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

Method for purifying AAV vectors by anion exchange chromatography Download PDF

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Publication number
CN116802308A
CN116802308A CN202180088674.XA CN202180088674A CN116802308A CN 116802308 A CN116802308 A CN 116802308A CN 202180088674 A CN202180088674 A CN 202180088674A CN 116802308 A CN116802308 A CN 116802308A
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raav vector
aex
solution
stationary phase
column
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CN202180088674.XA
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Chinese (zh)
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A·贝里尔
W·S·基什
J·R·莱特霍尔德
M·K·罗奇
W·B·韦尔伯恩
T·泽科维奇
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Pfizer Inc
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Pfizer Inc
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Priority claimed from PCT/IB2021/060095 external-priority patent/WO2022097008A1/en
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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 purifying AAV vectors by anion exchange chromatography
Cross Reference to Related Applications
The present application claims priority from U.S. provisional patent application No. 63/253,215 filed on 7 th 10 th 2021, U.S. provisional patent application No. 63/217,194 filed on 30 th 6 th 2021, and U.S. provisional patent application No. 63/109,049 filed on 3 th 11 2020, the entire contents of which are incorporated herein by reference.
Sequence listing
The present application comprises a sequence listing, which is electronically submitted in ASCII format and incorporated herein by reference in its entirety. The ASCII copy was created at 10 and 15 days 2021, named PC072555a_sequence_listing_st25.Txt, with a size of 1,048,576 bytes.
Technical Field
The present application relates to purification of AAV, particularly recombinant AAV (rAAV) vectors, by anion exchange chromatography.
Background
Gene therapy using recombinant AAV (rAAV) vectors to deliver therapeutic transgenes has the potential to treat a range of severe diseases that are incurable and in many cases have limited therapeutic approaches (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 the complete vector genome encoding the therapeutic transgene. In addition to developing purification methods that produce high purity and clinical grade rAAV vector compositions with 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 of removing host cell proteins and DNA and isolating viral capsids that are empty (i.e., do not contain vector genome), partially packaged (also referred to as "intermediate capsids", containing partial vector genome and/or non-transgene related DNA), or fully packaged (also referred to as "complete capsids", containing complete vector genome) (burn ham et al (2015) hum. Gene ter. 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 carrier production (Hermens et al hum. Gene ter. (1999) 10:1885-1891). Chromatographic methods, including affinity and/or ion exchange chromatography, have proven 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 the recombinant vector genome and package it in the viral capsid. In most mammalian expression systems, empty capsids are overproduced relative to full vector, and various systems produce 1-30% of full vector (Penaud-Budloo et al molecular Therapy, methods & Clinical Dev (2018) 8:166-180). The production of empty capsids can be attributed to the imbalance in the ratio of the plasmid encoding the transgene to the plasmid encoding the rep/cap gene. The presence of a hollow capsid in the drug product may cause an undesired immune response and/or compete with the recombinant vector for binding sites on the target cell.
Some use acetate buffers and resins such as POROS TM Anion exchange chromatography methods of 50HQ and Q-Sepharose XL have been used to separate empty capsids from rAAV2 vector libraries by relying on their slightly less anionic character than complete vectors (US 7,261,544;Qu et al (2007) j.virol.meth.140 (1): 183-192). A similar method uses a combination of affinity chromatography and ion exchange chromatography (IEX) and a 10mM to 300mM Tris acetate gradient at pH 8 with POROS TM 50HQ resin to enrich for all AAV vectors of various serotypes (less et al (2018) molecular.&Clin. Dev. 9:33-46). Other studiesBuffers and conditions have been identified that can be used to chromatographically separate empty capsids from whole AAV vectors. For example, urabe has determined that AAV1 material can be used with a material comprising MgCl 2 And glycerol in Tris-HCl buffer for dilution to load onto an anion exchange chromatography (AEX) column, and the solution containing counter-ions is an effective elution buffer for separating empty AAV1 capsids from the full carrier (uarabe et al (2006) molecular ter.13 (4): 823-828). Other people have described dilution of affinity chromatography eluate (e.g. 50-fold) and use of gentle gradient elution from monolithic support (e.g. 20mM to 180mM NaCl) in AEX methods 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 the rAAV vector and may lead to a decrease in therapeutic efficacy.
Processes have been developed that use a combination of methods, including for example, but not in particular order, tangential Flow Filtration (TFF) of host cell supernatants, precipitation of capsid materials (including rAAV vector and empty capsids) using ammonium sulfate, AEX chromatography, and size exclusion chromatography to separate rAAV from empty capsids (Tomono et al (2018) molecular.
There remains a need for methods of preparing clinical-grade rAAV vectors (e.g., rAAV 9) with optimal purity, potency, and consistency. These methods involve isolating rAAV comprising a vector genome with a therapeutic transgene from an empty AAV capsid on a scale necessary to meet the clinical needs of treating a disease, e.g., duchenne Muscular Dystrophy (DMD), friedreich Ataxia (FA).
Summary of The Invention
The present disclosure provides improved AEX methods of purifying rAAV vectors, including but not limited to isolating complete rAAV vectors (e.g., rAAV9 vectors) from empty capsids. Such purified complete rAAV vectors are suitable for the production of a pharmaceutical product for administration to a human subject (e.g., a subject with DMD). The present disclosure also provides a novel method of preparing a chromatographic eluate comprising a rAAV vector (e.g., from affinity chromatography) for further purification of the chromatographic eluate by AEX. The present disclosure also provides methods of regenerating an AEX stationary phase that allow the stationary phase to be used for multiple chromatographic runs while maintaining the integrity of the process (e.g., successful purification of rAAV vector, separation of complete vector 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 the following embodiment (E).
E1. 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 into a stationary phase in a column;
ii) gradient elution of material from the stationary phase in the column, wherein the percentage of the first gradient elution buffer varies in inverse proportion to the variation of the percentage of the second gradient elution buffer;
iii) At least one eluent fraction is collected from the column during gradient elution beginning when the absorbance of the column flow-through reaches an absorbance threshold.
E2. The method according to E1, wherein loading a solution comprising a rAAV vector onto the column comprises loading 2.5X10 15 To 3.0X10 17 Vector Genome (VG)/L column volume was applied to the column.
E3. The method of E1 or E2, wherein loading comprises loading 8.0X10 12 Up to 2.0X10 18 Total VG was applied to the column.
E4. A method according to any one of E1 to E3, wherein the loading includes loading the data to include 2.6X10 12 To 6.8X10 13 The diluted and optionally filtered solution of VG/mL column volume is applied to a column (e.g., a 6.4L column) as measured by qPCR analysis of the transgene sequences within the vector genome.
E5. The method according to any one of E1 to E4, wherein loading comprises loading the memory cells to comprise 5X 10 memory cells 13 To 1.3X10 14 The diluted and optionally filtered solution of VG/mL column volume is applied to a column (e.g., a 1.3L column) as measured by qPCR analysis of ITR sequences within the vector genome.
E6. The method of any one of E1 to E5, wherein the rAAV vector is produced in a container, and wherein the volume of the container is about 1L, about 50L, about 100L, about 250L, about 500L, about 1000L, about 2000L, or greater.
E7. The method according to any one of E1 to E6, wherein the vessel 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 the group consisting of an affinity eluate, a supernatant of a cell lysate, and a post-harvest solution that has been diluted and optionally filtered prior to 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. The method according to E1 to E9, wherein the solution has undergone at least one further purification or treatment step.
E11. The method according to E10, wherein the at least one other purification or treatment step is selected from the group consisting of cell lysis, flocculation, filtration, chromatography (e.g., 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 from the purification of the rAAV vector produced in a disposable bioreactor (SUB) by a volume of affinity chromatography.
E13. The method of E12 wherein the SUB has a volume of about 1L to about 2000L.
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 fixed phase is positively charged.
E16. The method according to any one of E1 to E15, wherein the stationary phase is polystyrene divinylbenzene particles with covalently bound quaternized polyethylenimine, and optionally wherein the stationary phase is POROS TM 50HQ。
E17. The method according to any one of E1 to E16, further comprising, optionally after loading a solution comprising rAAV carrier on the stationary phase, applying a chase solution to the AEX stationary phase in the column.
E18. The 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-BIS (hydroxyethyl) glycine (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 10mM to 30mM (e.g., about 20 mM) Tris, pH 8-10.
E20. The method according to any one of E1 to E19, further comprising washing the stationary phase in the column before use.
E21. The method according to E20, wherein washing the stationary phase in the column prior to use is performed prior to loading a solution comprising rAAV vector on the column.
E22. The method of E20 or E21, wherein flushing the stationary phase in the column prior to 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 a solution comprising rAAV vector to the column.
E25. The method of E23 or E24, wherein sterilizing the stationary phase in the column comprises applying a solution comprising NaOH to the stationary phase.
E26. The method of any one of E23 to E25, wherein sterilizing the stationary phase in the column comprises applying a solution comprising about 0.1M to about 1.0M (e.g., about 0.5M) NaOH to the stationary phase.
E27. The method of any one of E23 to E26, wherein disinfecting the stationary phase in the column comprises applying a solution comprising about 0.1M to about 1.0M mnoh of about 5CV to about 10CV, or about 14.4CV to about 17.6CV, 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 of E29, wherein regenerating the stationary phase in the column is performed prior to loading a solution comprising the rAAV vector on the column.
E31. The method of E29 or E30, wherein regenerating the stationary phase in the column comprises applying a solution comprising a component selected from the group consisting of salts, buffers, and combinations thereof to the stationary phase.
E32. The method according to any one of E29 to E31, wherein regenerating the stationary phase in the column comprises applying a solution comprising about 1M to about 3M (e.g., about 2M) NaCl, about 50mM to about 150mM (e.g., about 100 mM) Tris, 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 50mM to about 150mM (e.g., 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 5.5CV of a solution comprising about 100mM 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 of E36, wherein equilibrating the stationary phase in the column occurs before or after loading a solution comprising the rAAV vector onto the column.
E38. The method of 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 buffers, salts, amino acids, detergents, 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-di (hydroxyethyl) glycine and combinations thereof.
E40. The method according to E36 or E39, wherein the salt is selected from the group consisting of 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 the group consisting of poloxamer (poloxamer) 188 (P188), triton X-100, polysorbate 80 (PS 80), brij-35, nonylphenoxy polyethoxy ethanol (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 50mM to about 150mM (e.g., about 100mM Tris), pH about 8 to 10 (e.g., about 9) to the stationary phase.
E47. The method of any one of E36 to E46, wherein equilibrating the stationary phase in the column comprises applying an equilibration buffer comprising about 50mM to about 150mM (e.g., about 100mM Tris), about 250mM to about 750mM (e.g., about 500 mM) sodium acetate, about 0.005% to about 0.015% (e.g., 0.01%) P188, pH about 8 to about 10 (e.g., about 8.9) to the stationary phase.
E48. The method of any one of E36 to E47, wherein equilibrating the stationary phase in the column comprises applying an equilibration buffer comprising about 100mM to about 300mM (e.g., about 200 mM) histidine, about 100mM to about 300mM (e.g., about 200mM Tris), about 0.1% to about 1.0% (e.g., about 0.5%) P188, pH about 8 to about 10 (e.g., about 8.8) to the stationary phase.
E49. The method of any one of E36 to E48, wherein equilibrating the stationary phase in the column comprises applying an equilibration buffer comprising about 50mM to about 150mM (e.g., about 100 mM) Tris, 0.005% to about 0.015% (e.g., about 0.01%) P188, pH about 8 to about 10 (e.g., about 8.9) to the stationary phase.
E50. The method of any one of E36 to E49, wherein equilibrating the stationary phase in the column comprises applying greater than about 4.5CV 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 5.5CV of equilibration buffer to the stationary phase.
E52. The method according to any one of E36 to E51, wherein equilibrating the stationary phase in the column is performed more than once.
E53. The method according to any one of E36 to E52, wherein at least one equilibration buffer is applied to the stationary phase prior to loading a solution comprising rAAV vector on the column; and wherein at least one equilibration buffer is applied to the stationary phase after loading the solution comprising the rAAV vector on the column.
E54. The method according to any one of E1 to E53, comprising performing a gradient elution of material from the stationary phase in the column.
E55. The method according to E54, wherein the gradient elution comprises applying 10 to 60CV of at least two different solutions (e.g., gradient elution buffers) or a mixture of the two solutions to the stationary phase, and wherein during gradient elution the percentage of the first solution varies in an inversely proportional manner to the percentage of the second solution.
E56. The method of E54 or E55, wherein the at least two different solutions (e.g., first gradient elution buffer, second gradient elution buffer) comprise a component selected from the group consisting of buffers, salts, detergents, and combinations thereof.
E57. The method according to E56, wherein said buffer is selected from Tris, BIS-Tris propane, diethanolamine, diethylamine, tris (hydroxymethyl) methylglycine, triethanolamine and/or n, n-di (hydroxyethyl) glycine.
E58. The method according to E56 or E57, wherein the salt is selected from the group consisting of 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 (PS 80), brij-35, nonylphenoxy polyethoxy ethanol (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. The method according to any one of E55 to E60, wherein the first solution (e.g., buffer a) comprises about 50mM to about 150mM (e.g., about 100 mM) Tris, about 0.005% to about 0.015% (e.g., about 0.01%) P188, about pH 8.5 to 9.5 (e.g., about 8.9).
E62. The method according to any one of E55 to E61, wherein the second solution (e.g., buffer B) comprises about 400mM to about 600mM (e.g., about 500 mM) sodium acetate, about 50mM to about 150mM (e.g., about 100 mM) Tris, about 0.005% to about 0.015% (e.g., about 0.01%) P188, about pH 8.5 to 9.5 (e.g., about 8.9).
E63. The method of any one of E55 to E62, wherein performing the gradient elution comprises applying about 10 to 60CV, about 20 to 40CV, or about 20 to 24CV of the at least two different solutions to the stationary phase.
E64. The method according to any one of E55 to E63, wherein at the beginning of the gradient elution the percentage of the first solution (e.g. first gradient elution buffer, buffer a) is 50% to 100% and at the end of the gradient elution the percentage of the second solution (e.g. second gradient elution buffer, buffer B) is 50% to 100%, and wherein optionally 10 to 60CV of the first solution, the second solution or a mixture of both is applied to the stationary phase during the gradient elution.
E65. The method according to any one of E55 to E64, wherein at the beginning of the gradient elution the percentage of the first solution (e.g. first gradient elution buffer, buffer a) is 100% and at the end of the gradient elution the percentage of the second solution (e.g. second gradient elution buffer, buffer B) is 100%, and wherein optionally 10 to 60CV of the first solution, the second solution or a mixture of both is applied to the stationary phase during the gradient elution.
E66. The method according to any one of E55 to E65, wherein the concentration of the components of the first solution (e.g. the first gradient elution buffer) or the second solution (e.g. the second gradient elution buffer) continuously increases or decreases during said 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 concentration of the component varies from about 10mM/CV to 50mM/CV during the gradient elution.
E67. The method according to any one of E54 to E66, wherein the complete capsid is eluted from the stationary phase in a first portion of the first elution peak and/or the second elution peak of the gradient elution.
E68. The method according to any one of E54 to E67, wherein the empty capsid is 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 performing gradient maintenance.
E70. The method of E69, wherein the gradient maintaining comprises applying a gradient maintaining solution of 1 to 10CV to the stationary phase in the column, the gradient maintaining solution comprising a component selected from the group consisting of salts, buffers, detergents, and combinations thereof.
E71. The method according to E69 or E70, wherein performing gradient maintenance comprises applying a gradient maintenance solution of 1 to 10CV for the AEX stationary phase in the column, the gradient maintenance solution comprising about 5mM to about 1M (e.g., about 500 mM) sodium acetate, 50mM to 150mM (e.g., about 100 mM) Tris, about 0.005% to about 0.015% (e.g., about 0.01%) P188, pH about 8.5 to 9.5 (e.g., about 8.9).
E72. The method according to any one of E1 to E71, comprising step elution (e.g. isocratic elution) from the stationary phase in the column.
E73. The method of E72, wherein performing stepwise elution comprises applying at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more stepwise elution solutions to the column stationary phase.
E74. The method of 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 the group consisting of buffers, salts, detergents, and combinations thereof.
E75. The 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 comprise about 10mM to about 50mM (e.g., about 20 mM) Tris and about 5mM to about 600mM sodium acetate, at a pH of about 8 to 10 (e.g., about 8.9 to about 9.1).
E76. The method according to any one of E72 to E75, wherein a step-wise elution solution with an increased concentration of sodium acetate is sequentially applied to the column.
E77. The method according to any one of E72 to E76, wherein the final step elution solution comprises about 20mM Tris, about 500mM sodium acetate, and a pH of 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 a complete rAAV capsid, optionally during gradient elution.
E79. The method according to E78, wherein the volume of the at least one eluent 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 said at least one eluent fraction is measured at 280nm, and wherein the threshold value, optionally measured at 280nm, is ≡0.5mAU/mm path length.
E81. The method according to E80, wherein said at least one eluent fraction is collected when A280 of said eluent is ≡0.5mAU/mm path length.
E82. The method according to E80 or E81, 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 CV, and wherein optionally the A260/A280 ratio of the at least one eluent fraction is not less than 1.25.
E83. The 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 eluent fractions are collected.
E84. The method according to any one of E78 to E83, further comprising adjusting the pH of the at least one eluent fraction collected from the column, optionally during gradient elution.
E85. The method of E84, wherein adjusting the pH of the at least one eluent fraction comprises i) adding 14.3% to 15% eluent volume by weight of solution comprising buffer, pH about 3.5, to the at least one eluent fraction, or ii) collecting the at least one eluent fraction into a vessel comprising a solution comprising about 0.01CV to 0.1CV (e.g., about 0.066 CV) of buffer.
E86. The method according to E85, wherein the buffer is about 200mM to about 300mM (e.g., about 250 mM) sodium citrate.
E87. The method of E84 to E86, wherein the pH of the at least one eluent 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. The method of E84 to E87, wherein the pH of the at least one eluent 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 eluent fraction collected from the column, optionally during gradient elution.
E90. The method according to E89, wherein the absorbance is measured at 260nm (A260), 280nm (A280) or 260nm and 280nm, and optionally wherein the A260/A280 ratio is determined.
E91. The method according to E90, wherein the absorbance at 260nm and 280nm is measured by Size Exclusion Chromatography (SEC).
E92. The method according to E90 or E91, wherein the a260/a280 ratio of at least one eluent 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 1.279, 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 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 1.2, about 0.5 to about 0.5, about 0.8 to about 0.8, about 0.8 to about 0.6, about 1.8 to about 1.8, about 0.8, about 0.8.8, about 1.8.4, about 1.8, about 0.8.8, about 1.8, about 0.8.0.8, or about 1.0.8.0.8, about 1.8.0.0.0, about 1.8.0, 0, 0.8.0, optionally.
E93. The method according to any one of E90 to E92, wherein the a260/a280 ratio of at least one eluent fraction is at least 1.25.
E94. The method of any one of E78 to E93, further comprising combining at least two eluent fractions collected from the column, optionally during gradient elution, to form a combined eluent comprising the rAAV vector.
E95. The method according to E94, wherein 2 to 50 eluent fractions are combined to form a combined eluent.
E96. The method according to E94 or E95, wherein said at least two eluent fractions each have>A ratio a260/a280 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 (e.g. about 1.28 to 1.35).
E98. The method according to any one of E94 to E97, wherein the combined eluates have a pH of 6.8 to 7.6 (e.g. 7.0 to 7.4).
E99. The method of any one of E78 to E98, 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 at least one eluent fraction or combined eluents, and optionally wherein the capsid is measured by Analytical Ultracentrifugation (AUC).
E100. The method according to any one of E78 to E99, wherein the complete capsid in the at least one eluent fraction or combined eluents comprises about 55% (e.g. 55% +/-7%) of the total capsid.
E101. The method according to any one of E78 to E99, wherein the complete capsid in the at least one eluent fraction or combined eluents comprises about 49% (e.g. 49% +/-2%) of the total capsid.
E102. The method according to any one of E78 to E99, wherein the complete capsid in the at least one eluent fraction or combined eluents represents 52+/-7% of the total capsid.
E103. The method of any one of E78 to E99, wherein the at least one eluent fraction or pooled eluent comprises a complete rAAV capsid comprising 48% to 62% of the total capsid, and wherein the at least one eluent fraction or pooled eluent is produced by purification of the rAAV vector produced in 250L SUB.
E104. The method of any one of E78 to E99, wherein the at least one eluate fraction or pooled eluate comprises 47% to 51% of the total capsids of the complete rAAV capsids, and wherein the at least one eluate fraction or pooled eluate is produced from purifying a rAAV vector produced in 2000 LSUB.
E105. The method according to any one of E78 to E99, wherein the at least one eluate fraction or combined eluate comprises more than 30% (e.g., 40% to 55%,45% to 65%,40% to greater than 99%) of the complete capsids of the total capsids, and wherein the solution comprising the rAAV vector to be purified comprises less than 30% (e.g., 12% to 25%) of the complete capsids of the total capsids in the solution.
E106. The method of any one of E78 to E105, wherein the hollow capsid 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 in the at least one eluent fraction or combined eluent, and optionally wherein the capsid is measured by Analytical Ultracentrifugation (AUC).
E107. The method of any one of E78 to E106, wherein in the at least one eluent fraction or combined eluents, the empty capsids comprise 20% +/-7% (e.g., 21%) of the total capsids.
E108. The method according to any one of E78 to E106, wherein the at least one eluent fraction or combined eluent comprises 11% to 31% of empty capsids of the total capsids, and wherein the at least one eluent fraction or combined eluent is produced by purification of rAAV vector produced in 250L SUB.
E109. The method according to any one of E78 to E106, wherein the at least one eluent fraction or pooled eluent comprises from 18% to 22% of empty capsids of the total capsids, and wherein the at least one eluent fraction or pooled eluent is produced by purification of the rAAV vector produced in 2000L SUB.
E110. The method of any one of E78 to E106, wherein the at least one eluate fraction or combined eluate comprises less than 30% of empty capsids of the total capsids, and wherein the solution comprising the rAAV vector to be purified comprises 40% to 90% of empty capsids of the total capsids in the solution.
E111. The method of any one of E78 to E110, 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 at least one eluent fraction or combined eluents, and optionally wherein the capsid is measured by Analytical Ultracentrifugation (AUC).
E112. The method according to any one of E78 to E111, wherein the intermediate capsid comprises 28% +/-5% of the total capsid in the at least one eluent fraction or combined eluents.
E113. The method according to any one of E78 to E111, wherein the at least one eluent fraction or combined eluent comprises 21% to 27% of the total capsids of the intermediate capsids, and wherein the at least one eluent fraction or combined eluent is produced by purification of the rAAV vector produced in 250L SUB.
E114. The method according to any one of E78 to E111, wherein the at least one eluent fraction or combined eluent comprises 28% to 36% of the total capsids of the intermediate capsids, and wherein the at least one eluent fraction or combined eluent is produced by purification of a rAAV vector produced in 2000L SUB.
E115. The method of any one of E78 to E111, wherein the at least one eluent fraction or pooled eluent 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, and wherein the at least one eluent fraction or pooled eluent is produced by purifying rAAV vectors produced in 250L SUB.
E116. The method according to E115, wherein in the at least one eluent fraction or combined eluents, the complete capsid comprises 55% +/-7% of the total capsid.
E117. The method according to E115, wherein in the at least one eluent fraction or combined eluents, the intermediate capsid comprises 24% +/-3% of the total capsid.
E118. The method according to E115, wherein in the at least one eluent fraction or combined eluents, the empty capsids comprise 21% +/-10% of the total capsids.
E119. The method of any one of E78 to E118, wherein the at least one eluate fraction or combined eluate comprises 45% to 52% of the total capsids of a complete rAAV capsid, 27% to 37% of an intermediate capsid, and/or 18% to 22% of an empty capsid, and wherein the at least one eluate fraction or combined eluate is produced by purifying a rAAV vector produced in 2000 LSUB.
E120. The method according to E119, wherein in the at least one eluent fraction or combined eluents, the complete capsid comprises 49% +/-2% of the total capsid.
E121. The method according to E119, wherein in the at least one eluent fraction or combined eluents, the intermediate capsid comprises 32% +/-4% of the total capsid.
E122. The method according to E119, wherein in the at least one eluent fraction or combined eluents, the empty capsids comprise 20% +/-2% of the total capsids.
The method of any one of E94 to E122, wherein the combined eluates are enriched in complete capsids and/or depleted in empty capsids compared to the solution loaded on the column.
E124. The method according to any one of E78 to E123, wherein the at least one eluent fraction or combined eluent has a VG step yield percentage of 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 eluent fraction or combined eluent is 31% to 66% (e.g. 47% +/-11%).
E126. The method according to E124, wherein the VG step yield percentage of at least one eluent fraction or combined eluent produced in 250L SUB is 30% to 70% (e.g. 37% to 60%).
E127. The method according to E124, wherein the VG step yield percentage of the at least one eluent fraction or combined eluent produced in 250L SUB is 45% +/-8%.
E128. The method according to E124, wherein the VG step yield percentage of at least one eluent fraction or combined eluent produced in 2000L SUB is 25% to 75% (e.g. 31% to 66%).
E129. The method according to E124, wherein the VG step yield percentage of the at least one eluent fraction or combined eluent generated in 2000LSUB is 50% +/-13%.
E130. The method according to any one of E124 to E129, wherein the VG step yield percentage of the at least one eluent fraction or combined eluent is greater than the VG step yield percentage of an otherwise identical eluent fraction or combined eluent purified by ultracentrifugation and cation exchange chromatography.
E131. The method according to any one of E78 to E130, wherein the at least one eluent fraction or combined eluent produced in 250L SUB has an a260/a280 (SEC) of 1.29+/-0.03.
E132. The method according to any one of E78 to E130, wherein the at least one eluent fraction or combined eluent produced in 2000L SUB has an a260/a280 (SEC) of 1.30+/-0.01.
E133. The method according to any one of E78 to E132, wherein the at least one eluent fraction or combined eluent has a VG column yield percentage of 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 at least one eluent fraction or combined eluent has a VG column yield percentage of 20% to 100% (e.g. 63% +/-26%).
E135. The method according to E133, wherein the VG column yield percentage of the at least one eluent fraction or combined eluent produced in 250L SUB is 40% to 100%.
E136. The method according to E133, wherein the VG column yield percentage of the at least one eluent fraction or combined eluent produced in 2000L SUB is 10% to 70% (e.g. 20% to 61%).
E137. The method of any one of E78 to E136, wherein the at least one eluent fraction or combined eluents is further subjected to a filtration 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.
E138. The method of E137, wherein in the drug substance 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, and optionally wherein the capsid is measured by Analytical Ultracentrifugation (AUC).
E139. The method of E137 or E138, wherein the drug substance comprises 45% to 65% of the total capsids of a complete rAAV capsid, and optionally wherein the drug substance is produced by purifying a rAAV vector produced in 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 of E137 or E138, wherein the drug substance comprises 45% to 52% of the total capsids of a complete rAAV capsid, and wherein the drug substance is produced by purifying a rAAV vector produced in 2000L SUB.
E142. The method according to E137 or E138, wherein the drug substance comprises more than 30% (e.g., 40% to 55%, 45% to 65%, 40% to greater than 99%) of the total capsids in the drug substance, and wherein the solution comprising the rAAV vector to be purified comprises less than 30% (e.g., 12% to 25%) of the total capsids in the solution.
E143. The method of any one of E137 to E142, wherein the drug substance hollow capsid 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., +.ltoreq.29%) of the total capsid, and optionally wherein the capsid is measured by Analytical Ultracentrifugation (AUC).
E144. The method of E143, wherein the drug substance comprises 10% to 37% 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 hollow shell comprises 20% +/-7% of the total shell in the drug substance.
E146. The method of E143, wherein the drug substance comprises an empty capsid that is 18% to 22% of the total capsid, and optionally wherein the drug substance is produced by purifying a rAAV vector produced in 2000L SUB.
E147. The method of any one of E143 to E146, wherein the drug substance comprises less than 30% of the empty capsids of the total capsids in the drug substance, and wherein the solution comprising the rAAV vector to be purified comprises 40% to 90% of the empty capsids of the total capsids in the solution.
E148. The method of 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 capsids in the drug substance, and optionally wherein the capsids are measured by Analytical Ultracentrifugation (AUC).
E149. The method of any one of E137 to E148, wherein the drug substance comprises 19% to 37% of the intermediate capsid 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 of E148 or E149, wherein the intermediate capsid comprises 28% +/-5% of the total capsids in the drug substance.
E151. The method according to any one of E137 to E150, wherein the drug substance comprises 45% to 65% of the total capsids of a complete rAAV capsid, 19% to 28% of an intermediate capsid, and 10% to 37% of an empty capsid, wherein the drug substance is produced by purifying a rAAV vector produced in 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 hollow capsids comprise 21% +/-10% of the total capsids.
E155. The method according to any one of E137 to E150, wherein the drug substance comprises 45% to 52% of the total capsids of a complete rAAV capsid, 27% to 37% of an intermediate capsid, and/or 18% to 22% of an empty capsid, and wherein the drug substance is produced by purifying a rAAV vector produced in 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 capsids.
E158. The method according to any one of E155 to E157, wherein the hollow 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 Protein (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 minimum quantitative level (LLOQ) measured by ELISA.
E161. The method according to any one of E137 to E160, wherein the drug substance comprises an amount of HCP that is lower than LLOQ, as measured by ELISA, wherein the solution comprising the rAAV vector to be purified comprises about 1 to 500pg HCP/1 x 10 9 VG, and optionally wherein the solution is produced by affinity chromatography purification of the rAAV vector produced in 250L SUB.
E162. The method according to any one of E137 to E161, wherein the drug substance comprises an amount of HCP that is lower than LLOQ, as measured by ELISA, wherein the solution comprising the rAAV vector to be purified comprises about 100 to 1000pg HCP/1 x 10 9 VG, and optionally wherein the solution is produced by affinity chromatography purification of rAAV vector produced in 2000L SUB.
E163. The 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 as measured by reverse phase HPLC.
E164. The method according to E163, wherein the purity of the drug substance is about 98.6+/-0.6%, and optionally wherein the drug substance is produced by purifying a rAAV vector produced in 250L SUB.
E165. The method according to E164, wherein the purity of the drug substance is about 99.3+/-0.3%, and optionally wherein the drug substance is produced by purifying a rAAV vector produced in 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 the rAAV vector produced in 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 2000L SUB.
E169. The method of E137-E168, wherein the drug substance comprises about 7.0-25 pg/1 x 10 9 Residual HC-DNA of VG.
E170. The method according to E169, wherein the drug substance comprises about 17.4+/-6.7 pg/1X 10 9 HC-DNA of VG, 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/1X 10 9 HC-DNA of VG, 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 as 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 250L SUB.
E174. The method according to E172, wherein a260/a280 of the drug substance is from 1.28 to 1.31, optionally measured by SEC, and optionally wherein the drug substance is produced by purifying a rAAV vector produced in 2000L SUB.
E175. The method according to any one of E1 to E174, wherein the column volume is 1.0mL to 6.6L.
E176. The method according to any one of E1 to E175, wherein the column volume is about 1.0mL, about 5.1mL, about 6.67mL, about 1.256L, about 1.3L, about 6.3L, about 6.4L, or about 6.6L.
E177. The method according to any one of E1 to E176, wherein the rAAV vector comprises 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.
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 pharmaceutically acceptable excipient are combined to form a pharmaceutical product.
E181. The method according to any one of E178 to E180, wherein the purified rAAV vector, drug substance and/or pharmaceutical product is suitable for administration to a subject to treat a disease, disorder or condition.
E182. The method according to E181, wherein the 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 a human micro-dystrophin protein (mini-dynasty).
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 a human microdystrophin protein 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 the 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 the 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 the 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. The method according to E194, wherein the at least one ITR comprises 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 a VP1 polypeptide of AAV 9.
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 a rAAV vector purified by AEX.
E201. The method according to E200, wherein the solution comprising the rAAV vector is selected from the group consisting of an affinity eluate, a supernatant of a cell lysate, and a post-harvest solution, which has been diluted and optionally filtered prior to loading.
E202. The method according to E200 or E201, wherein the solution comprising 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 from affinity chromatography purification of the rAAV vector.
E205. The method according to any one of E200 to E204, wherein preparing comprises diluting a solution comprising the rAAV vector.
E206. The method of E205, wherein diluting the solution comprising the rAAV vector comprises diluting the solution about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, about 20-fold, about 25-fold to produce a diluted solution.
E207. The method of E205 or E206, wherein diluting the solution comprising the rAAV vector comprises diluting the solution about 15-fold to produce a diluted solution.
E208. The method according to any one of E205 to E207, wherein diluting the solution comprising the rAAV vector is performed in series (in-line) with the AEX column, and wherein the diluted solution is delivered to the Y-connector via a first tubing system and the solution comprising the rAAV vector is delivered to the Y-connector via a second tubing system.
E209. The method of E208, wherein the diluted solution is delivered at a flow rate of 1 to 5mL/min, and wherein the solution comprising the rAAV vector is delivered at a flow rate of 0.1 to 2mL/min, whereby the solution is diluted about 15-fold.
E210. The method of E208 or E209, wherein the diluted solution comprises at least one component selected from the group consisting of buffers, amino acids, detergents, and combinations thereof.
E211. The method according to E210, wherein the buffer is selected from the group consisting of Tris, BIS-Tris propane, diethanolamine, diethylamine, tris (hydroxymethyl) methylglycine, triethanolamine, n-di (hydroxyethyl) glycine 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. The method according to any one of E210 to E213, wherein the detergent is selected from poloxamer 188 (P188), triton X-100, polysorbate 80 (PS 80), brij-35, nonylphenoxy polyethoxy ethanol (NP-40), and combinations thereof.
E215. The method according to any one of E210 to E214, wherein the detergent is P188.
E216. The method according to any one of E208 to E215, wherein the diluted solution comprises about 100mM to about 300mM (e.g., about 200 mM) histidine, about 100mM to about 300mM (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 on the column.
E218. The method according to any one of E205 to E217, wherein the pH of the solution comprising the rAAV vector after dilution is increased compared to the pH of the solution prior to dilution.
E219. The method according to any one of E205 to E218, wherein the pH of the solution comprising the rAAV vector prior to dilution is from 3.0 to 4.4 and the pH of the solution after dilution is from 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 prior to dilution.
E221. The method according to any one of E205 to E220, wherein the conductivity of the solution comprising the rAAV vector prior to dilution is from 5.0 to 7.0mS/cm (e.g., from 5.5 to 6.5 mS/cm) and the conductivity of the solution after dilution is from 1.7 to 3.5mS/cm.
E222. The method according to any one of E205 to E221, further comprising filtering the diluted solution comprising the rAAV vector.
E223. The method of E222, wherein filtering the diluted solution comprises filtering through a 0.2 μm filter.
E224. The method of E222 or E223, wherein the filter is 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 on the column.
E226. The method according to any one of E205 to E225, wherein the vector genome percent (% VG) yield of the diluted and optionally filtered solution comprising rAAV vector is 60% to 100% compared to the amount of VG present in the solution prior to dilution and optionally filtration.
E227. The method according to any one of E205 to E226, wherein the diluted solution comprising rAAV vector has a%vg dilution yield of 88% +/-36%.
E228. 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 diluting solution; 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.
A method of preparing a solution comprising rAAV vector purified by AEX, e229.E228, 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 solution after dilution has a% VG dilution yield of 35% to 100%.
A method of preparing a solution comprising a rAAV vector purified by AEX, wherein the rAAV vector comprises AAV9 capsid protein, E228 or E229.
E231. A method of purifying a rAAV vector by AEX, the method comprising the steps of:
i) Loading a solution comprising a rAAV vector to be purified into an AEX stationary phase in a column;
ii) gradient elution of 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;
iii) When the absorbance of the column flow-through reaches the absorbance threshold, beginning to collect at least one eluent fraction from the column during the gradient elution;
iv) measuring the absorbance of the at least one eluent fraction collected from the column and determining the a260/a280 ratio.
E232. The method of 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 a rAAV vector by AEX according to E231 or E232, wherein the AEX stationary phase is POROS TM 50HQ。
E234. The method of 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 prior to loading on the stationary phase.
E235. The method of 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. The method of purifying a rAAV vector by AEX according to any one of E231 to E235, wherein the first gradient elution buffer comprises about 50mM to about 150mM (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. The method of purifying a rAAV vector by AEX according to any one of E231 to E236, wherein the second gradient elution buffer comprises about 400mM to about 600mM (e.g., about 500 mM) sodium acetate, about 50mM to about 150mM (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. The method of purifying a rAAV vector by AEX according to any one of E231 to E237, wherein the percentage of the first gradient elution buffer is 100% at the beginning of the gradient elution and the percentage of the second gradient elution buffer is 100% at the end of the gradient elution.
E239. The method of purifying a rAAV vector by AEX according to any one of E236 to E238, wherein about 15 to about 25CV (e.g., 20 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; wherein the concentration of sodium acetate varies between 0mM and 500mM during the gradient elution, whereby the rate of change of the concentration of sodium acetate during the gradient elution is about 25mM/CV.
E240. The method of purifying a rAAV vector by AEX according to any one of E231 to E239, wherein the complete capsid is eluted from the stationary phase in a first elution peak; wherein the complete capsid is eluted from the stationary phase in a first part of the second elution peak and/or wherein the complete capsid is recovered in the AEX column flow-through and/or in a last part of the second elution peak.
E241. The method for purifying a rAAV vector by AEX according to any one of E231 to E240, wherein the absorbance threshold measured at 280nm is ≡0.5mAU/mm path length.
E242. The method of purifying a rAAV vector by AEX according to any one of E231 to E241, wherein the volume of the at least one eluent fraction is equal to ≡1/3CV.
E243. The method of purifying a rAAV vector by AEX according to any one of E231 to E242, wherein collecting at least one eluent fraction comprises collecting at least 10 eluent fractions.
E244. The method of 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 (e.g., about pH 7.2).
E245. The method of purifying a rAAV vector by AEX according to any one of E231 to E244, wherein the at least one eluent fraction, the at least two eluent fractions, and/or the combined eluent has an a260/a280 ratio of ≡1.25 (e.g., about 1.28 to 1.35).
E246. The method of purifying a rAAV vector by AEX according to any one of E231 to E245, wherein the combined eluates have a%vg column yield of 20% to 100% (e.g., 63 +/-26%).
E247. The method of purifying a rAAV vector by AEX according to any one of E231 to E246, wherein the combined eluates have a%vg step yield of 31% to 66% (e.g., 47 +/-11%).
E248. The method of purifying a rAAV vector by AEX according to any one of E231 to E247, wherein at least one eluent fraction and/or combined eluent is enriched in complete capsids and/or depleted in empty capsids compared to diluted and filtered affinity eluent loaded on a column.
E249. The method of purifying a rAAV vector by AEX according to any one of E231 to E248, wherein a purified rAAV vector is produced.
E250. The method of purifying a rAAV vector by AEX according to E231 to E249, further comprising 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.
E251. The method of purifying a rAAV vector by AEX according to E250, wherein the drug substance comprises 45% to 65% (e.g., 52 +/-7%) of the complete capsid of the total capsid.
E252. The method of 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 capsid of the total capsids.
E253. The method of purifying a rAAV vector by AEX according to E250-E252, wherein the drug substance comprises 10% to 37% (e.g., 20 +/-7%) of the empty capsids of the total capsids.
E254. The method of purifying a rAAV vector by AEX according to any one of E231 to E253, wherein the rAAV vector comprises AAV9 capsid proteins.
E255. A method of purifying a rAAV vector by AEX, the method comprising the steps of:
i) Loading an AEX stationary phase (e.g., POROS) comprising an affinity eluate of the rAAV vector to be purified into a column TM 50 HQ), wherein the eluent has:
a) About 14.4 to 15.5 fold (e.g., about 15 fold) diluted with a buffer comprising about 200mM histidine, about 200mM Tris, about 0.5% P188, pH 8.7 to 9.0, and optionally
b) Filtration through a 0.2 μm filter before loading on the stationary phase;
ii) subjecting the material of the stationary phase in the column to gradient elution, wherein about 20CV of a first gradient elution buffer (e.g. 100mM Tris,0.01% P188, pH 8.9), a second gradient elution buffer (e.g. 500mM sodium acetate, 100mM 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 between 0mM and 500mM, whereby the rate of change of the concentration of sodium acetate during gradient elution is about 25mM/CV;
iii) Collecting about 10 eluent fractions from the column during gradient elution when a280>0.5mAU/mm path length of the eluent, wherein the volume of said at least one eluent fraction is equal to ≡1/3CV;
iv) adjusting the pH of about 10 eluent fractions from the column to 6.8 to 7.6 by adding 14.3% to 15% (eluent volume weight) of a solution containing about 250mM sodium citrate (pH 3.5);
v) measuring the absorbance of the about 10 eluent fractions collected from the column and determining the a260/a280 ratio; and/or
vi) combining at least two eluent fractions collected from the column to form a combined eluent,
wherein A260/A280 of each of the at least two eluent fractions is greater than or equal to 1.25;
wherein the combined eluate is enriched in complete capsids and/or depleted in empty capsids compared to diluted and optionally filtered affinity eluate loaded on the column; a kind of electronic device with high-pressure air-conditioning system
Wherein a purified rAAV vector is produced.
E256. The method of 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 of purifying a rAAV vector by AEX, the method comprising the steps of:
i) A pre-use rinse comprising applying about 5CV of water for injection to the AEX stationary phase in the column;
ii) sterilization comprising applying about 16CV of a solution containing about 0.5M NaOH to the AEX stationary phase in the column, optionally flowing upward;
iii) Regeneration comprising applying about 5CV of a solution comprising about 2M NaCl, 100mM Tris, pH 9 to the AEX stationary phase in the column;
iv) equilibration, comprising applying about 5CV of a solution comprising about 100mM Tris, pH 9 to the AEX stationary phase in the column;
v) equilibration, comprising applying about 5CV of equilibration buffer (comprising about 100mM Tris, 500mM sodium acetate, 0.01% p188, ph 8.9) to the AEX stationary phase in the column;
vi) equilibration, comprising applying about 5CV of equilibration buffer (comprising about 200mM histidine, 200mM Tris, 0.5% p188, ph 8.8) to the AEX stationary phase in the column;
vii) loading an affinity eluate comprising the rAAV vector to be purified to the AEX stationary phase of the column, optionally wherein the eluate has:
a) Diluted about 15-fold with buffer containing about 200mM histidine, 200mM Tris, 0.5% P188, pH 8.7 to 9.0, and optionally
b) Filtration through a 0.2 μm filter before loading on the stationary phase;
viii) equilibration, comprising applying about 5CV of equilibration buffer (comprising about 100mM Tris, 0.01% P188, pH 8.9) to the AEX stationary phase in the column;
ix) gradient elution of material from a stationary phase in the column, wherein about 20CV of a first gradient elution buffer (e.g. 100mM Tris, 0.01% P188, pH 8.9), a second gradient elution buffer (e.g. 500mM sodium acetate, 100mM 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 0mM to 500mM, whereby the rate of change of the concentration of sodium acetate during gradient elution is about 25mM/CV;
x) collecting about 10 eluent fractions from the column during gradient elution when A280+.0.5 mAU/mm path length of the eluent; and wherein the volume of the about 10 eluent fractions is about ≡1/3CV;
xi) adjusting the pH of the about 10 eluent fractions to 6.8 to 7.6 by adding 14.3% to 15% (eluent volume weight) of a solution comprising about 250mM sodium citrate (pH 3.5);
xii) measuring the absorbance of the about 10 eluent fractions collected from the column and determining the a260/a280 ratio; and/or
xiii) combining at least two eluent fractions collected from the column to form a combined eluent,
wherein A260/A280 of each of the at least two eluent fractions is greater than or equal to 1.25;
Wherein the combined eluate is depleted of empty capsids and/or enriched in complete capsids compared to the affinity eluate and/or diluted and optionally filtered affinity eluate;
wherein at least one of steps i) to ix) is performed at a linear velocity of 270 to 330cm/hr (e.g. about 300 cm/hr), a flow rate of 1.5 to 2.0L/min (e.g. about 1.8L/min) or about 314mL/min or a residence time of 1.3L column and/or about 3.5 to 4.5min/CV (e.g. 4 min/CV); and/or wherein a purified rAAV vector is produced.
E258. The method of 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 of preparing a solution comprising a rAAV vector purified by AEX, the method comprising the steps of:
i) Diluting the affinity eluate about 15-fold with a dilution solution containing about 200mM histidine, 200mM Tris, 0.5% P188, pH 8.8; and
ii) filtering the affinity eluate of 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) as compared to the pH of the affinity eluate; and wherein the conductivity of the diluted and filtered affinity eluate is reduced (e.g., to about 1.7mS/cm to 3.3 mS/cm) as compared to the conductivity of the affinity eluate.
E260. A method of preparing a solution comprising rAAV carrier purified by AEX according to E259, wherein the diluted and optionally filtered affinity eluate is loaded to an AEX stationary phase.
E261. A method of preparing a solution comprising a rAAV vector purified by AEX according to E259 or E260, wherein the affinity eluate is produced by purifying a rAAV vector produced in a vessel having a volume of 250L or 2000L based on affinity chromatography.
E262. The method of preparing a solution comprising a rAAV vector purified by AEX according to any one of E259 to E261, wherein the diluted affinity eluate has a% VG dilution yield of 88% +/-36%.
E263. A method of preparing a stationary phase for use in a method of purifying a rAAV vector by AEX, the method comprising the steps of:
i) A pre-use rinse comprising applying ≡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 containing about 0.1M to 1.0M (e.g., about 0.5M) 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 5 CV) 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 300 cm/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 4 min/CV).
E264. The method of preparing a stationary phase for use in a method of purifying a rAAV vector by AEX according to E263, further comprising equilibrating comprising applying to the AEX stationary phase in the column about 5CV of one or more 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 of preparing a stationary phase for use in a method of purifying a rAAV vector by AEX according to E263 or E264, wherein at least one step is performed before loading a 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 steps of:
i) Loading a solution comprising a rAAV vector to be purified into an AEX stationary phase in a column;
ii) subjecting the material of the stationary phase in the column to gradient elution, wherein about 20CV of a first gradient elution buffer (e.g. 100mM Tris, 0.01% P188, pH 8.9), a second gradient elution buffer (e.g. 500mM sodium acetate, 100mM Tris, 0.01% P188, pH 8.9) or a mixture of both is applied to the stationary phase; wherein the concentration of salt varies between 0mM and 500mM, whereby the rate of change of the concentration of salt during gradient elution is about 25mM/CV;
iii) Collecting at least one (e.g., about 10) eluent fractions from a column during a chromatography step (e.g., gradient elution) when the absorbance of the column flow-through reaches an absorbance threshold (e.g., a280>0.5mAU/mm path length);
iv) measuring the absorbance of the at least one eluent fraction collected from the column and determining the a260/a280 ratio; and/or
v) combining at least two eluent fractions collected from the column to form a combined eluent 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. The purified rAAV vector prepared according to the method of any one of E267 to E269, wherein the solution is an affinity eluate that has been a) diluted about 14.4 to 15.5-fold (e.g., about 15-fold) with a buffer comprising about 200mM histidine, 200mM Tris, 0.5% P188, pH 8.7 to 9.0, and optionally b) filtered through a 0.2 μm filter prior to loading on the stationary phase.
E271. The purified rAAV vector prepared according to the method of any one of E267 to E270, further comprising adjusting the pH of the at least one (e.g., about 10) eluate fraction from the column to 6.8 to 7.6 by adding 14.3% to 15% (by volume of eluate) of a solution containing about 250mM sodium citrate (pH 3.5).
E272. The purified rAAV vector according to any one of E267 to E271, wherein a260/a280 of each of the at least two eluent fractions is ≡1.25; and wherein the combined eluate is enriched in complete capsids and/or depleted in empty capsids compared to diluted and optionally filtered affinity eluate loaded on the column; and wherein a purified rAAV vector is produced.
E273. The purified rAAV vector according to any one of E267 to E272, wherein the rAAV vector comprises AAV9 capsid proteins.
E274. A purified rAAV vector prepared by a method comprising the steps of:
i) Loading a solution comprising a rAAV vector to be purified into an AEX stationary phase in a column;
ii) gradient elution of 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;
iii) Beginning to collect at least one eluent fraction from the column when the absorbance of the column flow-through reaches an absorbance threshold during the chromatographic step;
iv) measuring the absorbance of the at least one eluent fraction collected from the column and determining the a260/a280 ratio; and/or
v) combining at least two eluent fractions collected from the column to form a combined eluent 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 comprises 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 proteins.
E277. The purified rAAV vector prepared according to the method of any one of E274 to E276, 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.
E278. A purified rAAV vector prepared according to the method of E277, 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.
E279. A purified rAAV vector prepared according to the method of E278, wherein the disease, disorder or condition is DMD.
E280. A solution comprising rAAV vectors purified by AEX, the solution prepared by a method comprising the steps of:
i) Diluting the first solution (e.g., affinity eluate) 2 to 25-fold (e.g., about 15-fold) with a buffer comprising 200mM histidine, 200mM Tris, 0.5% P188, pH 8.8; 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. The solution comprising 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 (e.g., a disposable bioreactor) having a volume of 250L or 2000L.
E283. The solution comprising 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. The solution comprising rAAV vector purified by AEX according to any one of E280 to E283, wherein the diluted and optionally filtered solution has a conductivity of 1.7 to 3.3mS/cm.
E285. The solution comprising rAAV vector purified by AEX according to any one of E280 to E284, wherein the diluted and optionally filtered solution has a%vg dilution yield of 88% +/-36%.
E286. A solution comprising rAAV vectors purified by AEX, the solution prepared by a method comprising the steps of:
i) Diluting the first solution (e.g., affinity eluate) 2 to 25-fold (e.g., 15-fold) with a dilution solution comprising histidine, tris, and P188; optionally
ii) filtering the diluted 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.
E287. A solution comprising a rAAV vector purified by AEX prepared according to the method of E280, 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.
E288. A solution comprising a 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 prior to 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 a rAAV vector purified by AEX prepared according to the method of any one of E280 to E282, wherein the conductivity of the first solution is 5.0 to 7.0mS/cm (e.g., 5.5 to 6.5 mS/cm) prior to the diluting and optional filtering steps, and the conductivity of the first solution is 1.7 to 3.5mS/cm, 1.8 to 2.8mS/cm, or 2.2 to 2.6mS/cm after the diluting and optional filtering steps.
E290. A solution comprising rAAV vector purified by AEX prepared according to the method of any one of E280 to E283, wherein the diluted and optionally filtered first solution has a% VG dilution yield of 35% to 100%.
E291. A solution comprising a 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 proteins, and optionally wherein the diluted and optionally filtered solution is loaded on an AEX stationary phase.
E292. A method of regenerating an AEX stationary phase, the method comprising the steps of:
i) Post-use sterilization of the stationary phase comprising applying to the stationary phase a solution comprising about 0.1M to 1.0M (e.g., about 0.5M) NaOH, optionally flowing upward, at 14.4 to 17.6CV (e.g., about 16 CV);
ii) regenerating the stationary phase comprising applying to the stationary phase a solution comprising about 1 to 3M (e.g. about 2M) NaCl, 50 to 150mM (e.g. about 100 mM) Tris, pH 8.5 to 9.5 (e.g. about pH 9) at 4.5 to 5.5CV (e.g. about 5 CV);
iii) A stationary phase is equilibrated comprising applying 4.5 to 5.5CV (e.g., about 5 CV) of a solution comprising about 50mM to 150mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., about pH 9) to the stationary phase;
iv) post-use washing of the stationary phase, comprising applying ≡4.5CV (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 a 2.7 to 3.3CV (e.g., about 3 CV) 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 330cm/hr (e.g., about 300 cm/hr), a flow rate through a 6.0 to 6.6L (e.g., 6.4L) column of 1.5 to 2.0L/min (e.g., about 1.8L/min), or a flow rate through a 1.3L column of about 314mL/min and/or a residence time of 3.5 to 4.5min/CV (e.g., about 4 min/CV).
E293. The method of regenerating an AEX stationary phase according to E292, wherein any one of steps i) to v) follows the chromatographic elution step of the method of purifying a rAAV carrier by AEX.
E294. A regenerated AEX stationary phase prepared by a process comprising the steps of:
i) Post-use sterilization of the stationary phase, comprising applying to the stationary phase a solution containing about 0.1M to 1.0M (e.g., about 0.5M) NaOH at 14.4 to 17.6CV (e.g., about 16 CV), optionally by upward flow;
ii) regenerating the stationary phase, comprising applying to the stationary phase a solution of 4.5 to 5.5CV (e.g. about 5 CV) comprising about 1M to 3M (e.g. about 2M) NaCl, about 50mM to 150mM (e.g. about 100 mM) Tris, pH8.5 to 9.5 (e.g. about pH 9);
iii) A stationary phase comprising applying to the stationary phase a solution of 4.5 to 5.5CV (e.g., about 5 CV) comprising about 50mM to 150mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., pH 9);
iv) post-use washing of the stationary phase, including application of ≡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 containing about 17% to 17.5% ethanol to the stationary phase at 2.7 to 3.3CV (e.g., about 3 CV); optionally
Wherein at least one of steps i) to v) is performed at a linear velocity of 270 to 330cm/hr (e.g. about 300 cm/hr), at a flow rate of 1.5 to 2.0L/min (e.g. about 1.8L/min) through a 6.0 to 6.6L (e.g. 6.4L) column, or about 314mL/min through a 1.3L column and/or a residence time of 3.5 to 4.5min/CV (e.g. about 4 min/CV).
E295. The regenerated AEX stationary phase according to E288, wherein the regenerated AEX stationary phase is used to purify a rAAV carrier.
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 a purified rAAV vector according to E267 to E279.
E298. Use of a rAAV vector purified according to the method of any one of E1 to E227 or E231 to E258 in the manufacture of a medicament for the treatment and/or prevention of a disease, disorder or condition.
E299. The use according to E298, wherein the disease, disorder or condition is DMD.
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 transport ATPase2 (ATP 7B) protein.
E301. The method according to E300, wherein the modified nucleic acid comprises a nucleic acid sequence encoding a deleted copper transport ATPase2 (ATP 7B) protein comprising or consisting 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 a deletion of heavy metal related sites HMA 1, HMA 2, HMA 3 and HMA 4.
E303. The method according to any one of E300 to E302, wherein the vector genome further comprises an alpha 1-antitrypsin promoter, a polyadenylation (polyA) signal sequence, a 5'itr and a 3' itr.
E304. The method according to E303, wherein said alpha 1-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 a VP1 polypeptide of AAV 3B.
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 of purifying a rAAV vector by AEX, the method comprising the steps of:
i) Loading a solution comprising a rAAV vector to be purified into an AEX stationary phase in a column;
ii) gradient elution of 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 the percentage of the first gradient elution buffer is from about 75% to about 100% at the beginning of the gradient elution and the percentage of the second gradient elution buffer is from about 60% to about 100% at the end of the gradient elution; and wherein in gradient elution the percentage of the second elution buffer increases at a rate of about 2% to 5% per CV;
iii) When the gradient elution is initiated when the percentage of the second gradient elution buffer is about 30% to about 35%, at least one eluent fraction is collected from the column,
And wherein the at least one eluate fraction comprises the rAAV vector to be purified.
E310. The method of purifying a rAAV vector by AEX according to E309, 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.
E311. The method of purifying a rAAV vector by AEX according to E309 or E310, 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).
E312. The method of purifying a 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 comprising a solution comprising about 0.01CV to 0.1CV (e.g., about 0.066 CV) comprising 200mM to 300mM (e.g., about 250 mM) sodium citrate, pH 3.0 to 4.0 (e.g., about 3.5).
E313. According to any one of E310 to E312A method of purifying a rAAV vector by AEX, wherein the at least one eluent fraction is enriched in complete capsids and/or depleted in empty capsids as compared to an affinity eluent; optionally, wherein the rAAV vector is a rAAV3B vector; and optionally wherein the AEX stationary phase is POROS TM 50HQ。
E314. The method of purifying a rAAV vector by AEX according to any one of E309 to E313, wherein collecting at least one eluent fraction from the column at the time of gradient elution is ended when the percentage of second gradient elution buffer is about 50% to about 55%.
E315. A method of purifying a rAAV vector by AEX, the method comprising the steps of:
i) Loading a solution comprising a rAAV vector to be purified into an AEX stationary phase in a column;
ii) gradient elution of 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; a kind of electronic device with high-pressure air-conditioning system
iii) When the absorbance of the column flow-through reaches an absorbance threshold, collecting at least one eluent fraction from the column during gradient elution is started, and wherein the at least one eluent fraction comprises the rAAV vector to be purified.
E316. The method of purifying a rAAV vector by AEX according to E315, wherein the method further comprises measuring absorbance of the at least one eluent fraction collected from the column and determining the a260/a280 ratio.
E317. The method of purifying a rAAV vector by AEX according to E315 or E316, wherein the solution comprising the rAAV vector to be purified is diluted about 2-fold to 25-fold (e.g., 15-fold) with a diluted solution comprising histidine, tris and P188, and optionally filtered before application to the stationary phase.
E318. The method of purifying a rAAV vector by AEX according to any one of E315 to E317, wherein the solution is an affinity eluate.
E319. The method of purifying a rAAV vector 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 reduced compared to the conductivity of the solution.
E320. The method of purifying a rAAV vector by AEX according to any one of E315 to E319, 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 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 8.5 to 9.5; and wherein 10 to 60 Column Volumes (CVs) (e.g., about 20CV, about 37.5 CV) of a first gradient elution buffer, a second gradient elution buffer, or a mixture of both are applied to the stationary phase during gradient elution.
E321. The method of purifying a rAAV vector by AEX according to any one of E315 to E320, wherein the percentage of the first gradient elution buffer is 50% to 100% at the beginning of the gradient elution and the percentage of the second gradient elution buffer is 50% to 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% to 5% per CV.
E322. The method of purifying a rAAV vector 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 both, is continuously increased 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.
E323. The method of purifying a rAAV vector by AEX according to any one of E315 to E322, wherein during gradient elution, the complete capsid is eluted from the stationary phase in a first portion of a first elution peak and/or a second elution peak.
E324. The method of purifying a rAAV vector by AEX according to any one of E315 to E323, wherein the empty capsid is recovered in the column flow-through during gradient elution, in the first elution peak and/or in the last part of the second elution peak.
E325. The method of purifying a rAAV vector by AEX according to any one of E315 to E324, 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.
E326. The method of purifying a rAAV vector by AEX according to any one of E315 to E325, wherein the volume of the at least one eluent fraction is equal to 1/8CV to 10CV, e.g., 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.
E327. The method of 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. The method of 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 of ≡0.98 or ≡1.0, to form a combined eluate comprising the rAAV vector.
E329. The method of purifying a rAAV vector 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 of ≡1.0.
E330. The method of purifying a rAAV vector by AEX according to E328 or E329, wherein the combined eluate is enriched in complete capsids and/or depleted in empty capsids compared to the solution loaded on the column.
E331. The method of purifying a rAAV vector by AEX according to any one of E315 to E330, wherein a purified rAAV vector is produced.
E332. The method of purifying a rAAV vector by AEX according to any one of E328 to E331, further comprising 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.
E333. A method of purifying a rAAV vector by AEX according to E332, wherein the drug substance comprises: i) 45% to 65% (e.g., 52 +/-7%) of the total capsids are complete 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.
E334. The method of purifying a rAAV vector 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. The method of 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, 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.
E336. The method of 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 a nucleic acid of SEQ ID No. 1.
E337. The method of 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 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
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 reduced compared to the conductivity of the first solution.
E339. The method of preparing a solution comprising a rAAV vector purified by AEX according to E338, wherein the first solution comprising the rAAV vector is selected from the group consisting of an affinity eluate, a supernatant of a cell lysate, and a post-harvest solution.
E340. The method of preparing a solution comprising a rAAV vector purified by AEX according to E338 or E339, 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.
E341. The method of preparing a solution comprising rAAV vector purified by AEX according to any one of E338 to E340, 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%.
E342. The method of preparing a solution comprising a 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 steps of:
i) Loading a solution comprising a rAAV vector to be purified into an AEX stationary phase in a 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 salt is varied from 0mM to 500mM, whereby the rate of increase of the concentration of salt during gradient elution is about 10mM/CV to 50mM/CV (e.g. about 25 mM/CV);
iii) When the absorbance of the column flow-through reaches an absorbance threshold, beginning to collect at least one eluent fraction from the column during gradient elution; and/or
vi) measuring the absorbance of the at least one eluent fraction collected from the 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 to form a combined eluate comprising the purified rAAV vector when the a260/a280 ratio is ≡1.0.
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 an AAV9 capsid protein or an AAV3B capsid protein.
E347. The 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 that has been diluted and optionally filtered prior to loading on 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 a rAAV vector.
E349. The 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 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.
E350. A purified rAAV vector prepared according to the method of E349, 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.
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 Drawings
FIG. 1 depicts an exemplary POROS at 1mL TM The 50HQ column was run on an AEX chromatogram of the elution phase generated using four eluting salts. A is that 260 The trace is shown in dashed lines, A 280 And the conductivity trace is shown in solid lines. Solid bars represent the eluted fractions used to form the pool.
FIG. 2 depicts an example POROS at 1mL TM SEC a of AEX eluted fraction on 50HQ column using four eluting salts 260 /A 280 。
FIG. 3 depicts an exemplary POROS at 5.1mL TM AEX chromatograms generated using 9 steps of washing and elution with sodium acetate performed on a 50HQ column. A is that 260 The trace is shown in dashed lines, A 280 And the conductivity trace is shown in solid lines. Wash (W), elution (E), strip and regeneration (regen.) fractions are shown in agreement with tables 5 and 6.
FIG. 4A depicts an exemplary AEX chromatogram produced using a step elution with sodium acetate, the elution run being at 600cm/hr, 5.1X10 13 Vector genome/mL resin challenge (VG/mL resin, measured by qPCR of ITR sequence) and POROS at 5.1mL TM A57 mM sodium acetate wash was performed on a 50HQ column. A is that 260 The trace is shown in dashed lines, A 280 And the conductivity trace is shown in solid lines. Fig. 4B depicts an enlarged view of the chromatograms of the wash, elution and strip phases of the AEX run.
Fig. 5 depicts an exemplary AAV9 affinity eluate in-line mixing with 100mM Tris, pH 9 to produce AEX loading (also referred to herein as diluted affinity eluate). Fluid is delivered to a Y-connector with peristaltic pump.
FIG. 6 depicts pH, conductivity, Z-average and aggregation (given in +or-of) of an exemplary AAV9 affinity eluate diluted with 100mM Tris (pH 9).
FIGS. 7A and 7B depict exemplary% Vector Genome (VG) yields of 5, 9, or 25-fold dilutions of affinity eluents with 200mM histidine, 200mM Tris, X% (w/v) P188, pH 8.8, where X is 0.01%, 0.05%, 0.2%, and 0.5%, followed by filtration. Fig. 7A depicts a contour plot of the percent yield of VG (after dilution and filtration) as a function of conductivity (controlled by dilution factor) and P188 concentration. Figure 7B depicts one-way ANOVA analysis of percent yield of VG (after dilution and filtration) as a function of P188 concentration or conductivity. The data are also presented in Table 13.
FIG. 8A depicts an example chromatogram produced using an optimized AEX method. Fig. 8B depicts an enlarged view of AEX sodium acetate gradient elution, fraction numbers 1-14, consistent with table 15. A is that 260 The traces are shown in dashed lines, A 280 And the conductivity trace is shown in solid lines.
FIG. 9 depicts SEC A of chromatographic fractions produced on 0%, 20%, 40%, 60%, 80% and 100% ineffective affinity cells (Null affinity pool) using an exemplary optimized AEX method 260 /A 280 Values. Flow through is abbreviated as F/T.
FIG. 10 depicts an exemplary 10cm Inside Diameter (ID) by 16cm Bed Height (BH), 1.3L POROS TM Elution phase of the 250L SUB AEX chromatogram of batch 250L-4 run on a 50HQ column. A is that 260 The trace is shown in dashed lines, A 280 And the conductivity trace is shown in solid lines.
FIG. 11 depicts an exemplary 20cm ID by 20.5cm BH, 6.4L POROS TM The elution phase of the 2000L Scale AEX chromatogram of batch 2000L-4 run on a 50HQ column. A is that 260 The trace is shown in dashed lines, A 280 And the conductivity trace is shown in solid lines.
Fig. 12 depicts a chromatogram of an exemplary use-optimized AEX method for purifying an AAV3B vector. A is that 260 The traces are shown in solid lines, A 280 The traces are indicated by dashed lines.
Description of the invention
1. Definition of the definition
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the specification of the present invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. The following terms have the given meanings:
As used herein, the term "about" or "approximately" refers 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 usage index, the number of CpG dinucleotides, the dose, the time, the temperature, etc., and is meant to encompass a change of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or even 0.1% in any direction (greater or less) of the specified amount, unless otherwise indicated, as apparent 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 combinations when interpreted with the alternative ("or").
As used herein, the term "adeno-associated virus" and/or "AAV" refers to parvoviruses having a linear single stranded DNA genome and variants thereof. The term encompasses all subtypes as well as 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 packaging signals for the virus in cis form. The genome comprises two large open reading frames, called AAV replication ("AAV rep" or "rep") and capsid ("AAV-cap" or "cap") genes, respectively. AAV rep and cap may also be referred to herein as AAV "packaging genes". 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 are alternatively spliced resulting in the production of VP1, VPN and VP3 capsid proteins (Grieger and Samulski (2005) J.Virol.79 (15): 9933-9944). The single P40 promoter allows expression of all three capsid proteins at a ratio of VP1, VP2, VP3 of 1:1:10, respectively, which complements AAV capsid production. More specifically, VP1 is a full-length protein, and VP2 and VP3 are shorter and shorter due to increased N-terminal truncation. A well-known example is the capsid of AAV9 as described in U.S. Pat. No. 7,906,111, wherein 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, termed Assembly Activator Protein (AAP), which is critical to the capsid assembly process (Sonntag et al (2011) J.Virol.85 (23): 12686-12697).
At least four viral proteins are synthesized from AAV rep genes: rep 78, rep68, rep 52, and Rep 40 are named according to their apparent molecular weights. As used herein, "AAV Rep" or "Rep" refers to AAV replication proteins Rep 78, rep68, 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 polypeptides encoded thereby. In some embodiments, the nucleic acid encoding rep comprises nucleotides from more than one AAV serotype. For example, nucleic acids encoding rep proteins may comprise nucleotides from AAV2 serotypes and nucleic acids from AAV3 serotypes (Rabinowitz et al (2002) J.virology 76 (2): 791-801).
As used herein, the terms "recombinant adeno-associated viral vector," "rAAV" and/or "rAAV vector" refer to an AAV capsid comprising the vector genome. The vector genome comprises polynucleotide sequences that are at least partially not derived from a naturally occurring AAV (e.g., heterologous polynucleotides that are not present in a wild-type AAV), and 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 an AAV are removed (and/or ITRs of the AAV are added or retained), the nucleic acid within the AAV is referred to as the "vector genome". Thus, the term rAAV vector encompasses rAAV viral particles comprising a capsid but not the complete AAV genome; in contrast, recombinant viral particles may comprise heterologous nucleic acids, i.e. nucleic acids that are not initially present in the capsid, hereinafter referred to as vector genome. Thus, an "rAAV vector genome" (or "vector genome") refers to a heterologous polynucleotide sequence (including at least one ITR) that may, but need not, be contained within an AAV capsid. The rAAV vector genome may 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), a gene editing nucleic acid, and the like, which typically encodes a therapeutic transgene.
As used herein, the terms "rAAV vector," "rAAV viral particle," and/or "rAAV vector particle" refer to an AAV capsid that consists of at least one AAV capsid protein (but typically all capsid proteins of an AAV are present, such as VP1, VP2, and VP3, or variants thereof) and that contains a vector genome comprising a heterologous nucleic acid sequence. These terms are to be distinguished from non-recombinant "AAV viral particles" or "AAV viruses" in which the capsid contains the viral genome encoding the rep and cap genes and is capable of replication when present in a cell that also contains helper virus (e.g., adenovirus and/or herpes simplex virus) and/or helper genes required therefrom. Thus, production of a rAAV vector particle necessarily includes production of a recombinant vector genome using recombinant DNA technology, e.g., the vector genome is contained within a capsid to form a rAAV vector, rAAV viral particle, or rAAV vector particle.
The genomic sequences of the various serotypes of AAV, and the sequences of the Inverted Terminal Repeats (ITRs), rep proteins, and capsid subunits are known in the art. These sequences can be found in literature or public databases such as GenBank. See, e.g., genBank Accession Numbers NC _002077 (AAV 1), AF063497 (AAV 1), nc_001401 (AAV 2), AF043303 (AAV 2), nc_001729 (AAV 3), AF028705.1 (AAV 3B), nc_001829 (AAV 4), U89790 (AAV 4), nc_006152 (AAV 5), AF028704 (AAV 6), AF513851 (AAV 7), AF513852 (AAV 8), nc_006261 (AAV 8), AY530579 (AAV 9), AY631965 (AAV 10), AY631966 (AAV 11), and DQ813647 (AAV 12); the disclosure of which is incorporated herein by reference. See, e.g., 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 2013/063279, 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 the other. For example, if the presence, level, and/or form of a particular entity (e.g., polypeptide, genetic feature, metabolite, microorganism, etc.) is associated with the incidence and/or susceptibility of a disease, disorder, or condition (e.g., in a related population), the entity is considered to be associated with the particular disease, disorder, or condition. In some embodiments, two or more entities are physically "related" to each other if they interact directly or indirectly with each other in physical proximity and/or remain in physical proximity. In some embodiments, two or more entities that are physically related to each other are covalently linked to each other; in some embodiments, two or more entities that are physically related to each other are not covalently linked to each other, but are not covalently linked, such as by hydrogen bonding, van der waals interactions, hydrophobic interactions, magnetic properties, and combinations thereof.
As used herein, the term "coding sequence" or "coding nucleic acid" refers to a nucleic acid sequence encoding a protein or polypeptide, and refers to 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 generally determined by a start codon at the 5 '(amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. Coding sequences may include, but are 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 US6,491,907 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 to a;265 insert T;705N mutation to A;708V is mutated to A; and 716T is mutated to N. The nucleotide sequence encoding such a 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 (pulcherla et al (2011) Molecular Therapy (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 2015/01393, aavdj/8, aavdj/9, described in WO 2007/120542.
As used herein, the term "eluent" refers to a fluid (e.g., "eluting from a stationary phase") that is discharged from a chromatographic stationary phase (e.g., monolith, membrane, resin, medium) that consists of a mobile phase and a material that passes through or is removed from the stationary phase. In some embodiments, the stationary phase comprises, for example, a monolith, a membrane, a resin, or a medium. The mobile phase may be a solution that has been loaded onto the column and has flowed through the column (i.e., a "flow-through fraction"); equilibration solutions (e.g., equilibration buffers); isocratic elution of the solution; gradient elution of the solution; a solution for regenerating the stationary phase; a solution for disinfecting the 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 and means that there is 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 contiguous. For example, an insertion sequence may be present between the nucleic acid encoding the transgene and the flanking elements. Sequences (e.g., transgenes) that are "flanked" by two other elements (e.g., ITRs) represent that one element is located 5 'of the sequence and the other element is located 3' of the sequence, however, intervening sequences may be present therebetween.
As used herein, the term "flocculation" refers to the process of bringing together fine particles to form a floe. The fine particles may include proteins, nucleic acids, cell fragments produced by the lysis of host cells. In some embodiments, the flocs formed in the liquid phase may float to the top of the liquid (emulsion), settle to the bottom of the liquid (sediment), 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 the whole but lacks one or more portions found in the whole. In some embodiments, the fragments are composed of discrete portions. In some embodiments, the fragment comprises or consists of a characteristic structural element or part found in whole. In some embodiments, the 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 monomer units (e.g., amino acid residues, nucleotides) found throughout the polymer.
When the capsid contains the complete or substantially complete vector genome (including the transgene), the rAAV vector is referred to as a "complete", "complete capsid", "complete vector" or "fully packaged vector". During the production of rAAV vectors by host cells, vectors may be produced that have less packaged nucleic acid than a complete capsid and contain, for example, a partial or truncated vector genome. These vectors are referred to as "intermediates," intermediate capsids, "" partially "or" partially packaged vectors. The intermediate capsid may also be a capsid having an intermediate sedimentation rate (i.e. the sedimentation rate between a complete capsid and an empty capsid) when analyzed by analytical ultracentrifugation. The host cell may also produce a viral capsid that does not contain any detectable nucleic acid material. These capsids are referred to as "empty" or "empty capsids". The complete capsid can be distinguished from the empty capsid according to the SEC-HPLC determined a260/a280 ratio, wherein the a260/a280 ratio has been pre-calibrated for capsids separated by analytical ultracentrifugation (i.e. complete capsid, intermediate capsid and empty capsid). Other methods known in the art for capsid identification include CryoTEM, capillary isoelectric focusing, and charge detection mass spectrometry. The calculated isoelectric points of the empty and full 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 "null capsid" refers to an intentionally created capsid lacking the vector genome. Such null capsids may be produced by transfecting host cells with rep/cap and helper plasmids, rather than with plasmids comprising transgene cassette sequences (also known as vector plasmids).
As used herein, the term "functional" is a biological molecule that exhibits the identified property and/or activity. The biomolecules may have two functions (i.e., dual functions) or multiple functions (i.e., multi-functions).
As used herein, the term "gene" refers to a polynucleotide containing at least one open reading frame that, when transcribed and translated, is capable of encoding a particular polypeptide or protein. "Gene transfer" or "gene delivery" refers to a method or system for reliably inserting exogenous DNA into a host cell. Such methods can result in transient expression of non-integrated transferred DNA, expression of extrachromosomal replicas 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 the application of a mixture of at least two different solutions having different pH, conductivity and/or modifier concentrations to a chromatographic stationary phase (including, for example, monolithic columns, media, resins, membranes), which mixture gradually changes during elution. Gradient elution may 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 step-wise fashion. During the gradient elution, the percentage of the first solution varies continuously in inverse proportion 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., first gradient elution buffer) in the mixture is 100% and the percentage of gradient elution buffer B (e.g., second gradient elution buffer) in the mixture is 0%, thereby creating a continuously varying gradient (increasing or decreasing depending on the embodiment) of pH, conductivity, and/or modifier concentration as the solutions mix and flow through the stationary phase. In some embodiments, the concentration of the salt (e.g., sodium acetate) varies at a constant rate over the volume of the linear gradient. For example, for a 1mL column with a 20mL linear gradient (i.e., 20 CV), operating at a constant flow rate of 1 mL/min, the salt concentration will vary at a rate of 5% per minute. In some embodiments, the rAAV capsid (e.g., complete capsid, intermediate capsid, empty capsid) is bound to the stationary phase during loading of the solution comprising the rAAV capsid to be purified to the AEX stationary phase. During gradient elution, as the percentage of buffer B increases, such that the concentration of salt (e.g., sodium acetate) increases, the complete rAAV carrier is preferentially released (eluted) from the stationary phase and the empty capsid preferentially remains on the stationary phase. As the percentage of buffer B further increases, the empty capsids are released in larger amounts. During gradient elution, elution of the complete rAAV vector from the stationary phase can be monitored by measuring 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, whereas 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 eluent fraction is measured using a method such as analytical Size Exclusion Chromatography (SEC), online UV tracing, offline UV methods, and the like in a High Performance Liquid Chromatography (HPLC) system, and wherein absorbance is measured at one or more wavelengths (e.g., 260nm and/or 280 nm).
As used herein, the term "heterologous" refers to a nucleic acid inserted into a vector (e.g., a rAAV vector) that 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, the heterologous nucleic acid is non-native relative to viral (e.g., AAV) nucleic acids. Once transferred or delivered into the cell, the heterologous nucleic acid contained within the vector can be expressed (e.g., transcribed and translated, if appropriate). Alternatively, the heterologous nucleic acid contained within the vector for transfer or delivery in the cell need not be expressed. Although the term "heterologous" is not always used herein to refer to a nucleic acid, reference to a nucleic acid is intended to include a heterologous nucleic acid even in the absence of the modifier "heterologous". For example, the heterologous nucleic acid is a nucleic acid encoding a dystrophin polypeptide or fragment thereof, such as a codon optimized mini-dystrophin transgene described in WO 2017/221145, incorporated herein by reference, for use in treating Duchenne muscular dystrophy.
Another example heterologous nucleic acid comprises a wild-type coding sequence or fragment thereof (e.g., truncated, internal deletion) of one of the following genes, and may or may not be codon optimized:
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As used herein, the term "homology" or "homology" refers to the sharing of at least some identity between two or more of the mentioned entities (e.g., nucleotide or polypeptide sequences) 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. Notably, the homologous peptide will retain activity or function associated with the unmodified or reference peptide, and the modified peptide will typically 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 fragment thereof, "substantially homologous" or "substantially similar" means that there is at least about 95% to 99% sequence identity when properly inserted or deleted with another polypeptide, nucleic acid (or its complementary strand), or fragment thereof for optimal alignment. The degree of homology (identity) between two sequences may be determined using a computer program or a mathematical algorithm. Such algorithms for calculating percent sequence homology (or identity) typically consider sequence gaps and mismatches over the comparison region or regions. 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 a cell into which exogenous nucleic acid has been introduced, and include the progeny of such a cell. Host cells include "transfectants," "transformants," "transformed cells," and "transduced cells," including primary transfected, transformed or transduced cells and progeny derived thereof, regardless of the number of passages. In some embodiments, the host cell is a packaging cell for producing a rAAV vector.
As used herein, the term "host cell DNA" or "HCDNA" refers to residual DNA derived from a host cell culture that produces a rAAV vector, present in a chromatographic fraction (e.g., affinity eluate, AEX eluate, wash) or chromatographic loading (e.g., affinity loading, AEX loading). Host cell DNA can be measured by methods known in the art, such as qPCR, to detect sequences specific for host cells. Fluorescent dyes (e.g.Or->Green), absorbance measurements (e.g., at 260nm or 254 nm), or electrophoresis techniques (e.g., agarose gel electrophoresis or capillary electrophoresis) to estimate general DNA concentrations. The amount of HCDNA present in the eluate can be expressed relative to the amount of vg present in the eluate, e.g., ng HCDNA/1X 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 volume of eluate, e.g., pg HCDNA/mL eluate. />
As used herein, surgeryThe term "host cell protein" or "HCP" refers to residual protein derived from a host cell culture that produces a rAAV vector, present in a chromatographic fraction (e.g., affinity eluate, AEX eluate, wash) or chromatographic loading (e.g., affinity loading, AEX loading). Host cell proteins can be measured by methods known in the art, such as ELISA. Host cell proteins can be prepared by various electrophoretic staining methods (e.g., silver-stained SDS-PAGE, Ruby-stained SDS-PAGE and/or Western blot). The amount of HCP present in the eluate can be expressed relative to the amount of vg present, e.g., ng HCP/1X 10 14 vg or pg HCP/1X 10 9 vg。
As used herein, the term "identity" or "identical" refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymeric molecules are considered "substantially identical" to each other 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 percent identity of two nucleic acid or polypeptide sequences can be calculated by aligning the two sequences for optimal alignment purposes (e.g., gaps can be introduced in one or both of the first and second sequences to achieve optimal alignment, and non-identical sequences can be ignored for comparison purposes). In certain embodiments, the length of the sequences aligned 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 a residue (e.g., a nucleotide or amino acid) that is identical to the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between 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 that need to be introduced in order to achieve optimal alignment of the two sequences. Comparison of sequences and determination of percent identity between two sequences can be accomplished using mathematical algorithms.
To determine identity or homology, sequences can be aligned using methods and computer programs, including BLAST, which is available on the world Wide Web ncbi.nlm.nih.gov/BLAST. Another comparison algorithm is FASTA, available from Genetics Computing Group (GCG) software package, from Madison, wis. 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 gaps in the sequences to exist. Smith-Waterman is an algorithm that allows gaps in sequence alignment. See Meth.mol.biol.70:173-187 (1997). Furthermore, the GAP program using Needleman and Wunsch alignment methods 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. The 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. The 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 input sequences to be compared. Preferably, the sequence identity is determined using default parameters determined by the program. This program is also available from Genetics Computing Group (GCG) software package, from Madison, wis., 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; connection Penalty (join 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 a solution comprising the rAAV vector to be purified. Impurities include empty capsids, intermediate capsids, biological macromolecules such as DNA, RNA, non-AAV proteins (such as host cell proteins), AAV aggregates, damaged AAV capsids, molecules that are part of the absorber for chromatography (which may penetrate into the sample during previous purification steps), endotoxins, cell debris, and chemicals from cell culture, including media components, plasmid DNA from transfection, adventitious factors, bacteria, and viruses.
As used herein, the terms "inverted terminal repeat," "ITR," "terminal repeat," and "TR" refer to palindromic terminal repeats at or near the end of an AAV viral genome, including predominantly complementary, symmetrically arranged sequences. These ITRs can fold to form T-shaped hairpin structures, which act as primers at the beginning of DNA replication. They are also required for integration of the viral genome into the host genome, rescue from the host genome; and for encapsidation of viral nucleic acids into mature viral particles. ITR is required in cis for replication of the vector genome and its packaging in viral particles. "5' ITR" refers to the ITR at the 5' end of the AAV genome and/or at the 5' end of the recombinant transgene. "3' ITR" refers to ITRs at the 3' end of the AAV genome and/or at 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 portion of a wild-type AAV ITR sequence. One of ordinary skill in the art will appreciate that during successive rounds of DNA replication, ITR sequences can be interchanged such that a 5'ITR becomes a 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 an "rAAV vector particle" or "rAAV viral particle") comprising the vector genome.
ITR is required in cis for replication of the vector genome and its packaging in viral particles. "5' ITR" refers to the ITR at the 5' end of the AAV genome and/or at the 5' end of the recombinant transgene. "3' ITR" refers to ITRs at the 3' end of the AAV genome and/or at 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 portion of a wild-type AAV ITR sequence. One of ordinary skill in the art will appreciate that the ITR sequences may be interchanged during successive rounds of DNA replication such that a 5'ITR becomes a 3' ITR and vice versa.
As used herein, the term "isolated" refers to a substance or composition of matter that: 1) Is designed, produced, prepared and/or produced manually, and/or 2) is separated from at least one component associated therewith at the time of initial production (whether in nature or in an experimental setting). Typically, the isolated composition is substantially free of one or more materials, such as one or more proteins, nucleic acids, lipids, carbohydrates, and/or cell membranes, to which it is normally associated in nature. 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 formulations (e.g., such as, but not limited to, rAAV vector particles comprising AAV9 capsids). The term "isolated" also does not exclude additional physical forms of the composition, such as hybridosomes/chimeras, multimers/oligomers, modifications (e.g., phosphorylations, glycosylation, lipidations), variants or derived forms, or artificial forms expressed in host cells.
The isolated substance or composition may be 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 greater than about 99% separated from the other components with which it was originally associated. In some embodiments, the isolated formulation 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 greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, 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.), as understood by those skilled in the art; in such embodiments, the percent separation or purity of the material is calculated without the inclusion of such carriers or excipients.
As used herein, the term "loading the chase solution" refers to a solution that is applied to the column after the loading or loading solution (definition see below) has been applied. Loading a 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 "loaded" or "loaded solution" refers to any material (e.g., solution) loaded on a chromatographic stationary phase that contains a product of interest (e.g., a complete rAAV carrier). In some embodiments, the "loading solution" is exposed to a chromatographic stationary phase. In some embodiments, the loading solution is an affinity eluate. In some embodiments, the loading solution is a diluted and optionally filtered affinity eluate.
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 (e.g., an impurity). In some embodiments, the chromatographic stationary phase is a resin, medium, membrane adsorbent, or monolith. In some embodiments, the chromatographic stationary phase is a medium that binds to an AAV capsid under certain conditions. In some embodiments, the chromatographic stationary phase is an ion exchange medium (e.g., anion exchange medium, 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 alter the chromatography. Such changes in chromatography result in, for example, removal or washing of impurities from the stationary phase, or elution of the product or material of interest (e.g., rAAV carrier) 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), chaotropes (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 linkages. The nucleic acid may be an oligonucleotide or a polynucleotide. The nucleic acid sequence is presented herein in the 5 'to 3' direction. The nucleic acid sequences (i.e., polynucleotides) of the present disclosure may 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 RNAs or short hairpin RNAs (shRNA), micrornas, small interfering RNAs or short interfering RNAs (siRNA), trans-spliced RNAs, antisense RNAs, messenger RNAs, transfer RNAs, ribosomal RNAs. Where the polynucleotide is a DNA molecule, the molecule may be a gene, cDNA, antisense molecule or a fragment of any of the foregoing. Nucleotides are represented herein by single letter codes: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I) and uracil (U). The nucleotide sequence may be chemically modified or artificial. Nucleotide sequences include Peptide Nucleic Acids (PNAs), morpholino nucleic acids and Locked Nucleic Acids (LNAs), as well as ethylene Glycol Nucleic Acids (GNAs) and Threonine Nucleic Acids (TNAs). Each of these sequences is distinguished from naturally occurring DNA or RNA by changes in the molecular backbone. In addition, phosphorothioate nucleotides may be used. Other deoxynucleotide analogs include methylphosphonate, phosphoramidate, dithiophosphate, N3'-P5' -phosphoramidate and oligoribonucleotide phosphorothioate and 2 '-0-allyl analogs thereof and 2' -0-methylribonucleotide methylphosphonate, 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., a recombinant nucleic acid) produced using recombinant DNA technology. Nucleic acid constructs are single-or double-stranded nucleic acid molecules which have been modified to contain segments of nucleic acid sequences which are combined and arranged in a manner which is not found in nature. The nucleic acid construct may be a "vector" (e.g., a plasmid, rAAV vector genome, expression vector, etc.), i.e., the nucleic acid molecule is designed to deliver exogenously generated DNA into a host cell.
As used herein, the term "operably linked" refers to a linkage of nucleic acid sequence (or polypeptide) elements in a functional relationship. A nucleic acid is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or other transcriptional regulatory sequence (e.g., an enhancer) is operably linked to a coding sequence if it affects the transcription of the coding sequence. In some embodiments, operably linked means that the nucleic acid sequences being linked are contiguous. In some embodiments, operably linked does not mean that the nucleic acid sequences are linked consecutively, but rather that intervening sequences are present between those nucleic acid sequences that are linked.
As used herein, the term "percent Vector Genome (VG) dilution yield" or "% VG dilution yield" refers to the percentage of the amount of VG present in a diluted affinity cell (also referred to herein as diluted affinity eluate) to the amount of VG present in the affinity cell (also referred to herein as affinity eluate) prior to dilution. For example,% VG dilution yield= ((amount of VG in diluted affinity cell)/(amount of VG in affinity cell)) = ((amount of VG in diluted affinity cell)) =.
As used herein, the term "percent VG column yield" or "% VG column yield" refers to the percentage of the amount of Vector Genome (VG) present in the combined eluate collected from the AEX column (i.e., AEX pool) to the amount of VG in the diluted or diluted and filtered affinity eluate alone.
In some embodiments, the affinity eluate comprising the rAAV vector to be purified is diluted only and is referred to as a "diluted affinity pool". Optionally, the rAAV vector to be purified is harvested from a 250L or 2000L vessel (e.g., a disposable bioreactor (SUB)). For example,% VG column yield= ((amount of VG in AEX cell)/(amount of VG in diluted affinity cell)) = (-100.
In some embodiments, the affinity eluate comprising 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 250L) vessel (e.g., bioreactor). For example,% VG column yield= ((amount of VG in AEX cell)/(amount of VG in diluted and filtered affinity cell)) × 100.
As used herein, the term "percent VG step yield" or "% VG step yield" refers to the percentage of the amount of VG in the combined eluate collected from the AEX column (i.e., AEX pool) to the amount of VG present in the affinity pool (also referred to herein as affinity eluate) prior to dilution or filtration. For example,% VG step yield= ((amount of VG in AEX pool)/(amount of VG in affinity pool)) =.
As used herein, the terms "pharmaceutically acceptable" and "physiologically acceptable" refer to a biologically acceptable formulation, gas, liquid or solid, or mixtures thereof, suitable for one or more routes of administration, in vivo delivery or contact.
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 a full-length native sequence, a naturally occurring protein, and a functional subsequence, modified form, or sequence variant, so long as the subsequence, modified form, or variant retains some 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 may be, but are not required to be, identical to endogenous proteins that are deficient, or under-expressed, or 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 cloning, restricting, or ligating various combinations of steps (e.g., involving polynucleotides or polypeptides contained therein), and/or other methods of obtaining a construct that is 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 elution" refers to the application of a solution having a defined pH, conductivity, and/or modifier concentration to a chromatographic stationary phase (including, for example, monolithic columns, media, resins, membranes). A series of stepwise elutions (e.g. increasing conductivity or salt concentration) may be performed to optimise separation. The elution solution has a defined composition for each step, which does not change during its application. In a step elution process, as a series of solutions (e.g., loaded chase solution, pH stabilizing solution, wash buffer, elution buffer) is applied to the stationary phase, the pH, conductivity, and/or modifier concentration increases or decreases relative to the previous series of solutions. For example, at the beginning of the step elution series, the concentration of the modifier (e.g., salt such as sodium acetate) in the first solution is relatively low, e.g., 0 to 10mM, e.g., about 1mM, about 2mM, about 3mM, about 4mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM. In each subsequent solution of the series, the concentration of the salt increases, whereby the concentration of the salt increases, for example, to 50mM to 300mM (e.g., about 50mM, about 60mM, about 70mM, about 80mM, about 90mM, about 100mM, about 120mM, about 140mM, about 160mM, about 180mM, and about 200 mM) over the course of 2 to 20 solutions. The salt concentration in the 2 to 20 (or more) solution series is not necessarily varied in equal or proportional increments.
In some embodiments, the step elution comprises 2 to 20 solutions, 2 to 10 solutions, 10 to 20 solvents, e.g., 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, the rAAV capsid (e.g., complete capsid, intermediate capsid, empty capsid) binds to the stationary phase during loading of the solution comprising the rAAV capsid to the AEX stationary phase. During step elution, the complete rAAV vector is preferentially released (eluted) from the stationary phase, while the empty capsid is preferentially retained on the stationary phase, as a function of pH, conductivity, and/or modifier concentration. With increasing concentrations of modifier (e.g., salt), empty capsids are released in large amounts. During step elution, elution of the complete rAAV vector from the stationary phase can be monitored by measuring 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 eluent fraction is measured using a method such as analytical Size Exclusion Chromatography (SEC), online UV tracing, offline UV methods, and the like in a High Performance Liquid Chromatography (HPLC) system, and wherein absorbance is measured at one or more wavelengths (e.g., 260nm and/or 280 nm).
As used herein, the term "subject" refers to an organism, such as a mammal (e.g., human, non-human mammal, non-human primate, experimental animal, mouse, rat, hamster, gerbil, cat, dog). In some embodiments, the human subject is an adult, adolescent or pediatric subject. In some embodiments, the subject has a disease, disorder, or condition, e.g., a disease, disorder, or condition that can be treated as provided herein. In some embodiments, the subject has a disease, disorder, or condition associated with 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, a susceptible subject is predisposed to and/or exhibits an increased risk of developing a disease, disorder, or condition (as compared to the average risk observed in a reference subject or population). In some embodiments, the subject exhibits one or more symptoms of the disease, disorder, or condition. In some embodiments, the subject does not exhibit a particular symptom (e.g., clinical manifestation of the disease) or characteristic of the disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or features of the disease, disorder, or condition. In some embodiments, the subject is a human patient. In some embodiments, the subject is an individual who is 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 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 disease); urea cycle diseases or disorders (e.g., ornithine carbamoyltransferase deficiency); lysosomal storage diseases or disorders (e.g., metachromatic leukodystrophy, mucopolysaccharidosis); liver diseases or disorders (e.g., progressive familial intrahepatic cholestasis types 1-3); hematological diseases or disorders (hemophilia a, hemophilia B, alpha thalassemia); cancers (e.g., carcinoma, sarcoma, hematological cancer); genetic diseases or disorders (e.g., cystic fibrosis) or infectious diseases (e.g., HIV).
Diseases, disorders, and conditions that may be treated using rAAV vectors purified according to the methods described herein include, for example: 21-hydroxylase deficient congenital adrenocortical hyperplasia, type 1B cartilage hypoplasia, 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), alagille syndrome, urine black urine, alpha-1 antitrypsin deficiency, alpha-thalassemia, alport syndrome, alzheimer's disease, apert's syndrome, arginase deficiency, argininosuccinate lyase (ASL) deficiency, arginine succinate synthase (ASS 1) deficiency (type 1 citrullinemia), aromatic L-amino acid decarboxylase deficiency, autosomal recessive inherited congenital ichthyosis, becker muscular dystrophy, beta-thalassemia, carbamoylphosphatase synthetase I deficiency, ceroid lipofuscinosis, charcot-Marie Tooth neuropathy, choroiditis, chronic granulomatosis, vitamin P deficiency, crigler-Najjar syndrome types 1 and 2, severe limb ischemia, cystic fibrosis, cystine storage disorder, danon disease, diabetic macular retinopathy, dominant inherited short stature, dravet syndrome, duchenne muscular dystrophy, dysferlinopathy (such as Miyoshi myopathy, limb banding muscular dystrophy 2B), dystrophy bullous epidermolysis, fabry disease, familial hypercholesterolemia, familial lipoprotein deficiency, fanconia anemia (e.g., fanconia anemia type A), friedreich ataxia, frontotemporal dementia, gaucher's disease, glycogen storage disease type 1A and type 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 ganglioside deposition disease, cyclotron, hemophilia A, hemophilia B, hereditary angioedema type I-III, huntington's chorea, inclusion body myositis, junctional epidermolysis bullosa, kabuki syndrome, leber congenital black Mongolian, white cell adhesion deficiency type 1, limb banding muscular dystrophy type 2C (gamma-limb banding muscular dystrophy), limb banding 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 (Maroteux-Lamy), myotonic muscular dystrophy type 1, myotonic muscular dystrophy type 2, N-acetylglutamate synthase (NAGS) deficiency, netherton syndrome, neuronal ceroid lipofuscinosis, ornithine transposase deficiency, ornithine carbamoyltransferase deficiency, parkinson's disease, phenylketonuria, pompe, progressive familial intrahepatic cholestasis type 1-3, progressive myofibrillary myopathy, pyruvate kinase deficiency, retinitis pigmentosa, RPE 65-related Leber congenital amaurosis, sandhoff's disease, sickle cell disease, spinal muscular atrophy, tay-Sachs disease, wilson's disease, wiskott-Aldrich syndrome 2, X-linked adrenoleukodystrophy, X-linked chronic granulomatosis, X-linked myotubular myopathy, X-linked retinitis pigmentosa, X-linked retinal split disease, and X-linked severe combined immunodeficiency.
As used herein, the term "substantially" refers to a qualitative condition that exhibits all or nearly all of the range or degree of a feature or property of interest. Those of ordinary skill in the art will appreciate that biological and chemical phenomena are rarely, if ever, accomplished and/or proceed to a complete or realized or absolute result. Thus, the term "substantially" is used herein to represent the potential lack of integrity 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 reduce or reduce symptoms caused by a protein deficiency or defect in a target cell (e.g., an isolated cell) or organism (e.g., a subject). The therapeutic polypeptide or protein encoded by the transgene is a polypeptide or protein that confers a benefit to the subject, e.g., correcting a genetic defect associated with expression or function. Similarly, a "therapeutic transgene" is a transgene encoding a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide expressed in the host cell is an enzyme expressed from a transgene (i.e., an exogenous nucleic acid that has been introduced into the host cell). In some embodiments, the therapeutic polypeptide is a dystrophin protein or 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 upon administration. In some embodiments, the term refers to an amount sufficient to treat a disease, disorder, or condition when administered to a population suffering from or susceptible to the 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 and/or delays the onset of one or more symptoms of a disease, disorder, and/or condition. Those of ordinary skill in the art will appreciate that the term "therapeutically effective amount" does not actually require successful treatment in a particular individual. Conversely, a therapeutically effective amount may be an amount that provides a particular desired pharmacological response in a large number of subjects when administered to a patient in need of such treatment.
As used herein, the term "transgene" refers to any heterologous polynucleotide for delivery and/or expression in a host cell, target cell or organism (e.g., a subject). Such "transgenes" can be delivered to a host cell, target cell, or organism using a vector (e.g., a rAAV vector). The transgene may be operably linked to a control sequence, such as a promoter. Those skilled in the art will appreciate that the expression control sequences may be selected based on their ability to promote expression of the transgene in the host cell, target cell or organism. Typically, a transgene may be operably linked to an endogenous promoter associated with the transgene in nature, but more typically, a transgene is operably linked to a promoter not associated with the transgene in nature. One example of a transgene is a nucleic acid encoding a therapeutic polypeptide, such as a dystrophin polypeptide or fragment thereof, and an exemplary promoter is one that is not operably linked in nature to a nucleotide encoding a dystrophin protein. Such non-endogenous promoters may include CBh promoters or muscle-specific promoters, as well as many others known in the art.
The nucleic acid of interest may be introduced into the host cell by a variety of techniques known in the art, including transfection and transduction.
"transfection" is generally known as a technique for introducing foreign nucleic acid into cells without the use of viral vectors. As used herein, the term "transfection" refers to the transfer of a recombinant nucleic acid (e.g., an expression plasmid) into a cell (e.g., a host cell) without the use of a viral vector. Cells into which recombinant nucleic acid has been introduced are referred to as "transfected cells". The transfected cells may be host cells (e.g., CHO cells, pro10 cells, HEK293 cells) containing the expression plasmid/vector for the production of the recombinant AAV vector. In some embodiments, transfected cells (e.g., packaging cells) can comprise plasmids comprising transgenes (e.g., dystrophin transgenes), plasmids comprising AAV rep genes and AAV cap genes, and plasmids comprising helper genes. 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., vector genome) to a cell (e.g., a target cell such as a muscle cell) by a viral vector (e.g., rAAV vector). In some embodiments, gene therapy for Duchenne muscular dystrophy includes transducing a vector genome comprising a modified nucleic acid encoding a muscular dystrophy protein or fragment thereof into a muscle cell. Cells in 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 transduction occurs ex vivo. In some embodiments, the transduced cells are cells within an organism (e.g., a subject) and transduction occurs in vivo. The transduced cells can be target cells of an organism that have been transduced by the recombinant AAV vector such that the target cells of the organism express the polynucleotide (e.g., a transgene, 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., mesodermal, ectodermal, or endodermal). Non-limiting examples of cells include cells from the liver (e.g., hepatocytes, liver sinus endothelial cells), pancreas (e.g., beta-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 (heart), muscle or psoas, or intestinal tract (e.g., endocrine cells), adipose tissue (white, brown or beige), muscle (e.g., fibroblasts, myocytes), synovial cells, 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 multipotent progenitor cells, that develop or differentiate into liver (e.g., hepatocytes, liver sinus 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, muscle or psoas, or intestinal tract (e.g., endocrine cells), adipose tissue (white, brown or beige), muscle (e.g., fibroblasts, myocytes), synovial cells, chondrocytes, osteoclasts, epithelial cells, endothelial cells, salivary gland cells, inner ear nerve cells, or hematopoietic (e.g., blood or lymphocytes).
In some embodiments, a particular region of a tissue or organ (e.g., muscle) can be transduced by a rAAV vector (e.g., a rAAV vector with a dystrophin or partial dystrophin transgene) administered to the tissue or organ. In some embodiments, the muscle cells are transduced with a rAAV comprising a dystrophin transgene. In some embodiments, skeletal muscle cells are transduced with a rAAV comprising a dystrophin transgene. In some embodiments, cardiomyocytes are transduced with a 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 insertion or incorporation of nucleic acids (e.g., recombinant nucleic acids). Vectors can be used for a variety of purposes, including, for example, genetic manipulation (e.g., cloning vectors) to introduce/transfer nucleic acids into cells to transcribe or translate inserted nucleic acids in the cells. In some embodiments, the vector nucleic acid sequence contains at least an origin of replication for propagation in a cell. In some embodiments, the vector nucleic acid comprises a heterologous nucleic acid sequence, an expression control element (e.g., promoter, enhancer), a selectable marker (e.g., antibiotic resistance), a poly a sequence, and/or ITR. In some embodiments, the nucleic acid sequence proliferates when delivered to a host cell. In some embodiments, the cell expresses a polypeptide encoded by a heterologous nucleic acid sequence when delivered to the host cell in vitro or in vivo. In some embodiments, the nucleic acid sequence or a portion of the nucleic acid sequence is packaged into a capsid when delivered to a host cell. The host cell may be an isolated cell or a cell within a host organism. In addition to nucleic acid sequences encoding a polypeptide or protein (e.g., a transgene), additional sequences (e.g., regulatory sequences) may be present within the same vector (i.e., cis to the gene) and flanking the gene. In some embodiments, the regulatory sequences may be present on a separate (e.g., second) vector that acts in trans to regulate expression of the gene. Plasmid vectors may be referred to herein as "expression vectors".
As used herein, the term "vector genome" refers to a nucleic acid packaged/encapsidated in an AAV capsid to form a rAAV vector. Typically, the vector genome comprises a heterologous polynucleotide sequence (e.g., transgene, regulatory element, etc.) and at least one ITR. In the case of constructing or manufacturing a recombinant vector (e.g., a rAAV vector) using a recombinant plasmid, the vector genome does not include the entire plasmid, but only sequences intended to be delivered by the viral vector. This non-vector genomic portion of the recombinant plasmid is referred to as the "plasmid backbone," which is important for cloning, selection and amplification of the plasmid (which is the process required for the production and proliferation of the recombinant viral vector), but is not itself packaged or encapsidated into the rAAV vector. Typically, the heterologous sequence to be packaged into the capsid is flanked by ITRs, whereby it is packaged into the capsid when cleaved from the plasmid backbone.
As used herein, the term "viral vector" generally refers to a viral particle that functions as a nucleic acid delivery vehicle, comprising a vector genome (e.g., comprising transgenes that have replaced wild-type rep and cap) 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, the recombinant viral vector does 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 the transgene of interest.
The present disclosure provides methods of purifying rAAV vectors (e.g., complete rAAV vectors) from host cell harvests. In particular, the present disclosure provides methods of purifying rAAV vectors (e.g., complete rAAV vectors) from other nucleic acids and proteins (including empty capsids) produced by host cells. Furthermore, the present disclosure provides methods of isolating empty capsids from a complete rAAV vector (e.g., a rAAV vector comprising a vector genome). Each of these aspects of the disclosure is further discussed in the following sections.
AAV and rAAV vectors
AAV
As described above, "adeno-associated virus" and/or "AAV" refer to parvoviruses having a linear single stranded DNA genome and variants thereof. The term encompasses all subtypes as well as naturally occurring and recombinant forms, unless otherwise required. Parvoviruses, including AAV, are useful as gene therapy vectors because they can penetrate cells and introduce nucleic acids (e.g., transgenes) into the nucleus. In some embodiments, the introduced nucleic acid (e.g., rAAV vector genome) forms a circular concatemer that persists as an exosome in the nucleus of the transduced cell. 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 a predictable long-term expression profile. The insertion site of AAV in the human genome is referred to as AAVs1. Once introduced into a cell, the polypeptide encoded by the nucleic acid may be expressed by the cell. Because AAV is not associated with any pathogenic disease in humans, the nucleic acids delivered by AAV can be used to express therapeutic polypeptides for treating diseases, disorders, and/or conditions in a human subject.
There are a number of AAV serotypes in nature, and at least 15 wild-type serotypes (i.e., AAV1-AAV 15) have been identified to date in humans. Naturally occurring serotypes and variant serotypes are distinguished by having a protein capsid that is serologically distinct from other AAV serotypes. AAV serotype 1 (AAV 1), AAV serotype 2 (AAV 2), AAV serotype 3 (AAV 3), including AAV serotype 3A (AAV 3A) and AAV serotype 3B (AAV 3B), AAV serotype 4 (AAV 4), AAV serotype 5 (AAV 5), AAV serotype 6 (AAV 6), AAV serotype 7 (AAV 7), AAV serotype 8 (AAV 8), AAV serotype 9 (AAV 9), AAV serotype 10 (AAV 10), AAV serotype 12 (AAV 12), AAVrh10, AAVrh74 (see WO 2016/210170), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, and ovine AAV, as well as recombinantly produced variants (e.g., capsid variants with insertions, deletions, substitutions, etc.), such as variants known as AAV serotype 2i8 (AAV 2i 8), NP4, NP22, NP66, 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 (9): 1625-1634).
"primate AAV" refers to AAV that infects primates, and "non-primate AAV" refers to AAV that infects non-primates. "bovine AAV" refers to AAV that infects bovine mammals, and the like. Serotype distinctivity is determined based on the lack of cross-reactivity between antibodies of one AAV and another AAV. This cross-reactivity difference is typically due to differences in capsid protein sequences and antigenic determinants (e.g., VP1, VP2, and/or VP3 sequence differences due to AAV serotypes). However, some naturally occurring AAV or artificial AAV mutants (e.g., recombinant AAV) may not exhibit serological differences from any of the serotypes currently known. These viruses may be considered as a subgroup of the corresponding serotypes, or more simply variant AAV. Thus, as used herein, the term "serotype" refers to both serologically distinct viruses, such as AAV, and viruses that are serologically indistinguishable but may belong to a subset or variant of a serotype, such as AAV.
Comprehensive lists and alignments of amino acid sequences of capsids of known AAV serotypes are provided by Marsic et al (2014) Molecular Therapy (11): 1900-1909, particularly in supplemental fig. 1.
The genomic sequences of the various serotypes of AAV, as well as the sequences of the natural terminal repeats (ITRs), rep proteins, and capsid subunits, are known in the art. These sequences can be found in literature or public databases such as GenBank. See, e.g., genBank accession nos. nc_002077 (AAV 1), AF063497 (AAV 1), nc_001401 (AAV 2), AF043303 (AAV 2), nc_001729 (AAV 3), AF028705.1 (AAV 3B), nc_001829 (AAV 4), U89790 (AAV 4), nc_006152 (AAV 5), AF028704 (AAV 6), AF513851 (AAV 7), AF513852 (AAV 8), nc_006261 (AAV 8), AY530579 (AAV 9), AY631965 (AAV 10), AY631966 (AAV 11), and DQ813647 (AAV 12); the disclosure of which is incorporated herein by reference. See, e.g., 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.usa99:11854; moris et al (2004) Virology33:375-383; international patent publication WO 00/28061, WO 99/61601, WO 98/11244; WO 2013/06379; WO 2014/194132; WO 2015/121501, U.S. patent No. 6,156,303 and U.S. patent No. 7,906,111. For illustrative purposes only, wild-type AAV2 comprises the small (20-25 nm) icosahedral viral capsid of AAV, consisting of three proteins with overlapping sequences (VP 1, VP2, and VP3; 60 capsid proteins in total make up the AAV capsid). Proteins VP1 (735 amino acids; genbank accession number AAC 03780), VP2 (598 amino acids; genbank accession number AAC 03778) and VP3 (533 amino acids; genbank accession number AAC 03779) are present in the capsid in a ratio of about 1:1:10. That is, for AAV, VP1 is a full-length protein, VP2 and VP3 are progressively shorter versions of VP1, with an increasing degree of truncation at the N-terminus relative to VP 1. In one embodiment of the methods disclosed herein, the rAAV vector comprises AAV9 VP1, which comprises the amino acid sequence of SEQ ID NO. 11.
Recombinant AAV (rAAV)
As previously described, a "recombinant adeno-associated virus" or "rAAV" is distinguished from a wild-type AAV by replacement of all or part of the viral genome with a non-native sequence. Incorporation of non-native sequences into viruses defines the viral vector as a "recombinant" vector and thus as a "rAAV vector. A "rAAV vector can include a heterologous polynucleotide (e.g., a human codon-optimized gene encoding a human microdystrophin, such as SEQ ID NO: 1) encoding a desired protein or polypeptide (e.g., a dystrophin polypeptide or fragment thereof, such as SEQ ID NO: 2). The recombinant vector sequences may be encapsidated or packaged into AAV capsids, referred to as "rAAV vectors", "rAAV vector particles", "rAAV viral particles", or simply "rAAV".
The present disclosure provides methods of purifying rAAV vectors comprising polynucleotide sequences that are not AAV-derived (e.g., polynucleotides heterologous to AAV). The heterologous polynucleotide may be flanked by at least one, and sometimes two AAV terminal repeats (e.g., inverted Terminal Repeats (ITRs)). Heterologous polynucleotides flanked by ITRs, also referred to herein as "vector genomes", typically encode a polypeptide of interest or a gene of interest ("GOI"), e.g., a target for therapy (e.g., a nucleic acid encoding a dystrophin protein or fragment thereof for the treatment of Duchenne muscular dystrophy). Delivery or administration of the rAAV vector to a subject (e.g., a patient) provides the subject with the encoded proteins and peptides. Thus, 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 typically retains 145 base ITRs in cis with a heterologous nucleic acid sequence that replaces the 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 fully synthetic sequences, may also achieve this goal. The ITRs form hairpin structures and function, for example, as primers for host cell-mediated complementary DNA strand synthesis after infection. ITR also plays a role in viral packaging, integration, etc. ITR is the only cis AAV viral element required for AAV genome replication and packaging into a rAAV vector. The rAAV vector genome optionally comprises two ITRs, typically located at the 5 'and 3' ends of the vector genome, which comprises a heterologous sequence (e.g., a transgene encoding a gene or nucleic acid sequence of interest, including but not limited to antisense and siRNA, CRISPR molecules, etc.). The 5 'and 3' ITRs may both comprise the same sequence, or may comprise 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 can comprise ITRs from an AAV serotype (e.g., wild-type AAV2, fragment or variant thereof) that is different from the serotype of the capsid (e.g., AAV9 or otherwise). 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 us patent No. 7,172,893). AAV ITRs can 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 at the left and right (i.e., 5 'and 3' ends, respectively) of the vector genome (e.g., ITRs from AAV2, but may comprise ITRs from any wild-type AAV serotype, or variant thereof). In some embodiments, the left (e.g., 5' end) 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' end) 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' end) 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' end) 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 cis to each other or to other elements in the vector genome, but can be isolated by a nucleic acid sequence of variable length, for example a recombinant nucleic acid comprising a modified nucleic acid encoding a dystrophin protein or fragment thereof and a regulatory element. In some embodiments, the ITR is an AAV2 ITR or variant thereof and is flanking 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 the sequences set forth 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 ITRs. Prior to transcription and translation of the heterologous gene, the single stranded DNA genome of about 4700 nucleotides must be converted to a double stranded form, which is formed by a DNA polymerase (e.g., a DNA polymerase within a transduced cell) using the free 3' -OH of one of the self-priming ITRs to initiate second strand synthesis. In some embodiments, the full length single stranded vector genome (i.e., sense and antisense) anneals to produce a full length double stranded vector genome. This can occur when multiple rAAV vectors carrying opposite polarity (i.e., sense or antisense) genomes transduce the same cell at the same time. Regardless of how it is produced, once the double stranded vector genome is formed, the cell can transcribe and translate double stranded DNA and express heterologous genes.
The efficiency of transgene expression by rAAV vectors may be hindered by the need to convert single stranded rAAV genome (ssav) to double stranded DNA prior to expression. By circumventing this step using a self-complementary AAV genome (scAAV), scAAV can package inverted repeat genomes that can be folded into double stranded DNA without the need for DNA synthesis or base pairing between multiple vector genomes (McCarty, (2008) molecular 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 element, and IRT to be packaged in the capsid is about half the size of the ssav vector genome (i.e., about 4,900 nucleotides, including two ITRs) (i.e., about 2,500 nucleotides, where 2,200 nucleotides can be the transgene and regulatory element plus two copies of about 145 nucleotide ITRs).
The scAAV vector genome was prepared by deleting a terminal dissociation site (TRS) from one rAAV ITR of the expression plasmid, thereby preventing replication from that end (see us patent No. 8,784,799). Replication of AAV in host cells starts with a wild-type ITR of the genome and continues to replicate through the mutant ITR without terminal dissociation, then returns to the genome to produce dimers. The dimer is a self-complementary genome with a mutant ITR in the middle and a wild-type ITR at each end. In some embodiments, the mutant ITR with the deleted TRS is located 5' to the vector genome. In some embodiments, the mutant ITR with the deleted 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 exist within the capsid because many bases are in contact with amino acid residues of the capsid inner shell and the phosphate backbone chelates toward the center (McCarty, molec.Therapy (2008) 16 (10): 1648-1656). It is possible that after uncoating, both halves of the scAAV genome anneal to form dsDNA hairpin molecules with covalently blocked ITRs at one end and two open-ended ITRs at the other end. ITRs flank the double-stranded region encoding the transgene and its cis-regulatory elements.
Viral capsids of rAAV vectors may be from wild-type AAV or variant AAV, such as AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh74 (see WO 2016/210170), AAV12, AAV2i8, 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-S312N, AAV B-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 a number of AAV serotypes, disclosed in U.S. patent No. 7,906,111; gao et al (2004) j.virol.78:6381; morris et al (2004) Virol.33:375; WO 2013/06379; WO 2014/194132; and includes the true AAV (AAV-TT) variants disclosed in WO 2015/121501, and RHM4-1, RHM15-1 to RHM15-6, and variants thereof, disclosed in WO 2015/01393. The 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 (9): 1625-1634). One skilled in the art will appreciate that there may be other AAV variants that perform the same or similar functions that have not yet been identified. The complete 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 full complement of an AAV Cap protein, or may provide full complement of an AAV Cap protein.
In another embodiment, the present disclosure provides the use of an ancestral AAV vector for therapeutic in vivo gene therapy. In particular, computer-derived sequences can be synthesized de novo and their biological activity identified. In addition to assembly into rAAV vectors, prediction and synthesis of ancestral sequences can be accomplished using the methods described in WO 2015/054653, the contents of which are incorporated herein by reference. Notably, rAAV vectors assembled from ancestral viral sequences exhibit reduced susceptibility to preexisting immunity in humans as compared to contemporary viruses or portions thereof.
In some embodiments, a rAAV vector comprising a capsid protein encoded by a nucleotide sequence derived from more than one AAV serotype (e.g., wild-type AAV serotype, variant AAV serotype) is referred to as a "chimeric vector" or "chimeric capsid" (see U.S. patent No. 6,491,907, the entire disclosure of which is incorporated herein by reference). In some embodiments, the chimeric capsid protein is encoded by a nucleic acid sequence 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, AAV3A, AAV B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh74, AAVrs10, AAV2i8, or variants thereof, thereby producing a chimeric capsid protein comprising a combination of amino acids from any of the foregoing 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 can comprise an AAV1 cap protein or subunit and at least one AAV2 cap protein or subunit. Chimeric capsids may, for example, comprise AAV capsids having one or more B19 cap subunits, e.g., AAV cap proteins or subunits may be replaced with B19 cap proteins or subunits. For example, in one embodiment, the VP3 subunit of the AAV capsid may be replaced with the VP2 subunit of B19.
In some embodiments, the chimeric vector has been engineered to exhibit altered tropism or tropism for a particular tissue or cell type. The term "tropism" refers to preferential entry of viruses into certain cell or tissue types and/or preferential interaction with cell surfaces, 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 cellular receptors (Lykken et al (2018) j.neurodev. Discord.10:16). Preferably, once the virus or viral vector enters the cell, the sequence (e.g., heterologous sequence, such as a transgene) carried by the vector genome (e.g., rAAV vector genome) is expressed.
"tropism profile" refers to the transduction pattern of one or more target cells, tissues and/or organs. For example, AAV capsids may have a tropism profile characterized by efficient transduction of myocytes, whereas e.g. brain cells are only poorly transduced.
3. Recombinant nucleic acid
The methods of the present disclosure include purifying rAAV vectors comprising recombinant nucleic acids including modified nucleic acids, plasmids and vector genomes comprising modified nucleic acids. The recombinant nucleic acid, plasmid, or vector genome may comprise regulatory sequences to regulate proliferation (e.g., of a plasmid) and/or control expression of a modified nucleic acid (e.g., transgene). Recombinant nucleic acids can also be provided as components of viral vectors (e.g., rAAV vectors). Typically, a viral vector comprises a vector genome comprising a recombinant nucleic acid packaged in a capsid.
Modified nucleic acids
Modified form or variant form of a gene, nucleic acid or polynucleotide (e.g., transgene) refers to a nucleic acid that deviates from a reference sequence. The reference sequence may be a naturally occurring wild-type sequence (e.g., a gene) and may include naturally occurring variants (e.g., splice variants, variable reading frames). Those skilled in the art will appreciate that reference sequences can be found in publicly available databases, such as GenBank (ncbi.nlm.nih.gov/GenBank). The modified/variant nucleic acid may have substantially the same, greater or lesser activity, function or expression as the reference sequence. Preferably, a modified or variant nucleic acid, as used interchangeably herein, exhibits improved protein expression, e.g., the protein encoded thereby is expressed at a detectably higher level in a cell than the expression level of the protein provided by an endogenous gene (e.g., wild-type gene, mutant gene) in otherwise the same cell. In some embodiments, modified or variant nucleic acids, as used interchangeably herein, exhibit improved protein expression, e.g., the protein encoded thereby is expressed at a detectably higher level in a cell as compared to the level of expression of the protein provided by an endogenous gene comprising the mutation in an otherwise identical cell.
Modifications to a nucleic acid 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., additions 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 may have 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% identity to the reference sequence.
The modified nucleic acid may 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 that has 100% identity to a reference polypeptide.
In some embodiments, the modified nucleic acid (e.g., transgene) encodes a wild-type protein. Such modified nucleic acids may be codon optimized. "optimized" or "codon optimized" as referred to interchangeably herein refers to a coding sequence that has been optimized with respect to a wild-type coding sequence or reference sequence (e.g., a coding sequence for a microdystrophin polypeptide, such as SEQ ID NO:2, a coding sequence for a deleted copper-transporting ATPase2 polypeptide, such as SEQ ID NO: 15) to increase expression of the polypeptide, such as by minimizing the use of rare codons, reducing the number of CpG dinucleotides, removing cryptic splice donor or acceptor sites, removing Kozak sequences, removing ribosome entry sites, and the like. In some embodiments, the expression level of the protein from the codon optimized sequence is increased compared to the expression level of the protein from the wild-type gene in an otherwise identical cell. In some embodiments, the expression level of the protein from the codon optimized sequence is not increased (e.g., expression is substantially similar) as compared to the expression level of the protein from the wild-type gene in an otherwise identical cell. In some embodiments, the expression level of the protein from the codon optimized sequence is increased compared to the expression level of the protein from the mutant gene in an otherwise identical cell.
Examples of modifications include the elimination of one or more cis-acting motifs and the 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 may be eliminated include internal TATA boxes; a chi site; ribosome entry sites; ARE, INS and/or CRS sequence elements; repeat sequences and/or RNA secondary structures; (cryptic) splice donor and/or acceptor sites, branch points; and a restriction site.
In some embodiments, the modified nucleic acid encodes a modified or variant polypeptide. The modified polypeptide encoded by the modified nucleic acid (e.g., codon-optimized microdystrophin) may retain all or part of the function or activity of the polypeptide encoded by the wild-type encoding 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 demonstrated to have limited or no role 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 comprise fewer nucleotides than the wild-type coding sequence or reference sequence (e.g., a shortened microdystrophin transgene, see WO 2001/83695; b domain deleted human factor VIII transgene, see WO2017/074526, all of which are incorporated herein by reference), and also include truncated and codon optimized shortened transgenes (e.g., a codon optimized microdystrophin transgene described in WO 2017/221145; a deleted copper transport ATPase2 with a deletion of Metal Binding Sites (MBS) 1-4, 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, but at least a portion of, the polypeptide encoded by the reference sequence.
The modified nucleic acid may have a modified GC content (e.g., the number of G and C nucleotides present in the nucleic acid sequence), a modified (e.g., increased or decreased) CpG dinucleotide content, and/or a modified (e.g., increased or decreased) codon usage index (CAI) relative to the reference sequence and/or the wild-type sequence. See, e.g., WO 2017/07451 (discussing various considerations for codon optimization of nucleic acid sequences of interest, including publicly available software for analysis of nucleic acid sequences for optimization), as is well known in the art. 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 sequences of the present disclosure is increased relative to a reference sequence and/or a 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, GC content is expressed as a percentage of G (guanine) and C (cytosine) nucleotides in the sequence.
In some embodiments, the modified nucleic acid sequences of the present disclosure have a codon usage index of 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, i.e., about 10%, 20%, 30%, 50% or more, compared to the wild-type or reference nucleic acid sequence. 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 the reference sequence (e.g., wild-type sequence).
Methylation of CpG dinucleotides is known to play an important role in the regulation of gene expression in eukaryotic organisms. Specifically, methylation of CpG dinucleotides in eukaryotes essentially silence gene expression by interfering with the transcriptional machinery. Thus, nucleic acids and vectors with reduced numbers of CpG dinucleotides will provide high and durable transgene expression levels due to gene silencing caused by CpG motif methylation.
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, apaL and XmaI.
The present disclosure includes modified nucleic acids of SEQ ID NO. 1 encoding functionally active fragments of a dystrophin polypeptide. By "functionally active" or "functional dystrophin polypeptide" is meant that the fragment provides the same or similar biological function and/or activity as the full-length dystrophin polypeptide. That is, the fragments provide the same function, including but not limited to structural proteins of the myofilaments that are muscle fibers. The biological activity of a functional fragment of a dystrophin protein includes reversing or preventing the neuromuscular phenotype associated with Duchenne muscular dystrophy.
Thus, one embodiment of the invention relates to a method of purifying a rAAV vector comprising a modified nucleic acid encoding a microdystrophin protein, the nucleic acid comprising, consisting essentially of, or at least about 90% identical to the nucleic acid sequence of SEQ ID No. 1. 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 nucleic acid is in the length of viral vectors, such as parvoviral vectors, such as rAAV vectors, capacity range. In some embodiments, the nucleic acid is about 5000, 4900, 4800, 4700, 4600, 4500, 4400, 4300, 4200, 4100, or about 4000 nucleotides or less in length.
In some embodiments, the nucleic acid encodes a microdystrophin protein comprising, consisting essentially of, or consisting of at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID No. 2.
In some embodiments, the nucleic acid encodes a deleted copper transport ATPase2 protein comprising, consisting essentially of, or consisting of 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 regulatory elements for increasing 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 regulatory elements useful for increasing copper transport ATPase2 expression.
Adjusting element
The methods of the present disclosure include purifying rAAV vectors comprising recombinant nucleic acids including modified nucleic acids encoding polypeptides (e.g., microdystrophin) and various regulatory or control elements. Typically, a regulatory element is a nucleic acid sequence that affects the expression of an operably linked polynucleotide. The precise nature of the regulatory elements useful for gene expression will vary from organism to organism and also from cell type to cell type, including, for example, promoters, enhancers, introns, etc., in order to facilitate the transcription and translation of the appropriate heterologous polynucleotide. Regulatory control may be affected at the level of transcription, translation, splicing, information stability, etc. Typically, regulatory control elements that regulate transcription are juxtaposed near (i.e., upstream of) the 5' end of the transcribed polynucleotide. Regulatory control elements may also be located 3' to (i.e., downstream of) the transcribed sequence or within the transcript (e.g., in an intron). Regulatory control elements may be located at a distance from the transcribed sequence (e.g., 1 to 100, 100 to 500, 500 to 1000, 1000 to 5000, 5000 to 10,000 or more nucleotides apart). However, due to the length of the AAV vector genome, regulatory control elements are typically within 1 to 1000 nucleotides of the polynucleotide.
Promoters
As used herein, the term "promoter," such as "eukaryotic promoter," refers to a nucleotide sequence that initiates transcription of a particular gene or one or more coding sequences (e.g., microdystrophin coding sequences) in a eukaryotic cell (e.g., a muscle cell). Promoters may work with other regulatory elements or regions to direct the transcriptional level of a gene or coding sequence. These regulatory elements include, for example, transcription binding sites, repressors, and activator protein binding sites, as well as other nucleotide sequences known to act directly or indirectly to regulate the amount of transcription from a promoter, including, for example, attenuators, enhancers, and silencers. Promoters are typically located on the same strand near the transcription initiation site 5' to the gene or coding sequence to which they are operably linked. Promoters are typically 100-1000 nucleotides in length. Promoters generally increase gene expression relative to expression of the same gene in the absence of the promoter.
As used herein, "core promoter" or "minimal promoter" refers to the minimal portion of the promoter sequence required to properly initiate transcription. It may comprise any of the following sites: transcription initiation site, binding site for RNA polymerase and general transcription factor binding site. Promoters may also comprise proximal promoter sequences (5 'to the core promoter) containing other major regulatory elements (e.g., enhancers, silencers, border elements, insulators) as well as distal promoter sequences (3' to the core promoter). In some embodiments, the core or minimal promoter is an α1-antitrypsin core or minimal promoter, optionally comprising or consisting of the 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; the Rous Sarcoma Virus (RSV) promoter; an albumin promoter; inducible promoters, such as the Mouse Mammary Tumor Virus (MMTV) promoter; a metallothionein promoter; a heat shock promoter; an alpha-1-antitrypsin promoter; hepatitis b surface antigen promoter; a transferrin promoter; an apolipoprotein a-1 promoter; chicken beta-actin (CBA) promoter; an elongation factor 1a (EF 1 a) promoter; hybrid forms of the CBA promoter (CBh promoter); CAG promoter (cytomegalovirus early enhancer element and promoter, first exon and first intron of chicken β -actin gene, splice acceptor of rabbit β -globin gene) (alexopaloulu et al (2008) biomed.central Cell biol.9:2); a creatine kinase promoter; human dystrophin gene promoter.
In some embodiments, the promoter is a creatine kinase promoter, e.g., 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 disclosure, a eukaryotic promoter sequence (e.g., creatine kinase promoter) is operably linked to a modified nucleic acid encoding, for example, a microdystrophin protein or a deleted copper transport ATPase 2. In some embodiments, a promoter comprising the 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 a microdystrophin protein. 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 the nucleic acid sequence of SEQ ID NO. 16 (e.g., an. Alpha.1-antitrypsin promoter) is operably linked to a modified nucleic acid encoding copper transport ATPase2 with a deletion of MBS 1-4 (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 that is 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 that is at least 95% identical to the nucleic acid sequence of SEQ ID NO. 1 and induces expression of the polypeptide encoded by the nucleic acid sequence of SEQ ID NO. 1 in a muscle cell.
Promoters may be constitutive, tissue-specific, or regulated. Constitutive promoters are those that cause an operably linked gene to be expressed at substantially any time. In some embodiments, the constitutive promoter is active in most eukaryotic tissues under most physiological and developmental conditions.
Regulated promoters are those that can be activated or deactivated. Regulated promoters include inducible promoters, which are normally "off but can be induced to be" on ", and" repressed "promoters; the latter is typically "on" but may be "off. Many different regulatory factors are known, including temperature, hormones, cytokines, heavy metals and regulatory proteins. The distinction is not absolute; constitutive promoters can generally be regulated to some extent. In some cases, endogenous pathways may be utilized to provide for modulation of transgene expression, for example, using promoters that are naturally down-regulated as the pathological condition improves.
A tissue-specific promoter refers to a promoter that is active only in a particular type of tissue, cell or organ. Typically, a tissue-specific promoter is recognized by transcriptional activation elements specific to a particular tissue, cell, and/or organ. For example, a tissue-specific promoter may be more active in one or several specific tissues (e.g., two, three, or four) than in other tissues. In some embodiments, the expression of a gene regulated by a tissue specific promoter is much higher in the tissue specific for the promoter than in other tissues. In some embodiments, the promoter may have little or no activity in any tissue other than its specific tissue. The promoter may be a tissue specific promoter active in hepatocytes, such as the mouse albumin promoter or the transthyretin promoter (TTR). Other examples of tissue specific promoters include promoters from genes encoding skeletal muscle alpha-actin, myosin light chain 2A, dystrophin, 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 the alpha fetoprotein (Arbuthenot et al, (1996) hum.Gene.Ther.7:1503-1514).
Enhancers
In another aspect, the modified nucleic acid encoding the therapeutic polypeptide further comprises an enhancer to increase expression of the therapeutic polypeptide. Typically, the enhancer element is located upstream of the promoter element, but may also be located downstream of or within another sequence (e.g., transgene). Enhancers can be located 100 nucleotides, 200 nucleotides, 300 nucleotides or more upstream or downstream of the modified nucleic acid. Enhancers generally increase the expression of a modified nucleic acid (e.g., encoding a therapeutic polypeptide) over that provided by the separate promoter element.
Many enhancers are known in the art, including but not limited to the major immediate early enhancer of cytomegalovirus. More specifically, the Cytomegalovirus (CMV) MIE promoter comprises three regions: modulators, unique regions and enhancers (Isomura and Stinski (2003) J.Virol.77 (6): 3602-3614). The CMV enhancer region may be combined with another promoter or a portion thereof to form a hybrid promoter to further increase expression of the nucleic acid to which it is operably linked. For example, the chicken beta-actin (CBA) promoter or portion thereof may be combined with a CMV promoter/enhancer or portion thereof to produce a version of CBA known as the "CBh" promoter, which represents Chicken betaActin proteinHeterozygosityPromoters as described by Gray et al (2011,Human Gene Therapy 22:1143-1153). Like promoters, enhancers may be constitutive, tissue-specific, or regulated.
In some embodiments of the disclosure, the regulatory element comprises a hybrid enhancer and promoter, e.g., a synthetic hybrid enhancer or promoter derived from the Creatine Kinase (CK) gene, which serves as a muscle-specific transcriptional regulatory element (hCK) and is operably linked to a modified nucleic acid encoding a microdystrophin protein. In some embodiments, the synthetic hybrid enhancer and promoter comprising the nucleic acid sequence of SEQ ID NO. 5 is operably linked to a modified nucleic acid encoding a microdystrophin protein. In some embodiments, synthetic hybrid enhancers and promoters derived from the Creatine Kinase (CK) gene comprising 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 the nucleic acid sequence of SEQ ID No. 5 are operably linked to a nucleic acid comprising the nucleic acid sequence of SEQ ID No. 1.
In some embodiments, synthetic hybrid enhancers and promoters derived from the Creatine Kinase (CK) gene comprise a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NO. 5, operably linked to a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NO. 1, and induce expression of the polypeptide encoded by the nucleic acid sequence of SEQ ID NO. 1 in muscle cells.
Filling sequence, spacing sequence and filling sequence
As disclosed herein, recombinant nucleic acids for rAAV vectors may include additional nucleic acid elements to adjust the length of the nucleic acid to near or normal size (e.g., about 4.7 to 4.9 kilobases) for viral genomic sequences acceptable for AAV packaging into the rAAV vector (Grieger and Samulski (2005) j.virol.79 (15): 9933-9944). Such sequences may be interchangeably referred to as stuffer sequences, spacer sequences, or stuffer sequences. In some embodiments, the stuffer DNA is an untranslated (non-protein encoding) segment of a nucleic acid. In some embodiments, the filling 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 typically accept DNA insert sequences of about 4kb to about 5.2kb or about 4.1 to 4.9kb in size to optimally package the nucleic acids into the AAV capsid. In some embodiments, the rAAV vector comprises a vector genome that is 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 in total length. In some embodiments, the rAAV vector comprises a vector genome that is about 4.5kb in total length. In some embodiments, the rAAV vector comprises a self-complementing vector genome. Although the total length of the self-complementary (sc) vector genome in a rAAV vector is equal to the single-stranded (ss) vector genome (i.e., about 4kb to about 5.2 kb), the nucleic acid sequence encoding the sc vector genome (i.e., comprising the transgene, regulatory elements, and ITRs) must be only half the length of the nucleotide sequence encoding the ss vector genome in order to package the sc vector genome in the capsid.
Introns and exons
In some embodiments, the recombinant nucleic acid includes, for example, introns, exons, and/or portions thereof. Introns may be used as stuffer or stuffer polynucleotide sequences to achieve the appropriate length for packaging the vector genome into the rAAV vector. Introns and/or exonic sequences may also enhance expression of polypeptides (e.g., transgenes) compared to expression in the absence of introns and/or exonic elements (Kurachi et al (1995) J.biol. Chem.270 (10): 576-5281; WO 2017/074526). Furthermore, filling/stuffer 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.
The intronic elements may be derived from the same gene as the heterologous polynucleotide, or from a completely different gene or other DNA sequence (e.g., chicken β -actin gene, mouse adenovirus (MVM)). In some embodiments, the recombinant nucleic acid includes at least one element selected from the group consisting of an intron and an exon derived from a non-homologous gene (i.e., not derived from a modified nucleic acid such as a transgene).
Polyadenylation signal sequences (polyA)
Further regulatory elements may include stop codons, stop sequences, and polyadenylation (polyA) signal sequences, such as, but not limited to, bovine growth hormone polyA signal sequences (BHG-polyA). The polyA signal sequence drives the effective addition of a polyadenosine "tail" at the 3' end of eukaryotic mRNA, which directs 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 precursor mRNA at its 3 'end and as a signal for addition of an RNA sequence consisting of adenine bases only at this 3' end. The polyA tail region is important for nuclear export, translation and stability of mRNA. In some embodiments, the 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 other 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, a microdystrophin protein (e.g., SEQ ID NO: 2) or a deleted copper transport ATPase 2 (e.g., SEQ ID NO: 15). In some embodiments, the polyA sequence comprises or consists of the 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 the 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: promoter sequences (e.g., SEQ ID NO:3, SEQ ID NO: 4), hybrid enhancers and promoters (e.g., SEQ ID NO: 5) and polyA (SEQ ID NO: 6), and regulate expression of heterologous polypeptides 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: promoter sequences (e.g., SEQ ID NO: 16) and polyA (SEQ ID NO/17) and regulate expression of heterologous polypeptides comprising the amino acid sequence of SEQ ID NO: 15.
In some embodiments, a rAAV9 vector having tropism for a myocyte 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 microdystrophin and at least one of the following regulatory elements: promoters (e.g., human CK promoter), hybrid enhancers, and poly a signal sequences.
In some embodiments, a rAAV3B vector having 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 transport ATPase 2 (e.g., amino acid sequence of SEQ ID NO: 15) and at least one of the following regulatory elements: promoters (e.g., an alpha 1-antitrypsin promoter, e.g., a nucleic acid sequence having SEQ ID NO: 16) and a poly A signal sequence (e.g., a nucleic acid of SEQ ID NO: 17).
In some embodiments, a rAAV9 vector having tropism for muscle cells comprises a vector genome comprising an AAV ITR (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 a microdystrophin protein (e.g., SEQ ID NO: 1), and at least one regulatory element as follows: promoters (e.g., SEQ ID NO:3 or SEQ ID NO: 4), hybrid enhancers and promoters (e.g., SEQ ID NO: 5) and poly A (e.g., SEQ ID NO: 6).
4. Assembly of viral vectors
Viral vectors (e.g., rAAV vectors) carrying transgenes (e.g., encoding microdystrophin) are assembled from polynucleotides encoding the transgenes, appropriate regulatory elements, and elements necessary 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 (as described above).
The vector genome component of the rAAV vector produced according to the methods of the present disclosure includes at least one transgene, e.g., a codon-optimized microdystrophin transgene, and associated expression control sequences for controlling expression of a modified nucleic acid encoding a dystrophin protein or fragment thereof.
In an exemplary, non-limiting embodiment, the vector genome comprises a portion of a parvoviral genome, such as an AAV genome with rep and cap deletions and/or substitutions of modified nucleic acids (e.g., transgenes, such as codon-optimized dystrophin transgenes), and associated expression control sequences. Modified nucleic acids encoding muscular dystrophy proteins, or fragments thereof, are typically inserted adjacent to one or both (i.e., flanked by) AAV ITRs or ITR elements sufficient for viral replication (Xiao et al (1997) j.virol.71 (2): 941-948) in place of the nucleic acids encoding viral rep and cap proteins. Other regulatory sequences suitable for promoting tissue-specific expression of the codon-optimized micro-dystrophin transgene in a target cell (e.g., a muscle cell) may also be included.
Packaging cells
Those skilled in the art will appreciate that rAAV vectors comprising transgenes and lacking viral proteins (e.g., cap and rep) required for viral replication cannot replicate because these proteins are necessary for viral replication and packaging. The Cap and rep genes may be provided to the cell (e.g., host cell, such as a packaging cell) as part of a plasmid that is separate from the plasmid that provides the transgene to the vector genome.
"packaging cell" or "producer cell" refers to a cell or cell line that can be transfected with a vector, plasmid or DNA construct and which in trans provides all of the deleted functions required for complete replication and packaging of the viral vector. Genes required for rAAV vector assembly include vector genomes (e.g., codon optimized mini-dystrophin transgenes, regulatory elements, and ITRs), AAV rep genes, AAV cap genes, and certain helper genes from other viruses such as adenoviruses. Those skilled in the art will appreciate that genes required for AAV production may be introduced into packaging cells in a variety of ways, including, for example, transfection of one or more plasmids. However, in some embodiments, some genes (e.g., rep, cap, helper genes) may already be present in the packaging cell, either integrated into the genome, or carried on exosomes. In some embodiments, the packaging cell expresses one or more deleted viral functions in a constitutive or inducible manner.
Any suitable packaging cell known in the art may be used to produce the packaged viral vector. Mammalian cells or insect cells are preferred. Examples of cells that may be used in the practice of the present disclosure to produce packaging cells include, for example, human cell lines such as PER.C6, WI38, MRC5, A549, HEK293 (which expresses functional adenovirus E1 under the control of a constitutive promoter), B-50 or any other HeLa cell, 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 cell is capable of growing in suspension culture. In some embodiments, the packaging cells are capable of growing in serum-free medium. For example, HEK293 cells were grown in suspension in serum-free medium. In another embodiment, the packaging cell is a HEK293 cell, as described in U.S. patent No. 9,441,206, and is deposited with the American Type Culture Collection (ATCC) as PTA 13274. Many rAAV packaging cell lines are known in the art, including but not limited to those disclosed in WO 2002/46359.
Cell lines used as packaging cells include insect cell lines. Any insect cell that allows AAV to replicate and that can be maintained in culture may be used in accordance with the present disclosure. Examples include spodoptera frugiperda (Spodoptera frugiperda), such as Sf9 or Sf21 cell lines, drosophila (Drosophila spp.) cell lines, or mosquito cell lines, such as Aedes albopictus (Aedes albopictus) derived cell lines. A preferred cell line is the Spodoptera frugiperda Sf9 cell line. The following references, which are incorporated herein by reference, teach methods for expressing heterologous polypeptides using insect cells, introducing nucleic acids into such cells, and maintaining such cells in culture: methods in Molecular Biology, ed. Richard, humana Press, NJ (1995); o' Reilly et al Baculovirus Expression Vectors: ALaboratory Manual, oxford Univ. Press (1994); samulski et al (1989) J.Virol.63:3822-3828; kajigaya et al (1991) Proc.Nat' l.Acad.Sci.USA88:4646-4650; ruffing et al (1992) J.Virol.66:6922-6930; kimbauer et al (1996) Virol.219:37-44; zhao 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 may be produced in insect cells using baculovirus vectors to deliver the rep/cap genes and rAAV templates, for example as described by uarabe et al (2002) Human Gene Therapy 13:1935-1943. When baculoviruses are used to produce AAV, in some embodiments, the vector genome is self-complementing. In some embodiments, the host cell is a baculovirus-infected cell (e.g., an insect cell), optionally comprising additional nucleic acid encoding a baculovirus helper function, thereby facilitating production of the viral capsid.
Packaging cells typically include one or more viral vector functions, helper functions, and packaging functions sufficient to cause replication and packaging of the viral vector. These different functions may be provided to the packaging cell together or separately by using genetic constructs such as plasmids or amplicons, and they may be present extrachromosomal to the cell line or integrated into the chromosome of the host cell. In some embodiments, the packaging cells are transfected with 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: enhancers, promoters, exons, introns and poly a, and ii) plasmids containing the rep gene (e.g., AAV2 rep) and cap gene (e.g., AAV9 or other cap).
In some embodiments, the host cell is provided with one or more packaging or helper functions incorporated therein, e.g., the host cell line has one or more vector functions incorporated extrachromosomally or integrated into the chromosomal DNA of the cell.
Auxiliary function
AAV is a virus that cannot replicate in cells without co-infection of the cells by helper virus. Helper functions include helper viral elements required to establish active infection of the packaging cell, which is required to initiate viral vector packaging. Helper viruses typically include adenovirus or herpes simplex virus. Adenovirus helper functions typically include the adenovirus components adenovirus early region 1A (E1A), E1b, E2a, E4, and virus-associated (VA) RNA. Auxiliary functions (e.g., E1a, E1b, E2a, E4, and VARNA) may be provided to the packaging cells by transfecting the packaging cells with one or more nucleic acids encoding various auxiliary elements. Alternatively, the host cell (e.g., packaging cell) may comprise a nucleic acid encoding an accessory protein. HEK293 cells, for example, are produced by transforming human cells with adenovirus 5DNA 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). Thus, these helper functions can be provided by HEK293 packaging cells without the need to provide them to the cells by, for example, a plasmid 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: enhancers, promoters, exons, introns and poly a, and ii) plasmids containing rep genes (e.g., AAV2 rep) and cap genes (e.g., AAV9 or other cap), and iii) plasmids containing helper functions.
Any method of introducing a nucleotide sequence carrying an ancillary function into a cellular host for replication and packaging may be employed, including but not limited to electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, carrier molecules (e.g., polyethylenimine (PEI)), and liposomes that combine a nuclear localization signal. In some embodiments, standard methods of generating viral infections may be used by transfection with viral vectors or by providing helper functions through infection with helper viruses.
The vector genome may be any suitable recombinant nucleic acid, e.g. a DNA or RNA construct, and may be single-stranded, double-stranded or duplex (i.e. self-complementary as described in WO 2001/92551).
4. Production of packaged viral vectors
Viral vectors can be prepared by several methods known to the person skilled in the art (see e.g. WO 2013/06379). Grieger, et al (2015) Molecular Therapy (2): 28One exemplary, non-limiting method is described in 7-297, the contents of which are incorporated herein by reference for all purposes. Briefly, efficient transfection of HEK293 cells was used as a starting point, where adherent HEK293 cell lines from a qualified clinical master cell bank were used for growth in shake flasks and WAVE bioreactors in suspension without animal components, which allows for rapid and scalable rAAV production. Using triple transfection methods (e.g., WO 96/40240), HEK293 cell line suspensions can produce greater than 1X 10 when harvested 48 hours post-transfection 5 Particles (VG)/cells containing vector genome, or greater than 1X 10 14 VG/L cell culture. More specifically, triple transfection refers to a method of transfecting packaging cells with three plasmids: one plasmid encodes the AAV rep and cap (e.g., AAV9 cap) genes, another encodes various helper functions (e.g., adenovirus or HSV proteins, such as E1a, E1b, E2a, E4, and VA RNAs), and yet another encodes the transgene (e.g., dystrophin or fragments thereof) and various elements that control the expression of the transgene.
The single stranded vector genome is packaged into the capsid in approximately equal proportions as either a plus strand or a minus strand. In some embodiments of the rAAV vector, the vector genome is in the positive strand polarity (i.e., sense or coding sequence of the DNA strand). In some embodiments of the rAAV vector, the vector is in negative strand polarity (i.e., 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, which is the reverse complement of the positive strand nucleotide sequence, can be determined.
In order to obtain the desired yield, a number of variables are optimized, such as selection of compatible serum-free suspension medium supporting growth and transfection, selection of transfection reagents, transfection conditions and cell density.
The rAAV vector can be purified by methods standard in the art, for example by column chromatography or cesium chloride gradients. Methods of purifying rAAV vectors are known in the art and include 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 WO 98/09657 describe methods.
After the rAAV vectors of the present disclosure are produced and purified according to the methods disclosed herein, they can be subjected to titer assays (e.g., the amount of rAAV vector in a sample can be quantified) to prepare compositions for administration to a subject (e.g., a human subject suffering from Duchenne muscular dystrophy). rAAV vector titers can be accomplished using methods known in the art.
In some embodiments, the number of viral particles, including particles containing the vector genome and "empty" capsids not containing the vector genome, can be determined by electron microscopy, such as Transmission Electron Microscopy (TEM). Such TEM-based methods can provide the number of carrier particles (or viral particles in the case of wild-type AAV) in the sample. In some embodiments, the amount of particles containing the vector genome (complete capsids) and "empty" capsids not containing the vector genome can be determined by methods such as charge detection mass spectrometry, analytical Ultracentrifugation (AUC), and/or measuring absorbance at 260nm and 280nm to determine the a260/a280 ratio.
In some embodiments, the rAAV vector genome can be titred using quantitative PCR (qPCR) using primers directed to 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 a standard of known concentration (e.g., a plasmid containing the vector genome sequence), a standard curve can be generated and the concentration of rAAV vector can be calculated as vector genome number (VG) per unit volume (e.g., microliter or milliliter). The percentage of empty capsids can be estimated by comparing the number of vector particles measured by SEC or ELISA, for example, with the number of vector genomes in the sample. Because the vector genome contains a therapeutic transgene, vg/kg or vg/ml of the vector sample may be more indicative of the therapeutic amount of vector that the subject will receive than the number of vector particles (some of which may be empty and not contain the vector genome). Once the concentration of rAAV vector genome in the stock solution is determined, it can be diluted or dialyzed in a suitable buffer for use in preparing a composition (e.g., drug substance) for administration to a subject (e.g., a subject with Duchenne muscular dystrophy).
5. Purification of rAAV vectors by anion exchange chromatography (AEX)
A novel universal purification strategy based on ion exchange chromatography can be used to generate a variety of AAV serotypes and/or high purity rAAV vector preparations 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, primates AAV and ovine AAV, and recombinantly produced variants (e.g., capsid variants with insertions, deletions, substitutions, etc.), such as, for example, the variants known as AAV2i8, NP4, NP22, NP66, AAVDJ/8, AAVDJ/9, AAVLK03, RHM4-1, AAVHSC1, aafsc 2, AAVHSC 3, vhsc4, aasc 5, aavrs C6, AAVHs C7, aafsc 8, aasc 9, vhsc10, vhsc12, vhsc13, vhsc14, and vhsc 13). In some embodiments, this method can be completed in less than one week, resulting in a high complete to empty capsid ratio (up to 70% complete capsid), providing step yields of up to 70% and purity suitable for clinical use. In some embodiments, such methods are generic to the chimerism of AAV serotypes and/or capsids. As described herein, scalable production techniques can be used to produce GMP clinical and commercial grade rAAV vectors to treat diseases (e.g., DMD, friedreich ataxia, wilson disease, etc.).
Production of recombinant AAV vectors (rAAV) for gene therapy requires purification of the rAAV vector from host cells (e.g., host cell debris, including but not limited to host cell DNA, RNA, proteins, lipids, membranes, and organelles) that produce the vector, and removal of capsids that do not contain the complete vector genome (e.g., intermediate capsids and/or empty capsids) and thus do not contain a therapeutic transgene.
Such purification methods typically include a number of steps including, for example, lysis of host cells, 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 may include, for example, ion exchange chromatography (e.g., anion, cation), affinity chromatography, size exclusion chromatography, multimodal chromatography, and/or hydrophobic interaction chromatography. Centrifugation methods may include, for example, ultracentrifugation or low-speed centrifugation (e.g., for solids removal and clarification). Filtration methods may 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 their charge differences, and can separate rAAV capsids from impurities based on charge differences at moderately acidic to basic pH (e.g., greater than pH 6). AEX can also separate empty capsids from rAAV vectors containing the complete vector genome (i.e., complete capsids) by relying on charge differences between empty capsids compared to complete capsids.
Without wishing to be bound by theory, the tightness of the binding between AAV capsids and AEX chromatographic stationary phases is related to the strength of the negative charge of the capsids, including charge contribution of any nucleic acid within the capsids, solution pH and solution conductivity (Qu, g.et al, j.virologic methods (2007) 140:183-192). In some embodiments, the AEX chromatographic stationary phase is a resin comprising polystyrene divinylbenzene particles modified with covalently bound quaternized polyethylenimine and optionally bound OH groups (e.g., POROS TM 50HQ resin). The polystyrene divinylbenzene particles can include 500-10000 angstromsIs formed by a plurality of holes.
In some embodiments, the AEX chromatographic stationary phase is a resin (e.g., capto Q ImpRes, Q Sepharose High Performance) comprising agarose particles with cationic ligands. In some embodiments, the AEX chromatographic stationary phase is a resin selected from the group consisting of: capto Q, capto Q XP, Q Sepharose XL, STREAMLINE Q XL, capto HiRes Q, RESOURCE Q, SOURCE 15Q,SOURCE 30Q,Q Sepharose HP,QSepharose FF,Q Sepharose TM BB，POROS TM 20HQ，POROS TM 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)，TSKgel SuperQ-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，UNOsphere Q，Macro-Prep High Q，Macro-Prep 25Q，BioRad/>1-X2，WorkBeads TM 40Q，WorkBeads TM 100Q，Cellufine MAX Q-r，Cellufine MAX Q-h，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，TOYOPEARL GigaCap DEAE-650M，TOYOPEARL DEAE-650S，TOYOPEARL DEAE-650M，TOYOPEARL DEAE-650C，TSKgel DEAE-5PW(20)，TSKgel DEAE-5PW(30)，Ceramic HyperD DEAE，Hypercel Star 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 chromatographic stationary phase is a monolith comprising a porous polymethacrylate with cationic ligands (e.g., CIMmultus TM QA). In some embodiments, the AEX chromatographic stationary phase is a membrane adsorber (e.g., mustang Q, mustang E,Q、Sartobind/>PA)。
In some embodiments, the rAAV vector can be purified by AEX from a solution that comes out of an affinity chromatography stationary phase (e.g., "eluting from the stationary phase") that comprises a mobile phase and a material, such as a rAAV vector or capsid, that passes through or is removed from the stationary phase. Such a solution may be referred to as an affinity eluent or an "affinity pool".
In some embodiments, the rAAV vector can be purified by AEX from a "supernatant of a cell lysate" (also referred to as a "clarified lysate"), which as used herein refers to a solution collected from a host cell culture after precipitation of the lysed host cells.
In some embodiments, the rAAV vector can be purified by AEX from a "post-harvest solution," which as used herein refers to a solution produced by cell lysis through flocculation, depth filtration, and/or nominal filtration.
In some embodiments, the rAAV vector can be purified from solution that is subjected to at least one other purification or treatment step (e.g., cell lysis, flocculation, filtration, dilution, pH adjustment, chromatography). In some embodiments, the affinity eluate is diluted and optionally filtered prior to purifying the rAAV vector, e.g., prior to loading the affinity eluate onto the AEX column.
In some embodiments, the rAAV vector can be purified by AEX from an affinity eluate that is optionally subjected to at least one additional purification or treatment step (e.g., cell lysis, flocculation, filtration, dilution, pH adjustment, chromatography). In some embodiments, the rAAV vector can be purified by AEX from a cell lysate that optionally has undergone at least one additional purification or treatment step (e.g., cell lysis, flocculation, filtration, dilution, pH adjustment, chromatography). In some embodiments, the rAAV vector can be purified by AEX from a post-harvest solution that optionally has been subjected to at least one additional purification or treatment 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, the substance bound to the positively charged AEX stationary phase (e.g., negatively charged protein, e.g., AAV capsid or rAAV vector, etc.) is retained in the stationary phase. Unbound material is passed through the column and collected in the flow-through and/or in a subsequent washing step. The bound material may be eluted from the stationary phase by adjusting the salt concentration and/or pH within the column. For example, and without wishing to be bound by any particular theory of operation, the salt concentration of the elution buffer is gradually increased such that the salt (e.g., acetate (C) 2 H 3 O 2 - )、Cl - SO 4 -2 ) The anions of (a) compete with and displace (i.e., elute) the substance bound to the resin. In another embodiment, the pH of the solution in the column may be gradually lowered to reduce the negative charge of the bound material and allow it to be released (i.e., eluted) from the stationary phase. After release from the stationary phase, the material may be collected as a column eluate.
Without wishing to be bound by theory, the separation of a substance, such as a mixture of AAV capsids, or more specifically rAAV vectors (i.e., complete capsids), AAV capsids (e.g., empty capsids, intermediate capsids), and host cell proteins, will depend on the total charge differential of the substance. The charge composition of the ionizable side groups will determine the total charge of the protein at a particular pH. At the isoelectric point (pI), the total charge on the protein is 0 and will not bind to the matrix. If the pH is higher than the pI, the protein will have a negative charge and bind to the anion exchange column stationary phase.
The AEX protocol for separating a complete rAAV vector from an empty capsid includes a number of steps, e.g., pre-use flushing of the column medium to displace the storage solution, pre-use sterilization of the column stationary phase, post-use sterilization of the column stationary phase, equilibration of the column stationary phase, loading of the solution containing the rAAV vector (e.g., diluted affinity eluate) to the column stationary phase, eluting the substance to be purified from the stationary phase (e.g., by gradient elution, by stepwise elution), applying gradient hold to the column stationary phase, sterilization of the column stationary phase, regeneration of the column stationary phase, application of the storage solution to the column stationary phase. Those of skill in the art will appreciate that the AEX protocol for purifying the rAAV vector may include all or only some of these steps. Those of skill in the art will also appreciate that the order of the steps may vary and that certain steps may be performed more than once and not necessarily in order.
AEX column preparation
AEX methods of the present disclosure can be performed on various scales using columns ranging in volume from 1.0mL to 20L. In some embodiments, the AEX method includes using a column having a Column Volume (CV) of about 1.0mL, about 5.1mL, about 49mL, about 52mL, about 6.67mL, about 1.256L, about 1.3L, about 6.0L, about 6.1L, about 6.2L, about 6.3L, about 6.4L, about 6.5L, about 6.6L, about 6.7L, about 6.8L, about 6.9L, or about 7.0L. In some embodiments, the AEX methods of the present disclosure include using columns having a CV of 1.0mL to 20L, e.g., 1.0mL to 10mL, 30mL to 70mL, 10mL to 100mL, 100mL to 1000mL, 1L to 1.5L, 1.5L to 2.0L, 2.0L to 5L, 5L to 7.5L, 7.5L to 10L, 10L to 15L, or 15L to 20L. In some embodiments, the AEX methods of the present disclosure include using columns having a CV of 1.0mL to 10L, 10mL to 10L, 100mL to 20L, 100mL to 10L,1L to 20L,1L to 10L,1L to 5L, 1L to 2L, or 1L to 1.5L. In some embodiments, the AEX methods of the present disclosure include using a column having a CV of 6.0L to 6.6L (e.g., 6.4L).
For example, to equilibrate the stationary phase therein, the volume of solution applied to the column is typically expressed as "column volume" (CV), where one CV is equal to the volume of the column.
In some embodiments, the AEX chromatographic stationary phase (also referred to herein as a "resin" or "medium") of the present disclosure is polystyrene divinylbenzene particles (e.g., POROS) with covalently bound quaternized polyethylenimine TM 50HQ resin).
Typically, at least one solution is applied to the stationary phase, e.g., to rinse, disinfect, regenerate and/or equilibrate the stationary phase, prior to applying (i.e., loading) the solution to be purified (e.g., an affinity chromatography eluent, also referred to herein as an "affinity eluent" or "affinity cell") to a column comprising a chromatography stationary phase. In some embodiments, the "affinity eluate" or "affinity cell" has been diluted and optionally filtered prior to loading the solution into the AEX column.
As disclosed herein, a method of preparing an AEX stationary phase for a method of purifying a rAAV (e.g., rAAV9, rAAV3B, etc.) carrier from a solution (e.g., an affinity eluate) by AEX includes flushing the AEX stationary phase in a column prior to use. In some embodiments, the pre-use rinse of the AEX stationary phase is intended to displace a storage solution (e.g., a solution comprising ethanol) from the stationary phase. In some embodiments, the column is subjected to a pre-use rinse prior to loading the solution comprising the rAAV vector to be purified onto the column. In some embodiments, the pre-use rinse comprises applying water (e.g., water for injection) to the AEX stationary phase in the column. In some embodiments, the pre-use rinse comprises water flowing upward. During the upflow of the pre-use rinse, the flow direction is opposite to the flow direction of the chromatographic separation step (e.g., loading, washing or eluting) such that the solution (e.g., water) flows from the bottom of the column to the top of the column, while 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 rinse comprises applying 1 to 10 Column Volumes (CVs) (e.g., about 5 CVs) of water to the AEX stationary phase in the column at a linear velocity of 10cm/hr to 1000cm/hr and/or a flow rate of 0.2L/min to 3.0L/min. In some embodiments, the pre-use rinse comprises applying ≡4.5CV (e.g., about 5 CV) of water for injection to the AEX stationary phase in the column at a linear velocity of 270cm/hr to 330cm/hr (e.g., about 300 cm/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., contact time) of 3.5min/CV to 4.5min/CV (e.g., about 4 min/CV).
A method of preparing an AEX stationary phase for use in a method of purifying a rAAV (e.g., rAAV9, rAAV3B, etc.) carrier from a solution (e.g., an affinity eluate) by AEX, comprising sterilizing the AEX stationary phase in a column. The AEX stationary phase is sterilized for reducing bioburden (including but not limited to bacteria) and/or inactivating microorganisms and viruses within the column, and more generally removing contaminants such as proteins, particulates, and the like. In some embodiments, the sterilization is performed prior to loading the solution comprising the rAAV vector to be purified onto the column. In some embodiments, sterilization includes applying a solution comprising NaOH, ethanol, acetic acid, phosphoric acid, guanidine hydrochloride, urea, PAB (phosphoric acid, acetic acid, benzyl alcohol), peroxyacetic acid, etc., to the AEX stationary phase in the column. In some embodiments, disinfecting comprises 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, disinfecting comprises applying a solution comprising about 0.5M NaOH to the AEX stationary phase in the column using an upward flow (i.e., in a flow direction opposite to that of a chromatographic separation step such as loading, washing or eluting). In some embodiments, disinfecting comprises applying a solution comprising about 0.5M NaOH to the AEX stationary phase in the column using a downward flow (i.e., in the same direction as the chromatographic separation step, e.g., loading, washing, or eluting). In some embodiments, disinfecting comprises applying a solution comprising about 0.5M NaOH at 14.4CV to 17.6CV (e.g., about 16 CV) to the AEX stationary phase in the column. In some embodiments, disinfecting comprises applying 5CV to 10CV (e.g., about 8 CV) of a solution comprising about 0.5M NaOH to the AEX stationary phase in the column. In some embodiments, sanitizing comprises applying 5CV to 20CV of a solution comprising about 0.5M NaOH to an AEX stationary phase in a column at a linear velocity of 100cm/hr to 1000cm/hr and/or a flow rate of 0.2L/min to 3.0L/min. In some embodiments, disinfecting comprises applying 14.4 to 17.6 (e.g., about 16) CV of a solution comprising about 0.5M NaOH to the AEX stationary phase in the column at a line speed of 270 to 330cm/hr (e.g., about 300 cm/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 4 min/CV), i.e., the amount of time per column volume of the solution in contact with the in-column stationary phase, also referred to herein as contact time. In some embodiments, sanitizing comprises applying 5 to 10 (e.g., about 8) CV of a solution comprising about 0.5M NaOH to the AEX stationary phase in the column at a linear velocity of 270 to 330cm/hr (e.g., about 298 cm/hr) and/or a residence time of 1.5 to 2.5min/CV (e.g., about 2 min/CV).
A method of preparing an AEX stationary phase for use in a method of purifying rAAV (e.g., rAAV9, rAAV3B, etc.) carriers from a solution (e.g., an affinity eluate) by AEX, comprising regenerating the AEX stationary phase in a column (also referred to herein as a "rinse"). Those skilled in the art will appreciate that regenerating the ion exchange stationary phase serves to replace the ions admitted 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 prior to loading the solution comprising the rAAV vector to be purified 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 a salt and/or buffer having 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 salt (e.g., naCl) concentration in the solution ranges from 1M to 5M, e.gAbout 1M to about 4.5M, about 1M to about 4M, about 1M to about 3.5M, about 1M to about 3M, about 1M to about 2.5M, or about 1.5M to about 2.5M. In some embodiments, the salt (e.g., naCl) concentration 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-di (hydroxyethyl) glycine. In some embodiments, the buffer (e.g., tris) is present in the solution at a concentration ranging from 10mM to 500mM (e.g., from about 10mM to about 450mM, from about 10mM to about 400mM, from about 10mM to about 350mM, from about 10mM to about 300mM, from about 10mM to about 250mM, from about 10mM to about 200mM, from about 10mM to about 150mM, or from about 50mM to about 150 mM). In some embodiments, the concentration of buffer (e.g., tris) in the solution is about 10mM, about 20mM, about 50mM, about 100mM, about 150mM, about 200mM, about 300mM, about 400mM, or about 500mM. In some embodiments, regeneration comprises applying a solution comprising 50mM to 150mM (e.g., 100 mM) Tris to the stationary phase in the column.
In some embodiments, regenerating 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, 5, 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 100 mM) 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 a solution comprising about 2M NaCl, 100mM Tris, pH 9 of 4.5 to 5.5CV (e.g., about 5 CV) 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 a 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, regenerating comprises applying 4.5 to 5.5 (e.g., about 5) CV of a solution comprising about 2M NaCl, 100mM Tris, pH 9 to the AEX stationary phase in the column at a linear velocity of 270 to 330cm/hr (e.g., about 298cm/hr, about 300 cm/hr), a flow rate of 1.5 to 2.0L/min (e.g., about 1.8L/min), and/or a residence time (i.e., contact time) of 1.5 to 4.5min/CV (e.g., about 2min/CV, about 4 min/CV).
In some embodiments, the present disclosure provides a method of preparing an AEX stationary phase for use in a method of purifying a rAAV (e.g., rAAV9, rAAV3B, etc.) vector by AEX, the method comprising the steps of: i) A pre-use rinse comprising applying ≡4.5CV (e.g. about 5 CV) of water for injection to the AEX stationary phase in the column; ii) sterilization comprising applying about 5 to 10CV (e.g., about 8 CV) or about 14.4 to 17.6CV (e.g., about 16 CV) 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 a solution comprising 1M to 3M (e.g., about 2M) NaCl, 50mM to 150mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., about 9) to the AEX stationary phase in the column of 4.5 to 5.5CV (e.g., about 5 CV); wherein at least one of steps i) to iii) is performed at a linear velocity of 270cm/hr to 330cm/hr (e.g., about 298cm/hr, about 300 cm/hr), a flow rate of 1.5L/min to 2.0L/min (e.g., about 1.8L/min), and/or a residence time of 1.5min/CV to 4.5min/CV (e.g., about 2min/CV, about 4 min/CV); optionally, at least one of the steps is performed prior to loading the solution comprising the rAAV vector to be purified into the column; and optionally wherein the AEX stationary phase is POROS TM 50HQ. The skilled person will appreciate that the above steps may be performed in any order and may be performed more than once.
Balancing
A method of preparing an AEX stationary phase for use in a method of purifying a rAAV (e.g., rAAV9, rAAV3B, etc.) carrier from a solution (e.g., an affinity eluate) by AEX, comprising equilibrating the AEX stationary phase in a column. In some embodiments, the AEX stationary phase in the equilibrium column is used to adjust the pH, conductivity, modifier (e.g., salt, detergent, amino acid, etc.) concentration, or other conditions of the mobile and stationary phases such that some of the species loaded on the column will bind to the stationary phase while other species will flow through the stationary phase with the mobile phase. For example, conditions within the column may be adjusted by applying a series of equilibration buffers to the column such that the complete rAAV vector binds to the stationary phase and at least a portion of the empty capsids do not bind. In some embodiments, the AEX stationary phase in the column is equilibrated prior to applying a solution comprising the substance to be purified (e.g., rAAV vector) to the column. In some embodiments, the AEX stationary phase in the column is equilibrated by application of 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 "wash buffer", "post-sterilization rinse", "rinse" or "regeneration buffer". The reference to equilibration buffers as first, second, third, fourth, etc. equilibration buffers does not necessarily imply the order in which the buffers are applied to the column.
In some embodiments, the equilibration buffer (e.g., first equilibration buffer, second equilibration buffer, third equilibration buffer, fourth equilibration buffer, etc.) comprises at least one component selected from the group consisting of buffers, salts, amino acids, detergents, and/or combinations 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. One of ordinary skill in the art will appreciate that Tris Base, tris-HCl, or both, can be used to prepare a Tris buffer having the desired pH. 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 nonylphenoxy polyethoxy ethanol (NP-40).
In some embodiments, the equilibration buffer comprises 10mM to 350mM of a buffer selected from the group consisting of Tris (e.g., a mixture of Tris Base and Tris-HCl), BIS-Tris propane, diethanolamine, diethylamine, tris (hydroxymethyl) methylglycine, triethanolamine and n, n-di (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 10mM Tris, about 20mM Tris, about 30mM Tris, about 40mM Tris, about 50mM Tris, about 60mM Tris, about 70mM Tris, about 80mM Tris, about 90mM Tris, about 100mM Tris, about 110mM Tris, about 120mM Tris, about 130mM Tris, about 140mM Tris, about 150mM Tris, about 160mM Tris, about 170mM Tris, about 180mM Tris, about 190mM Tris, about 200mM Tris, about 220mM Tris, about 240mM Tris, about 250mM Tris, about 275mM Tris, about 300mM Tris, or about 350mM Tris. In some embodiments, the equilibration buffer comprises about 20mM Tris, 100mM Tris, or 200mM Tris.
In some embodiments, the equilibration buffer comprises 1mM to 1M salt, preferably about 500mM salt. In some embodiments, the equilibration buffer comprises from about 10mM to about 950mM, from about 10mM to about 900mM, from about 10mM to about 850mM, from about 10M to about 800mM, from about 10mM to about 750mM, from about 10mM to about 700mM, from about 10mM to about 650mM, from about 10mM to about 600mM, from about 10mM to about 550mM, from about 50mM to about 750mM, from about 50mM to about 700mM, from about 50mM to about 650mM, from about 50mM to about 600mM, from about 50mM to about 550mM, from about 100mM to about 600mM, from about 200mM to about 600mM, from about 300mM to about 600mM, or from about 400mM to about 600mM salt. In some embodiments, the equilibration buffer comprises about 500mM salt. In some embodiments, the equilibration buffer comprises a buffer 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 ) Is a salt of (a).
In some embodiments, the equilibration buffer comprises 5mM to 1M sodium acetate. In some embodiments, the equilibration buffer comprises from about 10mM to about 950mM, from about 10mM to about 900mM, from about 10mM to about 850mM, from about 10M to about 800mM, from about 10mM to about 750mM, from about 10mM to about 700mM, from about 10mM to about 650mM, from about 10mM to about 600mM, from about 10mM to about 550mM, from about 50mM to about 750mM, from about 50mM to about 700mM, from about 50mM to about 650mM, from about 50mM to about 600mM, from about 50mM to about 550mM, from about 100mM to about 600mM, from about 200mM to about 600mM, from about 300mM to about 600mM, or from 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 50mM, about 75mM, about 100mM, about 125mM, about 150mM, about 175mM, about 200mM, about 225mM, about 250mM, about 275mM, or about 300mM 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 100mM to 300mM amino acid (e.g., histidine, arginine, glycine, or citrulline). In some embodiments, the equilibration buffer comprises from about 10mM to about 600mM, from about 10mM to about 550mM, from about 10mM to about 500mM, from about 10mM to about 450mM from about 10mM to about 400mM, from about 10mM to about 350mM, from about 10mM to about 300mM, from about 50mM to about 600mM, from about 50mM to about 550mM, from about 50mM to about 500mM, from about 50mM to about 450mM, from about 50mM to about 400mM, from about 50mM to about 350mM, from about 50mM to about 300mM, from about 100mM to about 600mM, from about 100mM to about 500mM, from about 100mM to about 400mM, from about 100mM to about 300mM salt, or from about 150mM to about 250mM amino acid (e.g., histidine). In some embodiments, the equilibration buffer comprises about 200mM 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 from about 0.01% to about 1.5%, from about 0.01% to about 1.0%, from about 0.01% to about 0.75%, from about 0.05% to about 1.5%, from about 0.05% to about 1.0%, from about 0.05% to about 0.75%, from about 0.1% to about 1.5%, from about 0.1% to about 1.0%, from about 0.1% to about 0.75%, or from 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% of a 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 equilibration buffer is from 8 to 10. In some embodiments, the pH of the equilibration buffer is from 8.7 to 9.3. In some embodiments, the pH of the equilibration buffer is from 8.7 to 9.0. In some embodiments, the pH of the equilibration 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 equilibration buffer is about 8.8. In some embodiments, the pH of the equilibration buffer is about 8.9. In some embodiments, the pH of the equilibration buffer is about 9.0.
In some embodiments, the equilibration buffer comprises 20mM Tris, pH 9.0. In some embodiments, the equilibration buffer comprises 100mM Tris, pH 9.
In some embodiments, the equilibration buffer comprises 20mM Tris and 500mM NaCl, pH 9.0+/-0.3. In some embodiments, the equilibration buffer comprises 20mM Tris and 500mM NH 4 Acetate, pH 9.0+/-0.3. In some embodiments, the equilibration buffer comprises about 20mM Tris, 500mM sodium acetate, pH 9.0+/-0.3. In some embodiments, the equilibration buffer comprises 20mM Tris and 500mM 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 (Na) 2 SO 4 ) The pH was 9.0. In some embodiments, the equilibration buffer comprises 20mM Tris, 7mM sodium acetate, pH9.0. In some embodiments, the equilibration buffer comprises 20mM Tris, 14mM sodium acetate, pH9.0. In some embodiments, the equilibration buffer comprises 20mM Tris, 21mM sodium acetate, pH9.0. In some embodiments, the equilibration buffer comprises 20mM Tris, 42mM sodium acetate, pH9.0. In some embodiments, the equilibration buffer comprises 20mM Tris, 49mM sodium acetate, pH9.0. In some embodiments, the equilibration buffer comprises 20mM Tris, 57mM sodium acetate, pH9.0. In some embodiments, the equilibration buffer comprises 20mM Tris, 67mM sodium acetate, pH9.0.
In some embodiments, the equilibration buffer comprises 50mM to 150mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., about 9). In some embodiments, the equilibration buffer comprises 50mM to 150mM (e.g., about 100 mM) Tris, 400mM to 600mM (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). In some embodiments, the equilibration buffer comprises 100mM to 300mM histidine (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, pH 8.5 to 9.5 (e.g., about 8.8). In some embodiments, the equilibration 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., about 8.9).
In some embodiments, the equilibration buffer (e.g., first equilibration buffer) comprises 100mM Tris, pH 9. In some embodiments, the equilibration buffer (e.g., first and second equilibration buffers) comprise 100mM Tris, 500mM sodium acetate, 0.01% P188, pH 8.9. In some embodiments, the equilibration buffer (e.g., second or third equilibration buffer) comprises 200mM histidine, 200mM Tris, 0.5% P188, pH 8.8. In some embodiments, the equilibration buffer (e.g., third and fourth equilibration buffers) comprise 100mM Tris, 0.01% P188, pH 8.9.
In some embodiments, the equilibration buffer may be a first, second, third, and fourth equilibration buffer. In some embodiments, the first, second, third, or fourth equilibration buffer is applied sequentially to the column stationary phase. In some embodiments, a solution (e.g., an affinity eluate) is applied to the column between the application of two equilibration buffers. For example, the first, second and third equilibration buffers may be applied to the column, followed by application of the affinity eluate, followed by application of the fourth equilibration buffer. In another example, the first and second equilibration buffers are applied to the column, then the affinity eluate is applied, 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 from 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 equilibration buffer, is set to flow through the stationary phase at a particular rate (e.g., cm/hr, mL/min) such that the solution within the column contacts the stationary phase for a particular period of time (referred to herein as the "residence time" or "contact time"). In some embodiments, the residence time of the solution in the column is 0.1min/CV to 10min/CV, for example 0.1min/CV to 1min/CV, 1min/CV to 2min/CV, 2min/CV to 4min/CV, 4min/CV to 6min/CV, 6min/CV to 8min/CV or 8min/CV to 10min/CV. In some embodiments, the residence time of the solution in the column is 0.1min/CV, about 0.5min/CV, about 1.5min/CV, about 2min/CV, about 3min/CV, about 3.6min/CV or about 4min/CV, about 5min/CV, about 6min/CV, about 7min/CV, about 8min/CV, about 9min/CV or about 10min/CV. In some embodiments, the residence time of the solution in the column is from 1.5 to 4.5min/CV. In some embodiments, the residence time of the solution in the column is from 3.5 to 4.5min/CV.
In some embodiments, the residence time of the solution in a column having a height of about 5cm, a diameter of about 0.5cm, and a volume of about 1.0mL is about 0.5min/CV. In some embodiments, the residence time of the solution in a column having a height of about 15cm, a diameter of about 0.66cm, and a volume of about 5.1mL is about 0.5min/CV, about 1.5min/CV, or about 4min/CV. In some embodiments, the residence time of the solution in a column having a height of about 19.5cm, a diameter of about 0.66cm, and a volume of about 6.67mL is about 4min/CV. In some embodiments, the residence time of the solution in a column having a height of about 10cm, a diameter of about 2.5cm, and a volume of about 49mL is 1.5min/CV to 2.5min/CV (e.g., about 2 min/CV). In some embodiments, the residence time of the solution in a column having a height of about 16cm, a diameter of about 10cm, and a volume of about 1.256L to 1.3L is 3.5min/CV to 4.5min/CV (e.g., about 4 min/CV). In some embodiments, the residence time of the solution in a column having a height of about 20.5cm, a diameter of 20cm, and a volume of about 6.4L is about 3.6min/CV. In some embodiments, the residence time of the solution in the about 6.4L column is from 3.5min/CV to 4.5min/CV (e.g., about 4 min/CV). In some embodiments, the residence time of a solution including, but not limited to, equilibration buffer in a 6.0L to 6.6L (e.g., 6.4L) column comprising an AEX stationary phase is from 3.5min/CV to 4.5min/CV (e.g., about 4 min/CV).
Those skilled in the art will appreciate that the linear velocity of the solution through the 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, equilibration buffer through a stationary phase in a column is from 100cm/hr to 1800cm/hr, such as from 100cm/hr to 200cm/hr, from 200cm/hr to 400cm/hr, from 400cm/hr to 600cm/hr, from 600cm/hr to 800cm/hr, from 800cm/hr to 1000cm/hr, from 1000cm/hr to 1500cm/hr, or from 1500cm/hr to 1800cm/hr. In some embodiments, the linear velocity of the solution through the stationary phase in the column is about 100cm/hr, about 240cm/hr, about 298cm/hr, about 300cm/hr, about 600cm/hr, about 611cm/hr, or about 1790cm/hr.
In some embodiments, the linear velocity of the solution through the stationary phase in a column of about 5cm in height, about 0.5cm in diameter, and about 1.0mL in volume is about 611cm/hr. In some embodiments, the linear velocity of the solution through the stationary phase in a column of about 15cm in height, about 0.66cm in diameter, and about 5.1mL in volume is about 600cm/hr. In some embodiments, the linear velocity of the solution through the stationary phase in a column of about 15cm in height, about 0.66cm in diameter, and about 5.1mL in volume is about 1790cm/hr. In some embodiments, the linear velocity of the solution through the stationary phase in a column about 10cm high, about 2.5cm in diameter, and about 49mL in volume is about 298cm/hr. In some embodiments, the linear velocity of the solution through the stationary phase in a column of about 16cm in height, about 10cm in diameter, and about 1256mL in volume is about 240cm/hr. In some embodiments, the linear velocity of the solution through the stationary phase in a column of about 20.5cm in height, about 20cm in diameter, and about 6.4L in volume is 270cm/hr to 330cm/hr (e.g., 300 cm/hr). In some embodiments, the linear velocity of a solution including, but not limited to, equilibration buffer through the AEX stationary phase in a 6.0L to 6.6L (e.g., 6.4L) column is between about 270cm/hr and 330cm/hr (e.g., about 300 cm/hr).
In some embodiments, the flow rate (i.e., volumetric flow rate) of the solution (including but not limited to equilibration buffer) through the stationary phase in the column is 1.0mL/min to 3.0L/min, such as 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, or 2mL/min to 3L/min. In some embodiments, the flow rate of the solution through the stationary phase in the column is about 1mL/min, about 1.28mL/min, about 1.67mL/min, about 314mL/min, about 1.57L/min, about 1.8L/min, about 2L/min, about 3L/min.
In some embodiments, the flow rate of the solution through the stationary phase in a column having 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 having 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 having 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 having 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 a solution including, but not limited to, equilibration 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 of preparing an AEX stationary phase for use in a method of purifying a rAAV (e.g., rAAV9, rAAV3B, etc.) carrier from a solution (e.g., an affinity eluate) by AEX, comprising equilibrating the AEX stationary phase in a column. In some embodiments, equilibration is performed prior to loading the solution comprising the rAAV vector to be purified onto the column. In some embodiments, equilibration is performed after loading the solution comprising the rAAV vector to be purified onto the column.
In some embodiments, equilibrating comprises applying an equilibration buffer comprising 50mM to 150mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 to the AEX stationary phase in the column. In some embodiments, equilibration includes applying 4.5 to 5.5CV (e.g., about 5 CV) equilibration buffer comprising 100mM Tris, pH 9 to a 6.0L to 6.6L (e.g., 6.4L) column comprising an AEX stationary phase, a linear velocity of 270cm/hr to 330cm/hr (e.g., about 300 cm/hr), a flow rate of 1.5L/min to 2.0L/min (e.g., about 1.8L/min), and/or a residence time of 3.5min/CV to 4.5min/CV (e.g., about 4 min/CV).
In some embodiments, equilibration includes application of an equilibration buffer comprising 400mM to 600mM sodium acetate, 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, equilibrating comprises applying 4.5 to 5.5CV (e.g., about 5 CV) of an equilibration buffer comprising 100mM Tris, 500mM sodium acetate, 0.01% P188, pH 8.9 to a column comprising an AEX stationary phase at a linear velocity of 270 to 330cm/hr (e.g., about 298cm/hr, about 300 cm/hr), a flow rate of 1.5 to 2.0L/min (e.g., about 1.8L/min), and/or a residence time of 1.5 to 4.5min/CV (e.g., about 2min/CV, about 4 min/CV). In some embodiments, the column is 6.0L to 6.6L (e.g., 6.4L). In some embodiments, the column is 30mL to 70mL (e.g., about 49mL, about 52 mL).
In some embodiments, equilibration includes application of an equilibration buffer comprising 100mM to 300mM histidine, 100mM to 300mM 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 includes applying ≡4.5CV (e.g., about 5 CV) equilibration buffer comprising 200mM histidine, 200mM Tris, 0.5% P188, pH 8.8 to a column comprising AEX stationary phase at a linear velocity of 270cm/hr to 330cm/hr (e.g., about 298cm/hr, about 300 cm/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., about 2min/CV, about 4 min/CV). In some embodiments, the column is 6.0L to 6.6L (e.g., 6.4L). In some embodiments, the column is 30mL to 70mL (e.g., about 49mL, about 52 mL).
In some embodiments, equilibrating comprises applying an equilibration 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, equilibration includes applying 4.5 to 5.5CV (e.g., about 5 CV) equilibration buffer comprising 100mM Tris, 0.01% P188, pH 8.9 to a column comprising an AEX stationary phase at a linear velocity of 270 to 330cm/hr (e.g., about 298cm/hr, about 300 cm/hr), a flow rate of 1.5 to 2.0L/min (1.8L/min), and/or a residence time of 1.5 to 4.5min/CV (e.g., 2min, 4 min/CV). In some embodiments, the column is 6.0L to 6.6L (e.g., 6.4L). In some embodiments, the column is 30mL to 70mL (e.g., about 49mL, about 52 mL).
In some embodiments, the invention provides a method of preparing an AEX stationary phase for use in a method of purifying a rAAV (e.g., rAAV9, rAAV3B, etc.) vector by AEX, the method comprising the steps of: i) Flushing before use, including flushing with a temperature of 4.5C or higherV (e.g., about 5 CV) of water for injection was applied to the AEX stationary phase in the column; ii) sterilization comprising applying about 5 to 10CV (e.g., about 8 CV) or about 14.4 to 17.6CV (e.g., about 16 CV) of a solution comprising 0.1 to 1.0M (e.g., about 0.5M) NaOH to the AEX stationary phase in the column, optionally flowing upward; iii) Regeneration, comprising applying a solution comprising 1M to 3M (e.g., about 2M) NaCl, 50mM to 150mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., about 9) of 4.5CV to 5.5CV (e.g., about 5 CV) to the AEX stationary phase in the column; iv) equilibration, comprising applying a solution comprising 50mM to 150mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., about 9) of 4.5CV to 5.5CV (e.g., about 5 CV) to the AEX stationary phase in the column; v) equilibration, comprising applying 4.5 to 5.5CV (e.g., about 5 CV) of an equilibration buffer comprising 50mM to 150mM (e.g., about 100 mM) Tris, 400mM to 600mM (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) to the AEX stationary phase in the column; vi) equilibration, including ≡4.5CV (e.g., about 5 CV) equilibration buffer comprising 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, pH 8.5 to 9.5 (e.g., 8.8) applied to the AEX stationary phase in the column; and/or vii) equilibration, comprising applying an equilibration buffer comprising 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) to the AEX stationary phase in the column, at 4.5CV to 5.5CV (e.g., about 5 CV); optionally, wherein at least one of steps i) to vii) is performed at a linear velocity of 270cm/hr to 330cm/hr (e.g., about 298cm/hr, about 300 cm/hr) and/or a residence time of 1.5min/CV to 4.5min/CV (e.g., about 2min/CV, about 4 min/CV); optionally, wherein the AEX stationary phase is POROS TM 50HQ; optionally, wherein the rAAV vector is a rAAV9 vector or a rAAV3B vector, and optionally, wherein steps (e.g., loading steps) can be performed between any equilibration steps. In some embodiments, at least one of steps i) through vii) is performed at a flow rate of 1.5L/min to 2.0L/min (e.g., about 1.8L/min) through a 6L to 6.6L column (e.g., about 6.4L), or at a flow rate of about 314mL/min through a 1.3L column. The order of the above steps may be varied as will be appreciated by the skilled artisan.
Dilution and filtration
Methods of purifying rAAV (e.g., rAAV9, rAAV3B, etc.) vectors from solutions (e.g., affinity eluents) by AEX include preparing the solutions by dilution and optionally filtration of the solutions. The solution comprising the rAAV vector to be purified may be an affinity eluate, a supernatant from a cell lysate, and/or a post-harvest solution that has undergone at least one purification or processing step. The solution comprising the rAAV vector to be purified may be diluted and optionally filtered before loading to the AEX column to render the solution compatible with processing through the AEX column. In some embodiments, diluting and optionally filtering a solution comprising the rAAV vector to be purified results in a change in pH and/or conductivity of the solution. In some embodiments, the solution comprising 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 greater).
The method of preparing a solution comprising rAAV vector purified by AEX comprises: i) Diluting the 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 cell", "loading" or "AEX loading"). In some embodiments, the pH of the diluted and optionally filtered affinity eluate is increased compared to the pH of the affinity eluate prior to dilution. In some embodiments, the conductivity of the diluted and optionally filtered affinity eluate is reduced compared to the conductivity of the affinity eluate prior to dilution. In some embodiments, the diluted and optionally filtered affinity eluate is loaded to the AEX stationary phase.
In some embodiments, the affinity eluate is generated by affinity purification of a rAAV vector generated in a vessel (e.g., a disposable bioreactor (SUB)) having a volume of 1mL to 2000L or greater than 2000L. In some embodiments, the affinity eluate is produced by affinity purification of a rAAV vector produced in a vessel (e.g., SUB) having a volume of about 1mL, about 10mL, about 50mL, about 100mL, about 250mL, about 500mL, about 750mL, about 1L, about 50L, about 100L, about 250L, about 500L, about 1000L, about 2000L, or more. In some embodiments, the affinity eluate is produced by affinity purification of a rAAV vector produced in a vessel (e.g., SUB) having a volume of 1mL to 100mL, 100mL to 500mL, 500mL to 750mL, 750mL to 1L, 1L to 10L, 10L to 50L, 50L to 100L, 100L to 250L, 250L to 500L, 500L to 750L, 750L to 1000L, 1000L to 1500L, 1500L to 2000L, 2000L to 3500L, 3500L to 4000L, or 4500L to 5000L. In some embodiments, the affinity eluate is produced by affinity purification of a rAAV vector produced in a vessel (e.g., SUB) having a volume of 1mL to 5000L, 100mL to 4000L, 100mL to 2000L, 100mL to 1000L, 1L to 5000L, 1L to 4000L, 1L to 2000L, 1L to 1000L, 500mL to 5000L, 500mL to 2000L, or 500mL to 1000L.
In some embodiments, diluting the solution comprising the rAAV vector to be purified (e.g., the affinity eluate) comprises diluting the solution about 2 to 25-fold or about 5 to 20-fold, or about 10 to 20-fold (e.g., about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 20-fold, about 25-fold) to produce a diluted affinity eluate. In some embodiments, diluting the solution comprising the rAAV vector to be purified (e.g., an affinity eluate) comprises diluting the solution about 2-fold. In some embodiments, diluting the solution comprising the rAAV vector to be purified (e.g., an affinity eluate) comprises diluting the solution about 15-fold.
In some embodiments, diluting the solution comprising the rAAV vector to be purified (e.g., an affinity eluate) is performed "in-line" with the column, wherein the diluted solution (diluent) is delivered to the Y-connector through a first tubing and the solution comprising the rAAV vector to be purified is delivered to the Y-connector through a second tubing, and optionally wherein a static mixer is contained within a third tubing located after the Y-connector.
In some embodiments, diluting the solution comprising the rAAV vector to be purified (e.g., an affinity eluate) is performed "in series" and introduced into a containment vessel (e.g., a break tank). For example, a dilute solution (diluent) is delivered to the Y-connector via a first tubing and a solution comprising the rAAV vector to be purified is delivered to the Y-connector via a second tubing, wherein the end of the Y-connector is connected to a containment vessel optionally connected to a chromatography column (e.g., an AEX column).
In some embodiments, diluting comprises delivering a diluted solution through a first tubing to a Y-connector at a flow rate of 1 to 5mL/min (e.g., about 3.5 mL/min), and delivering a solution comprising the rAAV carrier to be purified (e.g., affinity eluate) through a second tubing at a flow rate of 0.1 to 2mL/min (e.g., about 0.25 mL/min).
In some embodiments, diluting includes delivering the diluting solution through the first tubing to the Y-connector at a flow rate of about 3.5mL/min and delivering the affinity eluate through the second tubing at a flow rate of about 0.25mL/min, thereby diluting the affinity eluate by a factor of about 15.
In some embodiments, diluting comprises diluting the solution comprising the rAAV vector to be purified (e.g., an affinity eluate) with a dilution solution comprising a buffer (Tris, BIS-Tris propane, diethanolamine, diethylamine, tris (hydroxymethyl) methylglycine, triethanolamine and/or n, n-di (hydroxyethyl) glycine). In some embodiments, a solution comprising the rAAV vector to be purified (e.g., an affinity eluate) is diluted with a dilution solution comprising 10mM to 500mM buffer (e.g., tris). In some embodiments, the diluted 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, or about 150mM to about 250mM Tris. In some embodiments, the diluted solution comprises about 200mM Tris.
In some embodiments, the diluted solution comprises an amino acid, such as histidine, arginine, glycine, or citrulline. In some embodiments, the diluted solution comprises about 50mM, about 75mM, about 100mM, about 125mM, about 150mM, about 175mM, about 200mM, about 225mM, about 250mM, about 275mM, about 300mM, about 350mM, about 400mM, about 450mM, about 500mM, about 550mM, or about 600mM of an amino acid (e.g., histidine, arginine, glycine, or citrulline).
In some embodiments, the diluted solution comprises an amino acid, such as histidine or arginine. In some embodiments, the diluted solution comprises 10mM to 600mM amino acids (e.g., histidine, arginine, glycine, or citrulline). In some embodiments, the equilibration buffer comprises from about 10mM to about 600mM, from about 10mM to about 550mM, from about 10mM to about 500mM, from about 10mM to about 450mM from about 10mM to about 400mM, from about 10mM to about 350mM, from about 10mM to about 300mM, from about 50mM to about 600mM, from about 50mM to about 550mM, from about 50mM to about 500mM, from about 50mM to about 450mM, from about 50mM to about 400mM, from about 50mM to about 350mM, from about 50mM to about 300mM, from about 100mM to about 600mM, from about 100mM to about 500mM, from about 100mM to about 400mM, from about 100mM to about 300mM, or from about 150mM to about 250mM amino acids (e.g., histidine). In some embodiments, the diluted solution comprises about 200mM histidine.
In some embodiments, the diluted solution comprises a detergent, such as P188, triton X-100, polysorbate 80, brij-35 or NP-40. In some embodiments, the diluted solution comprises 0.005% to 1.5% detergent (e.g., P188). In some embodiments, the diluted solution comprises 0.1% to 1.0% detergent (e.g., P188). In some embodiments, the diluted solution comprises from about 0.01% to about 1.5%, from about 0.01% to about 1.0%, from about 0.01% to about 0.75%, from about 0.05% to about 1.5%, from about 0.05% to about 1.0%, from about 0.05% to about 0.75%, from about 0.1% to about 1.5%, from about 0.1% to about 1.0%, from about 0.1% to about 0.75%, or from about 0.25% to about 0.75% detergent (e.g., P188). In some embodiments, the diluted solution comprises about 0.5% P188.
In some embodiments, the diluted solution has a pH of 8 to 10. In some embodiments, the diluted solution has a pH of 8.5 to 9.5. In some embodiments, the diluted solution has a pH of 8.7 to 9.0. In some embodiments, the pH of the diluted 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 diluted solution is about 8.8. In some embodiments, the pH of the diluted solution is about 8.9. In some embodiments, the pH of the diluted solution is about 9.0.
In some embodiments, diluting comprises diluting the solution comprising the rAAV vector to be purified (e.g., an affinity eluate) with a buffer selected from the group consisting of: 20mM Tris, pH 9;1M Tris Base, pH 11;100mM Tris, pH 9;100mM Tris, 0.01% P188, pH 9;100mM Tris, 0.1% P188, pH 9;100mM Tris, 1.0% P188, pH 9;1M Tris, pH 9;150mM acetate, 100mM glycine, 25mM MgCl 2 pH 4.2;5mM arginine, 2mM MgCl 2 0.1% P188, 100mM Tris, pH 8.9;50mM arginine, 2mM MgCl 2 0.1% P188, 100mM Tris, pH 9;500mM arginine, 2mM MgCl 2 0.1% P188, 400mM Tris, pH 9.1;200mM glycine, 5mM MgCl 2 200mM Tris, pH 8.9;200mM histidine, 200mM Tris, pH 8.9;200mM histidine, 200mM Tris, 5mM MgCl 2 pH 8.9;200mM histidine, 200mM Tris, 5mM MgCl 2 5% glycerol, pH 8.9;200mM histidine, 250mM Tris, 10mM MgCl 2 25% glycerol, pH 8.9;200mM histidine, 200mM Tris, 5mM MgCl 2 5% iodixanol pH 8.8;200mM histidine, 200mM Tris, 10mM MgCl 2 25% iodixanol, pH 8.8;200mM histidine, 200mM Tris, 0.5% Triton X-100, pH 8.9;200mM histidine, 200mM Tris, 0.5% P188, pH 8.8; and combinations thereof.
In some embodiments, diluting comprises diluting a solution comprising the rAAV vector to be purified (e.g., an affinity eluate) with a buffer comprising about 20mM Tris, pH 9, about 1M Tris base, pH 11, or both. In some embodiments, diluting comprises diluting the solution comprising the rAAV vector to be purified (e.g., the affinity eluate) 7-to 8-fold (e.g., about 7.1-fold) with a buffer comprising about 20mM Tris, pH 9, about 1M Tris base, pH 11, or both.
In some embodiments, diluting comprises diluting the solution comprising the rAAV vector to be purified (e.g., an affinity eluate) with a buffer comprising 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., 0.5%) P188, pH 8.7 to 9.0. In some embodiments, diluting comprises diluting the solution containing the rAAV vector to be purified (e.g., the affinity eluate) 10-to 20-fold (e.g., about 15-fold) with a buffer comprising about 200mM histidine, 200mM Tris, 0.5% p188, pH 8.7 to 9.0 (e.g., about 8.8). In some embodiments, diluting comprises diluting the affinity eluate comprising the rAAV vector to be purified 14.4 to 15.5 times (e.g., about 15 times) with a buffer comprising about 200mM histidine, 200mM Tris, 0.5% P188, pH 8.7 to 9.0 (e.g., about pH 8.8), thereby forming a diluted affinity eluate.
In some embodiments, the solution comprising the rAAV vector to be purified (e.g., an affinity eluate) is incorporated into 20mM MgCl prior to diluting the solution 2 So that MgCl in the diluted solution 2 Is about 1.7mM. In some embodiments, mgCl 2 Stabilizing the rAAV vector in solution.
In some embodiments, filtering comprises filtering the solution comprising the rAAV vector to be purified (e.g., an affinity eluate, diluted affinity eluate) prior to loading the solution to the AEX column. In some embodiments, the filter is pre-wetted with water for injection and/or a dilution solution prior to filtration. In some embodiments, filtering comprises filtering a solution comprising the rAAV vector to be purified (e.g., an affinity eluate, diluted affinity eluate) through a filter that collects aggregates, such as nucleic acid or protein aggregates or other high molecular weight species, but allows AAV capsids 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 comprises filtering the diluted affinity eluate comprising the rAAV vector to be purified through a 0.2 μm filter before loading to the AEX column. The filter used to filter the solution comprising the rAAV vector to be purified (e.g., affinity eluate, diluted affinity eluate) may be separate from the column or may be in series with the column or chromatographic instrument (also referred to as chromatography skin).
In some embodiments, filtering comprises filtering the diluted affinity eluate comprising the rAAV vector to be purified through a series of 0.2 μm filters before loading the eluate to the AEX column.
In some embodiments, the pH of the solution comprising the rAAV vector to be purified (e.g., the affinity eluate) is 3.0 to 4.4 prior to dilution and optionally filtration, and the pH of the solution comprising the rAAV vector to be purified (e.g., the affinity eluate) is 8.5 to 9.5, 8.7 to 9.0, or ≡8.6 (e.g., about pH 8.8, pH 9.0) after dilution and optionally filtration.
In some embodiments, the solution comprising the rAAV vector to be purified (e.g., the affinity eluate) has a conductivity of 5.0mS/cm to 7.0mS/cm (e.g., about 5.5mS/cm to 6.5 mS/cm) prior to dilution and optionally filtration, and the solution comprising the rAAV vector to be purified (e.g., the affinity eluate) has a conductivity of 1.7mS/cm to 3.5mS/cm, 1.8mS/cm to 2.8mS/cm, 2.2mS/cm to 2.6mS/cm, or +.2.5 mS/cm after dilution and optionally filtration. In some embodiments, the affinity eluate has a conductivity of about 1.8mS/cm to about 2.8mS/cm after dilution and optional filtration. In some embodiments, the affinity eluate has a conductivity of about 2.3+/-0.5mS/cm after dilution and optional filtration.
As used herein, the term "percent VG dilution yield" or "% VG dilution yield" refers to the percentage of the amount of VG present in a diluted affinity cell (also referred to herein as diluted affinity eluate) to the amount of VG present in the affinity cell (also referred to herein as affinity eluate) prior to dilution. For example,% VG dilution yield= ((amount of VG in diluted affinity cell)/(amount of VG in affinity cell)) = ((amount of VG in diluted affinity cell)) =.
In some embodiments, the percentage of VGs recovered in the diluted and optionally filtered solution (e.g., affinity eluate) comprising the rAAV vector to be purified (the% VG dilution yield) is 60% to 100% of the VGs present in the solution (e.g., affinity eluate) prior to dilution and optionally filtration. In some embodiments, the% VG yield of a diluted and optionally filtered solution (e.g., affinity eluate) comprising 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 optionally filtration. In some embodiments, the% VG yield of a diluted and optionally filtered solution (e.g., affinity eluate) comprising the rAAV vector to be purified 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 optionally filtration.
In some embodiments of diluting the affinity eluate according to the methods of the present disclosure, the% VG dilution yield is 88% +/-36%. In some embodiments of diluting the affinity eluate according to the methods of the present disclosure, the% VG dilution yield is 120% +/-12%. Dilution of the affinity eluate obtained by affinity chromatography purification of the rAAV vector produced in 250L SUB resulted in a dilution yield of%vg of 35% to 100% (e.g. 41% to 92%). Dilution of the affinity eluate obtained by affinity chromatography purification of rAAV vectors produced in 2000L SUB resulted in a dilution yield of%vg of 70% to >100% (e.g. 88% to 154%).
In some embodiments, the Z-average (given in nm and determined by Dynamic Light Scattering (DLS)) of a diluted and optionally filtered solution (e.g., affinity eluate) comprising the rAAV vector to be purified is measured. The Z average measures the aggregation level of rAAV capsids present in the solution. In some embodiments, the diluted and optionally filtered solution comprising the rAAV vector to be purified has a Z average of about 15nm to 40nm, 15nm to 20nm, 20nm to 30nm, or 30nm to 40nm. In some embodiments, the Z-average value of the diluted and optionally filtered solution comprising 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 of purifying a rAAV (e.g., rAAV9, rAAV3B, etc.) vector from a solution (e.g., an affinity eluate) by AEX comprising diluting the solution 14-16-fold (e.g., about 15-fold) with a buffer comprising 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, pH 8.7 to 9.0 (e.g., about pH 8.8); and optionally including filtration, including 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 an electrical conductivity of 1.8mS/cm to 2.8 mS/cm.
A method of preparing an affinity eluate comprising a rAAV vector purified by AEX chromatography, as disclosed herein, the method comprising i) diluting the affinity eluate 2 to 25-fold (e.g., about 15-fold) with a buffer comprising 200mM histidine, 200mM 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 reduced 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 a rAAV vector produced in a vessel (e.g., SUB) having a volume of 250L or 2000L.
Loading
Methods of purifying rAAV (e.g., rAAV9, rAAV3B, etc.) vectors by AEX disclosed herein include loading a solution comprising a substance to be purified (e.g., rAAV vector) into a column of an AEX stationary phase. The load may be applied to the column by gravity feed or pumped to the chromatography column. In some embodiments, the solution comprising the rAAV vector to be purified by AEX is selected from the group consisting of an affinity eluate, a supernatant from a cell lysate, and a post-harvest solution, each of which is subjected to at least one other purification or treatment step (e.g., cell lysis, flocculation, filtration, dilution, pH adjustment, chromatography). The solution comprising the rAAV vector to be purified may be diluted, filtered, and/or pH adjusted prior to loading the solution onto the AEX column to render the solution compatible with processing through the AEX column. In some embodiments, the solution comprising the rAAV vector to be purified is an eluate produced by affinity chromatography purification of the rAAV vector produced in a 100L to 500L (e.g., about 250L), 1000L to 3000L (e.g., about 2000L), or larger vessel (e.g., disposable bioreactor (SUB)), wherein the eluate has been diluted and filtered.
In some embodiments, loading comprises applying diluted and optionally for the AEX column A filtered solution (e.g., an affinity eluate) comprising about 2.0X10 12 Vector Genome (VG)/mL to 2.0X10 15 VG/mL, e.g. 2.0X10 12 VG/mL to 2.0X10 13 VG/mL、2.0×10 13 VG/mL to 2.0X10 14 VG/mL、1.0×10 14 VG/mL to 3.0X10 14 VG/mL、2.0×10 14 VG/mL to 2.0X10 15 VG/mL or more column volumes (also referred to as "column challenge VG/mL resin") were measured by qPCR analysis of sequences within the vector genome. In some embodiments, loading includes loading the memory will contain 6.3X10 13 To 9.4X10 13 The VG/mL column volume of diluted solution (e.g., affinity eluate) is applied to about 30mL to 70mL AEX column as measured by qPCR analysis of the transgene sequence in the vector genome (e.g., where the transgene is an ATP7B transgene). In some embodiments, loading includes loading will include 5×10 13 To 1.3X10 14 The diluted and optionally filtered solution (e.g., affinity eluate) of the VG/mL column volume is applied to about a 1.3L AEX column as measured by qPCR analysis of ITR sequences within the vector genome. In some embodiments, loading includes loading will contain 2.6X10 12 To 6.8X10 13 The diluted and optionally filtered solution (e.g., affinity eluate) of the VG/mL column volume is applied to an about 6.4L AEX column as measured by qPCR analysis of the transgene sequence within the vector genome.
In some embodiments, loading includes loading will contain 2.5X10 15 VG/L to 2.5X10 16 VG/L、2.5×10 16 VG/L to 2.5X10 17 VG/L、2.5×10 15 VG/L to 3.0X10 17 A diluted and optionally filtered solution of VG/L or more column volumes (e.g., affinity eluate) is applied to the AEX column.
In some embodiments, loading includes loading the memory will include 8.0X10 12 Total VG to 2.0X10 18 Total VG, e.g. 8.0X10 12 Total VG to 8.0X10 13 Total VG, 8.0X10 13 Up to 8.0X10 14 Total VG, 8.0X10 14 Total VG to 8.0X10 15 Total VG, 8.0X10 15 Total VG to 8.0X10 16 Total VG, 8.0X10 16 Total VG to 8.0X10 17 Total VG, 8.0X10 17 Total VG to 2.0X10 18 A diluted and optionally filtered solution of total VG or more (e.g. affinity eluate) is applied to the AEX column. In one embodiment, loading includes loading the memory with a content of 15×10 or less 16 A diluted and optionally filtered solution of VG/L column volume (e.g. affinity eluate) is applied to the AEX column, and optionally wherein VG is measured by quantitative polymerase chain reaction (qPCR) analysis of the transgene.
When a solution (e.g., affinity eluate) comprising the rAAV vector to be purified is loaded into a column, the solution flows through the column stationary phase at a particular rate (e.g., cm/hr, mL/min) and is contacted with the stationary phase for a particular period of time (i.e., residence time).
In some embodiments, the residence time of the solution comprising the rAAV vector loaded onto the column is 0.1min/CV to 5min/CV, e.g., 0.1min/CV to 1.0min/CV, 1.0min/CV to 2min/CV, 2min/CV to 3min/CV, 3min/CV to 4min/CV, 4min/CV to 5min/CV, or longer. In some embodiments, the residence time of the solution comprising the rAAV vector loaded onto the column is about 0.5min/CV. In some embodiments, the residence time of the solution comprising the rAAV vector loaded onto the column is about 1.5min/CV. In some embodiments, the residence time of the solution comprising the rAAV vector loaded onto the column is about 2.0min/CV. In some embodiments, the residence time of the solution comprising the rAAV vector loaded onto the column is 3.5min/CV to 4.5min/CV. In some embodiments, the residence time of the diluted and/or filtered affinity eluate comprising the rAAV vector loaded on a 6.0L to 6.6L (e.g., about 6.4L) AEX column is 3.0min/CV to 5.0min/CV (e.g., about 4 min/CV).
In some embodiments, the solution comprising the rAAV carrier is loaded onto the column at a linear velocity of 100cm/hr to 1800cm/hr, e.g., 100cm/hr to 200cm/hr, 200cm/hr to 400cm/hr, 400cm/hr to 600cm/hr, 600cm/hr to 800cm/hr, 800cm/hr to 1000cm/hr, 1000cm/hr to 1500cm/hr, 1500cm/hr to 1800cm/hr. In some embodiments, the linear velocity of loading the solution comprising the rAAV vector onto the column is 270cm/hr to 330cm/hr (e.g., about 298cm/hr, about 300 cm/hr). In some embodiments, the linear velocity of loading the solution comprising the rAAV vector onto the column is about 300cm/hr, about 600cm/hr, about 611cm/hr, or about 1790cm/hr. In some embodiments, the linear velocity of loading the diluted and optionally filtered affinity eluate comprising the rAAV vector onto a 6.0L to 6.6L (e.g., about 6.4L) AEX column is 270cm/hr to 330cm/hr (e.g., about 300 cm/hr).
In some embodiments, the solution comprising the rAAV vector is loaded onto the column at a flow rate of 1.0mL/min to 3.0L/min, e.g., 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 solution comprising the rAAV vector is loaded onto the column at a flow rate of about 1.28mL/min. In some embodiments, the solution comprising the rAAV vector is loaded onto the column at a flow rate of about 314mL/min. In some embodiments, the solution comprising the rAAV vector is passed through the stationary phase in the column at a flow rate of 1.5L/min to 2.0L/min. In some embodiments, the solution comprising the rAAV vector is loaded onto the column at a flow rate of about 1.8L/min. In some embodiments, the diluted and/or filtered affinity eluate comprising the rAAV vector is loaded onto a 6.0L to 6.6L (e.g., about 6.4L) column at a flow rate of 1.5L/min to 2.0L/min (e.g., about 1.8L/min).
In some embodiments, the method of 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 to a column comprising an AEX stationary phase, wherein the AEX stationary phase has been washed, sterilized, washed and/or equilibrated prior to loading, and optionally wherein the AEX stationary phase is POROS TM 50HQ。
In some embodiments, the method of purifying a rAAV vector (e.g., rAAV9, rAAV3B, etc.) from an affinity eluate comprises: i) Diluting the affinity eluate 14.4 to 15.5 times (e.g., about 15 times) with a buffer containing about 100mM to 300mM (e.g., about 200 mM) histidine, 100mM to 300mM (e.g., about 200 mM) Tris, 1.0% to 1.5% (e.g., about 0.5%) P188, pH 8.7 to 9.0; ii) any one ofOptionally filtering the diluted affinity eluate through a series of 0.1 to 0.45 μm (e.g., about 0.2 μm) filters; and iii) loading the diluted and filtered affinity eluate to a column comprising an AEX stationary phase; optionally, wherein at least one step is performed through the column and/or a residence time of 1.5min/CV to 4.5min/CV (e.g., about 2min/CV, about 4 min/CV) at a linear velocity of 270cm/hr to 330cm/hr (e.g., about 298cm/hr, about 300 cm/hr), a flow rate of 1.5L/min to 2.0L/min (e.g., about 1.8L/min), and optionally, wherein the AEX stationary phase is POROS TM 50HQ. In some embodiments, the chromatographic column is a 6.0L to 6.6L (e.g., about 6.4L) column.
In some embodiments, the invention provides a method of purifying a rAAV (e.g., rAAV9, rAAV3B, etc.) vector by AEX, the method comprising the steps of: i) A pre-use rinse comprising applying ≡4.5CV (e.g. about 5 CV) of water for injection to the AEX stationary phase in the column; ii) sterilization comprising applying a 5CV to 10CV (e.g., about 8 CV) or 14.4 to 17.6CV (e.g., about 16 CV) 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 a solution comprising 1M to 3M (e.g., about 2M) NaCl, 50mM to 150mM (e.g., about 100 mM) Tris, pH8.5 to 9.5 (e.g., about 9) of 4.5CV to 5.5CV (e.g., about 5 CV) to the AEX stationary phase in the column; iv) equilibration, comprising applying a solution comprising 50mM to 150mM (e.g., about 100 mM) Tris, pH8.5 to 9.5 (e.g., about 9) of 4.5CV to 5.5CV (e.g., about 5 CV) to the AEX stationary phase in the column; v) equilibration, comprising applying an equilibration buffer comprising 50mM to 150mM (e.g., about 100 mM) Tris, 400mM to 600mM (e.g., about 500 mM) sodium acetate, 0.005% to 0.015% (e.g., about 0.01%) P188, pH8.5 to 9.0 (e.g., about 8.9) to the AEX stationary phase in the column, of 4.5CV to 5.5CV (e.g., about 5 CV); vi) equilibration, comprising applying an equilibration buffer comprising 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, ph8.5 to 9.5 (e.g., about 8.8) to the AEX stationary phase in the column; vii) loading the affinity eluate into the AEX stationary phase in the column, optionally wherein the eluate has been a) purified using a solid phase comprising 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, pH8.7 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 0.1 μm to 0.45 μm (e.g., about 0.2 μm) filters prior to application to the stationary phase; and/or viii) equilibration, comprising applying an equilibration buffer comprising 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., about 8.9) to the AEX stationary phase in the column, at 4.5CV to 5.5CV (e.g., about 5 CV); optionally wherein at least one of steps i) to viii) is performed at a linear velocity of 270cm/hr to 330cm/hr (e.g., about 298cm/hr, about 300 cm/hr) and/or a residence time of 1.5min/CV to 4.5min/CV (e.g., about 2min/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) through vii) is passed through a 6L to 6.6L column (e.g., about 6.4L) at a flow rate of 1.5L/min to 2.0L/min (e.g., about 1.8L/min) or through a 1.3L column at a flow rate of about 314 mL/min. The order of the above steps may be varied as will be appreciated by the skilled artisan.
Loading the additional solution
A method of purifying a rAAV vector (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., an affinity eluate) includes applying a loading chase solution to a column stationary phase after applying the solution comprising the rAAV vector. The loading of the chase solution is used to complete the loading or application of the loading solution and remove unbound material from the column. In some embodiments, a chase solution is loaded for removing unbound material from the column. In some embodiments, the loading chase solution comprises 5mM to 50mM (e.g., about 20 mM) Tris, pH 8.5 to 9.5 (e.g., about 9). In some embodiments, 9 to 11CV (e.g., about 10 CV) of loading chase solution is applied to the column stationary phase. In some embodiments, the loading of the chase solution is applied to the column stationary phase at a rate of 200cm/hr to 2000cm/hr (e.g., about 1800 cm/hr) and/or a residence time of 0.5 min/CV. In some embodiments, a loading chase solution comprising 20mM Tris, pH 9, of 9CV to 11CV (e.g., about 10 CV) is applied to the AEX stationary phase in the column, optionally at a rate of 200cm/hr to 2000cm/hr (e.g., about 1800 cm/hr) and/or about 0.5min/CV residence time.
Gradient elution
Methods of purifying rAAV vectors (e.g., rAAV9, rAAV3B, etc.) from solutions (e.g., affinity eluents) include recovery of complete, intermediate, and/or empty capsids by gradient elution. Gradient elution may involve the use of at least 2 different solutions (e.g., gradient elution buffers) having different pH, conductivity, and/or modifier concentrations. During gradient elution, the percentage of the first solution changes in inverse proportion to the percentage change of the second solution, such that when the solutions mix and flow through the column stationary phase, a gradient of pH, conductivity, and/or modifier concentration is created. For example, at the beginning of the gradient elution, the percentage of the first solution (e.g., first gradient elution buffer, buffer a) is 100%, while the percentage of the second solution (e.g., second gradient elution, buffer B) is 0%, and at the end of the gradient elution, the percentage of the first solution is 0%, 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. first gradient elution buffer, buffer a) is 100%, the percentage of the second solution (e.g. second gradient elution, buffer B) is 0%, and at the end of the gradient elution the percentage of the first solution is 25%, the percentage of the second solution is 75%. The skilled artisan will appreciate that the percentage of each solution at the beginning of the gradient and at the end of the gradient may 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 the elution, at the end of the elution, or at any point during the 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 the elution, at the end of the elution, or at any point during the 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 application of 10 to 60CV of solution to the column, the percentage of buffer a (e.g., first gradient elution buffer) decreases and the percentage of buffer B (e.g., second gradient elution buffer) increases such 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 application of about 20CV of solution to the column, the percentage of buffer a (e.g., first gradient elution buffer) decreases, the percentage of buffer B (e.g., second gradient elution buffer) increases, such that the rate of increase of buffer B is 5% buffer B/CV, and the final percentage of buffer B in solution is 100%. In some embodiments, during application of about 37.5CV of solution to the column, the percentage of buffer a (e.g., first gradient elution buffer) decreases, the percentage of buffer B (e.g., second gradient elution buffer) increases, such that the rate of increase of buffer B is about 2% buffer B/CV, and such that the final percentage of buffer B in solution is 75%.
In some embodiments, during the application of 10 to 60CV of solution to the column, the percentage of buffer a (e.g., first elution buffer) increases and the percentage of buffer B (e.g., 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%. Those skilled in the art will recognize 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, methods of purifying a rAAV vector by AEX of the present disclosure include gradient elution of material from a stationary phase in a column, wherein the concentration of a component of the first gradient elution buffer or the second gradient elution buffer continuously increases or decreases during the gradient elution. In some embodiments, the material eluted from the stationary phase comprises the rAAV vector to be purified. The rate of increase or decrease in the concentration of the component of the first gradient elution buffer or the second gradient elution buffer may correspond to a change in the concentration of the component/total CV. In some embodiments, the rate of increase in sodium acetate concentration during gradient elution corresponds to the change in sodium acetate concentration/total CV applied to the stationary phase during elution. In some embodiments, the change in concentration of the component is relative to the concentration of the component at the beginning of the elution compared to the concentration of the component at the end of the elution. For example, the concentration of the component (e.g., salt such as sodium acetate) at the beginning of the gradient elution is 0mM to 100mM, and the concentration of the component at the end of the elution is 100mM to 1M. In some embodiments, the concentration of salt (e.g., sodium acetate) at the beginning of the gradient elution is 0mM and the concentration of salt at the end of the gradient elution is 400mM to 600mM (e.g., about 500 mM). In some embodiments, the component concentration varies from 2mM to 1M from the beginning of the gradient to the end of the gradient elution during the application of 2CV to 100CV elution buffer. In some embodiments, the salt concentration varies from about 0mM to about 500mM during the application of 10CV to 60CV, 10CV to 50CV, 10CV to 40CV, 10CV to 30CV, or 15CV to 25CV (e.g., 20 CV) elution buffer from the beginning of gradient elution to the end of gradient elution, so that the rate of change of sodium acetate concentration is about 500Mm/20CV or 25mM/CV when the elution gradient comprises a 20CV solution. In some embodiments, the salt concentration varies from about 0mM to about 375mM during elution with 10CV to 60CV, 10CV to 50CV, 10CV to 40CV, 10CV to 30CV, or 15CV to 25CV (e.g., 37.5 CV) elution buffer from the beginning of the gradient elution to the end of the gradient elution, so the rate of change of sodium acetate concentration is about 375mM/37.5CV or 10mM/CV when the elution gradient comprises a solution of 37.5 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 both increases continuously during gradient elution; wherein the rate of increase of sodium acetate is equal to the change in sodium acetate concentration/total CV applied to the stationary phase; wherein the concentration of sodium acetate during the gradient elution varies at a rate of 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 25 mM/CV).
In some embodiments, the component concentration during gradient elution varies from about 1mM/CV to 1M/CV, such as from 1mM/CV to 10mM/CV, from 1mM/CV to 25mM/CV, from 5mM/CV to 15mM/CV, from 10mM/CV to 50mM/CV, from 50mM/CV to 100mM/CV, from 100mM/CV to 500mM/CV, from 500mM/CV to 1M/CV, from 1mM/CV to 750mM/CV, from 1mM/CV to 500mM/CV, from 1mM/CV to 100mM/CV, from 10mM/CV to 750mM/CV, or from 50mM/CV to 500mM/CV.
In some embodiments, the concentration of salt in the gradient solution may vary during the gradient elution. In some embodiments, during gradient elution, the concentration of salts (e.g., sodium chloride, sodium acetate, ammonium acetate, magnesium chloride, sodium sulfate, and combinations thereof) in the gradient solution may be increased or decreased. For example, at the beginning of the gradient elution, the salt concentration in the gradient solution may be 0mM to 100mM and increased to 50mM to 1M during the elution, 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, 600mM to 700mM, 700mM to 800mM, 800mM to 900mM,900mM to 1M, 50mM to 750mM, 50mM to 500mM, 50mM to 400mM, 50mM to 200mM, 100mM to 1M, 100mM to 750mM, 100mM to 500mM, 100mM to 400mM, or 100mM to 200mM. In further examples, at the beginning of the gradient elution, the salt concentration in the gradient solution may be 50mM to 1M, for example 50mM to 100mM, 100mM to 150mM, 150mM to 200mM, 200mM to 250mM, 250mM to 300mM, 300mM to 400mM, 400mM to 500mM, 500mM to 600mM, 600mM to 700mM, 700mM to 800mM, 800mM to 900mM,900mM to 1M, 50mM to 750mM, 50mM to 500mM, 50mM to 400mM, 50mM to 200mM, 100mM to 1M, 100mM to 750mM, 100mM to 500mM, 100mM to 400mM, or 100mM to 200mM, and reduced to 0mM to 100mM during the gradient elution. In some embodiments, the concentration of sodium acetate in the gradient solution is about 0mM at the beginning of the gradient elution and about 500mM at the end of the gradient elution. In some embodiments, the concentration of sodium acetate in the gradient elution solution is about 0mM at the beginning of the gradient elution and about 375mM at the end of the gradient elution.
In some embodiments, the pH of the gradient solution may vary during the gradient elution. In some embodiments, the pH of the gradient solution may be increased or may be decreased during the gradient elution process. In some embodiments, at the beginning of the gradient elution, the pH of the gradient solution may 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 may 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, the conductivity of the gradient solution may vary during the gradient elution. In some embodiments, the conductivity of the gradient solution may increase or decrease during the gradient elution. In some embodiments, at the beginning of the gradient elution, the conductivity of the gradient solution may 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 the gradient elution, the conductivity of the gradient solution may be between 20mS/cm and 35mS/cm, e.g., 27mS/cm and 33mS/cm. In some embodiments, the conductivity of the gradient solution is about 1.6mS/cm at the beginning of the gradient elution and about 30mS/cm at the end of the gradient elution.
In some embodiments, the concentration of buffer in the gradient solution may vary during the gradient elution. In some embodiments, during gradient elution, the concentration of buffer (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-di (hydroxyethyl) glycine) in the gradient solution can be increased or decreased. For example, at the beginning of the gradient elution, the concentration of the buffer in the gradient solution may be in the range of 10mM to 500mM, such as from 10mM to 400mM, from 10mM to 300mM, from 10mM to 200mM, from 10mM to 50mM, from 50mM to 100mM, from 50mM to 150mM, from 100mM to 200mM, from 100mM to 400mM, from 200mM to 300mM, from 300mM to 400mM, from 400mM to 500mM or more. At the end of the gradient elution, the concentration of buffer in the gradient solution may be between 10mM and 500mM, for example from 10mM to 400mM, from 10mM to 300mM, from 10mM to 200mM, from 10mM to 50mM, from 50mM to 100mM, from 50mM to 150mM, from 100mM to 200mM, from 100mM to 400mM, from 200mM to 300mM, from 300mM to 400mM, from 400mM to 500mM, or more.
In some embodiments, the concentration of the detergent in the gradient solution may vary during the gradient elution process. In some embodiments, during gradient elution, the concentration of a detergent (e.g., poloxamer 188 (P188), triton X-100, polysorbate 80 (PS 80), brij-35, nonylphenoxy polyethoxy ethanol (NP-40), and combinations thereof) in the gradient solution may 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 may be in the range of 0.005% to 1.0%, e.g., 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%.
In some embodiments, at the end of the gradient elution, the concentration of the detergent (e.g., P188) in the gradient solution may 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 1.0.0%, from 0.5% to 1.0%.
During gradient elution, 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 remain constant throughout the gradient elution process (e.g., pH of 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 of buffer (e.g., tris, BIS-Tris propane, n, n-BIS (hydroxyethyl) glycine, and combinations thereof) in the gradient elution ranges from 10mM to 500mM, such as 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 100 mM) during the gradient elution. In some embodiments, the concentration of buffer such as Tris in the gradient elution is 50mM to 150mM. In some embodiments, the concentration of buffer, such as Tris, in the gradient elution is about 100mM.
In some embodiments, in gradient elution, the concentration of a detergent such as poloxamer 188 (P188), triton X-100, polysorbate 80 (PS 80), brij-35, nonylphenoxy polyethoxy ethanol (NP-40), and combinations thereof may 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 0.04%, 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%, and from 0.0.03% to 0.0.04%, but is constant during the gradient elution. 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, the substance 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 comprising the capsid to be purified, the AAV capsid (e.g., complete capsid, intermediate capsid, empty capsid) binds to the stationary phase. During gradient elution, as the percentage of gradient elution buffer increases, whereby the concentration of salt (e.g., sodium acetate) increases, the complete rAAV carrier is preferentially released (eluted) from the stationary phase and the empty capsid is preferentially retained on the stationary phase. As the percentage of gradient elution buffer further increases (and salt concentration), empty capsids are released in larger amounts. The empty capsids can also be recovered in the AEX column flow-through, i.e. unbound fraction. In some embodiments, the complete and/or intermediate capsids are recovered in a portion of the first eluting peak and the second eluting peak (e.g., the first 2/3 of the second eluting peak) of the AEX column. By measuring a260 and a280 of the eluate, the complete rAAV vector eluted from the stationary phase can be monitored during gradient elution, whereby an increase in the a260/a280 ratio indicates an increase in the complete rAAV vector present in the eluate.
In some embodiments, performing the gradient elution comprises applying about 20CV 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 wherein when buffer B comprises sodium acetate, the concentration of sodium acetate increases at a rate of 25 mM/CV.
In some embodiments, performing the gradient elution comprises applying about 37.5CV 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 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). In some embodiments, buffer B (e.g., the second gradient elution buffer) comprises about 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., about 8.9).
In some embodiments, gradient elution begins with 100% buffer a applied to the column and ends with 100% buffer B applied to the column during 20CV to 24CV (e.g., about 20 CV), thereby creating a gradient between buffer a and buffer B during the elution phase, wherein buffer a comprises about 100mM Tris, 0.01% p188, pH 8.9, and buffer B comprises about 500mM sodium acetate, 100mM Tris, 0.01% p188, pH 8.9. In some embodiments, the gradient elution begins with the application of 100% buffer a to the column, ending with the application of 75% buffer B and 25% buffer a to the column during the application of 30CV to 40CV (e.g., about 37.5 CV), thereby creating a gradient between buffer a and buffer B during the elution phase, wherein buffer a comprises about 100mM Tris, 0.01% p188, pH 8.9, and buffer B comprises about 500mM sodium acetate, 100mM Tris, 0.01% p188, pH 8.9.
In some embodiments, the gradient elution buffer comprises 5mM to 40mM (e.g., about 20 mM) Tris, pH9.0. In some embodiments, the gradient elution buffer comprises 5mM to 40mM (e.g., about 20 mM) Tris, 400mM to 600mM (e.g., about 500 mM) salts (e.g., naCl, sodium acetate, ammonium acetate, and Na) 2 SO 4 ) pH9.0. In some embodiments, the gradient elution buffer comprises about 20mM Tris, 500mM sodium acetate, pH9.0.
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 0.1min/CV to 15min/CV, such as 0.1min/CV to 1min/CV, 1min/CV to 2min/CV, 1.5min/CV to 2.5min/CV, 2min/CV to 4min/CV, 4min/CV to 6min/CV, 6min/CV to 8min/CV, or 8min/CV to 10min/CV, 10min/CV to 12min/CV, 12min/CV to 15min/CV. In some embodiments, the residence time of the solution in the column is 0.1min/CV, about 0.5min/CV, about 1.5min/CV, about 2.0min/CV, about 2.5min/CV, about 3min/CV, about 3.6min/CV or about 4min/CV, about 5min/CV, about 6min/CV, about 7min/CV, about 8min/CV, about 9min/CV or about 10min/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 3.6min/CV or 4min/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 2.0min/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 2.5min/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 4.5min/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 11min/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 1800cm/hr, e.g., 50cm/hr to 100cm/hr, 100cm/hr to 200cm/hr, 200cm/hr to 400cm/hr, 400cm/hr to 600cm/hr, 600cm/hr to 800cm/hr, 800cm/hr to 1000cm/hr, 1000cm/hr to 1500cm/hr, or 1500cm/hr to 1800cm/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 298cm/hr or about 300cm/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 75cm/hr, about 204cm/hr, about 298cm/hr, about 300cm/hr, about 597cm/hr, or about 600cm/hr. In some embodiments, the linear velocity of the AEX stationary phase in a gradient elution buffer (e.g., buffer a, buffer B, or a mixture of buffer a and buffer B) through a 6.0L to 6.6L (e.g., 6.4L) column is about 270cm/hr to about 330cm/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 314mL/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 1.8L/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 2.0L/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 the 1.3L column is about 314mL/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 1.8L/min.
In some embodiments, methods of purifying a rAAV vector (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., an affinity eluate) include applying a gradient elution buffer to a solution comprising POROS TM Column of 50HQ stationary phase. In some embodiments, the method of purifying a rAAV vector (e.g., AAV9, AAV3B, etc.) from an affinity eluate comprises starting with the application of 100% buffer a (e.g., comprising 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., about 8.9)), and ending with the application of 75% to 100% buffer B (e.g., 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., about 8.9)) to the stationary phase in the column during the application of 15 to 40CV (e.g., about 20CV, about 37.5 CV), 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 6.0L to 6.6L (e.g., 6.4L) column.
In some embodiments, the method of purifying a rAAV vector (e.g., AAV9, AAV3B, etc.) from an affinity eluate comprises performing a gradient elution beginning with the application of 100% first buffer (comprising about 100mM Tris,0.01% p188, ph 8.9), and ending with the application of 75% to 100% second buffer (comprising 500mM sodium acetate, 100mM Tris,0.01%P188,pH 8.9) to a column comprising an AEX stationary phase during the application of 15 to 40CV (e.g., about 20CV, about 37.5 CV), with a linear velocity of 270cm/hr to 330cm/hr (e.g., about 298cm/hr, about 300 cm/hr), a flow rate of 1.5L/min to 2.0L/min (e.g., about 1.8L/min), and/CV with a residence time of 1.5min/CV to 4.5min/CV (e.g., 2min/CV,4 min/CV), whereby a gradient is created between the first buffer and the second buffer during the elution, wherein the percentage of buffer B varies from 2% to 5% buffer B/CV during the elution. In some embodiments, the column is a 6.0L to 6.6L (e.g., 6.4L) column.
In some embodiments, the invention provides a method of purifying a rAAV (e.g., rAAV9, rAAV3B, etc.) vector by AEX, the method comprising the steps of: i) Flushing prior to use, including applying ≡4.5CV (e.g., about 5 CV) of water for injection to the columnA stationary phase of AEX; ii) sterilization comprising applying a 5CV to 10CV (e.g., about 8 CV) or 14.4 to 17.6CV (e.g., about 16 CV) 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, a solution comprising 4.5 to 5.5CV (e.g., about 5 CV) containing 1M to 3M (e.g., about 2M) NaCl, 50mM to 150mM (e.g., about 100) Tris, pH8.5 to 9.5 (e.g., about 9) is applied to the AEX stationary phase in the column; iv) equilibration, comprising applying a solution comprising 50mM to 150mM (e.g., about 100 mM) Tris, pH8.5 to 9.5 (e.g., about 9) of 4.5 to 5.5CV (e.g., about 5 CV) to the AEX stationary phase in the column; v) equilibration, comprising applying 4.5 to 5.5CV (e.g., about 5 CV) equilibration buffer comprising 50mM to 150mM (e.g., about 100 mM) Tris, 400mM to 600mM (e.g., about 500 mM) sodium acetate, 0.005% to 0.015% (e.g., about 0.01%) P188, pH8.5 to 9.5 (e.g., about 8.9) to the AEX stationary phase in the column; vi) equilibration, comprising applying an equilibration buffer comprising 100mM to 300mM (e.g., about 200 mM) histidine, 100mM to 300mM (e.g., 200 mM) Tris, 0.1% to 1.0% (e.g., about 0.5%) P188, pH8.5 to 9.5 (e.g., about 8.8) to the in-column AEX stationary phase; vii) loading an affinity eluate comprising the rAAV vector to be purified into 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 to 300mM (e.g., about 200 mM) histidine, 100 to 300mM (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) filtered through a tandem 0.2 μm filter prior to application to the AEX stationary phase; viii) equilibration, comprising applying 4.5 to 5.5CV (e.g., about 5 CV) of 100% primary buffer comprising 50mM to 150mM (e.g., about 100 mM) Tris,0.005% to 0.015% (e.g., about 0.01%) P188, pH8.5 to 9.5 (e.g., about 8.9) equilibration buffer to the AEX stationary phase in the column, and/or ix) gradient elution of material from the stationary phase in the column to apply to the stationary phase 100% primary buffer comprising 50mM to 150mM (e.g., about 100 mM) Tris,0.005% to 0.015% (e.g., about 0.01%) P188, pH8.5 to 9.0 (e.g., about 8.9), beginning with application of primary buffer comprising 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, pH8.5 to 9.5 (e.g., pH 10.9) during application of 20CV to 24CV (e.g., about 20 CV) Ending with 0% of the second buffer, optionally wherein at least one of steps i) to ix) is performed at a linear speed of 270cm/hr to 330cm/hr (e.g. about 298cm/hr, about 300 cm/hr) and/or a residence time of 1.5min/CV to 4.5min/CV (e.g. about 2min/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 through a 6L to 6.6L (e.g., about 6.4L) column at a flow rate of 1.5L/min to 2.0L/min (e.g., about 1.8L/min) or through a 1.3L column at a flow rate of about 314 mL/min. In some embodiments, the material eluted from the stationary phase during gradient elution comprises the rAAV vector to be purified. The skilled person will appreciate that the order of the above steps may be varied.
Gradient retention
In some embodiments, a method of purifying a rAAV vector (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., an affinity eluate) includes applying a gradient maintenance solution to a solid phase comprising AEX (e.g., POROS TM 50 HQ) to continue the volume to ensure complete gradient formation, preferably after gradient elution. In some embodiments, the gradient hold solution comprises at least one component selected from the group consisting of salts, buffers, detergents, amino acids, and combinations thereof. In some embodiments, the gradient hold solution comprises a salt selected from the group consisting of 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-BIS (hydroxyethyl) glycine, and combinations thereof. In some embodiments, the gradient retention solution comprises a detergent selected from the group consisting of poloxamer 188 (P188), triton X-100, polysorbate 80 (PS 80), brij-35, nonylphenoxy polyethoxy ethanol (NP-40), and combinations thereof. In some embodiments, the gradient hold solution comprises a salt, a buffer, and a detergent. In some embodiments, the gradient hold solution comprises an amino acid selected from the group consisting of histidine, arginine, glycine, citrulline, and combinations thereof. In some embodiments, the gradient hold solution comprises sodium acetate, tris, and P188.
In some implementationsIn embodiments, the gradient hold solution comprises 5mM to 1M (e.g., about 500 mM) sodium acetate, 1mM to 1M (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). In some embodiments, a gradient hold 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, a gradient hold solution comprising about 500mM sodium acetate, 100mM Tris, 0.01% P188, pH 8.9 at a linear velocity of 270cm/hr to 330cm/hr (e.g., about 300 cm/hr), a flow rate of 0.4mL/min to 2.0L/min (e.g., about 1.8L/min), and/or a residence time of 3.5 to 11min/CV is applied to an AEX column stationary phase (e.g., POROS) TM 50HQ)。
Step elution
Methods of purifying rAAV vectors (e.g., rAAV9, rAAV3B, etc.) from solutions (e.g., affinity eluents) include step elution (also referred to as "isocratic elution"). In some embodiments, step elution includes applying at least one step elution solution to the column stationary phase, however more often 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 elution solution comprises at least one component selected from the group consisting of salts, buffers, detergents, amino acids, and combinations thereof. In some embodiments, the step elution solution comprises a salt selected from the group consisting of sodium chloride, sodium acetate, ammonium acetate, magnesium chloride, sodium sulfate, and combinations thereof. In some embodiments, the step elution solution comprises a buffer selected from Tris, BIS-Tris propane, n-BIS (hydroxyethyl) glycine, and combinations thereof. In some embodiments, the step elution solution comprises an amino acid selected from the group consisting of histidine, arginine, glycine, citrulline, and combinations thereof. In some embodiments, the step solution comprises a detergent selected from the group consisting of poloxamer 188 (P188), triton X-100, polysorbate 80 (PS 80), brij-35, nonylphenoxy polyethoxyethanol (NP-40), and combinations thereof. In some embodiments, the step elution solution includes a salt, a buffer, and a detergent. In some embodiments, the step elution solution comprises sodium acetate and Tris.
In some embodiments, the concentration of buffer (e.g., tris) in the step elution solution is about 1mM to 500mM, e.g., 1mM to 10mM, 10mM to 50mM, 50mM to 100mM, 100mM to 200mM, 200mM to 300mM, 300mM to 400mM, 400mM to 500mM. In some embodiments, the concentration of Tris in the step elution solution is about 20mM.
In some embodiments, the concentration of salt (e.g., sodium acetate) in the step elution solution is about 5mM to 600mM, e.g., 5mM to 50mM, 50mM to 100mM, 100mM to 200mM, 200mM to 300mM, 300mM to 400mM, 400mM to 500mM, 500mM to 600mM. In some embodiments, the concentration of sodium acetate in the step elution solution is about 64mM, about 75mM, about 85mM, about 95mM, about 100mM, about 105mM, about 109mM, about 110mM, about 150mM, about 200mM, about 300mM, about 400mM, about 500mM, or more.
In some embodiments, the step elution solution comprises 10mM to 50mM (e.g., about 20 mM) Tris,5 to 600mM salt, pH 8.9 to 9.1. In some embodiments, the salt is sodium acetate. In some embodiments, the 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 20mM Tris,500mM sodium acetate, pH 9.0.
In some embodiments, at least one step elution solution of 1CV to 20CV, e.g., 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 50cm/hr to 2000cm/hr (e.g., about 75cm/hr, about 150cm/hr, about 204cm/hr, about 600cm/hr, and about 1800 cm/hr). In some embodiments, the residence time of the step ladder elution solution in the stationary phase of the column is from 1min/CV to 15min/CV (e.g., about 1.5min/CV, about 6min/CV, and about 12 min/CV).
In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fractional elution solutions are applied to the stationary phase in the column. In some embodiments, at least one step elution solution (e.g., 2, 3, 4, 5, etc.) comprising 20mM Tris, 5 to 600mM 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) at a linear velocity of 50cm/hr to 2000cm/hr and a residence time of 1min/CV to 15min/CV of 1CV to 20CV TM 50HQ)。
In some embodiments, the step elution solution may also be a strip solution, and is preferably applied to the column stationary phase as the final step elution step. The final step elution solution (i.e., strip solution) can be applied to the column stationary phase to cause release of the substance (e.g., rAAV carrier) from the stationary phase. In some embodiments, the final step elution solution may have a high salt concentration (e.g., >450 mM). In some embodiments, the final step elution solution may comprise 20mM Tris, 500mM salt (e.g., sodium acetate), pH 8.9 to 9.1.
A method of purifying a rAAV vector (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., an affinity eluate) includes 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 20mM Tris, 500mM sodium acetate, pH 8.9 to 9.1.
Fraction collection, neutralization and pooling
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 the AEX column to recover and enrich the complete capsid, optionally during gradient elution. In some embodiments, the complete capsid is 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). The empty capsids can be recovered in the AEX column flow-through, i.e. unbound fraction. Empty capsids can also be recovered in the elution peak, but are typically at a lower level than recovery in column flow-through. The intermediate shell may be recovered with either the complete shell or the empty shell.
During elution (e.g., gradient elution) of the AEX method of purifying the rAAV carrier, the eluate from the AEX column can be collected in a particular volume and/or discrete fraction having a particular property (e.g., absorbance at a particular wavelength). For example, the volume of eluent that can be collected from the AEX column during a chromatography step (e.g., gradient elution) is, e.g., 1mL to 4L, e.g., 1mL to 10mL, 1mL to 3L, 1mL to 2L, 1mL to 1L, 1mL to 100mL, 10mL to 50mL, 50mL to 100mL, 100mL to 250mL, 250mL to 500mL, 500mL to 1L, 1L to 1.5L, 1.5L to 2L, 2L to 3L, 3L to 4L, or more (e.g., about 1mL, 5mL, 10mL, 100mL, 500mL, 1L, 2L, 3L, 4L, etc.), or a particular CV equivalent such as 1/8CV to 10CV, e.g., 1/8CV to 1CV, 1CV to 2CV, 2CV to 5CV, 8CV to 10CV or more (e.g., 1/8CV, 1/4, 1/3, 1CV, 2CV, 3, 4, 5, 7, more CV, 9, or more). In some embodiments, during the chromatographic step, an eluent of > 1/3CV volume can be collected from the AEX column. In some embodiments, during the chromatography step, about 1/2CV volume of eluent can be collected from the AEX column. In some embodiments, collecting at least one eluent fraction from an AEX column during a chromatography step (e.g., gradient elution) comprises collecting the eluent when the absorbance of the column flow-through (e.g., absorbance at 260nm and/or 280 nm) reaches an absorbance threshold (e.g., ≡0.5mAU/mm path length, e.g., 10mAU/mm path length). In some embodiments, collecting at least a portion of the eluent from the AEX column during the chromatography step (e.g., gradient elution) comprises collecting the eluent when the gradient elution solution comprises a particular percentage of elution buffer, e.g., when the gradient elution solution comprises from 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 500mM sodium acetate, 100mM Tris, 0.01% P188, pH 8.9.
In some embodiments, the eluate is collected in multiple fractions (e.g., 5 fractions, 10 fractions, 20 fractions, or more) of a particular volume (e.g., 1/3CV,1/2 CV). In some embodiments, the eluate is collected as a single fraction. In some embodiments, when A280.gtoreq.0.5 mAU of the eluate, the eluate is collected in a single fraction, and optionally about 2.3CV.
In some embodiments, collecting at least one eluent fraction from the AEX column comprises measuring absorbance at 260nm (a 260) and/or absorbance at 280nm (a 280) of the eluent collected from the column, optionally during gradient elution. In some embodiments, the measurement of absorbance of the AEX eluate (e.g., at a260 or a 280) is performed in series with the collection of at least one eluate fraction. In some embodiments, at least one eluent fraction is collected when the eluent collected from the AEX column during chromatographic elution (e.g., gradient elution) has an a280 of path length of 0.5 to 10 mAU/mm. In some embodiments, collecting the eluent from the AEX column comprises collecting at least one eluent fraction having a volume of 1/3CV or more. In some embodiments, optionally during gradient elution, collecting at least one eluent fraction (e.g., a first eluent fraction) from the AEX column comprises collecting at least one eluent fraction when a280.gtoreq.0.5 mAU/mm path length of the eluent, wherein the volume of the at least one eluent fraction is gt1/3 CV.
In some embodiments, optionally during gradient elution, 1 to 25 eluent fractions, e.g., 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. 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 eluate fractions are collected from the AEX column. In some embodiments, optionally during gradient elution, at least 10 eluent fractions are collected from the AEX column, each fraction having a volume of ≡1/3CV. In some embodiments, optionally during gradient elution, at least 20 eluent fractions are collected from the AEX column, each eluent fraction having a volume of about 1/2CV.
In some embodiments, a method of purifying a rAAV vector (e.g., rAAV9, rAAV3B, etc.) from an affinity eluate by AEX comprises collecting a first fraction of about 10 eluate fractions from an AEX column, optionally during gradient elution, when a280 of the eluate is >0.5mAU/mm path length, and wherein the volume of each fraction is ≡1/3CV.
In some embodiments, a method of purifying a rAAV vector (e.g., rAAV3B, etc.) from an affinity eluate by AEX comprises, optionally during gradient elution, collecting a first fraction of about 20 eluate fractions from the AEX column when the percentage of a second elution buffer (e.g., buffer B) of the gradient elution solution is about 30% to about 35% (e.g., about 32%), 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 a rAAV vector (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., an affinity eluate) by AEX comprises adjusting the pH of at least one eluate fraction collected from the AEX column, optionally during gradient elution. In some embodiments, adjusting the pH of at least one eluent fraction is referred to as a neutralization step. In some embodiments, the pH of the at least one eluent fraction collected from the AEX column is from pH 8.5 to 9.1 prior to pH adjustment. In some embodiments, the pH of at least one eluent fraction is adjusted to 6.8 to 7.6 (e.g., about pH 7.2). In some embodiments, the pH of at least one eluent fraction is adjusted to 7.5 to 7.7 (e.g., about pH 7.6).
In some embodiments, the pH of at least one eluent fraction collected from the AEX column is adjusted to 6.8 to 7.6 by adding 14% to 16% (eluent 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 300mM, 200mM to 300mM, 300mM to 400mM, or 400mM to 500mM sodium acetate, pH3.0 to 4.0 (e.g., about 3.5). In some embodiments, optionally adjusting the pH of at least one eluent 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%) by volume weight of eluent solution, the added solution comprising about 250mM sodium citrate, pH 3.5. In some embodiments, the pH of at least one eluent fraction collected from the AEX column is adjusted by adding a solution comprising about 50mM citrate, pH 3.6.
In some embodiments, the pH of at least one eluent fraction collected from an AEX column is adjusted to about 7.5 to 7.7 by collecting the at least one fraction in a container comprising about 0.01CV to 0.1CV (e.g., about 0.066 CV) of a solution comprising 50mM to 500mM, e.g., about 50mM to 100mM, 50mM to 400mM, 50mM to 300mM, 50mM to 200mM, 100mM to 300mM, 200mM to 300mM, 300mM to 400mM, or 400mM to 500mM sodium citrate, pH3.0 to 4.0 (e.g., about 3.5). In some embodiments, optionally during gradient elution, adjusting the pH of at least one eluent 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 a solution comprising about 0.01CV to 0.1CV (e.g., about 0.066 CV) of about 250mM sodium citrate (pH 3.5).
In some embodiments, a method of purifying a rAAV vector (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., an affinity eluate) by AEX comprises measuring absorbance of at least one eluate fraction collected from the AEX column, optionally during gradient elution. In some embodiments, the absorbance of at least one eluent fraction is measured using analytical Size Exclusion Chromatography (SEC) in a High Performance Liquid Chromatography (HPLC) system, and the absorbance at one or more wavelengths (e.g., 260nm and/or 280 nm) is measured.
In some embodiments, measuring absorbance of at least one eluent fraction collected from the AEX column comprises measuring absorbance at 260nm (a 260) and 280nm (a 280), and optionally determining 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 the at least one eluent 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 higher. The at least one eluent fraction collected from the AEX column has 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, 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 at least one eluent fraction collected from the AEX column, optionally during gradient elution, is at least 1.25.
In some embodiments, a method of purifying a rAAV vector (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., an affinity eluate) by AEX comprises measuring the percentage of high molecular weight species (HMMS) of at least one eluate fraction collected from the AEX column, optionally during gradient elution. In some embodiments, the percentage of HMMS is measured by SEC. In some embodiments, the percentage of HMMS of the at least one eluent fraction collected during AEX purification of the rAAV vector produced in 250L SUB ranges from 0% to 10% (e.g., 0% to 3.2%). In some embodiments, the percentage of HMMS of the at least one eluent fraction collected during AEX purification of the rAAV vector produced in 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 comprises determining the purity percentage of at least one eluate fraction collected from the AEX column, optionally during gradient elution. In some embodiments, the percent purity is determined by RP-HPLC. In some embodiments, the percentage purity of the at least one eluent fraction collected during AEX purification of the rAAV vector produced in 250LSUB ranges from 95% to 100% (e.g., 99.1% to 99.4%). In some embodiments, the percentage purity of the at least one eluent fraction collected during AEX purification of the rAAV vector produced in 2000L SUB ranges from 75% to 100% (e.g., 79.6% to 98.7%).
In some embodiments, a method of purifying a rAAV vector (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., an affinity eluate) by AEX comprises measuring the amount of host cell DNA (HC-DNA) of at least one eluate fraction collected from the AEX column, optionally during gradient elution. In some embodiments, the amount of HC-DNA is measured by qPCR. In some embodiments, the amount of HC-DNA of at least one eluent fraction collected during AEX purification of the rAAV vector produced in 250L SUB is 0.1 pg/1X 10 9 VG to 20 pg/1X 10 9 VG (e.g. 1.0 pg/1X 10) 9 VG to 5.9 pg/1X 10 9 VG). In some embodiments, the HC-DNA amount of at least one eluent fraction collected during AEX purification of the rAAV vector produced in 2000L SUB is 0.1 pg/1X 10 9 VG to 50 pg/1X 10 9 VG (e.g. 2.7 pg/1X 10) 9 VG to 26.5 pg/1X 10 9 VG)。
In some embodiments, a method of purifying a rAAV vector (e.g., rAAV9, rAAV3B, etc.) from a solution (e.g., an affinity eluate) by AEX comprises measuring the amount of Host Cell Protein (HCP) of at least one eluate fraction collected from the AEX column, optionally during gradient elution. In some embodiments, the amount of HCP is measured by ELISA. In some embodiments, the HCP amount of the at least one eluent fraction collected during AEX purification of the rAAV vector produced in 250LSUB ranges from below the quantitative level (LLOQ) to 5.78pg/1 x 10 9 VG. In some embodiments, the HCP amount of at least one eluent fraction collected during AEX purification of the rAAV vector 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) includes combining at least two eluate fractions collected from an AEX column (e.g., during gradient elution) to form a combined eluate (also referred to herein as an "AEX pool"). In some embodiments, at least two fractions of the eluent from the AEX column are combined, each fraction having an a260/a280 ratio (e.g., as measured by SEC) of 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 higher. In some embodiments, at least two eluent fractions from an AEX column are combined to form a combined eluent, each fraction having an a260/a280 ratio (e.g., as measured by SEC) 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, 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, or at least 39.40. 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 eluent fraction having an a260/a280 ratio of ≡0.98, 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 eluent fraction 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 eluent fraction 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 eluent fraction having an a260/a280 ratio of ≡1.24, 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 eluent fraction having an a260/a280 ratio of ≡1.25, to form a combined eluent.
In some embodiments, combining at least two eluent fractions to form a combined eluent comprises combining 2 to 7, 2 to 10, 2 to 15, 2 to 20, or 2 to 50 eluent fractions collected from the AEX column, optionally during gradient elution. In some embodiments, the combined eluents have an a260/a280 ratio of 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 higher. In some embodiments, the combined eluate has 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, 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 combined eluents have an a260/a280 ratio >0.97. In some embodiments, the combined eluents have an a260/a280 ratio of 0.97 to 1.03. In some embodiments, the combined eluents have an a260/a280 ratio of 1.0 to 1.05. In some embodiments, the combined eluents have an a260/a280 ratio of 1.20 to 1.40. In some embodiments, the combined eluents have an A260/A280 ratio of ≡1.25, for example about 1.28 to 1.35, and are enriched for complete capsids compared to the affinity eluate prior to purification by AEX or 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 eluate contains only one fraction. In some embodiments, the combined eluate comprises only a single fraction, e.g., when a single fraction is collected during a gradient elution, a specific volume of eluate is collected starting at 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).
In some embodiments, the pH of the combined eluates 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 eluates is about 6.8 to 7.6.
In some embodiments, the 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) eluent fractions from the AEX column during a chromatography step (e.g., gradient elution) when a280 > 0.5mAU/mm path length of the eluent, and wherein the volume of the at least one eluent fraction is equal to 1/8CV to 2CV (e.g., about 1/3 CV); ii) adjusting the pH of at least one (e.g. about 10) eluate fraction from the column to 6.8 to 7.6 by adding 14.3% to 15% (by volume of eluate weight) of a solution comprising about 200mM to 300mM (e.g. about 250 mM) sodium citrate, the sodium citrate pH being 3.0 to 4.0 (e.g. 3.5); iii) Measuring the absorbance of at least one eluent fraction collected from the column and determining the a260/a280 ratio; and/or iv) combining at least two eluent fractions collected from the column to form a combined eluent, wherein A260/A280 of each of the at least two eluent fractions is greater than or equal to 1.25; wherein 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 combined eluate is from 6.8 to 7.6, and wherein the at least one eluate fraction or combined eluate is enriched in complete capsids and/or depleted in empty capsids compared to the affinity eluate prior to purification by AEX or diluted and optionally filtered affinity eluate.
In some embodiments, a method of purifying a rAAV vector (e.g., rAAV9, rAAV3B, etc.) from an affinity eluate comprises: i) During the 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 100mM Tris, about 0.01% P188, pH 8.9, and 30% to about 35% (e.g., about 32%) of a second elution buffer (e.g., buffer B) comprising about 500mM sodium acetate, about 100mM Tris, about 0.01% P188, pH 8.9, collecting a first fraction of at least one (e.g., about 20) of the eluent fractions from the AEX column, and continuing to collect all of the eluent 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 the at least one eluent fraction corresponds to 1/8CV to 2CV (e.g., about 1/2 CV); ii) adjusting the pH of at least one (e.g. about 20) eluent fraction from the column to 7.5 to 7.7 by collecting the at least one eluent fraction into a vessel containing 0.01CV to 0.1CV (e.g. about 0.066 CV) of a solution containing about 200mM to 300mM (e.g. about 250 mM) sodium citrate, pH 3.0 to 4.0 (e.g. about 3.5); iii) Measuring the absorbance of at least one eluent fraction collected from the column and determining the a260/a280 ratio; and/or iv) combining at least two eluent fractions collected from the column to form a combined eluent, wherein A260/A280 of each of the at least two eluent fractions is greater than or equal to 0.98; wherein A260/A280 of the combined eluate is ≡1.0, and wherein the at least one eluate fraction or combined eluate is enriched in complete capsids and/or depleted in empty capsids compared to the affinity eluate or diluted affinity eluate prior to purification by AEX.
In some embodiments, the invention provides a method of purifying a rAAV (e.g., rAAV9, rAAV3B, etc.) vector by AEX, the method comprising the steps of: i) A pre-use rinse comprising applying ≡4.5CV (e.g. about 5 CV) of water for injection to the AEX stationary phase in the column; ii) sterilization comprising applying a solution comprising 0.1M to 1.0M (e.g. about 0.5M) NaOH, of 14.4CV to 17.6CV (e.g. about 16 CV), optionally flowing upward, to the AEX stationary phase in the column; iii) Regeneration, comprising applying a solution comprising 1M to 3M (e.g., about 2M) NaCl,50mM to 150mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., about pH 9) of 4.5CV to 5.5CV (e.g., about 5 CV) to the AEX stationary phase in the column; iv) equilibration, comprising applying a solution comprising 50mM to 150mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., about 9) of 4.5CV to 5.5CV (e.g., about 5 CV) to the AEX stationary phase in the column; v) equilibration, comprising applying to the AEX stationary phase in the column an equilibration buffer of from 4.5CV to 5.5CV (e.g., about 5 CV) comprising from 50mM to 150mM (e.g., about 100 mM) Tris, from 400mM to 600mM (e.g., about 500 mM) sodium acetate, from 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 5 CV) equilibration buffer to the AEX stationary phase in the column, said buffer comprising 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, pH 8.5 to 9.5 (e.g., about 8.8); vii) loading an affinity eluate comprising the rAAV vector to be purified into 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 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, pH 8.7 to 9.0, and optionally b) filtered through a tandem 0.2 μm filter prior to application to the stationary phase; viii) equilibration, comprising applying to the column an equilibration buffer of 4.5CV to 5.5CV (e.g., about 5 CV) comprising 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., about 8.9); ix) gradient elution from the immobilized relative material in the column starting with 100% of a first buffer comprising 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 ending with 100% of a second buffer comprising 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) during application of 20CV to 24CV (e.g. about 20 CV). x) collecting a first fraction of at least one (e.g., about 10) eluent fractions from the column during gradient elution when A280.gtoreq.0.5 mAU/mm path length of the eluent, and wherein the volume of at least one eluent fraction is equal to 1/8 to 2CV (e.g., about 1/3 CV); xi) optionally, adjusting the pH of at least one (e.g. about 10) eluent fraction of the column to 6.8 to 7.6 by adding 14.3% to 15% (eluent volume weight) of a solution comprising 200mM to 300mM (e.g. about 250 mM) sodium citrate, pH 3.0 to 4.0 (e.g. about 3.5); xii) measuring absorbance of at least one eluent fraction collected from the column and determining the a260/a280 ratio (e.g. as measured by SEC); and/or xiii) combining at least two eluent fractions collected from the column to form A combined eluent, wherein A260/A280 of at least one eluent fraction is greater than or equal to 1.25, wherein A260/A280 of the combined eluent is greater than or equal to 1.25 (e.g., about 1.28 to 1.35), and optionally wherein the pH of at least one eluent fraction or combined eluent is from 6.8 to 7.6, and wherein the at least one eluent fraction or combined eluent is enriched in complete capsids and/or depleted in empty capsids as compared to an affinity eluent or diluted and optionally filtered affinity eluent; optionally wherein at least one of steps i) to ix) is operated at a linear velocity of 270cm/hr to 330cm/hr (e.g. about 300 cm/hr), at a flow rate of 1.5L/min to 2.0L/min (e.g. about 1.8L/min) through a 6.0L to 6.6L (e.g. about 6.4L) column or at a flow rate of about 314mL/min through a 1.3L column, and/or a residence time of 3.5min/CV to 4.5min/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 TM 50HQ. In some embodiments, the material eluted from the stationary phase during gradient elution comprises the rAAV vector to be purified.
In some embodiments, the invention provides a method of purifying a rAAV (e.g., rAAV9 or AAV3B, etc.) vector by AEX, the method comprising the steps of: i) Sterilization, comprising applying a 5CV to 10CV (e.g., about 8 CV) 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 to the AEX stationary phase in the column a solution of 4.5 to 5.5CV (e.g. about 5 CV) comprising 1 to 3M (e.g. about 2M) NaCl, 50 to 150mM (e.g. about 100 mM) Tris, pH 8.5 to 9.5 (e.g. about pH 9); iii) Equilibration, comprising applying to the AEX stationary phase in the column a 4.5CV to 5.5CV (e.g., about 5 CV) equilibration buffer comprising 50mM to 150mM (e.g., about 100 mM) Tris, 400mM to 600mM (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); iv) equilibration, comprising applying ≡4.5CV (e.g., about 5 CV) equilibration buffer to the AEX stationary phase in the column, said buffer comprising 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, pH 8.5 to 9.5 (e.g., about 8.8); v) loading an affinity eluate comprising the rAAV vector to be purified into the AEX stationary phase in the column, optionally wherein the eluate About 14.4 to 15.5 fold (e.g., about 15 fold) diluted with a buffer comprising 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, pH 8.7 to 9.0; vi) equilibration, comprising applying to the column an equilibration buffer of 4.5CV to 5.5CV (e.g., about 5 CV) comprising 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., about 8.9); vii) gradient elution from the immobilized relative material in the column, starting with 100% of a first buffer comprising 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), ending with 75% of a second buffer comprising 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) during application of 20 to 40CV (e.g., about 37.5 CV); viii) when the percentage of the first buffer is about 65% to about 70% (e.g., about 68%), when the percentage of the second buffer is about 30% to 35% (e.g., about 32%), collecting a first of the at least one (e.g., about 20) eluent fractions from the column during the gradient elution, continuing to collect all eluent 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 at least one eluent fraction is equal to 1/8 to 2CV (e.g., about 1/2 CV); ix) optionally, adjusting the pH of at least one (e.g., about 20) eluent fraction of the column to 7.5 to 7.7 by collecting the at least one eluent fraction into a container comprising about 0.01CV to 0.1CV (e.g., about 0.066 CV) of a solution comprising 200mM to 300mM (e.g., about 250 mM) sodium citrate, pH 3.0 to 4.0 (e.g., about 3.5); x) measuring the absorbance of at least one eluent fraction collected from the column and determining the a260/a280 ratio (e.g. by SEC); and/or xii) combining at least two eluent fractions collected from the column to form a combined eluent, wherein A260/A280 of at least one eluent fraction is greater than or equal to 0.98, wherein A260/A280 of the combined eluent is greater than or equal to 1.0, and wherein at least one eluent fraction or combined wash Enriching the whole capsid and/or depleting the empty capsid in comparison 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 270cm/hr to 330cm/hr (e.g. about 298 cm/hr) and/or a residence time of 1.5min/CV to 4.5min/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 TM 50HQ. In some embodiments, the material eluted from the stationary phase during gradient elution comprises the rAAV vector to be purified.
In some embodiments, the invention provides a method of purifying a rAAV (e.g., rAAV9, etc.) vector by AEX, the method comprising the steps of: i) A pre-use rinse comprising applying ≡4.5CV (e.g. about 5 CV) of water for injection to the AEX stationary phase in the column; ii) sterilization comprising applying a solution comprising 0.1M to 1.0M (e.g. about 0.5M) NaOH, of 14.4CV to 17.6CV (e.g. about 16 CV), optionally flowing upward, to the AEX stationary phase in the column; iii) Regeneration, comprising applying a solution of 4.5CV to 5.5CV (e.g., about 5 CV) to the AEX stationary phase in the column, the solution comprising 1M to 3M (e.g., about 2M) NaCl, 50mM to 150mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., about pH 9); iv) equilibration, comprising applying a solution comprising 50mM to 150mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., about 9) of 4.5CV to 5.5CV (e.g., about 5 CV) to the AEX stationary phase in the column; v) equilibration, comprising applying to the AEX stationary phase in the column an equilibration buffer of 4.5CV to 5.5CV (e.g., about 5 CV) comprising 50mM to 150mM (e.g., about 100 mM) Tris, 400mM to 600mM (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); vi) equilibration, comprising applying ≡4.5CV (e.g., about 5 CV) equilibration buffer to the AEX stationary phase in the column, said buffer comprising 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, pH 8.5 to 9.5 (e.g., about 8.8); vii) loading an affinity eluate comprising the rAAV vector to be purified into 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 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, 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 to the column an equilibration buffer of from 4.5CV to 5.5CV (e.g., about 5 CV) comprising from 50mM to 150mM (e.g., about 100 mM) Tris, from 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., about 8.9)); ix) gradient eluting the material of the stationary phase in the column starting with the application of 100% of a first buffer comprising 50mM to 150mM (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) ending with the application of 100% of a second buffer comprising 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) during the application of 20CV to 24CV (e.g. about 20 CV) to the stationary phase; x) collecting an eluent fraction from the column during gradient elution when A280.gtoreq.0.5 mAU/mm path length of the eluent, and wherein the volume of the eluent fraction corresponds to 1/8 to 2CV (e.g., about 1/3 CV); and/or xi) optionally, adjusting the pH of the eluate fraction from the column to 6.8 to 7.6 by adding 14.3% to 15% (by volume of eluate weight) of a solution comprising about 200mM to 300mM (e.g., about 250 mM) sodium citrate, pH 4.0 to 4.5 (e.g., about 3.5); and wherein the eluent fraction is enriched in complete capsids and/or depleted in empty capsids compared to an affinity eluent or diluted and optionally filtered affinity eluent; optionally, wherein at least one of steps i) to ix) is performed at a linear velocity of 270cm/hr to 330cm/hr (e.g. about 300 cm/hr), at a flow rate of 1.5L/min to 2.0L/min (e.g. about 1.8L/min) through a 6.0L to 6.6L (e.g. about 6.4L) column or at a flow rate of about 314mL/min through a 1.3L column, and/or at a residence time of 3.5min/CV to 4.5min/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 TM 50HQ. In some embodiments, the material eluted from the stationary phase during gradient elution comprises the rAAV vector to be purified.
In some embodiments, the invention provides a method of purifying a rAAV (e.g., rAAV9, AAV3B, etc.) by AEX) A method of supporting a body, the method comprising the steps of: i) Sterilization, comprising applying a 5CV to 10CV (e.g., about 8 CV) 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 to the AEX stationary phase in the column a solution of 4.5 to 5.5CV (e.g. about 5 CV) comprising 1 to 3M (e.g. about 2M) NaCl, 50 to 150mM (e.g. about 100 mM) Tris, pH 8.5 to 9.5 (e.g. about pH 9); iii) Equilibration, comprising applying a solution comprising 50mM to 150mM (e.g., 100 mM) Tris, pH 8.5 to 9.5 (e.g., about 9) of 4.5CV to 5.5CV (e.g., about 5 CV) to the AEX stationary phase in the column; iv) equilibration, comprising applying to the column an equilibration buffer of 4.5CV to 5.5CV (e.g., about 5 CV) comprising 50mM to 150mM (e.g., about 100 mM) Tris, 400mM to 600mM (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); v) equilibration, comprising adding ≡4.5CV (e.g., about 5 CV) equilibration buffer comprising 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, 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 into the AEX stationary phase in the column, optionally wherein the eluate has been diluted about 14.4 to 15.5 fold (e.g., about 15 fold) with a buffer comprising 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, pH 8.7 to 9.0; vii) equilibration, comprising applying to the column an equilibration buffer of from 4.5CV to 5.5CV (e.g., about 5 CV) comprising from 50mM to 150mM (e.g., about 100 mM) Tris, from 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 immobilized relative material in the column starting with 100% of a first buffer comprising 50mM to 150mM (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) starting with 75% of a second buffer comprising 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., during application of 20CV to 40CV (e.g., about 37.5 CV) stationary phase to the stationary phase About 0.01%) P188, pH 8.5 to 9.5 (e.g., pH 8.9); ix) during 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 eluent fraction from the column and continuing to collect all of the eluent fraction 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 eluent fraction corresponds to 1/8 to 2CV (e.g., about 1/2 CV); and/or x) optionally, adjusting the pH of the eluent fraction to 7.5 to 7.7 by collecting the eluent fraction into a container comprising about 0.01CV to 0.1CV (e.g., about 0.066 CV) of a solution comprising 200mM to 300mM (e.g., about 250 mM) sodium citrate, pH 3.0 to 4.0 (e.g., about 3.5); and wherein the eluent fraction is enriched in complete capsids and/or depleted in empty capsids compared to the affinity eluent or diluted affinity eluent; optionally, wherein at least one of steps i) to viii) is performed at a linear velocity of 270cm/hr to 330cm/hr (e.g., about 298 cm/hr) and/or a residence time of 1.5min/CV to 4.5min/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 gradient elution comprises the rAAV vector to be purified.
Identification of combined eluent and drug substance
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., gradient elution) and forming a combined eluate enriched in complete capsids as compared to the percentage of complete capsids in the solution. A method of purifying a rAAV vector from solution by AEX comprising collecting at least one eluent fraction from an AEX column during an elution step and forming a combined eluent, further comprising filtering by a filter selected from the group consisting of virus filtration, ultrafiltration/diafiltration (UF/DF), filtration through a 0.2 μm filter, and combinations thereof, resulting in a drug substance. In some embodiments, the mass properties of the combined eluate, including a260/a280 (e.g., as measured by SEC), the percentage of complete, intermediate, and empty capsids,% purity,% HMMS, the amount of HCP, and/or the amount of HC-DNA, are not substantially different from the same mass properties of the drug substance produced by the combined eluate.
In some embodiments, the percentage of complete capsids in the affinity eluate comprising the rAAV vector to be purified is less than 20% of the total capsids. In some embodiments, the combined eluate or drug substance prepared by the methods disclosed herein is enriched for complete capsids, whereby 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%, 40% to 50%, 40% to 60%, 40% to 70%, 40% to 80% (e.g., 44%,45%,50%, 53%) of the total capsids in the combined eluate or drug substance, and optionally wherein the capsids are measured by Analytical Ultracentrifugation (AUC) (burns b.et al human Gene Therapy Methods (2015) 26; 228-242). In some embodiments, the combined eluents or drug substances prepared by the methods disclosed herein are enriched in the complete capsid, whereby the complete capsid comprises 52+/-7% of the total capsid in the combined eluents or drug substances. In some embodiments, a method of purifying a rAAV vector from an affinity eluate comprises increasing the percentage of complete capsids from less than 30% (e.g., 12% to 25%) of total capsids in the affinity eluate to greater than 30% (e.g., 40% to 55%,45% to 65%,40% to greater than 99%) of total capsids in a combined AEX eluate or drug substance.
In some embodiments, the combined AEX eluate produced by the methods disclosed herein is enriched in complete capsids, whereby the complete capsids comprise 22.9+/-2.9% of the total capsids in the combined eluate. In some embodiments, a method of purifying a rAAV vector from an affinity eluate comprises increasing the percentage of complete capsids from less than 20% (e.g., 10% to 19%) of 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 total capsids in the combined AEX eluate. In some embodiments, the method of purifying a rAAV vector from an affinity eluate comprises increasing the percentage of complete capsids from 11.1±2.1 of total capsids in the affinity eluate to 22.9±2.9% of total capsids in the pooled AEX eluate.
A method of 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., gradient elution) and forming a combined eluate of empty capsid depletion percentage compared to empty capsid percentage 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 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 drug substance prepared by the methods disclosed herein is depleted of empty capsids, whereby empty 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 29% (e.g., +.29%) of the total capsids in the combined eluate or drug substance, and optionally wherein the capsids are measured by Analytical Ultracentrifugation (AUC). In some embodiments, the combined eluate or drug substance prepared by the methods disclosed herein depletes the empty capsids, whereby the empty capsids comprise 20% +/-7% of the total capsids in the combined eluate or drug substance. In some embodiments, a method of purifying a rAAV vector from an affinity eluate includes reducing the percentage of empty capsids to less than or equal to 30% of the combined AEX eluate or drug substance from 40% to 90% of the total capsids in the affinity eluate. In some embodiments, the 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 a combined AEX eluate or drug substance 67.5±3.8％。
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., gradient elution) and forming a combined eluate comprising an intermediate capsid, and wherein the combined eluate is further subjected to a filtration selected from the group consisting of virus filtration, ultrafiltration/diafiltration (UF/DF), filtration through a 0.2 μm filter, and combinations thereof, to produce a drug substance. In some embodiments, in the combined eluents or drug substances, 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 of purifying rAAV vectors (e.g., rAAV9, rAAV3B, etc.) from solution (e.g., affinity eluate) by AEX includes collecting at least one eluate fraction from an AEX column during an elution step (e.g., gradient elution) and forming a combined eluate or drug substance that enriches the complete capsid and depletes the empty capsid compared to the percentage of complete capsid and empty capsid in a solution comprising the rAAV vector to be purified. In addition to complete and empty capsids, in certain non-limiting example embodiments, capsids containing a portion of the vector genome (also referred to as a truncated or fragmented vector genome) and/or non-transgene-related DNA (i.e., intermediate capsids) may constitute a combined eluate (e.g., a combined AEX eluate) or balance of capsid species in the drug substance.
In some embodiments, the method of purifying a rAAV vector from an affinity eluate by AEX produces a combined 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 a rAAV vector from an affinity eluate by AEX produces a combined eluate or drug substance comprising about 44% complete rAAV capsids, about 27% intermediate capsids, and about 29% empty capsids.
In some embodiments, the method of purifying a rAAV vector from an affinity eluate by AEX produces a combined eluate or drug substance comprising about 20% to >99% of a complete rAAV capsid, about 5% to 65% of an intermediate capsid, and 10% to 65% of an empty capsid.
In some embodiments, the method of purifying a rAAV vector from an affinity eluate by AEX produces a combined 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 a rAAV vector produced in a vessel having a volume of 100L to 500L (e.g., about 250L), optionally wherein the vessel is SUB.
In some embodiments, the combined eluate or drug substance prepared by the methods disclosed herein is enriched in complete capsids, whereby complete capsids comprise 55% +/-7% of the total capsids in the combined eluate or drug substance. 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 SUB.
In some embodiments, the combined eluate or drug substance prepared by the methods disclosed herein comprises 24% +/-3% of the 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 SUB.
In some embodiments, the combined eluate or drug substance prepared by the methods disclosed herein depletes the empty capsids, whereby the empty capsids comprise 21% +/-10% of the total capsids in the combined eluate or drug substance. 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 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 capsid, and 18% to 22% empty capsid. In some embodiments, the affinity eluate is produced by affinity chromatography purification of a rAAV vector produced in a 1000L to 3000L (e.g., about 2000L) vessel, optionally wherein the vessel is SUB.
In some embodiments, the combined eluate or drug substance prepared by the methods disclosed herein is enriched in complete capsids, whereby complete capsids comprise 49% +/-2% of the total capsids in the combined eluate or drug substance. In some embodiments, the rAAV vector present in the combined eluate or drug substance is produced in a 1000L to 3000L (e.g., about 2000L) container, optionally wherein the container is SUB.
In some embodiments, the combined eluents or drug substances prepared by the methods disclosed herein comprise 32% +/-4% of the intermediate capsids of the total capsids in the combined eluents or drug substances. In some embodiments, the rAAV vector present in the combined eluate or drug substance is produced in a 1000L to 3000L (e.g., about 2000L) container, optionally wherein the container is SUB.
In some embodiments, the combined eluate or drug substance prepared by the methods disclosed herein depletes the empty capsids, whereby the empty capsids comprise 20% +/-2% of the total capsids in the combined eluate or drug substance. In some embodiments, the rAAV vector present in the combined eluate or drug substance is produced in a 1000L to 3000L (e.g., about 2000L) container, optionally wherein the container is SUB.
Methods of purifying rAAV vectors (e.g., rAAV9, rAAV3B, etc.) from solutions (e.g., affinity eluents) by AEX include collecting at least one eluate fraction from an AEX column during an elution step (e.g., gradient elution) and forming a combined eluate comprising the rAAV vector and optionally forming a drug substance, which can be quantified by quantitative polymerase chain reaction (qPCR) analysis of the vector genome (VG or VG). qPCR analysis can measure copies of ITR sequences, copies of transgene sequences, and/or copies of any other nucleotide sequences present in the complete vector genome.
The amount of VGs present in the combined eluate from the AEX column may be expressed as a percentage of the yield of the VG column, which refers to the percentage of the amount of VGs present in the combined eluate collected from the AEX column (i.e., AEX pool) to the amount of VGs present in the sample to be purified, e.g., an affinity eluate, which in some embodiments has been diluted only, or diluted and filtered and applied to the AEX column.
The method of purifying rAAV vectors according to the methods disclosed herein resulted in a 63% +/-26% yield of%vg column. Methods of purifying rAAV vectors according to the methods disclosed herein result in% VG column yields 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, purifying the rAAV vector produced in 250L SUB by the methods disclosed herein results in a%vg column yield of 40% -100%. In some embodiments, purification of the rAAV vector 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 percentage of VG present in the combined eluate collected from the AEX column (i.e., AEX pool) to 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 step yield of 47% +/-11% of%vg. Methods of purifying rAAV vectors according to the methods disclosed herein result in a step yield of%vg 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, purifying 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 the rAAV vector produced in 250L SUB by the methods disclosed herein results in a%vg step yield of 45% +/-8%.
In some embodiments, purification of the rAAV vector produced in 2000L SUB by the methods disclosed herein results in a step yield of 50% +/-13% of%vg. In some embodiments, purification of the rAAV vector produced in 2000L SUB by the methods disclosed herein results in a step yield of 25% to 75% (e.g., 31% to 66%) of VG.
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 the AEX column during an elution step (e.g., gradient elution) and forming a combined eluate or drug substance having a reduced amount of Host Cell Protein (HCP) compared to the amount of HCP in the solution. In some embodiments, the reduced amount of HCP in the combined eluate, at least one eluate fraction, or drug substance is below a quantitative level (LLOQ) as measured by ELISA. In some embodiments, the combined eluent, at least one eluent fraction, or reduced HCP amount in a drug substance is between 10ng and 2000 ng/1X 10 9 VG, 50ng to 200 ng/1X 10 9 VG, 100ng to 1000 ng/1X 109VG or 200 to 2000 ng/1X 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 increasing the amount of HCP in the affinity eluate from 1-500 pg/1X 10 9 VG (e.g. about 50 pg/1X 10) 9 ) The amount of LLOQ in the combined eluate, at least one eluate fraction, or drug substance is reduced, wherein the rAAV vector is produced in 250L SUB.
In some embodiments, a method of purifying a rAAV vector (e.g., rAAV9, rAAV3B, etc.) from an affinity eluate by AEX comprises increasing the amount of HCP in the affinity eluate from 100-500 pg/1X 10 9 VG (e.g. about 330 pg/1X 10) 9 ) The amount of LLOQ in the combined eluate, at least one eluate fraction, or drug substance is reduced, wherein the rAAV vector is produced in 2000L SUB.
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., gradient elution) and forming a combined eluate or drug substance comprising the rAAV vector, wherein the rAAV vector has a purity of 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, e.g., analytical reverse phase HPLC, capillary gel electrophoresis.
In some embodiments, the rAAV vector produced in 250L SUB is purified by the methods disclosed herein to yield a rAAV vector formulation (e.g., drug substance) having a purity of 98.6% +/-0.6%. In some embodiments, a rAAV vector produced in a 1000L to 3000L (e.g., about 2000L) vessel (e.g., SUB) is purified by the methods disclosed herein to yield a rAAV vector formulation (e.g., drug substance) having a purity of 99.3% +/-0.3%.
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., gradient elution) and forming a combined eluate or drug substance, the percentage of HMMS being 0% to 10%. In some embodiments, the percentage of HMMS is measured by Size Exclusion Chromatography (SEC). In some embodiments, rAAV vectors produced in 100L to 300L (e.g., about 250L) vessels (e.g., SUB) are purified by methods disclosed herein to obtain a rAAV vector preparation comprising 2.6% +/-0.8% HMMS, as measured by SEC. In some embodiments, a rAAV vector produced in 2000L of Sub is purified by the methods disclosed herein to obtain a rAAV vector preparation comprising 2.9% +/-0.4% HMMS.
The 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., gradient elution) and forming a purified DNA having about 7.0 to 25pg residual HC-DNA/1 x 10 9 Combined eluents or drug substances of VG. In some embodiments, the amount of HC-DNA is measured by qPCR. In some embodiments, the rAAV vector produced in 250L of SUB is purified by the methods disclosed herein to obtain a recombinant vector comprising 17.4+/-6.7pg HC-DNA/1X 10 9 rAAV vector preparations of VG (e.g., pooled eluents, drug substances). In some embodiments, purification in 2000L Sub by the methods disclosed hereinThe rAAV vector produced yielded a vector comprising 9.3+/-1.2pg HC-DNA/1X 10 9 rAAV vector preparations of VG (e.g., pooled eluents, drug substances).
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., gradient elution) and forming a combined eluate or 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, the rAAV vector produced in 250L of SUB is purified by the methods disclosed herein to obtain a rAAV vector preparation (e.g., combined eluate, drug substance) having an a260/a280 of 1.24 to 1.32 as measured by SEC. In some embodiments, the rAAV vector produced in 2000L Sub is purified by the methods disclosed herein to obtain a rAAV vector preparation (e.g., combined eluate, drug substance) having an a260/a280 of 1.28 to 1.31 as measured by SEC.
A method of 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., gradient elution) and forming a combined eluate, wherein the combined eluate is subjected to a filtration 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 suitable for the production of 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 eluting (e.g., gradient elution) and collecting at least one eluent fraction comprising the complete rAAV capsid from the AEX column, additional steps can be performed to prepare the column stationary phase for further rAAV purification runs. These steps may include, for example, sterilization, equilibration, regeneration, rinsing, and/or storage. Those of skill in the art will understand that one or more steps may be performed in a different order and frequency.
Methods of regenerating the AEX stationary phase in the column for further rAAV purification runs include post-use sterilization of the stationary phase. In some embodiments, post-use sterilization of the stationary phase follows the elution step (e.g., gradient elution). In some embodiments, disinfecting comprises 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 0.6M NaOH to the AEX stationary phase in the column. In some embodiments, disinfecting comprises applying a solution comprising about 0.5M NaOH to the AEX stationary phase in the column. In some embodiments, post-use sterilization 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 sterilization comprises applying a 14.4 to 17.6CV (e.g., about 16 CV) solution comprising 0.5M NaOH to the AEX stationary phase in the column. In some embodiments, post-use sterilization includes applying a solution comprising about 0.5M NaOH to the AEX stationary phase in the column at a line speed of 50 to 2000cm/hr, a flow rate of 0.2 to 3.0L/min, and/or a residence time of 2 to 15min/CV, 2 to 20CV, 5 to 15CV, 7 to 13CV (e.g., about 5, about 7.5, about 10, about 16CV, etc.). In some embodiments, post-use sterilization comprises applying a 14.4 to 17.6CV (e.g., about 16 CV) solution to the AEX stationary phase in the column at a linear velocity of 270 to 330cm/hr (e.g., about 300 cm/hr), at a flow rate of 1.5 to 2.0L/min (e.g., about 1.8L/min) through a 6.0 to 6.6L (e.g., about 6.4L) column, or at a flow rate of about 314mL/min through a 1.3L column and/or a residence time of 3.5 to 4.5min/CV (e.g., about 4 min/CV).
A method of regenerating a column stationary phase to further run rAAV purification includes regenerating the stationary phase (in some embodiments, this step may be referred to as "equilibration"). In some embodiments, the column stationary phase is regenerated after the elution step (e.g., gradient elution). In some embodiments, regeneration comprises adding a catalyst comprising a salt (e.g., naCl, sodium acetate, ammonium acetate (NH 4 Acetate)、MgCl 2 And Na (Na) 2 SO 4 ) Solutions of buffers (e.g., tris, BIS-Tris propane, diethanolamine, diethylamine, tris (hydroxymethyl) methylglycine, triethanolamine and/or n, n-di (hydroxyethyl) glycine) are applied to the stationary phase in the column. In some embodiments, regenerating comprises will comprise about 0.Solutions of 1M to 5M (e.g., 0.1M to 4M, 0.1M to 3.5M, 0.1M to 3M, 0.1M to 2.5M, 0.5M to 4M, 0.5M to 3.5M, 0.5M to 3.0M, 0.5M to 2.5M, 1M to 4M, 1M to 3.5M, 1M to 3M, 1M to 2.5M, or about 1.5M to 2.5M) salts are applied to the stationary phase. In some embodiments, regeneration comprises applying a solution comprising about 1mM to 500mM (e.g., 1mM to 450mM, 1mM to 400mM, 1mM to 350mM, 1mM to 300mM, 1mM to 250mM, 1mM to 200mM, 50mM to 450mM, 50mM to 400mM, 50mM to 350mM, 50mM to 300mM, 50mM to 250mM, 50mM to 200mM, or 50mM to 150 mM) buffer to the stationary phase.
In some embodiments, regenerating comprises applying a solution having a pH of about 7.0 and 11.0 (e.g., 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 a 2 to 15CV (e.g., about 5CV, about 10 CV) solution (e.g., regeneration solution) to the AEX stationary phase in the column. In some embodiments, regeneration comprises applying 4.5 to 5.5CV (e.g., about 5 CV) of a solution comprising 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, 100mM Tris, pH 9 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, 2 to 15CV (e.g., about 5CV, about 10 CV). In some embodiments, regeneration comprises applying a solution comprising 2M NaCl, 100mM Tris, pH 9 to the AEX stationary phase in a column at a linear velocity of 270 to 330cm/hr (e.g., about 300 cm/hr), a flow rate of 1.5 to 2.0L/min (e.g., about 1.8L/min), through a 6.0 to 6.6L (e.g., about 6.4L) column, or at about 314mL/min through a 1.3L column and/or a residence time of 3.5 to 4.5min/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., gradient elution). In some embodiments, equilibration of the medium in the column comprises applying a solution comprising about 100mM Tris, pH 9 to the AEX stationary phase in the column. In some embodiments, the equilibration of the column comprises applying a solution comprising 20mM Tris, pH 9 to the AEX stationary phase in the column. In some embodiments, the equilibration of the column comprises applying a 2 to 15CV (e.g., about 5CV,10 CV) solution (e.g., equilibration solution) to the AEX medium in the column. In some embodiments, the equilibration of the column comprises applying a solution comprising 100mM Tris, pH 9, of 4.5 to 5.5CV (e.g., about 5 CV) to the AEX stationary phase in the column. In some embodiments, the equilibration of the column comprises applying a solution comprising 100mM Tris, pH 9, at 2 to 15CV (e.g., about 5CV, about 10 CV) 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 15 min/CV. In some embodiments, the equilibration of the column comprises applying a solution comprising 100mM Tris, pH 9 to the AEX stationary phase in the column at a linear velocity of 270 to 330cm/hr (e.g., about 300 cm/hr), at a flow rate of 1.5 to 2.0L/min (e.g., about 1.8L/min) through a 6.0 to 6.6L (e.g., about 6.4L) column, or at 314mL/min through a 1.3L column and/or a residence time of 3.5 to 4.5min/CV (e.g., about 4 min/CV), 4.5 to 5.5CV (e.g., about 5 CV).
Methods of regenerating a column stationary phase for further rAAV purification runs include post-use washing (i.e., rinsing) of the stationary phase. In some embodiments, the post-use wash of the column follows the elution step (e.g., gradient elution). In some embodiments, post-use washing of the column includes applying water for injection (e.g., purified water) to the AEX stationary phase in the column. In some embodiments, post-use washing of the column includes applying ≡4.5CV (e.g., about 5 CV) of water for injection to the AEX stationary phase in the column. In some embodiments, post-use washing of the column includes applying a solution containing water for injection to the AEX stationary phase in the column at a line speed of 100 to 2000cm/hr, a flow rate of 0.2 to 3.0L/min, and/or a residence time of 2 to 15min/CV (e.g., about 5CV, about 10 CV). In some embodiments, post-use flushing of the column comprises applying 4.5 to 5.5CV (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 330cm/hr (e.g., about 300 cm/hr), at a flow rate of 1.5 to 2.0L/min (e.g., about 1.8L/min) through a 6.0 to 6.6L (e.g., about 6.4L) column, or at about 314mL/min through a 1.3L column and/or a residence time of 3.5 to 4.5min/CV (e.g., about 4 min/CV).
A method of regenerating a column stationary phase for further rAAV purification operations includes applying a storage solution to the stationary phase. In some embodiments, the storage solution is applied to the column after the elution step (e.g., 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, a storage buffer of 2 to 11CV (e.g., about 3CV, about 10 CV) is applied to the AEX stationary phase in the column. In some embodiments, 2.7 to 3.3CV (e.g., about 3 CV) of a storage solution comprising 17.5% ethanol is applied to the AEX stationary phase in the column. In some embodiments, a storage solution comprising 17.5% ethanol at 2 to 11CV (e.g., about 3 CV) 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 2 to 15 min/CV. In some embodiments, applying the storage solution to the column comprises applying a 2.7 to 3.3CV (e.g., about 3 CV) solution comprising 17.5% ethanol to the AEX stationary phase in the column at a linear velocity of 270 to 330cm/hr (e.g., about 300 cm/hr), a flow rate of 1.5 to 2.0L/min (e.g., about 1.8L/min) through a 6.0 to 6.6L (e.g., 6.4L) column, or at about 314mL/min through a 1.3L column and/or a residence time of 3.5 to 4.5min/CV (e.g., about 4 min/CV).
A method of regenerating a column stationary phase for further rAAV purification, the method comprising the steps of: i) Post-use sterilization, including application of a solution containing about 0.5M NaOH in a 14.4 to 17.6CV (e.g., about 16 CV) to the stationary phase; ii) regeneration, comprising applying a solution containing about 2M NaCl, 100mM Tris, pH 9 of 4.5 to 5.5CV (e.g., about 5 CV) to the stationary phase; iii) Equilibration, comprising applying a solution comprising about 100mM Tris, pH 9, of 4.5 to 5.5CV (e.g., about 5 CV) to the stationary phase; iv) post-use rinsing comprising applying 4.5 to 5.5CV (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 2.7 to 3.3CV (e.g., about 3 CV) of a storage solution comprising about 17.5% ethanol to the column; wherein steps i) to i)v) at least one step is carried out at a linear velocity of 270 to 330cm/hr (e.g. about 300 cm/hr), a flow rate of 1.5 to 2.0L/min (e.g. about 1.8L/min) through a 6.0 to 6.6L (e.g. 6.4L) column or about 314mL/min through a 1.3L column, and/or a residence time of 3.5 to 4.5min/CV (e.g. about 4 min/CV), wherein the stationary phase is an AEX stationary phase, optionally a POROS TM 50HQ stationary phase.
The method of regenerating the AEX stationary phase for further rAAV purification runs includes applying an ethanol rinse to the stationary phase prior to the first step of the method of purifying the rAAV carrier (i.e., prior to sterilization, prior to equilibration, etc.). In some embodiments, the ethanol rinse comprises about 20mM Tris, pH 9. In some embodiments, applying an ethanol rinse solution to the column stationary phase comprises applying 8 to 12CV (e.g., about 10 CV) of a solution comprising about 20mM 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 12CV (e.g., about 10 CV) of a solution comprising about 20mM Tris, pH 9 to the AEX stationary phase at a rate of 100 to 1000cm/hr (e.g., about 600 cm/hr) and/or at a residence time of 1 to 10min/CV (e.g., about 1.5 min/CV).
Equivalents (Equipped with)
The foregoing written description is considered to be sufficient to enable one skilled in the art to practice the disclosure. The foregoing description and examples detail certain exemplary embodiments of the disclosure. It will be appreciated, however, that no matter how detailed the foregoing appears in text, the disclosure may be practiced in many ways and that the disclosure should be interpreted in accordance with the appended claims and any equivalents thereof.
All references cited herein, including patents, patent applications, papers, textbooks, and the like, and the references cited therein, are hereby incorporated by reference in their entirety.
Example embodiments
The invention will be described in further detail by reference to the following experimental examples. These examples are only for illustrating the invention and are not intended to be limiting unless otherwise specified. Thus, the present invention should not be construed as limited to the following examples, but rather as embracing any and all changes apparent from the teachings provided herein.
Examples
Example 1: screening wash desalts by AEX chromatography to enrich complete AAV9 vector
Preparation of AEX loadings for eluted salt screening
HEK 293 cells were grown in suspension culture and transfected with 3 plasmids to produce AAV9 vectors (Grieger et al (2016) Molecular Therapy (2): 287-297) according to standard methods known in the art. HEK 293 cells were harvested, lysed, flocculated, and the resulting lysates filtered. AAV9 vector was purified from the clarified lysate by affinity chromatography. The affinity column was equilibrated, the clarified lysate loaded, washed, and the purified AAV9 vector eluted. AAV9 vector affinity cells (also known as affinity eluate) have a pH of 4.4 and a conductivity of 5.3mS/cm. The affinity cell was diluted 7.6-fold with 20mM Tris (pH 9), adjusted to pH 9 with 1M Tris base (pH 11), and filtered through a 0.2. Mu.M filter. The resulting solution had a pH of 9 and a conductivity of 1.9mS/cm and was loaded onto an AEX column for eluted salt screening studies.
At 1mL POROS TM Screening, washing and desalting by AEX chromatography on 50HQ column
Four wash desalts were studied for their ability to distinguish between AAV9 empty capsids (i.e., AAV capsids without recombinant vector genome) and complete capsids (i.e., AAV capsids with recombinant vector genome) during AEX chromatography. Consistent with Table 1, balance POROS TM A50 HQ column (inner diameter 0.5cm, bed height 5cm, column volume 1 mL) was loaded and washed. A50 CV gradient was produced from 0-50% buffer B, followed by a 10CV step elution with 100% buffer B. Four buffers B were used, consisting of 20mM Tris, 500mM salt, pH 9. The salt is NaCl, sodium acetate, ammonium acetate or Na 2 SO 4 One of them.
Table 1: at 1mL POROS TM AEX chromatography with 50HQ column screening gradient wash desalting
* Washing and desalting: naCl, sodium acetate, ammonium acetate, na 2 SO 4
# Vector Genome (VG) was measured by qPCR analysis of ITR sequences
Wash desalination vs elution peak shape sum A 260 /A 280 The effect of the ratio is shown in the chromatogram of fig. 1. A is that 260 /A 280 The ratio provides an estimate of the percentage of AAV capsids containing the recombinant vector genome (% complete), with higher ratios representing higher percentages of complete (Sommer et al molecular Therapy (2003) 7 (1): 122-128). Elution by NaCl results in a high A 260 /A 280 Main peak (indicating complete vector) and having low a 260 /A 280 Is indicated by the tight-fitting shoulder (indicating an empty capsid). Elution by sodium acetate and ammonium acetate yields a product with high a 260 /A 280 And has a low A 260 /A 280 Is a single peak. These results indicate that sodium acetate and ammonium acetate separated empty capsids from AAV9 vector better than NaCl. In contrast, na 2 SO 4 In medium A 260 /A 280 Is eluting AAV9 material, which means that there is little separation of AAV9 vector from empty capsids.
1mL of the eluate fraction was collected throughout the gradient elution and neutralized with 0.15mL of 50mM citrate pH 3.6. By HPLC-SEC A 260 /A 280 The neutralized fractions were analyzed. During the HPLC-SEC method, absorbance was monitored at 214, 260 and 280 nm. A is that 260 /A 280 The ratio provides an estimate of the percentage of complete capsids of AAV (Sommer et al molecular Therapy (2003) 7 (1): 122-128). AAV 9-characteristic SEC elution peaks integrated at 260 and 280nm and the ratio of these two values was reported as SEC A260 /A 280 As shown in fig. 2. Maximum SEC A of eluted fractions produced by sodium acetate gradient 260 /A 280 1.27 higher than NaCl (1.23), ammonium acetate (1.22) and Na 2 SO 4 (1.15) similar maxima. In addition, elution based on sodium acetate produced 7 consecutive eluent fractions, SEC a thereof 260 /A 280 More than or equal to 1.19, higher thanNaCl (3 fractions), ammonium acetate (4 fractions) and Na 2 SO 4 Similar results for (0 fractions). Merge SEC A 260 /A 280 Fractions of 1.19. Gtoreq., ITR was determined by qPCR to determine Vector Genome (VG) yield, and analyzed by Analytical Ultracentrifugation (AUC) to determine empty, complete and intermediate capsids (AAV capsids, which package less nucleic acid than complete capsids and contain, e.g., partially fragmented or truncated vector genome and/or non-transgene-related DNA).
Washing desalting screening studies showed that according to SEC A 260 /A 280 And AUC (table 2), sodium acetate was superior to NaCl, ammonium acetate, and Na in terms of VG yield and total percentage of AAV9 vector recovered 2 SO 4 . SEC A of sodium acetate AEX pool 260 /A 280 1.24, slightly higher than the use of NaCl (pool SEC A 260 /A 280 =1.23) and ammonium acetate (SEC a 260 /A 280 =1.21) and is significantly higher than Na 2 SO 4 Pool (SEC A) 260 /A 280 =1.15). AUC analysis of AEX pool showed that the percentage of sodium acetate gradient produced complete (43%) was slightly higher than the NaCl gradient (about 38%) and significantly higher than Na 2 SO 4 Gradient (20%). Notably, sodium acetate gradient elution reduced the percentage of empty capsids (% empty capsids) from 75% in AEX loading to 29% in AEX pool. To better describe the performance of the wash desalination, naCl, sodium acetate and ammonium acetate were chosen for 5.1mL POROS TM 50HQ column, as described in the following section.
Table 2: at 1mL POROS TM Results of eluted salt screening study on 50HQ column
The% VG column yield was determined as (VG in AEX pool)/(VG in AEX load); therefore, the loss generated at the time of load preparation is not considered. AUC data for NaCl gradient elution AEX pool is not yet defined and is therefore reported here as approximate (-). LLOQ-is below the lower limit of quantitation.
At 5.1mL POROS TM Screening, washing and desalting by AEX chromatography on 50HQ column
The ability of the eluting salts NaCl, sodium acetate and ammonium acetate to distinguish AAV9 empty capsids from complete carriers during AEX chromatography was investigated. Will POROS TM The 50HQ column (inner diameter 0.66cm, bed height 15cm, column volume 5.1 mL) was equilibrated, loaded, washed and eluted with a gradient of NaCl, sodium acetate or ammonium acetate (Table 3). 1ml of the eluted fraction was collected throughout the gradient, neutralized with 0.15ml of 50mM citrate pH 3.6 and purified by HPLC-SEC A 260 /A 280 Analysis was performed.
Elution based on sodium acetate produced 20 consecutive fractions of SEC a 260 /A 280 1.19, above similar results for NaCl (8 fractions) and ammonium acetate (11 fractions). Merge SEC A 260 /A 280 Fractions of 1.19. Gtoreq., ITR was determined by qPCR to determine VG yield, and analyzed by Analytical Ultracentrifugation (AUC) to determine% complete capsid.
Table 3: at 5.1mL POROS TM AEX chromatography with 50HQ column for desalting and screening
Washing desalting screening studies showed that according to SEC A 260 /A 280 And AUC, sodium acetate was superior to NaCl and ammonium acetate in terms of the percentage of complete capsids of the recovered AAV9 vector (table 4). SEC A of sodium acetate AEX pool 260 /A 280 Is 1.26, higher than that of NaCl (pool SEC A 260 /A 280 =1.24) and ammonium acetate (SEC a 260 /A 280 =1.19). AUC analysis of AEX pool showed that the sodium acetate gradient produced a slightly higher percentage of complete capsid (43%) than the NaCl gradient (39%) and NH4 acetate ammonium acetate gradient (36%).
Overall, AEX runs performed on column volume scales of 1mL and 5.1mL showed that sodium acetate resolved the complete AAV9 vector from empty capsids better than NaCl and ammonium acetate. Thus, in a further developed version of the AEX process, sodium acetate is used as the wash salt.
Table 4: at 5.1mL POROS TM Results of eluted salt screening study on 50HQ column
The% VG column yield was determined as (VG in AEX pool)/(VG in AEX load); therefore, the loss generated at the time of load preparation is not considered.
Example 2: enrichment of complete AAV9 vector by AEX chromatography with stepwise elution of sodium acetate
Based at least in part on the results in example 1, sodium acetate was selected to study the step elution operation of the AEX column to separate AAV9 empty capsids from the complete vector. Affinity eluate was generated as described in example 1, diluted with 20mM Tris, pH 9 and 1M Tris base, pH 11, and filtered through a 0.2. Mu.M filter.
Screening of optimized sodium acetate step elution conditions
For screening purposes, a nine step wash and elution AEX procedure was performed, increasing the sodium acetate concentration stepwise in 20mM Tris (pH 9). In accordance with Table 5, POROS is shown TM A50 HQ column (0.66 cm ID. Times.15 cm BH;5.1mL CV) equilibrates, loads, washes and elutes. By mixing buffer a:20mM Tris, pH 9 and buffer B: washing and elution buffers were formed in the FPLC system, 20mM Tris, 140mM sodium acetate, pH 9. The fractions were neutralized and passed through SEC a 260 /A 280 The ITR was determined by AUC and qPCR. FIG. 3 depicts a 9-step chromatogram showing a series A as the concentration of sodium acetate in the wash and elution buffers increases gradually 260 /A 280 Significant changes occurred.
Table 5: AEX screening method using 9-step sodium acetate elution (3 washes and 6 elutions)
Analysis of the nine-step run showed that AAV9 empty capsids could be separated from the complete AAV9 vector by stepwise washing and elution with sodium acetate (table 6). Washes 1 and 2 selectively removed bound empty capsids from the AEX column. Notably, washes 1 and 2 produced SEC A260/A280 values of 0.58 and 0.79, respectively, with empty capsid percentages (AUC) of 98% and 85%, respectively. Eluted fractions 1-4 were enriched for full AAV9 vector, SEC A260/A280 values were in the range of 1.27-1.30, full capsid percentage (AUC) was in the range of 29-53%, well above 12% in AEX load. Based on these findings, a stepwise washing and elution method based on sodium acetate was devised as follows.
Table 6: sodium acetate 9-step elution study results on 5.1mLPOROSTM 50HQ column
W-washing step; e-eluting; LLOQ-below quantitative limit
Enrichment of complete AAV9 vector by stepwise sodium acetate washing and elution
Based on the above results, the AEX method with different steps of sodium acetate wash and elution was tested for its ability to enrich the complete AAV9 vector capsid. In accordance with Table 7, POROSTM 50HQ column (0.66 cm ID. Times.15 cm BH,5.1mL CV) was equilibrated, washed and eluted. By mixing buffer a in the FPLC system: 20mM Tris, pH 9 and buffer B: washing, pH stabilization and elution buffers were prepared with 20mM Tris, 140mM sodium acetate, pH 9. In the method studied, the multiple parameters were different, i.e. elution linear velocity (75-600 cm/hr), loading challenge (5.1X10 13 Up to 1.1X10 15 VG/mL resin) and the concentration of sodium acetate (57 mM or 68 mM) during the washing step.
As shown in FIGS. 4A and 4B, chromatograms of stepwise washing and elution at 600cm/hr elution, 5.1X11013 VG/mL resin challenge and 57mM sodium acetate wash are presented. Table 8 reports the results and reveals that the developed stepwise sodium acetate wash and elution AEX method enriches the complete AAV9 vector and reduces Host Cell Protein (HCP) levels. The stepwise method developed increased the percentage of completion (judged by AUC) from 18% to 40-53% and SEC A260/A280 from 0.95 to 1.25-1.27. In addition to enriching AAV9 for complete capsids, the stepwise approach developed can also eliminate large amounts of HCP and moderate amounts of host cell DNA (HC-DNA) with low column challenges. The stepwise sodium acetate washing and elution method did not provide as high a% VG yield or% full AAV9 as ultracentrifugation or AEX chromatography by sodium acetate gradient elution (examples 6, 7 and 8 below). However, the step elution method avoids the complex operations associated with ultracentrifugation.
Table 7: AEX method of fractional sodium acetate elution was used. The runs utilized different loading challenges, wash conditions and elution residence times.
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Table 8: results of AEX runs with sodium acetate step elution on 5.1mL POROSTM 50HQ column
The% VG column yield was determined as (VG in AEX combined eluate)/(VG in AEX load); and thus does not take into account the losses that occur during load preparation.
Example 3: screening of AAV9 affinity eluate preparation method for AEX chromatography
One embodiment of the large-scale downstream processing of AAV9 involves cell lysis, filtration, and affinity chromatography, with the product eluting at low pH and moderate conductivity. Studies on various AAV serotype viral proteins reported calculated isoelectric points for AAV9 empty and complete capsids of-6.2 and-5.8, respectively (Venkatakrishnan et al., j.virology (2013) 87.9:4974-4984). Screening of various conditions showed that AAV9 bound to AEX resin only in a relatively alkaline, low conductivity environment (data not shown). Thus, preparing an acidic AAV9 affinity eluate for AEX chromatography requires increasing the pH and decreasing the conductivity of the carrier-containing buffer. This method crosses the AAV9 isoelectric point, an unstable transition, which may lead to vector loss.
This example details various methods of treating an acidic affinity cell to AEX chromatographic loading. The methods of load preparation for dilution, serial mixing (fig. 5) and Tangential Flow Filtration (TFF) were investigated. The results indicate that treatment of AAV9 affinity eluate to AEX chromatographic loadings with high product yields and low aggregation requires the specific development of novel and inventive methods and procedures.
Dilution method for preparing AAV9 affinity cell for AEX chromatography
The pH of the affinity elution pool is about 3.8-4.4, the conductivity is about 5.5-6.5mS/cm, and the pH comprises 7X 10 13 -1.4×10 14 AAV9 VG/mL. The affinity cell for AEX chromatography can be prepared by diluting the affinity cell with alkaline buffer as described in example 1. In agreement with table 9, AAV9 affinity cells were diluted in PETG containers with a combination of alkaline buffers to raise pH and lower conductivity. The resulting solution was passed through a 0.2 μm filter pre-wetted with diluent buffer. The diluted sample was filtered through 0.2 μm to simulate large scale downstream processing, with the filter placed at the inlet of the chromatographic column. The pH of the resulting filtrate was 8.7-9.0, conductivity was in the range of 1.8-2.1mS/cm, which would allow for high binding of AAV9 to AEX resin. Samples were collected after dilution and after filtration and ITR was determined by qPCR to determine VG titer.
Table 9 reports the results and shows that a large amount of AAV9 was lost during dilution and filtration. Serial dilutions were performed with 20mM Tris (pH 9) and pH was adjusted with 1M Tris base (pH 11) followed by 0.2 μm filtration, resulting in 39% loss of VG. Reversing the order of these dilutions produced similar results with 37% loss of carrier after filtration. The greater the dilution, the higher the amount of VG lost. The VG yield after filtration was only 36% after dilution 25-fold with 100mM Tris, pH 9, followed by pH adjustment with 1M Tris, pH 9. Dilution with the same buffer was 15-fold and the yield after filtration was 65%.
To reduce non-specific binding of AAV9 to dilution vessel and filter surfaces, 0.01% (v/v) poloxamer 188 (P188) was added to dilution buffer 100mM Tris (pH 9). This method only slightly increases the VG yield after filtration from 65% to 74%, with no and 0.01% P188, respectively. These dilution techniques provided lower than desired% VG yields, and other buffer replacement techniques were therefore investigated.
Table 9: screening of AAV9 affinity eluate preparation method for AEX chromatography
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The% VG yield was determined as (VG in AEX load)/(VG in affinity cell); 1M Tris base, pH 11 and 1M Tris, pH 9 were used for pH adjustment and applied at a dilution factor of less than 1 fold relative to the affinity cell.
Serial dilution to prepare AAV9 affinity cells for AEX chromatography
Serial dilution of AAV9 affinity pools was investigated to generate AEX loading while reducing the surface area and time of exposure of the vector to the surface. The serial dilution unit is shown in fig. 5.
Three platinum cured silicone tubes are connected together by a Y-shaped connector. Peristaltic pumps deliver 100mM Tris, pH 9 to the Y connector at a flow rate of 3.5 mL/min. The second peristaltic pump delivered AAV9 affinity cells to the Y connector at a flow rate of 0.25 mL/min. The ratio of these flow rates (14 parts dilution to 1 part affinity cell) was chosen to achieve 15-fold dilution and can be directly compared to similar dilution factors (tables 9 and 12). The connected fluid was passed through a platinum cured silicone tube having an inner diameter of 0.16cm and a length of 100 cm. The mixing tube dimensions were designed according to buffer mixing studies that showed that these conditions could result in stable, well mixed solutions.
The serially mixed solutions were collected, neutralized with 250mM sodium citrate (pH 3.5) and analyzed for ITR by qPCR. 79% of the VG pumped into the apparatus was recovered at the outlet of the mixing tube. Although this result showed a slight increase in the% VG yield compared to the batch dilution experiment, the yield was still lower than expected. Thus, further AEX load preparation experiments were performed by tangential flow filtration.
Tangential Flow Filtration (TFF) to prepare AAV9 affinity cells for AEX chromatography
To avoid VG loss during AEX loading preparation, TFF was used to maintain VG in high concentration and arginine was temporarily incorporated into the carrier-containing solution. In agreement with Table 10, fresh 20cm was used 2 The mPES hollow fiber membrane was subjected to two TFF runs. Balanced membranes, loaded with AAV9 affinity pool, and directed against 150mM acetate, 100mM glycine, 25mM MgCl 2 Diafiltration was performed at pH 4.2 (same buffer as AAV9 affinity cell). At the end of each step, the TFF system was suspended and the retentate vessel was sampled. Samples were analyzed for VG titer by qPCR on ITR and the results are shown in tables 9 and 10.
In run #1, diafiltration directly from affinity cell analogue buffer to 100mM Tris, 2mM MgCl 2 VG yield was 66% in 0.01% P188, pH 9. In run #2, 500mM arginine, a co-solvent known to reduce protein aggregation (Arakawa et al biophysical Chemistry (2007) 127 (1): 1-8), was added to diafiltration buffer 2 to increase VG yield to 93%, while the yield obtained in the absence of arginine was 66%. However, removal of arginine from the system in run #2 resulted in a significant loss of VG yield of 78%. 500mM arginine was not included in the final diafiltration buffer as it would give rise to a high conductivity (-20 mS/cm) AEX load, which would interfere with binding to the AEX resin. However, this finding has inspired a low solvent conductance at alkaline pH for other AAV9 stabilizing agents Investigation of the amino acid co-solvent at the rate. 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 AAV9 affinity cells upon dilution with 100mM Tris, pH 9
The Z-average (Z-AVG) was measured using Dynamic Light Scattering (DLS) to estimate capsid aggregation in AAV9 affinity cells diluted with 100mM Tris (pH 9). The Z-average is a reliable measure of the average size of particles in solution. As shown in table 11, AAV9 affinity pools were diluted with 100mM Tris, pH 9 (0 to 30 fold) in polypropylene tubes and immediately analyzed by DLS. For each dilution, a separate experiment was performed with fresh AAV9 affinity pools in new polypropylene tubes. After DLS analysis was completed, the pH and conductivity of the solution were measured.
The results are summarized in table 11 and fig. 6 and show that dilution of AAV9 affinity pools resulted in aggregation and increased Z-averages. The pH of the undiluted AAV9 affinity cell was 4.1, the conductivity was 6.0mS/cm, the Z average value was 15nm, and there was no aggregation. Double dilution with 100mM Tris (pH 9) raised the pH of the solution to 7.2, maintaining the conductivity at 5.8mS/cm, resulting in a 5-fold increase in aggregation compared to AAV9 affinity cells, with a Z average of 77nm. This result means that increasing the solution pH by estimating the AAV9 isoelectric point (calculated as-5.8 and-6.2 (Venkatakrishnan et al. (2013) j. Virology 87 (9): 4974-4984) for AAV9 complete and empty capsids, respectively) destabilizes the vector, resulting in product loss. Dilution factors of 5 and 10 times resulted in aggregation and very high Z-averages, 395nm and 221nm, respectively. Larger dilutions in the 15 to 30 fold range showed a mean value of 46-66nm and aggregation.
Overall, the results given in table 9 and table 10 indicate that a large amount of AAV9 vector was lost during AEX payload production. TFF loading preparation methods diluted in series and in the presence of 0.01% P188 at high VG concentrations did not prevent VG loss. The data in table 11 indicate 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: AAV9 affinity cell pH, conductivity, Z-average and aggregation diluted with 100mM Tris, pH 9
# aggregation present (+) or aggregation absent (-)
Example 4: screening of diluent cosolvents for preparing AAV9 affinity eluate for AEX
AAV9 affinity eluate was diluted 15-fold with various diluent cosolvents to determine the conditions that maximize the% VG yield during AEX loading preparation. The selected cosolvents include detergents, iodixanol, glycerol, magnesium chloride and amino acids. To investigate the effect of dilution alone, AAV9 affinity eluate was diluted with an affinity eluate pool buffer, i.e., 150mM acetate, 100mM glycine, 25mM MgCl, without pH or conductivity changes 2 pH 4.2. 14mL of the dilution was added to the polypropylene tube, followed by 1mL of AAV9 affinity eluate. The resulting solution was gently mixed by end-to-end stirring, pH and conductivity were measured, and a pre-filtered sample was taken. The diluted sample was then filtered through a 0.2 μm filter previously wetted with the diluent. The diluted and filtered samples were neutralized with 250mM sodium citrate (pH 3.5). The neutralized samples were analyzed by dynamic light scattering to estimate the particle Z-AVG and the relative amounts of aggregation, and ITR was analyzed by qPCR to determine VG titres.
The results of the diluent co-solvent screen showed that some co-solvents reduced aggregation, maintained Z-AVG around 30nm, and increased% VG yield compared to baseline diluent 100mM Tris, pH 9 (Table 12). The Z-AVG of the undiluted AAV9 affinity elution pool was 29nm, apparently without aggregation. Dilution of AAV9 affinity cells with affinity eluate pool buffer resulted in no increase in Z-AVG, no aggregation, but only 69% VG yield. This data shows that aggregation does not occur under conditions of the affinity cell (pH 4.2,7 mS/cm), but VG loss occurs through increased surface area by non-specific binding (by dilution).
Consistent with the results of example 3 (above), dilution of AAV9 affinity pool with 100mM Tris, pH 9 resulted in an increase in Z-AVG, high aggregation, with VG yield of only 59%. The addition of 0.01-1% P188 in 100mM Tris, pH 9 did not significantly improve performance compared to baseline buffer, with similar Z-AVG and aggregation. With 50mM arginine, 2mM MgCl 2 Dilution of AAV9 affinity eluate with 0.1% P188, 100mM Tris, pH 9, resulted in 33nm Z-AVG with no aggregation and a VG yield of about 80%, but with a conductivity of 4.5mS/cm, which interfered with AAV9 binding to AEX resin. Various histidine-containing dilutions provided desirable results. For example, 200mM histidine, 200mM Tris, 10mM MgCl 2 Dilution of AAV9 with 25% iodixanol (pH 8.8) can yield 99% VG yield. Iodixanol has potent uv activity and can interfere with uv readings in chromatographic systems, so this buffer and similar buffers are not used in AEX runs.
Importantly, AAV9 affinity eluate was diluted 15-fold with 0.5% P188, 200mM histidine, 200mM Tris, pH 8.8, and then filtered through a prewetted filter to give a yield of 35nm Z-AVG and 101% VG. The resulting diluted filtered solution had a pH of 8.8 and a conductivity of 2.5mS/cm, and the conditions may be suitable for incorporation with AEX resin. Thus, this dilution scheme is optimized in the examples below.
AAV2 aggregation studies involved screening various co-solvents by dilution stress assays in combination with DLS, and found that AAV2 aggregation was prevented by dilution to buffers containing various salts having an ionic strength of 200mM or more (Fraser et al molecular Therapy (2005) 12 (1): 171-178). A similar method for preparing AAV9 for AEX chromatography was not suitable for this example, as a solution with an ionic strength of 200mM or more reduced the binding of the carrier to the AEX resin (data not shown). Interestingly, the addition of the amino acids histidine, arginine or glycine to the dilutions did not inhibit AAV2 aggregation (Fraser et al molecular Therapy (2005) 12 (1): 171-178).
In this example, a mixture of histidine, arginine and glycineSubstance and MgCl 2 The combination of P188 and/or glycerol reduced aggregation of AAV9, but provided only a% VG yield of 69-80% (see diluent results R2, H3 in table 12). High% VG yields were obtained only when histidine was used in conjunction with the detergents P188 and Triton X-100 (see diluent results H7 and H8 in Table 12). These results indicate that high VG-yielding diluent co-solvents for AAV capsid (e.g., AAV 9) AEX payload preparation should contain detergents to reduce non-specific binding to surfaces (e.g., dilution vessel and filter) 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 dilution, filtered, analyzed by dynamic light scattering to determine the Z-average of estimated capsid aggregation (Z-AVG), and analyzed by qPCR to determine% VG yield.
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NR-a value not reported due to interference in DLS readout. NT-was not tested.
Example 5: optimization of dilution co-solvent for preparing recombinant AAV9 vector affinity eluate for AEX chromatography
The concentration of P188 in the dilution and the resulting conductivity of the diluted sample were optimized to achieve maximum recovery of AAV9 vector. The test dilutions P188 were 0.01%, 0.05%, 0.2% and 0.5% in concentration, while the diluted samples had conductivities of 2, 2.5 and 3mS/cm. In order to obtain different conductivities, different dilution factors were used. The diluted AAV9 vector affinity eluate was passed through a 0.2 μm filter and prewetted with each respective diluent. The resulting filtrate was assayed by qPCR on the transgenes to determine VG titers, the results are shown in table 13 and fig. 7A and 7B. The data shown in fig. 7A and 7B indicate that 0.5% P188 is required to obtain maximum VG yield, and that using a lower concentration (i.e., less than 0.5%) of P188 results in lower VG yield. Within the scope of the study, the final conductivity (or by combining dilution factors) did not significantly affect the% VG yield.
Table 13: optimization of P188 concentration, dilution factor and final conductivity
Example 6: enrichment of full AAV9 by optimized 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 form an optimized AEX chromatography that was able to enrich the complete AAV9 vector capsid with high% VG yield.
AAV9 affinity eluate was diluted 15-fold with a novel buffer comprising amino acids and a detergent co-solvent (200 mM histidine, 200mM tris,0.5% P188, pH 8.9) and filtered with a 0.2 μm filter. The 15-fold dilution is lower than that used in other methods (see us 2019-0002841; us 2019-0002842; us 2018-0002844), is easier to implement in mass production and gives high VG yields.
The pH of the resulting filtrate was 8.8 and the conductivity was 2.3mS/cm. In agreement with Table 14, the filtrate was loaded into POROS TM On a 50HQ column, elution was performed by a sodium acetate gradient, and fractions corresponding to 0.39CV were collected during the gradient elution.
The loaded and chromatographed fractions were neutralized with 250mM sodium citrate (pH 3.5) and purified by SEC A 260 /A 280 ITR was determined by AUC and qPCR. To test the repeatability of the AEX method, a second run was performed using the same materials and methods as the first run. The first run AEX chromatograms are shown in fig. 8A and 8B. SEC a of gradient elution fractions are provided in table 15 260 /A 280 And shows that the optimized AEX method is enriched compared to the loadWhole AAV9 vector. The percentage of complete, intermediate and empty capsids in the affinity cell (loaded on column), flow-through fraction and elution cell 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 conferring a high VG yield percentage.
Table 14: at 5.1mL POROS TM AEX chromatography optimized on 50HQ column
Table 15: SEC a from two duplicate fractions of optimized AEX method 260 /A 280 Analysis
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F/T: flow through (unbound fraction); the gradient elution fractions were numbered 1-14.
Table 16: determining the performance of an optimized AEX method by analyzing the intermediate and chromatographic fractions of the method
The% VG yield is calculated as (amount of VG per step or fraction)/(amount of VG in affinity cell) and thus the loss in load preparation is taken into account. Dash (-) indicates no measurement was performed.
Dilution and filtration of AAV9 affinity eluate produced 93% VG yield, confirming the results in examples 3 and 4. The flow-through fraction contained 10% VG, SEC A from the affinity cell starting material 260 /A 280 The population of capsids was 0.70 with 1% complete capsids, 14% intermediate capsids and 85% empty capsids. This junctionThe results indicate that the developed AEX method aided in further enrichment by sodium acetate gradient elution by column partitioning some empty capsids.
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% and consisted mainly of empty capsids. The broad cell contained 53% VG yield, average SEC A 260 /A 280 1.26, and 47% full AAV9 vector capsids, thus representing a 2.6-fold enrichment of full capsid percentage. The narrow elution pool contained an average yield of VG of 38%, SEC A of 1.28-1.29 260 /A 280 The ratio, and average AAV9 vector capsid population, was 53% full capsid, 23% intermediate capsid, and 24% empty capsid. Thus, the optimized AEX method enriched 2.9-fold complete AAV9 vector and consumed 2.8-fold empty capsids.
The results obtained from the narrow pool represent an ideal balance between% complete capsid and% VG yield. Therefore, to obtain similar results in most of the examples that follow, we have adopted a SEC A-based 260 /A 280 Fraction merge threshold of 1.25 (similar to minimum 1.25 obtained in narrow pool fractions 2-6, table 16). Thus, in 10 large-scale AEX runs in the examples below, there are 8 runs of SEC A 260 /A 280 Fractions 1.25 are included in the pool and SEC A is excluded 260 /A 280 All fractions of 1.24 or less.
The above data and policy examples illustrate one powerful feature of the optimized AEX method: flexibility of the merge strategy. High resolution gradient elution chromatography with SEC A 260 /A 280 Analysis of the collected fractions ensures a high percentage of complete capsids of the recovered product, irrespective of slight variations in the 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 of the present disclosure utilize lower dilution factors, histidine-containing dilution buffers, steeper elution gradients, sodium acetate as wash desalination, 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 tomno et al (molecular. Ter. Meth. Clin. Dev. (2018) 11:180-190), wherein the disclosed methods do not use AEX loading formulations having a conductivity of about 7mS/cm, do not use ammonium sulfate precipitation, nor do they use size exclusion chromatography. The novel and inventive methods disclosed herein can be carried out on a large scale and produce high yields of VG from affinity chromatography eluents.
To further illustrate the flexibility and robustness of the optimized AEX process, example 7 tested feed streams with different% complete carrier capsids.
Example 7: effect of percentage of complete capsid in affinity eluate on optimized AEX method performance
The optimized AEX method was tested for its ability to enrich complete AAV9 vector from feed materials with different percentages of AAV9 empty capsids. Briefly, HEK293 cells were grown in suspension culture and transfected with adenovirus helper plasmid and Rep2Cap9 plasmid (excluding plasmids containing the transgene cassette). Cells were cultured for three days after transfection, harvested, lysed, flocculated, depth filtered and absolute filtered. The resulting filtrate was subjected to affinity chromatography to generate an affinity pool containing AAV9 particles without vector genome (null transfected AAV9 affinity eluate, referred to herein as null capsid). To generate AEX starting material with different percentages of complete capsids, null transfected AAV9 affinity eluate was mixed with standard AAV9 affinity eluate at volume ratios of 0%, 20%, 40%, 60%, 80% and 100% null capsids.
The mixture was diluted 15-fold with 200mM histidine, 200mM Tris, 0.5% P188, pH 8.8, and filtered through 0.2 μm to give an AEX load of pH 8.8 with a conductivity of 2.6mS/cm. Consistent with table 17, the optimized AEX method was performed on 6 loads containing different percentages of capsids resulting from null transfection. For each AEX of the 6 AEX runs, 1.5X10 15 Individual total Viral Particles (VP) or 2.2X10 14 VP/mL resin uniformity challenge 6.67mL POROS TM 50HQ column. The chromatographic loading, flow-through and elution fractions were neutralized with 250mM sodium citrate (pH 3.5) and purified by SEC A 260 /A 280 Analysis was performed.
SEC A 260 /A 280 The data are reported in table 18 and fig. 9 and show that the optimized AEX method produced SEC a when 0%, 20% and 40% null capsid transfection starting materials were used 260 /A 280 The respective elution fractions were 1.25 or more. SEC A with column loads of 0%, 20% and 40% null capsids 260 /A 280 1.16, 1.10 and 1.01, respectively. The optimized AEX method uses these materials to produce 6 or 7 consecutive elution fractions, SEC a for each elution fraction 260 /A 280 ≥1.25。
SEC a containing 60%, 80% and 100% null capsid loading 260 /A 280 The values were 0.90, 0.77 and 0.62, respectively. The optimized AEX method enriches 60%, 80% and 100% of the starting material of the null capsid to produce the eluted fraction, maximum SEC a 260 /A 280 The values were 1.23, 1.16 and 0.83, respectively.
AAV9 upstream methods were performed on a 250L and 2000L disposable bioreactor (SUB) scale, and the resulting SEC A was harvested in combination 260 /A 280 AAV9 affinity eluate (n=7) of 1.10±0.1 and performing downstream manipulations of affinity chromatography. Thus, upstream and downstream processing (including the optimized AEX method described herein) resulted in AAV9 capsids, the full capsid percentage of which was available for gene therapy applications. Based on these results, the optimized AEX process was scaled up to achieve production at 250L and 2000L SUB scale, as described in the examples below.
Table 17: using POROS TM 50HQ column optimized AEX chromatography for starting materials with different percentages of complete capsids. All operations involved a 2.2X10 14 VP/mL resin evenly challenges the column, using transfection starting materials with different percentages of ineffective capsids.
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* A 2mm path length UV monitor was applied.
N Standard AAV9 affinity eluate and null capsid transfection AAV9 affinity eluate mixture at 0%, 20%, 40%, 60%, 80% and 100% null capsids by volume.
Table 18: SEC a of chromatographic fractions produced using optimized AEX method on feed of mixture with different% inactive capsids and standard affinity cell 260 /A 280
F/T: flow through (unbound) fraction. The elution fractions are given in digital form. The ratio is greater than or equal to 1.25 shown in bold.
Example 8: scaling up of optimized AEX process for enrichment of complete AAV9 vectors
The AEX method optimized for full AAV9 vector enrichment is scaled up for downstream processing of AAV vectors produced in 250L and 2000L disposable bioreactors (SUB). POROS (Power on demand) TM The size of the 50HQ column is based on 3×10 14 The scale-independent maximum challenge for VG/mL resins. Tables 19 and 20 provide the optimized AEX method implemented for 250L and 2000L SUB, respectively. On a 250L scale, a 1.3LAEX column with an Inner Diameter (ID) of 10cm and a bed height of 16cm was used. The AEX process was carried out on a 2000L SUB scale using a 6.4L AEX column with an ID of 20cm and a bed height of 20.5 cm. The residence time in both scales was fixed at 4.+ -. 0.5 min/CV, resulting in volumetric flow rates of 314mL/min and 1.8L/min for all steps of the AEX process performed in 250L and 2000L SUB, respectively. These flow rates are within acceptable ranges so that the pump and mixer on the chromatographic grid form a smooth-shaped linear sodium acetate gradient during product elution. At both scales, each chromatography step uses the same buffer for the same CV length The group was run except that the 2000L SUB-AEX process included an injection water rinse and disinfection and regeneration steps prior to use. Fig. 10 and 11 provide chromatograms of representative AEX runs performed on a 250L SUB scale and a 2000L SUB scale. In all scales tested (various small scale runs, 250L and 2000L SUB scale), the optimized AEX process produced similar A 260 /A 280 And (5) a chromatogram.
On a scale of 250L and 2000L, elution fractions of 1/3 Column Volume (CV) were collected. The fractions were neutralized with 250mM sodium citrate (pH 3.5) and assayed by various analytical techniques. SEC to determine A 260 /A 280 Ratio and percentage of high molar mass material (% HMMS). The residual amounts of Host Cell Protein (HCP) and host cell DNA (HC-DNA) were determined by ELISA and qPCR, respectively. qPCR was used to measure ITR copies to quantify VG on a 250L scale. qPCR was used to measure transgenic copies to quantify VG on a 2000L scale.
Table 19: at 1.3L POROS TM Optimized AEX chromatography on 50HQ column on 250L SUB scale
Table 20: at 6.4L POROS TM Optimized AEX chromatography with 50HQ column on 2000L SUB scale
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* The resin challenge was determined using qPCR method to measure transgenic copies.
Table 21 provides SEC A of the eluted fractions produced by the 250L and 2000L SUB AEX method, respectively 260 /A 280 Ratio. The method demonstrates the robustness of a broad range of column challenges: 2.7X10 12 -6.8×10 13 VG/mL resin. Nine AEX of ten AEX runs produced at least 6 fractions of SEC a thereof 260 /A 280 The ratio is more than or equal to 1.25. Batch 250L-1 used the affinity cell (0.94) with the lowest SEC ratio in this study and was the only AEX run that did not produce fractions with a ratio of > 1.25. For each AEX run, fraction 5 produced the highest SEC a among 7 out of 10 runs 260 /A 280 The ratios, therefore, show high consistency in chromatographic and fraction collection procedures at two different scales and various VG/mL resin challenges.
Tables 22 and 23 provide impurity profiles,% HMMS and SEC A for the individual AEX fractions of 250L-4 and 2000L-4 batches, respectively 260 /A 280 . The optimized AEX method clears a large number of HCPs from the affinity cell. For example, the 250L-4 affinity pool contains 51pg of HCP/1X 10 9 The VG, optimized AEX method clears HCP in eluted fractions 2-8 to LLOQ for AEX pool formation. 2000L-4 affinity pool contained 331pg HCP/1X 10 9 VG, which is cleared to LLOQ in elution fractions 2-9, is used to form the AEX pool.
AEX methods do not significantly reduce HC-DNA levels. The AEX method uses a sodium acetate elution gradient for HMMS, respectively. Early elution fractions contained <3% HMMS relative to the consumption of HMM (e.g., fractions 1-5 in 250L-4 and 2000L-4 runs). In contrast, the later eluted fractions contained higher relative levels of HMMS (e.g., fractions 8-10 from 2000L-4 run contained >7% HMMS).
Table 21: SEC a of affinity eluent reservoirs and AEX chromatographic fractions produced at 250L and 2000L SUB scale 260 /A 280
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Elution fractions are given in numbers. The ratio ≡1.25 is shown in bold. The 250L SUB VG/mL resin challenge was determined using qPCR method to measure ITR copies. 2000L SUB VG/mL resin challenges were determined using qPCR method to measure transgenic copies.
Table 22: SEC a from chromatographic fractions of AEX run at 250L SUB scale 260 /A 280 % HMMS and impurity profile (batch 250L-4)
The elution fractions are given in digital form. SEC A eluting fraction 2-8 260 /A 280 1.24 (shown in bold), so merge and continue processing. The LLoQ-detection amount is below the quantitative limit of the assay.
Table 23: SEC a from chromatographic fractions of AEX run at 2000L SUB scale 260 /A 280 % HMMS and impurity Profile (batch 2000L-4)
Elution fractions are given in numbers. SEC A eluting fraction 2-9 260 /A 280 1.25 (shown in bold), so merge and continue processing. The LLoQ assay was below the quantitative limit of the assay.
250L AEX run Using SEC A-based 260 /A 280 The various fractions of the ratio combine thresholds. Fractions 250L-1 and 250L-4 from the 250LAEX run were pooled based on SEC A therein 260 /A 280 Not less than 1.22 and not less than 1.24 respectively. Fractions 250L-2, 250L-3 and 250L-5 from the 250L AEX run and all five 2000L AEX run fractions were combined, all based on SEC A therein 260 /A 280 ≥1.25。
AEX product pools obtained from 2000L batches 2000L-2, 2000L-3, 2000L-4 and 2000L-5 were treated by a virus filtration step, while 250L batches and 2000L-1 batches were not virus filtered. AEX and/or virus filtration ponds obtained by ultrafiltration/diafiltration (UF/DF) and 0.2 μm filtration treatment to produce the Drug Substance (DS). None of the steps after AEX chromatography significantly affected the AAV9 empty capsid/full capsid ratio. Affinity cell and AEX cellqPCR was performed to determine the VG yield percentage for the AEX step. By AUC and SEC A 260 /A 280 The drug substance material was analyzed to determine the percent complete capsid of the purified AAV9 vector.
Table 24 reports the results and shows that the upscaled AEX method increases the percentage of complete capsids of the recovered AAV9 vector in high yield. The scale-up AEX method reduced the enriched total carrier to 45-65% of the total capsids and the empty capsids in 9 out of 10 drug substance batches to 28% total capsids as measured by AUC. During all 10 runs, the AEX method will SEC a 260 /A 280 The ratio increased from 0.94-1.25 in the affinity cell to 1.24-1.32 in the drug substance. The average VG step yield of the AEX process performed at 250L and 2000L scales was 47+/-11%.
Table 24: AEX performance at 250L and 2000L SUB scale.
1 "DS" means AUC and SEC A 260 /A 280 Is carried out on the raw material medicine (DS). None of the steps used to produce the drug substance after AEX affected the AAV9 vector empty capsid/full capsid ratio.
2 The% VG dilution yield was calculated as ((VG in diluted affinity cell)/(VG in affinity cell)) ×100%. "affinity cell" refers to the material collected from an affinity column; and are also referred to herein as "affinity eluents"
3 The% VG column yield was calculated as ((VG in AEX cell)/(VG in diluted affinity cell)) ×100%. "diluted affinity cell" is also referred to herein as "diluted affinity eluate". In this embodiment, the diluted affinity eluate is not filtered.
4 The% VG step yield was calculated as ((VG in AEX pool)/(VG in affinity pool)) ×100%.
The qPCR method of measuring ITR copies was used to determine 250LSUB VG/mL resin challenge and% VG yield.
* The 2000L SUB VG/mL resin challenge and% VG yield were determined using qPCR methods to measure transgenic copies.
Example 9: ultracentrifugation and cation exchange chromatography to separate empty capsids from complete AAV vector capsids
In another embodiment of the downstream processing of AAV9 vector in 250L SUB, density gradient Ultracentrifugation (UC) is used to separate empty capsids from complete vector. Bands containing 40% and 60% iodixanol were formed in UC tubes similar to the previously described method (Grieger et al molecular Therapy (2016) 24 (2): 287-297). The affinity eluate containing 25% iodixanol was added to the UC tube and ultracentrifuged. Fractions enriched for AAV capsids containing full length vector genomes were collected from the interface of 40% and 60% iodixanol bands. To remove iodixanol, the fractions are diluted and loaded onto a Cation Exchange (CEX) chromatographic column. AAV9 vector capsids bind to the CEX column, most iodixanol passes through the column in unbound fractions. AAV9 vector capsids eluted from the CEX column in a fraction substantially free of iodixanol. The CEX pool was treated forward by UF/DF and 0.2 μm filtration to produce a Drug Substance (DS) comprising AAV9 vector capsids.
Table 25 provides a comparison of the process performance of the uc+cex and optimized AEX methods, as well as the results of analysis of the drug substance produced by these methods. The optimized AEX process performed at 250L and 2000L scales provided average VG yields of 45±8% and 50±13%, respectively. These values are higher than the average 33+ -9% VG yield provided by the UC+CEX method. SEC a produced by all three methods 260 /A 280 The average DS readings for (1.26-1.30),% complete capsid (49-55%) and% empty capsid (20-25%) are very similar. The average percentage of intermediate capsids in the 2000L AEX method produced is slightly higher (32±4%) compared to the 250L AEX (24±3) and 250L uc+cex (23±4) methods produced DS.
The DS produced by these three methods has a high degree of similarity in terms of percentage HMMS, percentage purity, and HCP and HC-DNA levels. Overall, this data shows that AEX processes implemented at 250L and 2000L SUB scale provide process performance and product quality that are highly similar to the 250L uc+cex process.
Table 25: drug identification of AAV9 purified by ultracentrifugation and downstream processing of complete vector enrichment by cation exchange chromatography (UC+CEX) or optimized AEX method
The% VG step yield of UC+CEX for alpha through UC and CEX running methods.
AEX% VG step yield of β by dilution and AEX chromatographic procedures.
Gamma% VG step yield in 250L SUB as determined using qPCR method to measure ITR copies.
The% VG step yield in 2000L SUB was determined using qPCR method to measure transgenic copies.
Delta HCP levels for three UC+CEX drug substance batches were LLOQ, while HCP levels for one drug substance batch were 2.6 pg/1X 10 9 VG。
The LLOQ-detection amount is below the quantitative limit of the assay.
Example 10: optimized AEX method for enrichment of full AAV3B vectors
HEK 293 cells were grown in suspension culture and transfected with 2 plasmids to generate AAV3B vectors according to standard methods known in the art. HEK 293 cells were harvested, lysed, flocculated, and the resulting lysates filtered. AAV3B vector was purified from the clarified lysate by affinity chromatography. The affinity column was equilibrated, clarified lysate loaded, washed, and purified AAV3B vector eluted. 25mM MgCl 2 AAV3B vector affinity cell (also referred to as affinity eluate) was incorporated to achieve about 1.7mM of final MgCl in the diluted affinity cell 2 Concentration. The pH of the affinity cell was pH 7.6. The affinity cell was diluted about 15-fold (14-fold to 17.8-fold depending on the run) with a buffer containing 200mM histidine, 200mM Tris, 0.5% P188, pH 8.9. The pH of the diluted affinity eluate was 8.6 or more, the conductivity was 2.5mS/cm or less (target 2.3 mS/cm), and it was loaded on an AEX column.
Using POROS TM A50 HQ column, which had a volume of 49mL, had a bed height of 10cm and a diameter of 2.5cm. The target column loading challenge is 1×10 14 Up to 3X 10 14 vg/mL resin (e.g., about 2.4X10) 14 vg/mL). Table 26 provides the optimized AEX process conditions. The residence time for all steps in the AEX process was fixed at 2 minutes/CV to accommodate the lower elution gradient (compared to the previous examples) and the relatively smaller column. The elution gradient was 2.5 times lower than in the previous examples to maximize empty/full capsid discrimination. Empty capsids eluted first, followed by full AAV3B capsids (fig. 12). In this example, the full AAV3B capsid contains the vector genome with the transgene encoding amino acid sequence of SEQ ID NO. 15 (copper transport ATPase 2, deleted metal binding sites 1-4). Consistent with the lower gradient and wider elution peak, the fraction volume increased to 0.5CV (as opposed to 0.33CV for the previous example).
Table 26: optimized AEX chromatography for purification of complete rAAV3B vectors
Gradient elution was performed from 100% buffer A to 25% buffer A/75% buffer B at 37.5CV with a slope of 2% buffer B/CV. When the percentage of buffer B was 32% to 52% over the whole gradient, a total of 20 elution fractions were collected. Fractions were collected into containers pre-filled with 13.2% v/v (0.066 CV) 250mM sodium citrate (pH 3.5) to neutralize the fractions and reduce exposure of the capsids to alkaline pH. The pH of the neutralized fraction was in the range of pH 7.5 to 7.7. Will A 260 /A 280 The first elution fraction of ≡ 0.98 (determined by SEC) was combined with the successive elution fractions, but no more than 11 total fractions (table 27).
Table 27: elution fraction
The fractions shown in bold and underlined are combined.
The pooled fractions were assayed by various analytical techniques. Practical vg/mL resin challenges range from 6.3E13 to 9.4E13, on average 7.4E13 ±1.2e13. SEC to determine A 260 /A 280 Ratio. A with affinity cell 260 /A 280 In contrast, A of AEX pool 260 /A 280 Increased in all operating conditions (table 28). The percent complete, intermediate and empty capsids of the affinity and AEX elution cells were determined by analytical ultracentrifugation. An affinity cell with an average percentage of complete carrier of 11.2±2.1% was enriched to 22.9±2.9% in the AEX cell. The same affinity cell consumed empty capsids from 79.7±2.5% to 67.5±3.8% in the AEX cell.
Vg titers were determined by transgenic QPCR (table 28). Average percent vg dilution yield was 120% ± 12%. The average percentage of vg column yield was 47% ± 11%.
Table 28: AEX performance characteristics for purification of AAV3B vectors
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The method described in this example was used to create a purified rAAV3B vector pool that was enriched in complete capsids and depleted in empty capsids compared to the starting material (i.e., affinity eluate).
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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%.
CN202180088674.XA 2020-11-03 2021-11-01 Method for purifying AAV vectors by anion exchange chromatography Pending CN116802308A (en)

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