of the sodium acetate AEX pool was 1.24, slightly higher than similar values obtained using NaCl (pool SEC A ₂₆₀ /A ₂₈₀ =1.23) and ammonium acetate (SEC A ₂₆₀ /A ₂₈₀ =1.21), and significantly higher than the Na ₂ SO ₄ pool (SEC A ₂₆₀ /A ₂₈₀ =1.15). AUC analysis of the AEX pools showed that the sodium acetate gradient produced a slightly higher complete percentage (43%) than the NaCl gradient (approximately 38%) and significantly higher than the Na ₂ SO ₄ gradient (20%). Notably, sodium acetate gradient elution reduced the percentage of empty capsids (% empty capsids) from 75% in the AEX load to 29% in the AEX pool. To better characterize the performance of elution salts, NaCl, sodium acetate and ammonium acetate were selected for use with the 5.1 mL POROS ^™ 50HQ column as described in the following section.

Table 2: Results of elution salt screening study on 1 mL POROS ^™ 50HQ column

% VG column yield was determined as (VG in AEX pool)/(VG in AEX load); therefore, losses incurred during load preparation were not taken into account. AUC data for NaCl gradient elution AEX pools are inconclusive, so approximate values (~) are reported here. LLOQ - below the lower limit of quantitation.

Screening of eluted salts by AEX chromatography on a 5.1 mL POROS ^™ 50HQ column

The ability of the elution salts NaCl, sodium acetate, and ammonium acetate to distinguish between empty AAV9 capsids and complete vectors during AEX chromatography was investigated. A POROS ^™ 50HQ column (0.66 cm inner diameter, 15 cm bed height, 5.1 mL column volume) was equilibrated, loaded, washed, and eluted with a NaCl, sodium acetate, or ammonium acetate gradient (Table 3). 1 mL fractions were collected throughout the gradient, neutralized with 0.15 mL of 50 mM citrate at pH 3.6, and analyzed by HPLC-SEC A ₂₆₀ / A ₂₈₀ .

Sodium acetate-based elution produced 20 consecutive fractions with SEC _A260 / _A280 ≥ 1.19, which was higher than similar results with NaCl (8 fractions) and ammonium acetate (11 fractions). Fractions with SEC _A260 / _A280 ≥ 1.19 were pooled, and ITRs were determined by qPCR to determine VG yield and analyzed by analytical ultracentrifugation (AUC) to determine % complete capsids.

Table 3: AEX Chromatography for Elution Salt Screening on a 5.1 mL POROS ^™ 50HQ Column

Elution salt screening studies showed that sodium acetate was superior to NaCl and ammonium acetate in terms of the percentage of intact capsids of recovered AAV9 vectors, as judged by SEC _A260 / _A280 and AUC (Table 4). The SEC _A260 / _A280 of the sodium acetate AEX pool was 1.26, which was higher than similar values obtained using NaCl (pool SEC _A260 / _A280 = 1.24) and ammonium acetate (SEC _A260 / _A280 = 1.19). AUC analysis of the AEX pools showed that the sodium acetate gradient produced a slightly higher percentage of intact capsids (43%) than the NaCl gradient (39%) and the NH4 acetate ammonium acetate gradient (36%).

Overall, AEX runs performed at 1 mL and 5.1 mL column volume scales showed that sodium acetate resolved intact AAV9 vector from empty capsids better than NaCl and ammonium acetate. Therefore, sodium acetate was used as the elution salt in further developed versions of the AEX method.

Table 4: Results of elution salt screening study on 5.1 mL POROS ^™ 50HQ column

% VG column yield was determined as (VG in AEX pool)/(VG in AEX load); therefore, losses incurred during load preparation were not taken into account.

Example 2: Enrichment of complete AAV9 vector by AEX chromatography with sodium acetate step elution

Sodium acetate was selected to investigate the step elution procedure of the AEX column to separate empty AAV9 capsids from complete vectors based at least in part on the results in Example 1. Affinity eluate was generated as described in Example 1, diluted with 20 mM Tris, pH 9 and 1 M Tris base, pH 11, and filtered through a 0.2 μM filter.

Screening of optimized sodium acetate step elution conditions

For screening purposes, a nine-step wash and elution AEX method was performed with increasing sodium acetate concentrations in 20 mM Tris, pH 9. A POROS ^™ 50HQ column (0.66 cm ID×15 cm BH; 5.1 mL CV) was equilibrated, loaded, washed, and eluted in accordance with Table 5. Wash and elution buffers were formed in an FPLC system by mixing buffer A: 20 mM Tris, pH 9 and buffer B: 20 mM Tris, 140 mM sodium acetate, pH 9. Fractions were neutralized and ITRs were determined by SEC _A260 / _A280 , AUC, and qPCR. Figure 3 depicts the nine-step chromatogram and shows that the tandem _A260 / _A280 changed significantly as the sodium acetate concentration of the wash and elution buffers increased.

Table 5: AEX screening method using 9-step sodium acetate elution (3 washes and 6 elutions)

The results of the analysis of the nine-step run showed that AAV9 empty capsids can be separated from the complete AAV9 vector by sodium acetate step-by-step washing and elution (Table 6). Washes 1 and 2 selectively remove bound empty capsids from the AEX column. It is worth noting that the SEC A260/A280 values produced by washes 1 and 2 were 0.58 and 0.79, respectively, and the empty capsid percentages (AUC) were 98% and 85%, respectively. Elution fractions 1-4 enriched the complete AAV9 vector, with SEC A260/A280 values ranging from 1.27-1.30 and complete capsid percentages (AUC) ranging from 29-53%, much higher than 12% in the AEX load. Based on these findings, a sodium acetate-based step-by-step washing and elution method was designed, as shown below.

Table 6: Results of a 9-step sodium acetate elution study on a 5.1 mL POROSTM 50HQ column

W-washing step; E-elution step; LLOQ-below limit of quantitation

Enrichment of complete AAV9 vector by stepwise sodium acetate washing and elution

Based on the above results, the ability of AEX methods with different steps of sodium acetate washing and elution to enrich complete AAV9 vector capsids was tested. A POROSTM 50HQ column (0.66 cm ID×15 cm BH, 5.1 mL CV) was equilibrated, washed, and eluted in accordance with Table 7. Washing, pH stabilization, and elution buffers were prepared in an FPLC system by mixing buffer A: 20 mM Tris, pH 9 and buffer B: 20 mM Tris, 140 mM sodium acetate, pH 9. In the methods studied, multiple parameters were different, namely the elution linear velocity (75-600 cm/hr), the load challenge (5.1×10 ¹³ to 1.1×10 ¹⁵ VG/mL resin), and the concentration of sodium acetate in the wash step (57 mM or 68 mM).

As shown in Figures 4A and 4B, chromatograms of step washing and elution with 600cm/hr elution, 5.1×1013VG/mL resin challenge and 57mM sodium acetate wash are given. Table 8 reports the results and reveals that the developed step-by-step sodium acetate wash and elution AEX method enriches the complete AAV9 vector and reduces the host cell protein (HCP) level. The developed step-by-step method increased the complete percentage (judged by AUC) from 18% to 40-53% and increased SEC A260/A280 from 0.95 to 1.25-1.27. In addition to enriching the complete AAV9 capsid, the developed step-by-step method can also remove a large amount of HCP and a moderate amount of host cell DNA (HC-DNA) at low column challenge. The step-by-step sodium acetate wash and elution method does not provide a high % VG yield or % complete AAV9 as high as ultracentrifugation or AEX chromatography eluted by sodium acetate gradient (Examples 6, 7 and 8 below). However, the step-elution method avoids the complex manipulations associated with ultracentrifugation.

Table 7: AEX method with stepwise sodium acetate elution. Runs utilized different load challenges, wash conditions, and elution residence times.

Table 8: Results of AEX run on a 5.1 mL POROSTM 50HQ column using sodium acetate step elution

% VG column yield was determined as (VG in AEX pooled eluate)/(VG in AEX load); and therefore did not take into account losses incurred in load preparation.

Example 3: Screening of AAV9 Affinity Eluate Preparation Methods for AEX Chromatography

One embodiment of large-scale downstream processing of AAV9 involves cell lysis, filtration, and affinity chromatography, with the product eluted at low pH and moderate conductivity. Studies on viral proteins of various AAV serotypes report calculated isoelectric points of ~6.2 and ~5.8 for AAV9 empty capsids and complete capsids, respectively (Venkatakrishnan et al., J. Virology (2013) 87.9: 4974-4984). Screening of various conditions showed that AAV9 only binds to AEX resins in relatively alkaline, low conductivity environments (data not shown). Therefore, preparing an acidic AAV9 affinity eluent for AEX chromatography requires increasing the pH and reducing the conductivity of the buffer containing the vector. This method crosses the isoelectric point of AAV9, which is an unstable transition that may result in vector loss.

This example details various methods for processing the acidic affinity pool into an AEX chromatography load. Dilution, serial mixing (Figure 5), and tangential flow filtration (TFF) load preparation methods were investigated. The results show that processing the AAV9 affinity eluate into an AEX chromatography load with high product yield and low aggregation requires the dedicated development of novel and creative methods and procedures.

Dilution method for preparing AAV9 affinity pool for AEX chromatography

The affinity elution pool has a pH of approximately 3.8-4.4, a conductivity of approximately 5.5-6.5 mS/cm, and contains 7×10 ¹³ -1.4×10 ¹⁴ AAV9 VG/mL. As described in Example 1, an affinity pool for AEX chromatography can be prepared by diluting the affinity pool with an alkaline buffer. Consistent with Table 9, the AAV9 affinity pool was diluted in a PETG container with a combination of alkaline buffers to increase the pH and reduce the conductivity. The resulting solution was passed through a 0.2 μm filter pre-wetted with dilution buffer. The diluted sample was filtered through 0.2 μm to simulate large-scale downstream processing, where the filter was placed at the entrance of the chromatography column. The resulting filtrate had a pH of 8.7-9.0 and a conductivity in the range of 1.8-2.1 mS/cm, which would allow AAV9 to bind highly to the AEX resin. Samples were collected after dilution and filtration, and ITR was measured by qPCR to determine VG titer.

Table 9 reports the results and shows that a large amount of AAV9 is lost during dilution and filtration. Serial dilutions with 20mM Tris (pH 9) and pH adjustment with 1M Tris base (pH 11) followed by 0.2μM filtration resulted in 39% VG loss. Reversing the order of these dilutions produced similar results, with a 37% loss of vector after filtration. The greater the dilution, the greater the amount of VG loss. A 25-fold dilution with 100mM Tris, pH 9, followed by pH adjustment with 1M Tris, pH 9, resulted in only 36% VG yield after filtration. A 15-fold dilution with the same buffer resulted in a 65% yield after filtration.

To reduce nonspecific binding of AAV9 to the dilution vessel and filter surface, 0.01% (v/v) poloxamer 188 (P188) was added to the dilution buffer 100 mM Tris (pH 9). This approach only slightly increased the post-filtration VG yield from 65% to 74% without and with 0.01% P188, respectively. These dilution techniques provided lower than desired % VG yields, so other buffer exchange techniques were investigated.

Table 9: Screening of AAV9 affinity eluate preparation methods for AEX chromatography

% VG yield was determined as (VG in AEX load)/(VG in affinity pool); 1 M Tris base, pH 11 and 1 M Tris, pH 9 were used for pH adjustment and were applied at a dilution factor of less than 1 relative to the affinity pool.

Serial dilution to prepare AAV9 affinity pool for AEX chromatography

Serial dilution of an AAV9 affinity pool was investigated to generate an AEX load while reducing the surface area to which the vector is exposed and reducing the time that the vector is exposed to the surface. The serial dilution setup is shown in FIG5 .

Three platinum-cured silicone tubes were connected together by a Y-connector. A peristaltic pump delivered 100 mM Tris, pH 9 to the Y-connector at a flow rate of 3.5 mL/min. A second peristaltic pump delivered the AAV9 affinity pool to the Y-connector at a flow rate of 0.25 mL/min. The ratio of these flow rates (14 parts diluent to 1 part affinity pool) was selected to achieve a 15-fold dilution and enable direct comparison with similar dilution factors (Tables 9 and 12). The connecting fluids passed through platinum-cured silicone tubing with an inner diameter of 0.16 cm and a length of 100 cm. The mixing tube dimensions were designed based on buffer mixing studies that showed that these conditions can obtain stable, well-mixed solutions.

The serially mixed solutions were collected, neutralized with 250 mM sodium citrate (pH 3.5), and analyzed for ITR by qPCR. 79% of the VG pumped into the device was recovered at the outlet of the mixing tube. Although this result indicated a slight improvement in % VG yield compared to the batch dilution experiments, the yield was still lower than expected. Therefore, further AEX load preparation experiments were performed by tangential flow filtration.

Tangential flow filtration (TFF) to prepare AAV9 affinity pools for AEX chromatography

To avoid VG loss during AEX load preparation, TFF was used to maintain a high VG concentration and arginine was temporarily spiked into the vector-containing solution. Two TFF runs were performed using fresh 20 cm ² mPES hollow fiber membranes, consistent with Table 10. The membranes were equilibrated, the AAV9 affinity pool was loaded, and diafiltration was performed against 150 mM acetate, 100 mM glycine, 25 mM MgCl ₂ , pH 4.2 (same buffer for the AAV9 affinity pool). At the end of each step, the TFF system was paused and the retentate container was sampled. The samples were analyzed for VG titer by qPCR on ITRs, and the results are shown in Tables 9 and 10.

In run #1, diafiltration directly from the affinity elution pool analog buffer into 100 mM Tris, 2 mM _MgCl2 , 0.01% P188, pH 9 gave a VG yield of 66%. In run #2, the addition of 500 mM arginine, a cosolvent known to reduce protein aggregation (Arakawa et al. Biophysical Chemistry (2007) 127(1): 1-8), to diafiltration buffer 2 increased the VG yield to 93%, compared to the 66% yield obtained in the absence of arginine. However, removal of arginine from the system in run #2 resulted in significant losses, with a VG yield of 78%. 500 mM arginine was not included in the final diafiltration buffer because it would have resulted in a high conductivity (~20 mS/cm) AEX load that would have interfered with binding to the AEX resin. However, this finding encouraged the investigation of other amino acid cosolvents that could stabilize AAV9 but have lower solvent conductivity at alkaline pH. These studies are described in Example 6.

Table 10: Tangential flow filtration to prepare AAV9 affinity eluate for AEX chromatography

Dynamic light scattering analysis of the AAV9 affinity pool when diluted in 100 mM Tris, pH 9

Dynamic light scattering (DLS) was used to measure the Z-average value (Z-AVG) to estimate the capsid aggregation in the AAV9 affinity pool diluted with 100 mM Tris (pH 9). The Z-average value is a reliable measure of the average size of particles in solution. As shown in Table 11, the AAV9 affinity pool was diluted (0 to 30 times) in a polypropylene tube with 100 mM Tris, pH 9 and immediately analyzed by DLS. For each dilution factor, a separate experiment was performed with a fresh AAV9 affinity pool in a new polypropylene tube. After the DLS analysis was completed, the pH and conductivity of the solution were measured.

The results are summarized in Table 11 and Figure 6 and show that dilution of the AAV9 affinity pool resulted in aggregation and increased Z-means. The undiluted AAV9 affinity pool had a pH of 4.1, a conductivity of 6.0 mS/cm, a Z-mean of 15 nm, and no aggregation. Compared to the AAV9 affinity pool, a two-fold dilution with 100 mM Tris (pH 9) increased the solution pH to 7.2, with the conductivity remaining at 5.8 mS/cm, resulting in a 5-fold increase in aggregation with a Z-mean of 77 nm. This result means that increasing the solution pH by the estimated isoelectric point of AAV9 (calculated to be ∼5.8 and ∼6.2 for AAV9 complete capsid and empty capsid, respectively (Venkatakrishnan et al. (2013) J. Virology 87(9):4974-4984)) destabilizes the vector, resulting in product loss. Dilutions of 5 and 10 fold resulted in aggregation and very high Z-mean values of 395 nm and 221 nm, respectively. Larger dilutions in the 15 to 30 fold range showed Z-mean values of 46-66 nm and aggregation.

Overall, the results presented in Tables 9 and 10 indicate that a significant amount of AAV9 vector is lost during AEX load preparation. TFF load preparation methods at high VG concentrations, serial dilutions, and dilutions in the presence of 0.01% P188 were unable to prevent VG loss. The data in Table 11 suggest that the mechanism behind VG loss is aggregation. Based on this observation, a series of dilution experiments were performed using diluents that prevented aggregation and are described in Example 6.

Table 11: pH, conductivity, Z-mean and aggregation of AAV9 affinity pools diluted with 100 mM Tris, pH 9

# Aggregation exists (+) or aggregation does not exist (-)

Example 4: Screening of diluent co-solvents for preparing AAV9 affinity eluate for AEX

The AAV9 affinity eluate was diluted 15-fold with various diluent co-solvents to determine the conditions that maximized the %VG yield during AEX load preparation. The co-solvents screened included detergent, iodixanol, glycerol, magnesium chloride, and amino acids. To investigate the effect of dilution alone, the AAV9 affinity eluate was diluted with affinity eluate pool buffer, 150 mM acetate, 100 mM glycine, 25 mM MgCl ₂ , pH 4.2, without changing the pH or conductivity. 14 mL of the diluent was added to a polypropylene tube, followed by 1 mL of the AAV9 affinity eluate. The resulting solution was gently mixed by end-to-end stirring, the pH and conductivity were measured, and a pre-filtered sample was taken. The diluted sample was then filtered through a 0.2 μm filter pre-wetted with the diluent. The diluted and filtered sample was neutralized with 250 mM sodium citrate, pH 3.5. Neutralized samples were analyzed by dynamic light scattering to estimate particle Z-AVG and relative aggregation, and ITRs were analyzed by qPCR to determine VG titers.

The results of the diluent co-solvent screen showed that some co-solvents reduced aggregation, maintained Z-AVG near 30 nm, and increased % VG yield compared to the baseline diluent 100 mM Tris, pH 9 (Table 12). The Z-AVG of the undiluted AAV9 affinity elution pool was 29 nm and clearly not aggregated. Dilution of the AAV9 affinity pool with the affinity elution pool buffer resulted in no increase in Z-AVG, no aggregation, but only a 69% VG yield. This data suggests that aggregation does not occur under the conditions of the affinity pool (pH 4.2, 7 mS/cm), but that VG loss occurs through increased surface area (via dilution) through non-specific binding.

Consistent with the results of Example 3 (above), dilution of the AAV9 affinity pool with 100 mM Tris, pH 9 resulted in increased Z-AVG, high aggregation, and only 59% VG yield. Addition of 0.01-1% P188 in 100 mM Tris, pH 9 did not significantly improve performance compared to the baseline buffer, with similar Z-AVG and aggregation. Dilution of the AAV9 affinity eluate with 50 mM arginine, 2 mM _MgCl2 , 0.1% P188, 100 mM Tris, pH 9 produced 33 nm Z-AVG, no aggregation, and a VG yield of approximately 80%, but a conductivity of 4.5 mS/cm, which interfered with AAV9 binding to the AEX resin. Various histidine-containing dilutions provided ideal results. For example, dilution of AAV9 with 200 mM histidine, 200 mM Tris, ₁₀ mM MgCl2, 25% iodixanol, pH 8.8, yielded a 99% yield of VG. Iodixanol is strongly UV active and interferes with UV readings in chromatography systems, so this and similar buffers were not used in AEX runs.

Importantly, the AAV9 affinity eluate was diluted 15-fold with 0.5% P188, 200 mM histidine, 200 mM Tris, pH 8.8 and then filtered through a pre-wet filter to obtain 35 nm Z-AVG and a VG yield of 101%. The resulting diluted filtered solution had a pH of 8.8 and a conductivity of 2.5 mS/cm, conditions that may be suitable for binding to the AEX resin. Therefore, this dilution scheme was optimized in the following examples.

Studies on AAV2 aggregation involved screening various co-solvents by dilution stress assays combined with DLS, and found that AAV2 aggregation could be prevented by dilution to a buffer containing various salts with an ionic strength ≥ 200 mM (Fraser et al. Molecular Therapy (2005) 12 (1): 171-178). A similar approach to preparing AAV9 for AEX chromatography was not applicable to this example because solutions with an ionic strength ≥ 200 mM reduced the binding of the vector to the AEX resin (data not shown). Interestingly, the addition of the amino acids histidine, arginine, or glycine to the diluent did not inhibit AAV2 aggregation (Fraser et al. Molecular Therapy (2005) 12 (1): 171-178).

In this example, a mixture of histidine, arginine, and glycine in combination with _MgCl2 , P188, and/or glycerol reduced AAV9 aggregation, but provided only 69-80% %VG yields (see dilution results R2, H2, H3 in Table 12). High %VG yields were only achieved when histidine was used in conjunction with detergents P188 and Triton X-100 (see dilution results H7 and H8 in Table 12). These results suggest that high VG yield dilution cosolvents for AEX load preparation of AAV capsids (e.g., AAV9) should contain detergents to reduce nonspecific binding to surfaces (e.g., dilution vessels and filters), and use histidine or similar moieties to modulate charge interactions and/or hydrogen bonding between AAV capsid particles (e.g., AAV9 vector particles).

Table 12: Screening of diluent co-solvents for AEX loading preparation of AAV9 vector capsids. AAV9 affinity eluate was diluted 15-fold with diluent, filtered, analyzed by dynamic light scattering to determine the Z-average value (Z-AVG) for estimated capsid aggregation, and analyzed by qPCR to determine % VG yield.

NR - value not reported due to interference in DLS readout. NT - experiment not performed.

Example 5: Optimization of dilution co-solvent for preparation of recombinant AAV9 vector affinity eluate for AEX chromatography

The concentration of P188 in the diluent and the resulting conductivity of the diluted sample were optimized to achieve maximum recovery of the AAV9 vector. The diluent P188 concentrations were tested at 0.01%, 0.05%, 0.2% and 0.5%, while the diluted sample conductivity was 2, 2.5 and 3 mS/cm. Different dilution factors were used to obtain different conductivity. The diluted AAV9 vector affinity eluate was passed through a 0.2 μm filter and pre-wetted with each corresponding dilution. The resulting filtrate was assayed by qPCR for the transgene to determine the VG titer, and the results are shown in Table 13 and Figures 7A and 7B. The data shown in Figures 7A and 7B indicate that 0.5% P188 is required to obtain maximum VG yield, and the use of lower concentrations (i.e., less than 0.5%) of P188 results in lower VG yields. Within the range studied, the final conductivity (or by combining the dilution factor) did not significantly affect the % VG yield.

Table 13: Optimization of P188 concentration, dilution factor and final conductivity

Example 6: Enrichment of complete AAV9 by optimizing dilution and AEX chromatography techniques

The best performing dilutions from Examples 4 and 5 were combined with the best performing eluate from Example 1 to create an optimized AEX chromatography method that enriched intact AAV9 vector capsids with high %VG yields.

The AAV9 affinity eluate was diluted 15-fold with a novel buffer containing amino acids and detergent cosolvents (200 mM histidine, 200 mM Tris, 0.5% P188, pH 8.9) and filtered through a 0.2 μm filter. The 15-fold dilution is lower than the dilution used in other methods (see US 2019-0002841; US 2019-0002842; US 2018-0002844), making it easier to implement in large-scale production and obtain high VG yields.

The pH value of the obtained filtrate was 8.8 and the conductivity was 2.3 mS/cm. Consistent with Table 14, the filtrate was loaded onto a POROS ^™ 50HQ column, eluted by a sodium acetate gradient, and fractions corresponding to 0.39 CV were collected during the gradient elution.

The load and chromatographic fractions were neutralized with 250 mM sodium citrate (pH 3.5) and ITR was determined by SEC A ₂₆₀ / A ₂₈₀ , AUC and qPCR. In order to test the repeatability of the AEX method, a second run was performed using the same materials and methods as the first run. The AEX chromatograms of the first run are shown in Figures 8A and 8B. Table 15 provides the SEC A ₂₆₀ / A ₂₈₀ of the gradient elution fractions, and shows that the optimized AEX method enriches the complete AAV9 vector compared to the load. The percentage of complete capsids, intermediate capsids and empty capsids in the affinity pool (loaded on the column), the flow-through fraction and the elution pool was determined using analytical ultracentrifugation. The data are provided in Table 16, and show that the optimized AEX method enriches the percentage of complete AAV9 vector capsids while giving a high VG yield percentage.

Table 14: Optimized AEX chromatography on a 5.1 mL POROS ^™ 50HQ column

Table 15: SEC _A260 / _A280 analysis of two replicate fractions from the optimized AEX method

F/T: flow-through (unbound fraction); gradient elution fractions are numbered 1-14.

Table 16: Performance of the optimized AEX method as judged by analysis of intermediate and chromatographic fractions of the method

% VG yield was calculated as (amount of VG per step or fraction)/(amount of VG in affinity pool), thus accounting for losses during load preparation. Dashes (-) indicate that no determination was made.

Dilution and filtration of the AAV9 affinity eluate resulted in a 93% VG yield, confirming the results in Examples 3 and 4. The flow-through fraction contained 10% of the VG from the affinity pool starting material, with a SEC _A260 / _A280 of 0.70 and a capsid population of 1% complete capsids, 14% intermediate capsids, and 85% empty capsids. This result suggests that the developed AEX method partitions some empty capsids through the column, facilitating further enrichment by sodium acetate gradient elution.

For two AEX runs, three virtual elution pools were formed, namely a wide pool consisting of fractions 1-8, a narrow pool consisting of fractions 2-6, and a second peak pool consisting of fractions 8-13. The VG yield of the second peak pool was 20%, mainly composed of empty capsids. The wide pool contained 53% VG yield, an average SEC A ₂₆₀ /A ₂₈₀ of 1.26, and 47% complete AAV9 vector capsids, thus representing an enrichment of 2.6 times the complete capsid percentage. The narrow elution pool contained an average VG yield of 38%, a SEC A ₂₆₀ /A ₂₈₀ ratio of 1.28-1.29, and an average AAV9 vector capsid population of 53% complete capsids, 23% intermediate capsids, and 24% empty capsids. Therefore, the optimized AEX method enriched 2.9 times the complete AAV9 vector and consumed 2.8 times the empty capsids.

The results obtained from the narrow pool represent an ideal balance between % complete capsid and % VG yield. Therefore, in order to obtain similar results in most subsequent examples, we adopted a fraction pooling threshold based on SEC _A260 / _A280 ≥1.25 (similar to the minimum value of 1.25 obtained in narrow pool fractions 2-6, Table 16). Therefore, in 8 of the 10 large-scale AEX runs in the following examples, fractions with SEC _A260 / _A280 ≥1.25 were included in the pool, and all fractions with SEC _A260 / _A280 ≤1.24 were excluded.

The data and strategy examples above illustrate a powerful feature of the optimized AEX method: flexibility in the pooling strategy. Analysis of the collected fractions using high-resolution gradient elution chromatography with SEC _A260 / _A280 ensures that a high percentage of intact capsids are recovered, regardless of slight variations in feed stream or process operation.

In contrast to other chromatographic methods for purifying recombinant AAV vectors, particularly separating empty capsids from complete AAV vectors (US 2019-0002841; US 2019-0002842; US 2018-0002844), the methods disclosed herein utilize lower dilution factors, dilution buffers containing histidine, steeper elution gradients, sodium acetate as elution salts, and lower alkaline conditions (pH 9). The methods disclosed herein and illustrated in Examples 1-9 are also different from other reported methods, such as the method of Tomono et al. (Molec. Ther. Meth. Clin. Dev. (2018) 11: 180-190), in which the disclosed method does not use an AEX loading preparation with a conductivity of about 7 mS/cm, does not use ammonium sulfate precipitation, and does not use size exclusion chromatography. The novel and inventive methods disclosed herein can be implemented on a large scale and produce high yields of VG from affinity chromatography eluates.

To further illustrate the flexibility and robustness of the optimized AEX process, Example 7 tested feed streams with varying % complete vector capsids.

Example 7: Effect of the percentage of intact capsids in the affinity eluate on the performance of the optimized AEX method

The ability of the optimized AEX method to enrich complete AAV9 vectors from feed materials with different percentages of AAV9 empty capsids was tested. In brief, HEK293 cells were grown in suspension culture and transfected with adenovirus helper plasmids and Rep2Cap9 plasmids (excluding plasmids containing transgenic boxes). After transfection, the cells were cultured for three days, harvested, lysed, flocculated, deeply filtered and absolutely filtered. The resulting filtrate was subjected to affinity chromatography to produce an affinity pool containing AAV9 particles without vector genomes (ineffective transfection AAV9 affinity eluate, referred to herein as ineffective capsid). In order to produce AEX starting materials with different percentages of complete capsids, the ineffective transfection AAV9 affinity eluate was mixed with the standard AAV9 affinity eluate at a volume ratio of 0%, 20%, 40%, 60%, 80% and 100% ineffective capsids.

The mixture was diluted 15-fold with 200 mM histidine, 200 mM Tris, 0.5% P188, pH 8.8, and filtered through 0.2 μm to produce an AEX load at pH 8.8 with a conductivity of 2.6 mS/cm. Consistent with Table 17, the optimized AEX method was performed on 6 loads containing different percentages of capsids produced by ineffective transfection. For each of the 6 AEX runs, a 6.67 mL POROS ^™ 50HQ column was uniformly challenged with 1.5×10 ¹⁵ total viral particles (VP) or 2.2×10 ¹⁴ VP/mL resin. The chromatographic load, flow-through, and elution fractions were neutralized with 250 mM sodium citrate (pH 3.5) and analyzed by SEC A ₂₆₀ /A ₂₈₀ .

The SEC _A260 / _A280 data are reported in Table 18 and Figure 9 and show that the optimized AEX method produces individual elution fractions with SEC _A260 / _A280 ≥1.25 when using 0%, 20% and 40% null capsid transfection starting material. The SEC _A260 / _A280 for column loads containing 0%, 20% and 40% null capsid were 1.16, 1.10 and 1.01, respectively. The optimized AEX method produces 6 or 7 consecutive elution fractions with SEC _A260 / _A280 ≥1.25 for each elution fraction using these materials.

The SEC _A260 / _A280 values for loads containing 60%, 80% and 100% abortive capsids were 0.90, 0.77 and 0.62, respectively. The optimized AEX method enriched starting material with 60%, 80% and 100% abortive capsids to produce elution fractions with maximum SEC _A260 / _A280 values of 1.23, 1.16 and 0.83, respectively.

The AAV9 upstream process was implemented at 250L and 2000L disposable bioreactor (SUB) scales, and the resulting AAV9 affinity eluate (n=7) with a SEC _A260 / _A280 of 1.10±0.1 was harvested and subjected to downstream operations of affinity chromatography. Thus, upstream and downstream processing (including the optimized AEX process described herein) produced AAV9 capsids with a complete capsid percentage that can be used for gene therapy applications. Based on these results, the optimized AEX process was scaled up to achieve production at 250L and 2000L SUB scales, as described in the following examples.

Table 17: Optimized AEX chromatography using POROS ^™ 50HQ column for starting materials with different percentages of complete capsids. All runs involved uniformly challenging the column at 2.2 x ¹⁰¹⁴ VP/mL resin using transfection starting materials with different percentages of ineffective capsids.

*Applies 2mm path length UV monitor.

^N is a mixture of standard AAV9 affinity eluate and null capsid transfection AAV9 affinity eluate, with volume ratios of 0%, 20%, 40%, 60%, 80% and 100% null capsid.

Table 18: SEC _A260 / _A280 of chromatographic fractions generated using the optimized AEX method for feeds with mixed % void capsids and standard affinity pools

F/T: flow-through (unbound) fraction. Elution fractions are given in numerical form. Ratios ≥ 1.25 are shown in bold.

Example 8: Scale-up of an optimized AEX process for enrichment of complete AAV9 vectors

The optimized AEX method for enriching complete AAV9 vectors was scaled up for downstream processing of AAV vectors produced in 250L and 2000L disposable bioreactors (SUBs). The size of the POROS ^™ 50HQ column was based on the scale-independent maximum challenge of 3×10 ¹⁴ VG/mL resin. Tables 19 and 20 provide the optimized AEX methods implemented for 250L and 2000L SUBs, respectively. At the 250L scale, a 1.3L AEX column with an inner diameter (ID) of 10 cm and a column bed height of 16 cm was used. The AEX method was implemented at the 2000L SUB scale, using a 6.4L AEX column with an ID of 20 cm and a column bed height of 20.5 cm. The residence time in both scales was fixed at 4±0.5 minutes/CV, resulting in volume flow rates of 314 mL/min and 1.8 L/min for all steps of the AEX method in 250L and 2000L SUBs, respectively. The flow rates are within acceptable limits to form a linear sodium acetate gradient of smooth shape with the pump and mixer on the chromatographic skid during product elution. At these two scales, each chromatographic step is carried out using the same buffer set for the same CV length, except that the 2000L SUB-AEX method includes the sterilization and regeneration steps before flushing with water for injection and use. Figures 10 and 11 provide the chromatograms of the representative AEX runs carried out at 250L SUB scale and 2000L SUB scale. In all tested scales (various small-scale runs, 250L and 2000L SUB scales), the AEX method of optimization produces similar A ₂₆₀ / A ₂₈₀ chromatograms.

At 250L and 2000L scale, elution fractions of 1/3 column volume (CV) were collected. The fractions were neutralized with 250mM sodium citrate (pH 3.5) and measured by various analytical techniques. SEC was performed to determine the A ₂₆₀ / A ₂₈₀ ratio and the percentage of high molar mass substances (% HMMS). The residual amounts of host cell proteins (HCP) and host cell DNA (HC-DNA) were determined by ELISA and qPCR, respectively. At 250L scale, qPCR was used to measure ITR copies to quantify VG. At 2000L scale, qPCR was used to measure transgenic copies to quantify VG.

Table 19: Optimized AEX chromatography performed on a 1.3L POROS ^™ 50HQ column at 250L SUB scale

Table 20: Optimized AEX chromatography on a 6.4 L POROS ^™ 50HQ column at 2000 L SUB scale

*Resin challenge determined using qPCR method measuring transgene copies.

Table 21 provides the SEC _A260 / _A280 ratios of eluted fractions produced with the 250L and 2000L SUB AEX methods, respectively. The method demonstrated robustness across a wide range of column challenges: 2.7×10 ¹² -6.8×10 ¹³ VG/mL resin. Nine of the ten AEX runs produced at least 6 fractions with SEC _A260 / _A280 ratios ≥1.25. Batch 250L-1 used the affinity pool with the lowest SEC ratio in the study (0.94) and was the only AEX run that did not produce fractions with ratios ≥1.25. For each AEX run, fraction 5 produced the highest SEC _A260 / _A280 ratio in 7 of the 10 runs, thus showing a high degree of consistency in the chromatography and fraction collection operations for two different scales and various VG/mL resin challenges.

Tables 22 and 23 provide the impurity profiles, %HMMS, and SEC _A260 / _A280 of the individual AEX fractions of the 250L-4 and 2000L-4 batches, respectively. The optimized AEX method removed a large amount of HCP from the affinity pool. For example, the 250L-4 affinity pool contained 51 pg HCP/1×10 ⁹ VG, and the optimized AEX method removed HCP to LLOQ in elution fractions 2-8 for forming the AEX pool. The 2000L-4 affinity pool contained 331 pg HCP/1×10 ⁹ VG, which was removed to LLOQ in elution fractions 2-9 for forming the AEX pool.

The AEX method does not significantly reduce HC-DNA levels. The AEX method uses a sodium acetate elution gradient to separate HMMS. Early elution fractions are relatively depleted of HMMS (e.g., fractions 1-5 in the 250L-4 and 2000L-4 runs contain <3% HMMS). In contrast, later elution fractions contain higher relative levels of HMMS (e.g., fractions 8-10 from the 2000L-4 run contain >7% HMMS).

Table 21: SEC _A260 / _A280 of affinity eluate pools and AEX chromatography fractions generated at 250L and 2000L SUB scale

Elution fractions are given as numbers. Ratios ≥ 1.25 are shown in bold. 250L SUB VG/mL resin challenge determined using qPCR method measuring ITR copies. 2000L SUB VG/mL resin challenge determined using qPCR method measuring transgene copies.

Table 22: SEC _A260 / _A280 , %HMMS and impurity profiles of chromatographic fractions from AEX run at 250L SUB scale (Batch 250L-4)

Elution fractions are given numerically. Elution fractions 2-8 had SEC _A260 / _A280 ≥ 1.24 (in bold) and were therefore pooled and processed further. LLoQ - the amount detected was below the limit of quantitation of the assay.

Table 23: SEC _A260 / _A280 , %HMMS and impurity profiles of chromatographic fractions from AEX run at 2000L SUB scale (Batch 2000L-4)

Elution fractions are given as numbers. Elution fractions 2-9 had SEC _A260 / _A280 ≥ 1.25 (in bold) and were therefore pooled and processed further. The LLoQ detected amount was below the limit of quantitation of the assay.

The 250L AEX runs used various fraction pooling thresholds based on the SEC _A260 / _A280 ratio. Fractions 250L-1 and 250L-4 from the 250L AEX run were pooled based on where the SEC _A260 / _A280 was ≥1.22 and ≥1.24, respectively. Fractions 250L-2, 250L-3, and 250L-5 from the 250L AEX run and all five 2000L AEX run fractions were pooled based on where the SEC _A260 / _A280 was ≥1.25.

The AEX product pools obtained from 2000L batches 2000L-2, 2000L-3, 2000L-4 and 2000L-5 were processed through a virus filtration step, while the 250L batch and 2000L-1 batch were not virus filtered. The AEX pools and/or virus filtered pools obtained were processed by ultrafiltration/diafiltration (UF/DF) and 0.2μm filtration to produce the bulk drug substance (DS). None of the steps after AEX chromatography significantly affected the AAV9 empty capsid/complete capsid ratio. qPCR was performed on the affinity pool and the AEX pool to determine the VG yield percentage of the AEX step. The bulk drug material was analyzed by AUC and SEC _A260 / _A280 to determine the complete capsid percentage of the purified AAV9 vector.

Table 24 reports the results and shows that the scaled-up AEX process increased the percentage of intact capsids of recovered AAV9 vectors with high yields. The scaled-up AEX process enriched intact vectors to 45-65% of total capsids and reduced the amount of empty capsids in 9 of 10 bulk drug batches to ≤28% total capsids as measured by AUC. In all 10 runs, the AEX process increased the SEC _A260 / _A280 ratio from 0.94-1.25 in the affinity pool to 1.24-1.32 in the bulk drug. The average VG step yield of the AEX process implemented at 250L and 2000L scales was 47+/-11%.

Table 24: AEX performance at 250L and 2000L SUB scale.

¹ "DS" indicates that AUC and SEC _A260 / _A280 were performed on drug substance (DS). None of the steps after AEX used to produce drug substance affected the AAV9 vector empty capsid/full capsid ratio.

² % VG dilution yield is calculated as ((VG in diluted affinity pool)/(VG in affinity pool))*100%. "Affinity pool" refers to the material collected from the affinity column; and is also referred to herein as "affinity eluate"

³ % VG column yield was calculated as ((VG in AEX pool)/(VG in diluted affinity pool))*100%. "Diluted affinity pool", also referred to herein as "diluted affinity eluate". In this example, the diluted affinity eluate was not filtered.

⁴ % VG step yield was calculated as ((VG in AEX pool)/(VG in affinity pool))*100%.

A 250LSUB VG/mL resin challenge and % VG yield were determined using a qPCR method measuring ITR copies.

*2000L SUB VG/mL resin challenge and % VG yield were determined using a qPCR method that measures transgene copies.

Example 9: Ultracentrifugation and cation exchange chromatography to separate empty capsids from complete AAV vector capsids

In another embodiment of downstream processing of the AAV9 vector in the 250L SUB, density gradient ultracentrifugation (UC) is used to separate empty capsids from complete vectors. Similar to the previously described method (Grieger et al. Molecular Therapy (2016) 24 (2): 287-297), bands containing 40% and 60% iodixanol are formed in the UC tube. Affinity eluate containing 25% iodixanol is added to the UC tube and ultracentrifuged. Fractions enriched for AAV capsids containing full-length vector genomes are collected from the interface of the 40% and 60% iodixanol bands. To remove iodixanol, the fractions are diluted and loaded onto a cation exchange (CEX) chromatography column. The AAV9 vector capsid binds to the CEX column, and most of the iodixanol passes through the column in the unbound fraction. The AAV9 vector capsid is eluted from the CEX column in a fraction that is substantially free of iodixanol. The CEX pool was processed forward through UF/DF and 0.2 μm filtration to produce drug substance (DS) containing AAV9 vector capsids.

Table 25 provides the process performance of UC+CEX and optimized AEX methods, as well as a comparison of the analytical results of the raw drug substances produced by these methods. The optimized AEX methods implemented at 250L and 2000L scales provide an average VG yield of 45±8% and 50±13%, respectively. These values are higher than the average 33±9% VG yield provided by the UC+CEX method. The average DS readings of SEC A ₂₆₀ /A ₂₈₀ (1.26-1.30), % complete capsid (49-55%) and % empty capsid (20-25%) produced by all three methods are very similar. Compared with the DS produced by 250L AEX (24±3) and 250L UC+CEX (23±4) methods, the average percentage of intermediate capsids in the DS produced by the 2000L AEX method is slightly higher (32±4%).

The DS produced by the three methods were highly similar in terms of HMMS percentage, purity percentage, and HCP and HC-DNA levels. Overall, this data suggests that the AEX method implemented at 250L and 2000L SUB scales provides highly similar process performance and product quality to the 250L UC+CEX method.

Table 25: Drug identification of AAV9 purified by ultracentrifugation and cation exchange chromatography (UC+CEX) or downstream processing of complete vector enrichment by optimized AEX method

α % VG step yield of UC+CEX for the process run through UC and CEX.

βAEX % VG step yield by dilution and AEX chromatography protocol.

γ % VG step yield in 250L SUB determined using qPCR method measuring ITR copies.

% VG step yield in 2000L SUB determined using qPCR method measuring transgene copies.

δ Three UC+CEX bulk drug substance batches had HCP levels at LLOQ, while one bulk drug substance batch had an HCP level of 2.6 pg/1×10 ⁹ VG.

LLOQ - the amount detected is below the limit of quantitation of the assay.

Example 10: Optimized AEX method for enrichment of complete AAV3B vectors

HEK 293 cells were grown in suspension culture and transfected with two plasmids to produce AAV3B vectors according to standard methods known in the art. HEK 293 cells were harvested, lysed, flocculated, and the resulting lysate was filtered. The AAV3B vector was purified from the clarified lysate by affinity chromatography. The affinity column was balanced, the clarified lysate was loaded, washed, and the purified AAV3B vector was eluted. 25 mM _MgCl2 was spiked into the AAV3B vector affinity pool (also referred to as affinity eluate) to achieve a final _MgCl2 concentration of about 1.7 mM in the diluted affinity pool. The pH of the affinity pool was pH 7.6. The affinity pool was diluted approximately 15-fold (14-fold to 17.8-fold depending on the run) with a buffer containing 200 mM histidine, 200 mM Tris, 0.5% P188, pH 8.9. The resulting diluted affinity eluate had a pH ≥ 8.6 and a conductivity ≤ 2.5 mS/cm (target 2.3 mS/cm) and was loaded onto the AEX column.

A POROS ^TM 50HQ column was used, which had a volume of 49 mL, a column bed height of 10 cm, and a diameter of 2.5 cm. The target column load challenge was 1×10 ¹⁴ to 3×10 ¹⁴ vg/mL resin (e.g., about 2.4×10 ¹⁴ vg/mL). Table 26 provides optimized AEX process conditions. The residence time of all steps in the AEX process was fixed at 2 minutes/CV to accommodate a lower elution gradient (compared to the previous embodiment) and a relatively small column. The elution gradient was 2.5 times lower than the previous embodiment to maximize the resolution of empty capsids/complete capsids. The empty capsids were eluted first, followed by the complete AAV3B capsids (Figure 12). In this embodiment, the complete AAV3B capsids contained a vector genome having a transgene encoding SEQ ID NO: 15 amino acids (copper transport ATPase 2, missing metal binding sites 1-4). Consistent with the lower gradient and broader elution peak, the fraction volume was increased to 0.5CV (as opposed to 0.33CV in the previous embodiment).

Table 26: Optimized AEX chromatography for purification of complete rAAV3B vector

Gradient elution was performed from 100% buffer A to 25% buffer A/75% buffer B in 37.5 CV with a slope of 2% buffer B/CV. A total of 20 elution fractions were collected when the percentage of buffer B was 32% to 52% over the entire gradient. Fractions were collected into a container pre-filled with 13.2% v/v (0.066 CV) 250 mM sodium citrate (pH 3.5) to neutralize the fractions and reduce exposure of the capsid to alkaline pH. The pH range of the neutralized fraction was pH 7.5 to 7.7. The first elution fraction with _A260 / _A280 ≥ 0.98 (determined by SEC) was combined with successive elution fractions, but no more than 11 fractions in total (Table 27).

Table 27: Elution fractions

Bold and underlined fractions were combined.

The combined fractions were determined by various analytical techniques. The actual vg/mL resin challenge range was 6.3E13 to 9.4E13, averaging 7.4E13 ± 1.2E13. SEC was performed to determine the A ₂₆₀ /A ₂₈₀ ratio. Compared with the A ₂₆₀ /A ₂₈₀ of the affinity pool, the A ₂₆₀ /A ₂₈₀ of the AEX pool increased in all operating conditions (Table 28). The complete capsid, intermediate capsid and empty capsid percentages of the affinity pool and the AEX elution pool were determined by analytical ultracentrifugation. The average complete carrier percentage was 11.2 ± 2.1% of the affinity pool enriched to 22.9 ± 2.9% in the AEX pool. The same affinity pool consumed empty capsids from 79.7 ± 2.5% to 67.5 ± 3.8% in the AEX pool.

The vg titer was determined by transgenic QPCR (Table 28). The average percent vg dilution yield was 120% ± 12%. The average percent vg column yield was 47% ± 11%.

Table 28: AEX performance characteristics for purification of AAV3B vectors

The method described in this example was used to generate a purified rAAV3B vector pool that was enriched for complete capsids and depleted for empty capsids compared to the starting material (ie, affinity eluate).

Sequence Listing

Claims (55)

1. A method of purifying a rAAV vector by AEX, the method comprising the steps of:

i) Loading a solution comprising the rAAV vector to be purified onto the AEX stationary phase into a column;

ii) subjecting the material from the stationary phase in the column to gradient elution, wherein the percentage of the first gradient elution buffer varies inversely with the variation of the percentage of the second gradient elution buffer; and

iii) Collecting at least one eluent fraction from the column during gradient elution beginning when absorbance of the column flow-through reaches an absorbance threshold, and wherein the at least one eluent fraction comprises the rAAV vector to be purified.

2. The method of purifying a rAAV vector by AEX of claim 1, wherein the method further comprises measuring absorbance of the at least one eluate fraction collected from the column and determining an a260/a280 ratio.

3. The method of purifying a rAAV vector by AEX according to claim 1 or 2, wherein the solution comprising the rAAV vector to be purified is diluted about 2-fold to 25-fold (e.g. 15-fold) with a dilution solution comprising histidine, tris and P188, and optionally filtered before application to the stationary phase.

4. A method of purifying a rAAV vector by AEX according to any one of claims 1 to 3, wherein the solution is an affinity eluate.

5. The method of purifying a rAAV vector by AEX of claim 5, wherein the pH of the diluted and optionally filtered affinity eluate is increased compared to the pH of the solution; and wherein the conductivity of the diluted and optionally filtered affinity eluate is reduced compared to the conductivity of the solution.

6. The method of purifying a rAAV vector by AEX of any one of claims 1 to 5, wherein the first gradient elution buffer comprises about 50mM to about 150mM Tris, about 0.005% to about 0.015% P188, and a pH of about pH 8.5 to 9.5; wherein the second gradient elution buffer comprises about 400mM to about 600mM sodium acetate, about 50mM to about 150mM Tris, about 0.005% to about 0.015% P188 and a pH of about pH 8.5 to 9.5; and wherein 10 to 60 Column Volumes (CVs) (e.g., about 20CV, about 37.5 CV) of the first gradient elution buffer, the second gradient elution buffer, or a mixture of both are applied to the stationary phase during gradient elution.

7. The method of purifying a rAAV vector by AEX according to any one of claims 1 to 6, wherein the percentage of the first gradient elution buffer is 50% -100% at the beginning of the gradient elution and the percentage of the second gradient elution buffer is 50% -100% at the end of the gradient elution, and wherein the percentage of the second elution buffer increases in the gradient elution at a rate of about 2% -5%/CV.

8. The method of purifying a rAAV vector by AEX of any one of claims 1 to 7, wherein the sodium acetate concentration of the first gradient elution buffer, second gradient elution buffer, or mixture of both continues to increase during the gradient elution; and wherein the concentration of sodium acetate increases at a rate of about 10mM/CV to 50mM/CV (e.g., about 10mM/CV, about 25 mM/CV) during the gradient elution.

9. The method of purifying a rAAV vector by AEX according to any one of claims 1 to 8, wherein during the gradient elution, complete capsids elute from the stationary phase in a first elution peak and/or in a first portion of a second elution peak.

10. The method of purifying a rAAV vector by AEX according to any one of claims 1 to 9, wherein during the gradient elution, empty capsids are recovered in the column flow-through, in the first elution peak and/or in the last part of the second elution peak.

11. The method of purifying a rAAV vector by AEX according to any one of claims 1 to 10, wherein the absorbance of the at least one eluent fraction is measured at 280nm, and wherein optionally the absorbance threshold measured at 280nm is ≡0.5mAU/mm path length.

12. The method of purifying a rAAV vector by AEX according to any one of claims 1 to 11, wherein the volume of the at least one eluent fraction is equal to 1/8CV to 10CV, such as 1/8CV, 1/4CV, 1/3CV, 1/2CV, 3/4CV, 1CV, 2CV, 3CV, 4CV, 5CV, 6CV, 7CV, 8CV, 9CV, 10CV or more, and optionally wherein the a260/a280 ratio of the at least one eluent fraction is ≡1.25.

13. The method of purifying a rAAV vector by AEX according to any one of claims 1 to 12, wherein at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more eluent fractions are collected.

14. The method of purifying a rAAV vector by AEX of any one of claims 1 to 13, wherein the method further comprises combining at least two eluate fractions collected from the column, each fraction having an a260/a280 ratio of ≡0.98 or ≡1.0, to form a combined eluate comprising the rAAV vector.

15. The method of purifying a rAAV vector by AEX according to claim 14, wherein the combined eluate has a%vg column yield of 20% to 100% (e.g. 63 +/-26%), a%vg step yield of 31% to 66% (e.g. 47 +/-11%) and/or an a260/a280 ratio of ≡1.0.

16. The method of purifying a rAAV vector by AEX according to claim 14 or 15, wherein the combined eluate is enriched in complete capsids and/or depleted in empty capsids compared to the solution loaded to the column.

17. The method of purifying a rAAV vector by AEX of any one of claims 1 to 16, wherein a purified rAAV vector is produced.

18. The method of purifying rAAV vectors by AEX according to any one of claims 14 to 17, further comprising filtering the combined eluate by a method selected from viral filtration, ultrafiltration/diafiltration (UF/DF), filtration by a 0.2 μm filter, and combinations thereof, to produce a drug substance.

19. The method of purifying a rAAV vector by AEX of claim 18, wherein the drug substance comprises: i) 45% to 65% (e.g., 52 +/-7%) of the total capsids; ii) 19% to 37% (e.g. 28 +/-5%) of the total capsids; and/or iii) 10% to 37% (e.g., 20 +/-7%) of the total capsids.

20. The method of purifying a rAAV vector by AEX of claim 18 or 19, wherein the drug substance is enriched in complete capsids and/or depleted in empty capsids compared to the solution loaded to the column.

21. The method of purifying a rAAV vector by AEX of any one of claims 1 to 20, wherein the rAAV vector comprises AAV capsid proteins from an AAV serotype selected from the group consisting of: AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh74, AAV12, AAV2i8, NP4, NP22, NP66, AAVDJ/8, AAVDJ/9, aavlk03, aav1.1, aav2.5, aav6.1, aav6.3.1, aav9.45, RHM4-1 (SEQ ID NO of WO 2015/01353: 5), RHM15-1, RHM15-2, RHM15-3/RHM15-5, RHM15-4, RHM15-6,AAV hu.26,AAV1.1,AAV2.5,AAV6.1,AAV6.3.1,AAV9,45,AAV2i8,AAV29G,AAV2,8G9,AVV-LK03, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N, AAVHSC1, AAVHSC2, AAVHSC3, AAVHSC4, AAVHSC5, AAVHSC6, AAVHSC7, AAVHSC8, AAVHSC9, AAVHSC10, AAVHSC11, AAVHSC12, AAVHSC13, AAVHSC14 and AAVHSC15.

22. The method of purifying a rAAV vector by AEX according to any one of claims 1 to 21, wherein the rAAV vector comprises AAV9 capsid protein and a transgene comprising a nucleic acid set forth in SEQ ID No. 1.

23. The method of purifying a rAAV vector by AEX according to any one of claims 1 to 21, wherein the rAAV vector comprises an AAV3B capsid protein and a transgene comprising a nucleic acid encoding the amino acid sequence shown in SEQ ID No. 15.

24. A method of preparing a solution comprising a rAAV vector purified by AEX, the method comprising the steps of:

i) Diluting the first solution 2 to 25-fold (e.g., 15-fold) with a dilution solution comprising histidine, tris and P188; optionally, a plurality of

ii) filtering the first solution from step i) through a filter to produce a diluted and optionally filtered solution;

wherein the pH of the diluted and optionally filtered solution is increased compared to the pH of the first solution; and wherein the conductivity of the diluted and optionally filtered solution is reduced compared to the conductivity of the first solution.

25. The method of claim 24, wherein the first solution comprising the rAAV vector is selected from the group consisting of an affinity eluate, a cell lysate supernatant, and a post-harvest solution.

26. The method of making a solution comprising rAAV vector purified by AEX according to claim 24 or 25, wherein the diluted solution comprises about 100mM to 300mM (e.g., about 200 mM) histidine, 100mM to 300mM (e.g., about 200 mM) Tris, 0.1% to 1.0% (e.g., about 0.5%) P188, and a pH of 8.5 to 9.5.

27. The method of preparing a solution comprising rAAV vector purified by AEX according to any one of claims 24 to 26, wherein i) the diluted and optionally filtered solution has a pH of 8.5 to 9.5; ii) the conductivity of the diluted and optionally filtered solution is 1.7 to 3.3mS/cm; and/or iii) the diluted solution has a% VG dilution yield of 35% to 100%.

28. The method of preparing a solution comprising a rAAV vector purified by AEX according to any one of claims 24 to 27, wherein the rAAV vector comprises an AAV9 capsid protein or an AAV3B capsid protein.

29. A purified rAAV vector prepared by a method comprising the steps of:

i) Loading a solution comprising the rAAV vector to be purified onto an AEX stationary phase in a column;

ii) subjecting the material from the stationary phase in the column to gradient elution, wherein a first gradient elution buffer, a second gradient elution buffer or a mixture of both is applied to the stationary phase and the concentration of salt is varied from 0mM to 500mM such that the rate of increase of salt concentration during gradient elution is about 10mM/CV to 50mM/CV (e.g. about 25 mM/CV);

iii) Beginning to collect at least one eluent fraction from the column when absorbance of column flow-through reaches an absorbance threshold during gradient elution; and/or

iv) measuring the absorbance of the at least one eluent fraction collected from the column and determining the a260/a280 ratio.

30. The purified rAAV vector prepared by the method of claim 29, wherein the method further comprises: when the a260/a280 ratio is ≡1.0, at least two eluent fractions collected from the column are combined to form a combined eluent comprising the purified rAAV vector.

31. The purified rAAV vector prepared by the method of claim 29 or 30, wherein the salt is sodium acetate.

32. The purified rAAV vector prepared by the method of any one of claims 29 to 31, wherein the rAAV vector comprises an AAV9 capsid protein or an AAV3B capsid protein.

33. The purified rAAV vector prepared by the method of any one of claims 29 to 32, wherein the solution comprising the rAAV vector is an affinity eluate that has been diluted and optionally filtered prior to loading onto the stationary phase.

34. The purified rAAV vector prepared by the method of any one of claims 29 to 33, wherein the material eluted from the stationary phase comprises the rAAV vector.

35. The purified rAAV vector prepared by the method of any one of claims 30 to 34, wherein the method further comprises filtering the combined eluate by a method selected from the group consisting of viral filtration, ultrafiltration/diafiltration (UF/DF), filtration through a 0.2 μm filter, and combinations thereof to produce a drug substance.

36. The purified rAAV vector prepared by the method of claim 35, wherein the drug substance is used to prepare a pharmaceutical product suitable for administration to a human subject to treat a disease, disorder, or condition.

37. The purified rAAV vector produced by the method of claim 36, wherein the disease, disorder, or condition is DMD or Wilson disease, and optionally wherein the rAAV vector comprises a nucleic acid encoding the amino acid sequence of SEQ ID No. 2 or SEQ ID No. 15.

38. A solution comprising rAAV vectors purified by AEX, the solution prepared by a method comprising the steps of:

i) Diluting the affinity eluate 2 to 25-fold (e.g., 15-fold) with a dilution solution comprising histidine, tris and P188; optionally, a plurality of

ii) filtering the affinity eluate from step i) through a 0.2 μm filter to produce a diluted and optionally filtered affinity eluate;

wherein the pH of the diluted and optionally filtered affinity eluate is increased compared to the affinity eluate; and wherein the conductivity of the diluted and optionally filtered affinity eluate is reduced compared to the affinity eluate.

39. The solution comprising rAAV vector purified by AEX prepared by the method of claim 38, wherein the diluted solution comprises about 100mM to 300mM (e.g., about 200 mM) histidine, 100mM to 300mM (about 200 mM) Tris, 0.1% to 1.0% (about 0.5%) P188, pH 8.5 to 9.5.

40. The solution comprising rAAV vector purified by AEX prepared by the method of claim 38 or 39, wherein the pH of the affinity eluate is 3.0 to 4.4 prior to the step of diluting and optionally filtering, and the pH of the affinity eluate is 8.5 to 9.5 or 8.7 to 9.0 (e.g., 8.8, 9.0) after the step of diluting and optionally filtering.

41. The solution comprising rAAV carrier purified by AEX prepared by the method of any one of claims 38 to 40, wherein the affinity eluate has a conductivity of 5.0 to 7.0mS/cm (e.g., 5.5 to 6.5 mS/cm) prior to the step of diluting and optionally filtering, and the affinity eluate has a conductivity of 1.7 to 3.3mS/cm, 1.8 to 2.8mS/cm, and/or 2.2 to 2.6mS/cm after the step of diluting and optionally filtering.

42. The solution comprising rAAV vector purified by AEX prepared by the method of any one of claims 38 to 41, wherein the diluted and optionally filtered affinity eluate has a%vg dilution yield of 35% to 100%.

43. The solution comprising a rAAV vector purified by AEX prepared by the method of any one of claims 38 to 42, wherein the rAAV vector comprises AAV9 or AAV3B capsid protein.

44. The solution comprising rAAV carrier purified by AEX prepared by the method of any one of claims 38-43, wherein the diluted and optionally filtered affinity eluate is loaded onto an AEX stationary phase.

45. A method of preparing a stationary phase for use in a method of purifying a rAAV vector by AEX according to any one of claims 1 to 23, the method comprising at least one of the following steps:

i) Pre-use rinse, including application of >4.5CV (e.g., about 5 CV) of water for injection to the AEX stationary phase in the column;

ii) sterilization comprising applying about 14.4 to 17.6CV (e.g., about 16 CV) of a solution comprising about 0.1M to 1.0M NaOH to the AEX stationary phase in the column, optionally flowing upward; and/or

iii) Regeneration, comprising applying about 4.5 to 5.5CV (e.g., about 5 CV) of a solution comprising about 1M to 3M NaCl, 50mM to 150mM Tris, pH 8.5 to 9.5 to the AEX stationary phase in the column.

46. A method of regenerating an AEX stationary phase, the method comprising the steps of:

i) Disinfecting the stationary phase after use, comprising applying a solution comprising about 0.1M to 1.0M NaOH, optionally flowing upward, of 14.4 to 17.6CV (e.g., about 16 CV) to the stationary phase;

ii) regenerating the stationary phase comprising applying a solution comprising about 1M to 3M NaCl, 50mM to 150mM Tris, pH 8.5 to 9.5 of 4.5 to 5.5CV (e.g., about 5 CV) to the stationary phase;

iii) Equilibrating the stationary phase, comprising applying 4.5 to 5.5CV (e.g., about 5 CV) of a solution comprising about 50mM to 150mM Tris, pH 8.5 to 9.5 to the stationary phase;

iv) flushing the stationary phase after use, comprising applying ≡4.5 (e.g. about 5 CV) of water for injection to the stationary phase; and/or

v) applying a storage solution to the stationary phase, including applying a solution comprising about 17% to 17.5% ethanol to the stationary phase of 2.7 to 3.3CV (e.g., about 3 CV).

47. The method of regenerating an AEX stationary phase of claim 46, wherein any of steps i) to v) is after a chromatographic elution step of the method of purifying the rAAV carrier by AEX.

48. A regenerated AEX stationary phase prepared by a process comprising the steps of:

i) Disinfecting the stationary phase after use, comprising applying a solution comprising about 0.1M to 1.0M NaOH, optionally flowing upward, of 14.4 to 17.6CV (e.g., about 16 CV) to the stationary phase;

ii) regenerating the stationary phase comprising applying a solution comprising about 1M to 3M NaCl, 50mM to 150mM Tris, pH 8.5 to 9.5 of 4.5 to 5.5CV (e.g., about 5 CV) to the stationary phase;

iii) Equilibrating the stationary phase, comprising applying to the stationary phase a solution comprising about 50mM to 150mM Tris, pH 8.5 to 9.5 at 4.5 to 5.5CV (e.g., about 5 CV);

iv) flushing the stationary phase after use, comprising applying ≡4.5 (e.g. about 5 CV) of water for injection to the stationary phase; and/or

v) applying a storage solution to the stationary phase, including applying a solution comprising about 17% to 17.5% ethanol to the stationary phase of 2.7 to 3.3CV (e.g., about 3 CV).

49. The regenerated AEX stationary phase of claim 48, wherein the regenerated AEX stationary phase is used to purify a rAAV carrier.

50. A method of purifying a rAAV vector by AEX, the method comprising the steps of:

i) Loading a solution comprising the rAAV vector to be purified onto an AEX stationary phase in a column;

ii) gradient eluting material from the stationary phase in the column, wherein the percentage of the first gradient elution buffer varies inversely with the percentage variation of the second gradient elution buffer; wherein at the beginning of the gradient elution the percentage of the first gradient elution buffer is about 75% to about 100% and at the end of the gradient elution the percentage of the second gradient elution buffer is about 60% to about 100%; and the percentage of the second elution buffer increases at a rate of about 2% -5%/CV during the gradient elution;

iii) When gradient elution is performed, when the percentage of the second gradient elution buffer is about 30% to about 35%, collecting at least one eluent fraction from the column is started,

And wherein the at least one eluent fraction comprises the rAAV vector to be purified.

51. The method of purifying a rAAV vector by AEX of claim 50, wherein the solution comprising the rAAV vector is an affinity eluate that has been diluted about 15-fold with a buffer comprising histidine, tris, and P188.

52. The method of purifying a rAAV vector by AEX of claim 50 or 51, wherein the first gradient elution buffer comprises 50mM to 150mM (e.g., about 100 mM) Tris,0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., 8.9), and/or the second gradient elution buffer comprises 400mM to 600mM (e.g., about 500 mM) sodium acetate, 50mM to 150mM (e.g., about 100 mM) Tris,0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., pH 8.9).

53. The method of purifying a rAAV vector by AEX of any one of claims 50 to 52, wherein collecting at least one eluate fraction from the column comprises collecting the at least one eluate fraction into a container comprising a solution of about 0.01CV to 0.1CV (e.g., about 0.066 CV) comprising 200mM to 300mM (e.g., about 250 mM) sodium citrate at a pH of 3.0 to 4.0 (e.g., about 3.5).

54. The method of purifying a rAAV vector by AEX according to any one of claims 51 to 53, wherein the at least one eluent fraction is enriched in complete capsids and/or depleted in empty capsids compared to the affinity eluent; optionally, wherein the rAAV vector is a rAAV3B vector; and optionally wherein the AEX stationary phase is POROS ^TM 50HQ。

55. The method of purifying a rAAV vector by AEX of any one of claims 50 to 54, wherein the collecting at least one eluate fraction from the column while performing gradient elution is ended when the percentage of the second gradient elution buffer is about 50% to about 55%.