Adeno-associated virus purification method

Abstract

Provided herein are methods for producing adeno-associated virus (AAV) products and methods for purifying adeno-associated viruses. The AAV is loaded onto an affinity resin, a washing step is performed, and the AAV is eluted from the affinity resin. Various buffers for use in the washing step and elution are disclosed.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 611,709, filed on December 29, 2017, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to materials and methods for purifying adeno-associated viruses (AAV). Background Art

[0004] Adeno-associated virus (AAV) is a small, non-enveloped virus that packages a linear single-stranded DNA genome. AAV belongs to Parvoviridae and Dependovirus, because the productive infection of AAV occurs only in the presence of a helper virus (e.g., adenovirus or herpes virus). Even in the absence of a helper virus, AAV virus (serotype 2) can also be latent by being integrated into chromosome 19q13.4 of the host human genome. The AAV virus is the only known mammalian DNA virus capable of site-specific integration (Daya and Berns, Clinical Microbiology Reviews, 583–593 pages (2008)).

[0005] In order to make AAV safe for clinical use, AAV has been genetically modified at several locations in its genome. For example, in many viral vectors, the Rep gene (which is required for viral replication) and the elements required for site-specific integration have been eliminated from the AAV genome. This recombinant AAV (rAAV) exists in an extrachromosomal state and is very inefficiently integrated into genomic DNA. Therefore, the possibility of rAAV inducing random mutagenesis in host cells is reduced, if not completely eliminated. Due to these characteristics and lack of pathogenicity, rAAV shows great promise as a gene therapy vector in many aspects of preclinical and clinical applications. New serotypes and self-complementary vectors are in clinical testing. In addition to these ongoing vector developments, continued efforts are also focused on scalable manufacturing processes that can efficiently produce high titers of rAAV vectors with high purity and high potency.

[0006] Although much effort has been put into designing efficient, large-scale methods to purify AAV products suitable for human administration, there remains a need for better AAV purification methods. During the purification of AAV, various other proteins and materials in the host cell culture matrix can be removed more efficiently. Therefore, there is a need for AAV purification methods that include a step to remove host cell materials from the final AAV product. Summary of the invention

[0007] AAV vector production in cell culture is characterized by the formation of a complex matrix that contains material from disrupted cells. Specifically, host cell proteins, proteasomes, cell debris, and potential virus-specific receptors are often present in the material from disrupted cells. The disclosed method includes the steps of removing host cell material from the final AAV product under conditions that result in higher purity at a physiologically suitable pH.

[0008] In one aspect, a method for purifying an adeno-associated virus (AAV) is provided, the method comprising:

[0009] (a) loading the solution onto an affinity resin for AAV under conditions that allow binding between AAV in the solution and the affinity resin;

[0010] (b) performing at least two washing steps; and

[0011] (c) Elution of AAV from the affinity resin.

[0012] In some embodiments, the method further comprises contacting the AAV-containing solution with an anion exchanger and eluting the AAV-containing solution from the anion exchanger prior to loading the AAV-containing solution onto the affinity resin.

[0013] In some embodiments, at least one step in the washing step includes applying a buffer comprising an organic solvent or a detergent to the affinity resin. In some embodiments, the buffer comprises TrisHCl and salt. In some embodiments, the buffer comprises one or more of histidine, histidine-HCl, arginine-HCl, lysine-HCl, glycine, taurine, MES-Na, Bis-Tris and N-acetyl-D, L-tryptophan. In some embodiments, the salt is NaCl. In some embodiments, the buffer comprises sodium acetate. In some embodiments, the buffer comprises magnesium chloride. In some embodiments, the buffer comprises TrisHCl and ethylene glycol. In some embodiments, the buffer comprises one of sucrose and glycerol and arginine-HCl. In some embodiments, the buffer comprises taurine and ethylene glycol. In some embodiments, the buffer comprises arginine-HCl, lysine-HCl and histidine-HCl. In some embodiments, the buffer comprises TrisHCl and DMSO.

[0014] In some embodiments, at least three washing steps are performed. In some embodiments, three washing steps are performed. In some embodiments, three washing steps are performed consecutively.

[0015] In some embodiments, the organic solvent or detergent is polysorbate 80, ethylene glycol, sorbitol, mannitol, xylitol, DMSO, sucrose or trehalose. In some embodiments, the detergent includes one or more of Triton X100, polysorbate 80 and tri(n-butyl) phosphate (TNBP). In some embodiments, the buffer comprises Bis-Tris.

[0016] In some embodiments, the first wash step comprises applying a first buffer to the affinity resin, the first buffer comprising about 50 to about 2000 mM sodium acetate and about 0.05% to about 0.2% polysorbate 80, and the pH of the first buffer is about 5.2 to about 6.8;

[0017] The second wash step comprises applying a second buffer to the affinity resin, the second buffer comprising about 30 to about 200 mM TrisHCl and about 75 to about 500 mM salt, and the pH of the second buffer is about 7.5 to about 9.2; and

[0018] The third washing step includes applying a third buffer to the affinity resin, the third buffer comprising about 30 to about 200 mM TrisHCl and about 30 vol% to about 75 vol% ethylene glycol, and the third buffer has a pH of about 7.3 to about 8.8.

[0019] In some embodiments, the first wash step comprises applying a first buffer to the affinity resin, the first buffer comprising about 50 to about 500 mM sodium salt of 2-(N-morpholino)ethanesulfonic acid (MES-Na), about 3 to about 30 mM EDTA, and a solvent / detergent mixture comprising polysorbate 80, DMSO, and tri(n-butyl) phosphate (TNBP), and the pH of the first buffer is about 5.2 to about 6.8;

[0020] The second washing step comprises applying a second buffer to the affinity resin, the second buffer comprising about 30 to about 200 mM TrisHCl or arginine-HCl and about 75 to about 500 mM salt, and the pH of the second buffer is about 7.5 to about 9.2; and

[0021] The third washing step includes applying a third buffer to the affinity resin, the third buffer comprising about 20 to about 80 mM arginine-HCl and about 50 to about 200 mM salt, and the pH of the third buffer is about 7.3 to about 8.8.

[0022] In some embodiments, the first wash step comprises applying a first buffer comprising about 50 to about 200 mM taurine and 0.2% to 1.5% PEG (e.g., PEG6000) to the affinity resin, wherein the pH of the first buffer is about 5.2 to about 6.8;

[0023] a second wash step comprising applying a second buffer to the affinity resin, the second buffer comprising about 30 to about 300 mM glycine, and the pH of the second buffer being about 7.5 to about 9.2; and

[0024] The third wash step comprises applying a third buffer to the affinity resin, the third buffer comprising about 20 to about 150 mM taurine, about 30% to about 75% by volume ethylene glycol, and 0.05% to 0.2% octylpyranoglucoside, and the third buffer has a pH of about 7.3 to about 8.8.

[0025] In some embodiments, the first wash step comprises applying a first buffer to the affinity resin, the first buffer comprising about 80 to about 400 mM Bis-Tris and about 10 to about 20 grams of a solvent / detergent mixture comprising about Triton-X100, polysorbate 80, and TNBP in a ratio of about 11:3:3 (by weight), wherein the first buffer has a pH of about 5.2 to about 6.8;

[0026] A second wash step comprises applying a second buffer to the affinity resin, the second buffer comprising about 5 to about 20 mmol of sodium citrate, wherein the second buffer has a pH of about 7.5 to about 9.2; and

[0027] The third washing step comprises applying a third buffer to the affinity resin, wherein the third buffer comprises about 50 to about 200 mM arginine-HCl, about 50 to about 200 mM lysine-HCl, about 50 to about 200 mM histidine-HCl, and about 1 mM to about 4 mM N-acetyl-D,L-tryptophan and about 10% (w / w) to about 40% (w / w) polysorbate 80, and wherein the pH of the third buffer is about 7.3 to about 8.8.

[0028] In some embodiments, the first wash step comprises applying a first buffer to the affinity resin, the first buffer comprising about 50 nM to about 200 mM sodium acetate and about 0.05% to about 0.2% polysorbate 80, wherein the pH of the first buffer is about 5.2 to about 6.8;

[0029] A second wash step comprises applying a second buffer to the affinity resin, the second buffer comprising about 20 nM to about 100 mM TrisHCl and about 50 nM to about 200 nM salt, wherein the second buffer has a pH of about 7.5 to about 8.8; and

[0030] The third wash step includes applying a third buffer to the affinity resin, the third buffer comprising about 20 mM to about 100 mM TrisHCl and about 40% (w / w) to about 60% (w / w) ethylene glycol, and the third buffer has a pH of about 7.5 to about 8.8.

[0031] In some embodiments, the first wash step comprises applying a first buffer to the affinity resin, the first buffer comprising about 50 nM to about 200 mM sodium acetate and about 0.05% to about 0.2% polysorbate 80, wherein the pH of the first buffer is about 5.2 to about 6.8;

[0032] A second wash step comprises applying a second buffer to the affinity resin, the second buffer comprising about 20 nM to about 100 mM TrisHCl and about 50 nM to about 200 nM salt, wherein the second buffer has a pH of about 7.5 to about 8.8; and

[0033] The third wash step includes applying a third buffer to the affinity resin, the third buffer comprising about 20 mM to 100 mM TrisHCl, about 40% (w / w) to about 60% (w / w) ethylene glycol, and the third buffer has a pH of about 7.5 to about 8.8.

[0034] In some embodiments, the salt is selected from the group consisting of NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl, sodium acetate, and a combination of one or more of NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl, sodium acetate. In some embodiments, the salt is NaCl.

[0035] In some embodiments, the concentration of the salt does not exceed 500 mM. In some embodiments, the concentration of the salt does not exceed 200 mM. In some embodiments, the concentration of the salt in the third buffer does not exceed 500 mM. In some embodiments, the concentration of the salt in the third buffer does not exceed 200 mM.

[0036] In some embodiments, the method further comprises a fourth washing step, which is performed before the first washing step and comprises applying a fourth buffer comprising about 10 to about 30 mM TrisHCl and about 75 to about 250 mM NaCl to the affinity resin, and the pH of the fourth buffer is about 6.5 to about 8.0.

[0037] In some embodiments, the first buffer comprises about 100 mM sodium acetate, about 0.1% polysorbate 80, and the pH of the first buffer is about 6.0. In some embodiments, the second buffer comprises about 50 mM TrisHCl and about 125 mM NaCl, and the pH of the second buffer is about 8.5. In some embodiments, the third buffer comprises about 50 mM TrisHCl and about 50% by volume ethylene glycol, and the pH of the third buffer is about 8.5.

[0038] In some embodiments, the first wash step comprises applying a first buffer to the affinity resin, the first buffer comprising about 50 to about 200 mM sodium acetate and about 0.05% to about 0.2% polysorbate 80, and the pH of the first buffer is about 5.5 to about 6.5;

[0039] The second washing step comprises applying a second buffer to the affinity resin, the second buffer comprising about 10 to about 70 mM TrisHCl and about 75 to about 250 mM NaCl, and the pH of the second buffer is about 8.0 to about 9.0; and

[0040] The third washing step comprises applying a third buffer to the affinity resin, the third buffer comprising about 10 to about 70 mM TrisHCl and about 30% to about 75% by volume of ethylene glycol, and the pH of the third buffer is about 8.0 to about 9.0. In some embodiments, the method further comprises a fourth washing step, which is performed before the first washing step and comprises applying a fourth buffer comprising about 10 to about 30 mM TrisHCl and about 75 to about 250 mM NaCl to the affinity resin, and the pH of the fourth buffer is about 6.5 to about 8.0.

[0041] In some embodiments, the first buffer comprises about 100 mM sodium acetate and about 0.1% polysorbate 80, and the pH of the first buffer is about 6.0. In some embodiments, the second buffer comprises about 50 mM TrisHCl and about 125 mM NaCl, and the pH of the second buffer is about 8.5. In some embodiments, the third buffer comprises about 50 mM TrisHCl and about 50% by volume ethylene glycol, and the pH of the third buffer is about 8.0.

[0042] In some embodiments, the acidic component is removed. In some embodiments, the acidic component is host cell DNA, such as HEK293 DNA, and wherein the acidic component is reduced to a value below 250 pg / microgram of AAV antigen as measured by qPCR. In some embodiments, the acidic component is host cell DNA, such as HEK293 DNA, and wherein the acidic component is reduced to a value below 250 pg / microgram of AAV antigen as measured by ELISA.

[0043] In some embodiments, elution includes applying an elution buffer with a continuous linear increase in conductivity by gradient elution. In some embodiments, elution includes applying an organic solvent with a continuous linear increase in concentration by gradient elution. In some embodiments, elution includes contacting an affinity resin with an elution buffer, the elution buffer comprising ethylene glycol, a salt such as NaCl and a buffer such as TrisHCl, wherein the pH is at least 7.0. In some embodiments, the salt concentration is about 750mM, the buffer concentration is about 50mM, and the ethylene glycol is 50-60% (w / w). In some embodiments, the pH is about 8.0. In some embodiments, the salt is NaCl and the buffer is TrisHCl.

[0044] In some embodiments, elution comprises contacting the affinity resin with an elution buffer comprising about 750 mM NaCl and 50-60% (w / w) ethylene glycol at a pH of at least 7.0. In some embodiments, the elution buffer comprises at least about 55% (w / w) ethylene glycol. In some embodiments, elution comprises contacting the affinity resin with an elution buffer comprising about 50 mM Tris HCl, about 750 mM to about 1250 mM NaCl, and about 60% (w / w) ethylene glycol at a pH of at least 7.8.

[0045] In some embodiments, eluting comprises contacting the affinity resin with an elution buffer comprising about 2 mM magnesium chloride, about 50 mM arginine-HCl, about 750 mM to about 1000 mM NaCl, and at least about 55% (w / w) sucrose at a pH of at least about 8.0.

[0046] In some embodiments, elution further comprises

[0047] (a) contacting the affinity resin with a fifth buffer, the fifth buffer comprising about 20 to about 100 mM arginine-HCl and about 75 to about 250 mM NaCl, and the pH of the fifth buffer is about 7.5 to about 8.5; and

[0048] (b) contacting the affinity resin with a second elution buffer, wherein the second elution buffer comprises about 20 to about 100 mM arginine-HCl, about 40 to about 60% (w / w) glycerol, and about 500 to 1000 mM salt, and the pH of the second elution buffer is about 7.5 to about 8.5.

[0049] In some embodiments, the steps are performed continuously.

[0050] In some embodiments, eluting comprises contacting the affinity resin with an elution buffer comprising about 2 mM magnesium chloride, about 50 mM arginine-HCl, about 750 mM to about 1000 mM NaCl, and at least about 50% (w / w) glycerol, at a pH of at least about 8.0.

[0051] In some embodiments, eluting comprises contacting the affinity resin with an elution buffer comprising about 2 mM magnesium chloride, about 50 mM taurine, about 600 mM to about 1000 mM NaCl, about 0.05% to about 0.2% octyl glucopyranoside, and about 60% (w / w) ethylene glycol, at a pH of at least about 7.8. In some embodiments, eluting further comprises

[0052] (a) contacting the affinity resin with a fifth buffer, the fifth buffer comprising about 20 to about 100 mM Tris-HCl and about 75 to about 250 mM NaCl, and the pH of the fifth buffer is about 8.0 to about 8.8; and

[0053] (b) contacting the affinity resin with a second elution buffer comprising about 1 M ammonium sulfate, about 50 mM Tris HCl, and about 50% (v / v) ethylene glycol at a pH of at least about 6.8.

[0054] In some embodiments, the steps are performed continuously.

[0055] In some embodiments, elution comprises contacting the affinity resin with an elution buffer comprising about 1 M ammonium sulfate, about 50 mM Tris HCl, and about 50% (v / v) ethylene glycol, at a pH of at least about 6.8. In some embodiments, elution comprises contacting the affinity resin with an elution buffer comprising about 20% (w / w) sucrose, about 10% (w / w) sorbitol, about 5% (w / w) mannitol, or about 5% (w / w) sucrose, about 15% (w / w) glycerol, about 50 mM histidine, and about 750 to about 1000 mM NaCl, at a pH of at least about 7.8.

[0056] In some embodiments, elution further comprises

[0057] (a) contacting the affinity resin with a fifth buffer, the fifth buffer comprising about 20 to about 100 mM histidine, about 80 to about 120 mM NaCl, and the pH of the fifth buffer being about 8.0 to about 8.8; and

[0058] (b) contacting the affinity resin with a second elution buffer comprising about 20 to about 100 mM histidine, about 600 to about 900 mM NaCl, and about 5% to 60% (w / w) DMSO, and the pH of the fifth buffer is about 6.5 to about 8.5.

[0059] In some embodiments, the steps are performed continuously.In some embodiments, eluting comprises contacting the affinity resin with an elution buffer comprising about 100 mM glycine-HCl, about 200 mM NaCl, and a pH of about 2.5.

[0060] In some embodiments, the pH of the elution buffer is about 8.0. In some embodiments, the pH of the elution buffer is 8.0.

[0061] In some embodiments, elution comprises a stepwise increase in counterion concentration. In some embodiments, elution comprises a stepwise increase in organic solvent concentration. In some embodiments, the salt in the elution buffer is selected from monovalent, divalent or multivalent anions such as chloride, acetate, sulfate and citrate.

[0062] In some embodiments, the first wash step comprises applying a first buffer to the affinity resin, the first buffer comprising about 50 to about 200 mM sodium acetate, about 0.05% to about 0.2% polysorbate 80, and the pH of the first buffer is about 5.2 to about 6.8;

[0063] A second wash step comprises applying a second buffer to the affinity resin, the second buffer comprising about 25 to about 100 mM TrisHCl and about 50 to about 200 mM NaCl, and the pH of the second buffer is about 7.5 to about 9.2; and

[0064] The third washing step comprises applying a third buffer to the affinity resin, the third buffer comprising about 20 to about 100 mM TrisHCl and about 75 to about 250 mM NaCl, and the pH of the third buffer is about 7.5 to about 8.8. In some embodiments, the method further comprises eluting by applying purified water to the affinity resin, followed by applying about 20 to about 50 mM HCl at a pH of about 1.7 to about 2.5 to the affinity resin. In some embodiments, the method further comprises eluting by applying a gradient of 0% to 100% 20-50 mM hydrochloric acid / 800-1200 mM NaCl in 0.5-2.0 mM hydrochloric acid.

[0065] In some embodiments, the HC impurity level of the AAV obtained from the elution step is ≤ 99.9%. In some embodiments, the HC impurity level of the AAV obtained from the elution step is ≤ 99.0%.

[0066] In some embodiments, the AAV is AAV8 and the affinity resin is POROS TM CaptureSelect TM AAV8, the elution buffer is acidic and does not contain ethylene glycol. In some embodiments, the AAV is AAV9 and the affinity resin is POROS TM CaptureSelect TM For AAV9, the elution buffer was acidic and did not contain ethylene glycol.

[0067] In some embodiments, the AAV is AAV8 and the affinity resin is an immunoaffinity resin consisting of an immobilized monoclonal antibody against AAV8 from ADK type 8 or ADK type 8 / 9 immobilized on a chromatography matrix. In some embodiments, the AAV is AAV9 and the affinity resin is an immunoaffinity resin consisting of an immobilized monoclonal antibody against AAV9 from ADK type 9 or ADK type 8 / 9 immobilized on a chromatography matrix.

[0068] In some embodiments, the method further comprises contacting the AAV-containing solution with a filter comprising positively charged groups that effectively deplete the AAV-containing solution of acidic charged contaminants.

[0069] In some embodiments, the method further comprises nanofiltration of the AAV fraction to remove viruses larger than 35 nm.

[0070] In some embodiments, the method further comprises a polishing step comprising AEX chromatography using a column comprising a whisker gel.

[0071] In some embodiments, the method further comprises testing the AAV fraction by an AAV-specific ELISA.

[0072] In some embodiments, the AAV-specific ELISA is a sandwich ELISA specific for AAV.

[0073] In another aspect, an AAV product produced by a method according to any one of claims 1 to 83 is provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 Depicted is a Western blot showing significantly less heat shock protein 70 kDa (HSP70) according to the purification protocol with multiple wash steps as described herein (lane 4) compared to a comparative purification protocol without such wash steps (lane 6).

[0076] Figure 2 Depicted is a SDS-PAGE silver-stained gel showing AAV8 protein in lane 2, AAV8-containing eluate from a purification scheme with multiple wash steps as described herein (lane 4) and a comparative purification scheme without such wash steps (lane 6).

[0077] Figure 3 A Western blot is depicted showing that there is more AAV8 in the eluate prepared according to the purification scheme with multiple wash steps described herein (lane 4) than in the eluate of the comparative purification scheme without the wash steps (lane 6). An AAV8 reference sample is in lane 2.

[0078] Figure 4 Depicted is a SDS-PAGE silver-stained gel showing proteins present in various fractions from the initial loading (L) of cell culture medium from HEK 293 cells expressing AAV8 to the eluate (E) enriched with AAV8. There is little loss of AAV8 in the flow-through of the wash steps (W1, W2, and W3), and very little AAV8 is eluted from the column during the back extraction (S).

[0079] Figure 5 Western blots for AAV8 antigen are depicted. AAV8 was expressed in the initial load (L) of cell culture medium from HEK 293 cells expressing AAV8. Significantly less AAV8 was present in the effluent from the initial load (FT) and subsequent wash steps (W1, W2, and W3). AAV8 was present in the eluate (E, E2). Likewise, significantly less AAV8 was eluted from the column during the back extraction (S).

[0080] Figure 6 A chromatogram of the separation procedure according to Example 3 is depicted.

[0081] Figure 7 A chromatogram according to the separation procedure of Example 11 is depicted.

[0082] Figure 8 Depicted is a SDS-PAGE silver stained gel according to Example 11 showing the proteins present in the various fractions from each wash step and each eluate.

[0083] Fig. 9 A chromatogram of the separation procedure according to Example 12 is depicted.

[0084] Fig.10 Depicted is a SDS-PAGE silver stained gel according to Example 12 showing the proteins present in the various fractions from each wash step and each eluate.

[0085] Fig.11 A chromatogram of the separation process according to Example 13 is depicted.

[0086] Fig.12 Depicted is a SDS-PAGE silver stained gel according to Example 13 showing the proteins present in the various fractions from each wash step and each eluate.

[0087] Fig.13 A chromatogram of the separation procedure according to Example 14 is depicted.

[0088] Fig.14 Depicted is a SDS-PAGE silver stained gel showing the proteins present in the various fractions obtained from the wash steps and eluates as described in Example 14.

[0089] Fig.15 Depicted are Western blots for AAV antigens from various fractions obtained from the wash steps and eluates as described in Example 14.

[0090] Fig.16 A chromatogram according to the separation procedure of Example 15 is depicted.

[0091] Fig.17 A chromatogram according to the separation procedure of Example 16 is depicted.

[0092] Fig.18 Depicted are Western blots for AAV antigens from various fractions obtained from the wash steps and eluates as described in Example 16.

[0093] Fig.19 A chromatogram according to the separation procedure of Example 17 is depicted.

[0094] Fig. 20Depicted is a SDS-PAGE silver stained gel showing the proteins present in the various fractions obtained from the wash steps and eluates as described in Example 17.

[0095] Fig.21 Depicted is a chromatogram showing the results from the elution screen described in Example 18. DETAILED DESCRIPTION

[0096] Provided herein are methods for producing adeno-associated virus (AAV) products, methods for purifying AAV, and methods for purifying full AAV capsids from a concentrated AAV fraction comprising empty AAV capsids and full AAV capsids.

[0097] Unless otherwise indicated herein or clearly contradicted by context, in the context of describing the present disclosure (especially in the context of the following claims), the terms "a", "an", and "the", and similar referents are interpreted to cover both the singular and the plural. Unless otherwise indicated, the terms "comprising", "having", "including", and "containing" should be interpreted as open-ended terms (i.e., meaning "including but not limited to"),

[0098] Recitation of ranges of values ​​herein are merely intended to serve as a shorthand method of referring individually to each separate value and endpoint falling within the range, unless otherwise indicated herein, and each separate value and endpoint is incorporated into the specification as if it were individually recited herein.

[0099] Unless otherwise specified herein or clearly contradictory to the context, all methods described herein can be performed in any suitable order. Unless otherwise claimed, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present disclosure and will not limit the scope of the present disclosure. The language in the specification should not be interpreted as indicating any non-claimed essential elements for implementing the present disclosure.

[0100] Preferred embodiments of the present disclosure are described herein, including the best mode known to the inventor for implementing the present disclosure. For those of ordinary skill in the art, after reading the foregoing description, variations of those preferred embodiments will become apparent. The inventor expects that skilled technicians use such variations where appropriate, and the inventor intends to implement the disclosure in a manner different from that specifically described herein. Therefore, where applicable law permits, the present disclosure includes all modifications and equivalents of the subject matter cited in the attached claims. In addition, unless otherwise specified herein or clearly contradictory to the context, any combination of the above-mentioned elements in all possible variations thereof is encompassed in the present disclosure.

[0101] AAV vector production in cell culture is characterized by the formation of a complex matrix that contains material from disrupted cells. Specifically, host cell proteins, proteasomes, cell debris, and potential virus-specific receptors are often present in the material from disrupted cells. The disclosed method includes the steps of removing host cell material from the final AAV product under conditions that result in higher purity at a physiologically suitable pH.

[0102] In one aspect, a method for purifying adeno-associated virus (AAV) is provided. The method comprises (a) loading the solution containing AAV onto an affinity resin for AAV under conditions that allow binding between the AAV in the solution and the affinity resin; (b) performing at least two washing steps; and (c) eluting the AAV from the affinity resin.

[0103] In some embodiments, the AAV purified by the methods described herein is of AAV1 serotype, AAV2 serotype, AAV3 serotype, AAV4 serotype, AAV5 serotype, AAV6 serotype, AAV7 serotype, AAV8 serotype, AAV9 serotype, AAV10 serotype, AAV11 serotype, AAV12 serotype, AAV13 serotype, AAAV serotype, BAAV serotype, AAV (VR-195) serotype, and AAV (VR-355) serotype. In some embodiments, the AAV is modified by genetic engineering and / or is chemically modified.

[0104] In some embodiments, the method further comprises contacting a solution containing AAV with an anion exchanger, and eluting a solution containing AAV from the anion exchanger, after which the solution containing AAV is loaded onto an affinity resin. Anion exchangers can be operated in flow-through mode. In some embodiments, at least one step in the washing step comprises applying a buffer comprising an organic solvent or a detergent to an affinity resin. In some embodiments, the buffer comprises TrisHCl and a salt, such as NaCl. In some embodiments, the buffer comprises sodium acetate. In some embodiments, the buffer comprises magnesium chloride. In some embodiments, the buffer comprises TrisHCl and ethylene glycol. In some embodiments, the buffer comprises arginine-HCl and one of sucrose and glycerol. In some embodiments, the buffer comprises taurine and ethylene glycol. In some embodiments, the buffer comprises arginine-HCl, lysine-HCl and histidine-HCl. In some embodiments, the buffer comprises TrisHCl and DMSO.

[0105] In some embodiments, the buffer comprises arginine-HCl and a salt, such as NaCl. In some embodiments, the buffer comprises histidine and a salt, such as NaCl. In some embodiments, the buffer comprises TrisHCl and ethylene glycol. In some embodiments, the organic solvent is ethylene glycol. In some embodiments, the organic solvent is DMSO. In some embodiments, the salt is selected from the combination of one or more of NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl, sodium acetate and NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl and sodium acetate. In some embodiments, the concentration of salt is no more than 500mM. In some embodiments, the concentration of salt is no more than 200mM.

[0106] In some embodiments, at least three washing steps are performed. In some embodiments, three washing steps are performed. In some embodiments, three washing steps are performed consecutively.

[0107] In some embodiments, the organic solvent or detergent is polysorbate 80, ethylene glycol, sorbitol, mannitol, xylitol, DMSO, sucrose or trehalose. In some embodiments, the detergent includes one or more of Triton X100, polysorbate 80 and tri(n-butyl) phosphate (TNBP). In some embodiments, the buffer comprises Bis-Tris.

[0108] In some embodiments, at least three washing steps are performed; the first washing step comprises applying to the affinity resin a first buffer comprising about 50 to about 2000 mM sodium acetate and about 0.05% to about 0.2% polysorbate 80, and wherein the pH of the first buffer is about 5.2 to about 6.8; the second washing step comprises applying to the affinity resin a second buffer comprising about 30 to about 200 mM TrisHCl and about 75 to about 500 mM salt, and wherein the pH of the second buffer is about 7.5 to about 9.2; and the third washing step comprises applying to the affinity resin a third buffer comprising about 30 to about 200 mM TrisHCl and about 30% to about 75% by volume ethylene glycol, and wherein the pH of the third buffer is about 7.3 to about 8.8. In some embodiments, the salt is selected from NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl, sodium acetate and a combination of one or more of NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl and sodium acetate. In some embodiments, the salt is NaCl. In some embodiments, the concentration of the salt does not exceed 500mM. In some embodiments, the concentration of the salt does not exceed 200mM. In some embodiments, the salt concentration in the third buffer does not exceed 500mM. In some embodiments, the salt concentration in the third buffer does not exceed 200mM. In some embodiments, the method further includes a fourth washing step, which is performed before the first washing step and includes applying a fourth buffer comprising about 10 to about 30mM TrisHCl and about 75 to about 250mM NaCl to the affinity resin, and the pH of the fourth buffer is about 6.5 to about 8.0. In some embodiments, the first buffer comprises about 100mM sodium acetate, about 0.1% polysorbate 80, and wherein the pH of the first buffer is about 6.0. In some embodiments, the second buffer comprises about 50 mM TrisHCl and about 125 mM NaCl, wherein the pH of the second buffer is about 8.5. In some embodiments, the third buffer comprises about 50 mM TrisHCl and about 50% by volume ethylene glycol, and wherein the pH of the third buffer is about 8.5.

[0109] In some embodiments, the organic solvent or detergent is polysorbate 80, ethylene glycol, sorbitol, mannitol, xylitol, sucrose, or trehalose.

[0110] In some embodiments, at least three washing steps are performed; the first washing step comprises applying to the affinity resin a first buffer comprising about 50 to about 200 mM sodium acetate and about 0.05% to about 0.2% polysorbate 80, wherein the pH of the first buffer is about 5.5 to about 6.5; the second washing step comprises applying to the affinity resin a buffer comprising about 10 to about 70 mM TrisHCl and about 75 to about 250 mM NaCl, wherein the pH of the second buffer is about 8.0 to about 9.0; and the third washing step comprises applying to the affinity resin a third buffer comprising about 10 to about 70 mM TrisHCl and about 30% to about 75% by volume ethylene glycol, wherein the pH of the third buffer is about 8.0 to about 9.0. In some embodiments, the method further comprises a fourth washing step, which is performed before the first washing step and comprises applying to the affinity resin a fourth buffer comprising about 10 to about 30 mM TrisHCl and about 75 to about 250 mM NaCl, and the pH of the fourth buffer is about 6.5 to about 8.0. In some embodiments, the first buffer comprises about 100 mM sodium acetate and about 0.1% polysorbate 80, and the pH of the first buffer is about 6.0. In some embodiments, the second buffer comprises about 50 mM TrisHCl and about 125 mM NaCl, and wherein the pH of the second buffer is about 8.5. In some embodiments, the third buffer comprises about 50 mM TrisHCl and about 50% by volume ethylene glycol, and wherein the pH of the third buffer is about 8.0.

[0111] In more embodiments, at least three washing steps are performed; the first washing step comprises applying a first buffer to the affinity resin, the first buffer comprising about 50 to about 500 mM sodium salt of 2-(N-morpholino)ethanesulfonic acid (MES-Na), about 3 to about 30 mM EDTA, and a solvent / detergent mixture comprising polysorbate 80, DMSO, and tri(n-butyl) phosphate (TNBP), and wherein the pH of the first buffer is about 5.2 to about 6.8; the second washing step comprises applying a second buffer comprising about 30 to about 200 mM TrisHCl or arginine-HCl and about 75 to about 500 mM salt to the affinity resin, and wherein the pH of the second buffer is about 7.5 to about 9.2; and the third washing step comprises applying a third buffer comprising about 20 to about 80 mM arginine-HCl and about 50% (w / w) to about 60% (w / w) sucrose to the affinity resin, and wherein the pH of the third buffer is about 7.3 to about 8.8. In some embodiments, the salt is selected from NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl, sodium acetate and one or more combinations of NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl and sodium acetate. In some embodiments, the salt is NaCl. In some embodiments, the concentration of the salt does not exceed 500mM. In some embodiments, the concentration of the salt does not exceed 200mM. In some embodiments, the salt concentration in the third buffer does not exceed 500mM. In some embodiments, the salt concentration in the third buffer does not exceed 200mM. In some embodiments, the method also includes a fourth washing step, which is performed before the first washing step, and includes applying a fourth buffer comprising about 20 to about 100mM arginine-HCl and about 75 to about 250mM NaCl to the affinity resin, and wherein the pH of the fourth buffer is about 7.5 to about 8.0. In some embodiments, the first elution step comprises applying to the affinity resin a first elution buffer comprising about 20 to about 100 mM arginine-HCl and about 40% to about 60% (w / w) sucrose, wherein the pH of the first elution buffer is about 7.5 to about 8.5. In some embodiments, the method further comprises a fifth washing step performed after the first elution step and before the second elution step, the fifth washing step comprising applying to the affinity resin a fifth buffer comprising about 20 to about 100 mM arginine-HCl and about 75 to about 250 mM NaCl, and wherein the pH of the fifth buffer is about 7.5 to about 8.5.In some embodiments, the second elution step comprises applying a second elution buffer to the affinity resin, the second elution buffer comprising about 20 to about 100 mM arginine-HCl, about 40% (w / w) to about 60% (w / w) glycerol, and about 500 to 1000 mM salt (e.g., NaCl), and wherein the pH of the second elution buffer is about 7.5 to about 8.5. The fifth washing step can effectively minimize the fronting effect between the first elution step and the second elution step, for example, providing an elution triggered by only the first elution buffer and the second elution buffer themselves, rather than by a fronting that may be caused by a mixture of the first elution buffer and the second elution buffer. In some embodiments, the method further comprises a sixth washing step performed after the fifth washing step and the second elution step, the sixth washing step comprising applying a sixth buffer comprising about 20 to about 100 mM arginine-HCl and about 75 to about 250 mM NaCl to the affinity resin, and wherein the pH of the fifth buffer is about 7.5 to about 8.5.

[0112] In certain embodiments, the first buffer comprises about 100mM of 2-(N-morpholino)ethanesulfonic acid sodium salt (MES-Na), about 10mM EDTA and about 11g / kg of solvent / detergent mixture, the solvent / detergent mixture comprises about 18 grams of polysorbate 80, 3.5 grams of DMSO and 3.5 grams of tri(n-butyl) phosphate (TNBP), and wherein the pH of the first buffer is about 6.0. In certain embodiments, the second buffer comprises about 50mM arginine-HCl and about 100mM NaCl, and wherein the pH of the second buffer is about 8.0. In certain embodiments, the third buffer comprises about 50mM arginine-HCl and about 50% (w / w) sucrose, and wherein the pH of the third buffer is about 8.5. In certain embodiments, the fourth buffer comprises about 50mM arginine-HCl and about 100mM NaCl, and wherein the pH of the fourth buffer is about 8.0. In certain embodiments, the fifth buffer comprises about 50 mM arginine-HCl and about 100 mM NaCl, and wherein the pH of the fifth buffer is about 8.0. In certain embodiments, the sixth buffer comprises about 50 mM arginine-HCl and about 100 mM NaCl, and wherein the pH of the sixth buffer is about 8.0.

[0113] In some embodiments, at least three washing steps are performed; the first washing step comprises applying to the affinity resin a first buffer comprising about 50 to about 200 mM taurine and 0.2% to 1.5% PEG (e.g., PEG 6000), and wherein the pH of the first buffer is about 5.2 to about 6.8; the second washing step comprises applying to the affinity resin a second buffer comprising about 30 to about 300 mM glycine, and wherein the pH of the second buffer is about 7.5 to about 9.2; and the third washing step comprises applying to the affinity resin a third buffer comprising about 20 to about 150 mM taurine, about 30% to about 75% by volume ethylene glycol, and 0.05% to 0.2% octylpyranoglucoside, and wherein the pH of the third buffer is about 7.3 to about 8.8. In some embodiments, the salt is selected from NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl, sodium acetate and one or more combinations of NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl and sodium acetate. In some embodiments, the salt is NaCl. In some embodiments, the concentration of the salt is no more than 500mM. In some embodiments, the concentration of the salt is no more than 200mM. In some embodiments, the method further comprises a fourth washing step, which is performed before the first washing step, and comprises applying a fourth buffer comprising about 20 to about 100mM TrisHCl and about 75 to about 250mM NaCl to the affinity resin, and the pH of the fourth buffer is about 7.5 to about 8.8.

[0114] In some embodiments, the first elution step comprises applying a first elution buffer to the affinity resin, the first elution buffer comprising about 30 to about 70 mM taurine, about 50% to about 70% by volume ethylene glycol, 0.05% to 0.2% octylpyranoglucoside, and about 600 to about 900 mM NaCl, and wherein the pH of the first elution buffer is about 7.5 to about 8.5. In some embodiments, the method further comprises a fifth washing step performed after the first elution step and before the second elution step, the fifth washing step comprising applying a fifth buffer comprising about 20 to about 100 mM TrisHCl and about 75 to about 250 mM NaCl to the affinity resin, and wherein the pH of the fifth buffer is about 7.5 to about 8.8. The fifth washing step can effectively minimize the front effect. In some embodiments, the second elution step comprises applying a second elution buffer to the affinity resin, the second elution buffer comprising about 30 to about 70 mM TrisHCl, 0.05 to 0.2 M ammonium sulfate, and about 40% to about 60% by volume ethylene glycol, and wherein the pH of the second elution buffer is about 6.5 to about 7.5. The fifth washing step can effectively minimize the pre-effect between the first elution step and the second elution step, for example, providing an elution triggered only by the first elution buffer and the second elution buffer themselves, rather than by a pre-effect that may be caused by a mixture of the first elution buffer and the second elution buffer.

[0115] In some embodiments, the method further comprises a sixth wash step performed after the fifth wash step and the second elution step, the sixth wash step comprising applying a sixth buffer comprising about 20 to about 100 mM TrisHCl and about 75 to about 250 mM NaCl to the affinity resin, and wherein the pH of the fifth buffer is about 7.5 to about 8.8.

[0116] In certain embodiments, the first buffer comprises about 100 mM taurine and about 0.5% PEG 6000, wherein the pH of the first buffer is about 6.0. In certain embodiments, the second buffer comprises about 100 mM glycine, and the pH of the second buffer is about 7.5 to about 9.2. In certain embodiments, the third buffer comprises about 50 mM taurine, about 50% (w / w) ethylene glycol, and 0.1% octylpyranoglucoside, and wherein the pH of the third buffer is about 8.5. In certain embodiments, the fourth buffer comprises about 50 mM TrisHCl and about 125 mM NaCl, and wherein the pH of the fourth buffer is about 8.5. In certain embodiments, the fifth buffer comprises about 50 mM TrisHCl and about 125 mM NaCl, and wherein the pH of the fifth buffer is about 8.5. In certain embodiments, the sixth buffer comprises about 50 mM TrisHCl and about 125 mM NaCl, and wherein the pH of the sixth buffer is about 8.5.

[0117] In some embodiments, at least three washing steps are performed; the first washing step comprises applying a first buffer to the affinity resin, the first buffer comprising about 80 to about 400 mM Bis-Tris, and about 10 to about 20 grams of a solvent / detergent mixture comprising Triton-X100, polysorbate 80, and TNBP in a ratio of 11:3:3 (by weight), and wherein the pH of the first buffer is about 5.2 to about 6.8; a second wash step comprises applying to the affinity resin a second buffer comprising about 5 to about 20 mmol of sodium citrate, and wherein the pH of the second buffer is about 7.5 to about 9.2; and a third wash step comprises applying to the affinity resin a third buffer comprising about 50 to about 200 mM arginine-HCl, about 50 to about 200 mM lysine-HCl, about 50 to about 200 mM histidine-HCl, about 1 mM to about 4 mM N-acetyl-D,L-tryptophan, and about 10% (w / w) to about 40% (w / w) polysorbate 80, and wherein the pH of the third buffer is about 7.3 to about 8.8. In some embodiments, the salt is selected from NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl, sodium acetate and one or more combinations of NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl and sodium acetate. In some embodiments, the salt is NaCl. In some embodiments, the concentration of the salt is no more than 500mM. In some embodiments, the concentration of the salt is no more than 200mM. In some embodiments, the method also includes a fourth washing step, which is performed before the first washing step, and includes applying a fourth buffer comprising about 20 to about 100mM histidine and about 75 to about 250mM NaCl to the affinity resin, and wherein the pH of the fourth buffer is about 7.5 to about 8.8.

[0118] In some embodiments, the first elution step comprises applying a first elution buffer to the affinity resin, the first elution buffer comprising about 15% to 25% sucrose, 5% to 15% (w / w) sorbitol, 3% to 7% (w / w) mannitol, 10% to 20% (w / w) glycerol, 40 to 60 mM histidine, and 700 to 900 mM salt (e.g., NaCl), and wherein the pH of the first elution buffer is about 7.5 to about 8.5. In some embodiments, the method further comprises a fifth washing step performed after the first elution step and before the second elution step, the washing step comprising applying a first elution buffer comprising about 20 to about 100 mM histidine and about 75 to about 250 mM NaCl to the affinity resin, and wherein the pH of the fifth buffer is about 7.5 to about 8.8. In some embodiments, the second elution step comprises applying a second elution buffer to the affinity resin, the second elution buffer comprising about 30 to about 70 mM TrisHCl, about 700 to about 900 mM salt (e.g., NaCl) and 40% (w / w) to 60% (w / w) DMSO, and wherein the pH of the second elution buffer is about 7.5 to about 8.5. The fifth washing step can effectively minimize the pre-effect between the first elution step and the second elution step, for example, providing an elution triggered by only the first elution buffer and the second elution buffer themselves, rather than by a pre-triggered elution that may be caused by a mixture of the first elution buffer and the second elution buffer. In some embodiments, the method further comprises a sixth washing step performed after the fifth washing step and the second elution step, the sixth washing step comprising applying a sixth buffer comprising about 20 to about 100 mM histidine and about 75 to about 250 mM NaCl to the affinity resin, and wherein the pH of the fifth buffer is about 7.5 to about 8.8.

[0119] In certain embodiments, the first buffer comprises about 200 mM Bis-Tris, about 16 to about 17 grams of a solvent / detergent mixture, the solvent / detergent mixture comprising about Triton-X100, polysorbate 80, and TNBP in a ratio of about 11:3:3 (by weight), wherein the pH of the first buffer is about 6.0. In some embodiments, the second buffer comprises about 10 mM sodium citrate, and the pH of the second buffer is about 8.5. In some embodiments, the third buffer comprises about 100 mM arginine-HCl, about 100 mM lysine-HCl, about 100 mM histidine-HCl, about 2 mM N-acetyl-D, L-tryptophan, and about 20% (w / w) polysorbate 80, and the pH of the third buffer is about 8.5. In certain embodiments, the fourth buffer comprises about 50 mM histidine and about 100 mM NaCl, and wherein the pH of the fourth buffer is about 8.5. In certain embodiments, the fifth buffer comprises about 50 mM histidine and about 100 mM NaCl, and wherein the pH of the fifth buffer is about 8.5. In certain embodiments, the sixth buffer comprises about 50 mM histidine and about 100 mM NaCl, and wherein the pH of the sixth buffer is about 8.5.

[0120] In some embodiments, at least three washing steps are performed; the first washing step comprises applying a first buffer to the affinity resin, the first buffer comprising about 50 nM to about 200 nM sodium acetate and about 0.05% to about 0.2% polysorbate 80, wherein the pH of the first buffer is about 5.2 to about 6.8; the second washing step comprises applying a second buffer comprising about 20 nM to about 100 nM TrisHCl and about 50 nM to about 200 nM salt to the affinity resin, wherein the pH of the second buffer is about 7.5 to about 8.8; and the third washing step comprises applying a third buffer comprising about 20 mM to 100 mM TrisHCl, about 40% (w / w) to 60% (w / w) ethylene glycol to the affinity resin, and wherein the pH of the third buffer is about 7.5 to about 8.8. In some embodiments, the salt is selected from NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl, sodium acetate and one or more combinations of NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl and sodium acetate. In some embodiments, the salt is NaCl. In some embodiments, the concentration of the salt is no more than 500mM. In some embodiments, the concentration of the salt is no more than 200mM. In some embodiments, the method also includes a fourth washing step, which is performed before the first washing step, and includes applying a fourth buffer comprising about 10 to about 50mM TrisHCl and about 75 to about 250mM NaCl to the affinity resin, and the pH of the fourth buffer is about 6.5 to about 8.0. In some embodiments, the method further comprises a fifth washing step, which is performed after the third washing step and comprises applying to the affinity resin a fifth buffer comprising about 20 mM to about 100 mM TrisHCl, about 50% (w / w) to about 70% (w / w) ethylene glycol and about 500 to about 900 mM NaCl, and wherein the pH of the fifth buffer is about 7.5 to about 8.5. In some embodiments, the method further comprises a sixth washing step, which is performed after the fifth washing step and comprises applying to the affinity resin a sixth buffer comprising about 10 mM to about 50 mM TrisHCl and about 75 mM to about 250 mM NaCl, and wherein the pH of the sixth buffer is about 7.0 to about 8.0.

[0121] In certain embodiments, the first buffer comprises about 100 mM taurine and about 0.5% PEG 6000, wherein the pH of the first buffer is about 6.0. In certain embodiments, the second buffer comprises about 100 mM glycine, and the pH of the second buffer is about 7.5 to about 9.2. In certain embodiments, the third buffer comprises about 50 mM taurine, about 50% by volume ethylene glycol, and 0.1% octylpyranoglucoside, and wherein the pH of the third buffer is about 8.5. In certain embodiments, the fourth buffer comprises about 20 mM TrisHCl and about 150 mM NaCl, and wherein the pH of the fourth buffer is about 7.4. In certain embodiments, the fifth buffer comprises about 50 mM TrisHCl, 60% (w / w) ethylene glycol, and 750 mM NaCl, and wherein the pH of the fifth buffer is about 8.0. In certain embodiments, the sixth buffer comprises about 20 mM TrisHCl and about 150 mM NaCl, and wherein the pH of the sixth buffer is about 7.4.

[0122] In some embodiments, at least three washing steps are performed; the first washing step comprises applying a first buffer to the affinity resin, the first buffer comprising about 50 nM to about 200 nM sodium acetate and about 0.05% to about 0.2% polysorbate 80, wherein the pH of the first buffer is about 5.2 to about 6.8; the second washing step comprises applying a second buffer comprising about 20 nM to about 100 nM TrisHCl and about 50 nM to about 200 nM salt to the affinity resin, wherein the pH of the second buffer is about 7.5 to about 8.8; and the third washing step comprises applying a third buffer comprising about 20 mM to 100 mM TrisHCl, about 40% (w / w) to 60% (w / w) ethylene glycol to the affinity resin, and wherein the pH of the third buffer is about 7.5 to about 8.8. In some embodiments, the salt is selected from NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl, sodium acetate and one or more combinations of NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl and sodium acetate. In some embodiments, the salt is NaCl. In some embodiments, the concentration of the salt is no more than 500mM. In some embodiments, the concentration of the salt is no more than 200mM. In some embodiments, the method also includes a fourth washing step, which is performed before the first washing step, and includes applying a fourth buffer comprising about 10 to about 50mM TrisHCl and about 75 to about 250mM NaCl to the affinity resin, and the pH of the fourth buffer is about 6.5 to about 8.0. In some embodiments, the method further comprises a fifth washing step, which is performed after the third washing step and comprises applying to the affinity resin a fifth buffer comprising about 20 mM to about 100 mM TrisHCl, about 50% (w / w) to about 70% (w / w) ethylene glycol and about 500 to about 900 mM NaCl, and wherein the pH of the fifth buffer is about 7.5 to about 8.5. In some embodiments, the method further comprises a sixth washing step, which is performed after the fifth washing step and comprises applying to the affinity resin a sixth buffer comprising about 10 mM to about 50 mM TrisHCl and about 75 mM to about 250 mM NaCl, and wherein the pH of the sixth buffer is about 7.0 to about 8.0.

[0123] In certain embodiments, the first buffer comprises about 100 mM taurine and about 0.5% PEG 6000, wherein the pH of the first buffer is about 6.0. In certain embodiments, the second buffer comprises about 100 mM glycine, and the pH of the second buffer is about 7.5 to about 9.2. In certain embodiments, the third buffer comprises about 50 mM taurine, about 50% by volume ethylene glycol, and 0.1% octylpyranoglucoside, and wherein the pH of the third buffer is about 8.5. In certain embodiments, the fourth buffer comprises about 20 mM TrisHCl and about 150 mM NaCl, and wherein the pH of the fourth buffer is about 7.4. In certain embodiments, the fifth buffer comprises about 50 mM TrisHCl, 60% (w / w) ethylene glycol, and 750 mM NaCl, and wherein the pH of the fifth buffer is about 8.0. In certain embodiments, the sixth buffer comprises about 20 mM TrisHCl and about 150 mM NaCl, and wherein the pH of the sixth buffer is about 7.4.

[0124] In some embodiments, at least three washing steps are performed; the first washing step comprises applying to the affinity resin a first buffer comprising about 50 to about 200 mM sodium acetate and 0.05% to 0.2% polysorbate 80, and wherein the pH of the first buffer is about 5.2 to about 6.8; the second washing step comprises applying to the affinity resin a second buffer comprising about 25 to about 100 mM TrisHCl and about 50 to 200 mM salt, and wherein the pH of the second buffer is about 7.5 to about 9.2. In some embodiments, the salt is selected from NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl, sodium acetate, and a combination of one or more of NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl, and sodium acetate. In some embodiments, the salt is NaCl. In some embodiments, the method further comprises a third washing step, which is performed before the first washing step and comprises applying a third buffer comprising about 20 to about 100 mM TrisHCl and about 75 to about 250 mM NaCl to the affinity resin, and wherein the pH of the third buffer is about 7.5 to about 8.8. In some embodiments, the first elution step comprises applying purified water to the affinity resin, followed by applying 0.5 to 2 mM HCl at a pH of 3.0 to 3.5, followed by applying a buffer comprising about 25 to about 100 mM TrisHCl, about 500 to about 1000 mM NaCl, and about 25% (w / w) to about 75% (w / w) DMSO, and wherein the pH of the buffer is about 7.5 to about 8.5. In some embodiments, the method further comprises a fourth washing step performed after the first elution step and before the second elution step, the fourth washing step comprising applying purified water to the affinity resin. In some embodiments, the second elution step comprises applying about 20 to about 50 mM HCl at a pH of about 1.7 to about 2.5 to the affinity resin.

[0125] In certain embodiments, the first buffer comprises about 100mM sodium acetate and about 0.1% polysorbate 80, wherein the pH of the first buffer is about 6.0. In certain embodiments, the second buffer comprises about 50mMTrisHCl, about 125mM NaCl, and the pH of the second buffer is about 8.5. In certain embodiments, the third buffer comprises about 50mM TrisHCl and about 125mM NaCl, wherein the pH of the third buffer is about 8.5. In certain embodiments, the first elution step comprises applying purified water to the affinity resin, followed by applying 1mM HCl having a pH of about 3.2, followed by applying a buffer comprising about 50mM TrisHCl, about 750mM NaCl and about 50% (w / w) DMSO to the affinity resin, and wherein the buffer comprises a pH of about 8.0. In certain embodiments, the second elution step comprises applying about 33mM HCl having a pH of about 2.0 to the affinity resin.

[0126] In some embodiments, at least three washing steps are performed; the first washing step comprises applying to the affinity resin a first buffer comprising about 50 to about 200 mM sodium acetate and 0.05% to 0.2% polysorbate 80, and wherein the pH of the first buffer is about 5.2 to about 6.8; the second washing step comprises applying to the affinity resin a second buffer comprising about 25 to about 100 mM TrisHCl and about 50 to 200 mM salt, and wherein the pH of the second buffer is about 7.5 to about 9.2. In some embodiments, the salt is selected from NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl, sodium acetate, and a combination of one or more of NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl, and sodium acetate. In some embodiments, the salt is NaCl. In some embodiments, the method further comprises a third washing step, which is performed before the first washing step and comprises applying to the affinity resin a third buffer comprising about 20 to about 100 mM hydrochloric acid and about 75 to about 250 mM NaCl, and wherein the pH of the third buffer is about 7.5 to about 8.8. In some embodiments, the first elution step comprises applying to the affinity resin a gradient of 0% to 100% 20-50 mM hydrochloric acid / 800-1200 mM NaCl in 0.5-2.0 mM hydrochloric acid. In certain embodiments, the gradient is 0% to 100% 33 mM hydrochloric acid / 1000 mM NaCl in 1 mM hydrochloric acid. In certain embodiments, washing with purified water is performed prior to the first elution step.

[0127] In certain embodiments, the first buffer comprises about 100 mM sodium acetate and about 0.1% polysorbate 80, wherein the pH of the first buffer is about 6.0. In certain embodiments, the second buffer comprises about 50 mM TrisHCl, about 125 mM NaCl, and the pH of the second buffer is about 8.5. In certain embodiments, the third buffer comprises about 50 mM TrisHCl and about 125 mM NaCl, wherein the pH of the third buffer is about 8.5. In certain embodiments, the first elution step comprises applying a buffer comprising about 50 mM TrisHCl, about 750 mM NaCl and about 50% (w / w) DMSO to the affinity resin, and wherein the buffer comprises a pH of about 8.0.

[0128] In some embodiments, the organic solvent or detergent is polysorbate 80, ethylene glycol, sorbitol, mannitol, xylitol, sucrose, or trehalose.

[0129] In some embodiments, the acidic component is removed. In some embodiments, the acidic component is host cell DNA, such as HEK293 DNA, and the acidic component is reduced to a value below 250 pg / microgram of AAV antigen as measured by qPCR. In some embodiments, the acidic component is host cell DNA, such as HEK293 DNA, and wherein the acidic component is reduced to a value below 250 pg / microgram of AAV antigen as measured by ELISA.

[0130] In some embodiments, elution includes contacting the affinity resin with an elution buffer comprising ethylene glycol, a salt such as NaCl and a buffer such as TrisHCl, wherein the pH is at least 7.0. In some embodiments, the salt concentration is about 750 mM, the buffer concentration is about 50 mM, and the ethylene glycol is 50-60% (w / w). In some embodiments, the concentration of ethylene glycol is at least 55% (w / w). In some embodiments, the salt is NaCl and the buffer is TrisHCl. In some embodiments, the pH of the elution buffer is about 8.0. In some embodiments, the pH of the elution buffer is 8.0.

[0131] In some embodiments, elution includes contacting the affinity resin with an elution buffer comprising a sugar such as sucrose, a salt and a buffer such as arginine-HCl, wherein the pH is at least 8.0. In some embodiments, the salt concentration is about 800mM. In some embodiments, the buffer concentration is about 50mM. In some embodiments, the sugar is sucrose, and the elution buffer comprises 50-60% (w / w) sucrose. In some embodiments, the elution buffer comprises 1-3mM MgCl2 or about 2mM MgCl2. In some embodiments, the salt is NaCl and the buffer is arginine-HCl. In some embodiments, the pH of the elution buffer is about 8.0. In some embodiments, the pH of the elution buffer is 8.0.

[0132] In some embodiments, elution comprises contacting the affinity resin with an elution buffer comprising ethylene glycol, a salt such as NaCl and taurine, wherein the pH is at least 7.0. In some embodiments, the pH is 8.0. In some embodiments, the salt concentration is about 750 mM. In some embodiments, the buffer concentration is about 50 mM. In some embodiments, the concentration of ethylene glycol is 50-70% (w / w). In some embodiments, the concentration of ethylene glycol is at least 55% (w / w). In some embodiments, the elution buffer further comprises 0.05% to 0.2% octylpyranoglucoside. In some embodiments, the pH of the elution buffer is about 8.0. In some embodiments, the pH of the elution buffer is 8.0.

[0133] In some embodiments, elution includes contacting the affinity resin with an elution buffer comprising (i) a mixture of sucrose, sorbitol, mannitol, and glycerol, (ii) a salt such as NaCl, and (iii) a buffer such as histidine, wherein the pH is at least 7.8. In some embodiments, the salt concentration is about 800 mM. In some embodiments, the buffer concentration is about 50 mM. In some embodiments, the concentration of sucrose is about 20% (w / w). In some embodiments, the concentration of sorbitol is about 10% (w / w). In some embodiments, the concentration of mannitol is about 5% (w / w). In some embodiments, the concentration of glycerol is about 15% (w / w). In some embodiments, the salt is NaCl and the buffer is histidine. In some embodiments, the pH of the elution buffer is about 8.0. In some embodiments, the pH of the elution buffer is 8.0.

[0134] In some embodiments, a second elution step is performed, which comprises contacting the affinity resin with an elution buffer comprising (i) a buffer, (ii) a salt such as NaCl, and (iii) 50-60% (w / w) ethylene glycol, wherein the pH is at least 8.0. In some embodiments, the salt concentration is about 1000 mM. In some embodiments, the buffer concentration is about 50 mM. In some embodiments, the concentration of ethylene glycol is 60% (w / w). In some embodiments, the buffer is TrisHCl. In some embodiments, the salt is NaCl and the buffer is histidine. In some embodiments, the pH of the elution buffer is about 8.0. In some embodiments, the pH of the elution buffer is 8.0.

[0135] In some embodiments, a second elution step is performed, the step comprising contacting the affinity resin with an elution buffer, the elution buffer comprising (i) a buffer, (ii) a salt such as NaCl, and (iii) 50-60% (v / v) glycerol, wherein the pH is at least 8.0. In some embodiments, the salt concentration is about 800mM, the buffer concentration is about 50mM, and the glycerol concentration is 50% (v / v). In some embodiments, the salt is NaCl and the buffer is arginine-HCl. In some embodiments, the pH of the elution buffer is about 8.0. In some embodiments, the pH of the elution buffer is 8.0. In various embodiments, the fifth washing step is performed after the first elution step and before the second elution step. The fifth washing step comprises contacting the affinity resin with a fifth buffer, the fifth buffer comprising 40-60mM arginine-HCl and 80-120mM NaCl, and the pH is 7.5 to 8.5. The fifth wash step can effectively minimize the pre-effect between the first elution step and the second elution step, for example, providing an elution triggered only by the first elution buffer and the second elution buffer themselves, rather than by a pre-effect that may be caused by a mixture of the first elution buffer and the second elution buffer.

[0136] In some embodiments, a second elution step is performed, the step comprising contacting the affinity resin with an elution buffer, the elution buffer comprising (i) a buffer, (ii) a salt such as (NH4)2SO4, and (iii) 40-60% (v / v) ethylene glycol, wherein the pH is at least 8.0. In some embodiments, the salt concentration is about 1M, the buffer concentration is about 50mM, and the ethylene glycol concentration is 50% (v / v). In some embodiments, the salt is (NH4)2SO4 and the buffer is TrisHCl. In some embodiments, the pH of the elution buffer is about 7.0. In some embodiments, the pH of the elution buffer is 7.0. In various embodiments, the fifth washing step is performed after the first elution step and before the second elution step. The fifth washing step comprises contacting the affinity resin with a fifth buffer comprising 40-60 mM taurine, 50-70% (w / w) ethylene glycol, 600-900 mM NaCl and 0.05% to 0.2% octylpyranoglucoside, with a pH of 7.5 to 8.5. The fifth washing step can effectively minimize the pre-effect between the first elution step and the second elution step, for example, providing an elution triggered by only the first elution buffer and the second elution buffer themselves, rather than by a pre-effect that may be caused by a mixture of the first elution buffer and the second elution buffer.

[0137] In some embodiments, a second elution step is performed, the step comprising contacting the affinity resin with an elution buffer, the elution buffer comprising (i) a buffer, (ii) a salt such as NaCl, and (iii) 50-60% (w / w) DMSO, wherein the pH is at least 8.0. In some embodiments, the salt concentration is about 1000 mM. In some embodiments, the buffer concentration is about 50 mM. In some embodiments, the concentration of DMSO is 50% (w / w). In some embodiments, the salt is NaCl and the buffer is TrisHCl. In some embodiments, the pH of the elution buffer is about 8.0. In some embodiments, the pH of the elution buffer is 8.0. In various embodiments, the elution buffer containing DMSO effectively elutes AAV9 from the CaptureSelect AAV8 resin, but not AAV8. In various embodiments, the elution buffer containing DMSO effectively elutes AAV8 and AAV9 from the CaptureSelect AAVx resin. In various embodiments, an elution buffer containing DMSO is effective to elute one or more of AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV10, AAV11, AAV12, AAV13, AAAV, BAAV, AAV(VR-195), and AAV(VR-355) from CaptureSelect AAV8 resin, but not AAV8. In various embodiments, an elution buffer containing DMSO is effective to elute any of AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAAV, BAAV, AAV(VR-195), and AAV(VR-355) from CaptureSelect AAVx resin.

[0138] In various embodiments, the fifth washing step is performed after the first elution step and before the second elution step. The fifth washing step comprises contacting the affinity resin with a fifth buffer, the fifth buffer comprising 40-60 mM histidine and 70-130 mM NaCl, with a pH of 8.0 to 8.8. The fifth washing step can effectively minimize the pre-effect between the first elution step and the second elution step, for example, providing an elution triggered by only the first elution buffer and the second elution buffer themselves, rather than by a pre-triggered elution that may be caused by a mixture of the first elution buffer and the second elution buffer.

[0139] In some embodiments, elution comprises increasing the conductivity of the elution buffer continuously and linearly by gradient elution. In some embodiments, elution comprises increasing the concentration of the organic solvent continuously and linearly by gradient elution. In some embodiments, elution comprises contacting the affinity resin with an elution buffer containing about 750 mM NaCl and 50-60% (w / w) ethylene glycol at a pH of at least 7.0. In some embodiments, the concentration of ethylene glycol is at least 55% (w / w). In some embodiments, the pH of the elution buffer is about 8.0. In some embodiments, the pH of the elution buffer is 8.0.

[0140] In some embodiments, elution comprises a stepwise increase in counterion concentration. In some embodiments, elution comprises a stepwise increase in organic solvent concentration. In some embodiments, the salt in the elution buffer is selected from monovalent, divalent, or multivalent anions, such as chloride, acetate, sulfate, and citrate. In some embodiments, the HC impurity level of the AAV obtained from the elution step is ≤99.9%. In some embodiments, the HC impurity level of the AAV obtained from the elution step is ≤99.0%.

[0141] In some embodiments, the AAV is AAV8 and the affinity resin is POROS TM CaptureSelect TM AAV8, the elution buffer is acidic and does not contain ethylene glycol. In some embodiments, the AAV is AAV9 and the affinity resin is POROS TM CaptureSelect TM AAV9, and wherein the elution buffer is acidic and does not contain ethylene glycol. In some embodiments, the AAV is AAV8, and wherein the affinity resin is an immunoaffinity resin consisting of an immobilized monoclonal antibody against AAV8 from ADK8 or ADK8 / 9 immobilized on a chromatographic matrix. In some embodiments, the AAV is AAV9, and wherein the affinity resin is an immunoaffinity resin consisting of an immobilized monoclonal antibody against AAV9 from ADK9 or ADK8 / 9 immobilized on a chromatographic matrix.

[0142] In some embodiments, the method further comprises contacting the solution containing AAV with a filter comprising a positively charged group that effectively depletes the solution containing AAV of acidic charged contaminants. In some embodiments, the method further comprises nanofiltration of the AAV fraction to remove viruses greater than 35 nm. In some embodiments, the method further comprises a fine purification step comprising AEX chromatography with a column comprising a tentacle gel. In some embodiments, the method further comprises testing the AAV fraction by an AAV-specific ELISA, such as an ELISA specific for AAV8 or for AAV9. The AAV-specific ELISA can be a sandwich ELISA specific for AAV (e.g., AAV8 or AAV9).

[0143] In another aspect, an AAV product produced by any of the methods described herein is provided.

[0144] The affinity step is used to purify AAV particles, such as AAV8 and AAV9 particles, by performing multiple defined washing steps, such as by applying a wash buffer to the affinity matrix that binds the AAV and eluting at near neutral pH to maintain the infectivity of the virus. In certain embodiments, an anion exchange step is included prior to the affinity step. The anion exchange step can be performed in a flow-through mode. In certain embodiments, contaminants that affect the cycle time of the affinity resin used in the affinity step can be depleted.

[0145] The methods of the invention can result in increased purity of AAV virus, removal of host cell proteins, depletion of host cell DNA, removal of potential viral receptors, partial to complete inactivation of lipid enveloped viruses in which AAV binds to a ligand (e.g., during a washing step), and partial to complete inactivation of lipid enveloped viruses in a liquid phase (e.g., during an elution step). Without wishing to be bound by theory, ethylene glycol, by itself or in combination with another additive, can inactivate such lipid enveloped viruses. Exemplary additives include nonionic detergents, aliphatic agents (e.g., TnBP), and detergents (e.g., polysorbates (e.g., Tween), Triton X100, TnBP). For example, the solvent-detergent mixture can include 1% Triton X100, 0.3% tri-n-butyl phosphate, and 0.3% TWEEN 80.

[0146] When using the Baculo transfection system, inactivation of enveloped viruses can be particularly important. Elution according to the various embodiments described herein can prevent low pH exposure and maintain high potency of AAV. Further improvements can be seen when the washing step and the elution step are performed consecutively according to the various embodiments and examples described herein.

[0147] In Examples 11, 12 and 13 above, inactivation of lipid enveloped viruses "on-column" was tested in various affinity chromatography runs, as summarized in Table 1 below.

[0148] Table 1: Solvent detergent treatments used in variants A, B and C

[0149]

[0150] The DMSO-containing buffer Wash X buffer was surprisingly effective in triggering the elution of AAV9 but not AAV8 on CaptureSelect AAV8 resin at near neutral pH (e.g., pH 8.0). The DMSO-containing buffer Wash X buffer was surprisingly effective in triggering the elution of various AAVx on CaptureSelect AAVx resin at near neutral pH (e.g., pH 8.0), including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAAV, BAAV, AAV (VR-195), and AAV (VR-355). Without wishing to be bound by theory, Wash X buffer is expected to have the activity of washing the column and / or inactivating or decomposing lipid enveloped viruses. It was not expected that Wash X buffer would differentially elute AAV9 and AAV8.

[0151] The affinity purification step includes one or more washing steps. The one or more washing steps may be followed by one or more elution steps. In certain embodiments, the method of the present disclosure includes a filtration step, which is performed before the affinity purification step.

[0152] After culturing host cells (e.g., HEK293 cells) to produce AAV particles (e.g., AAV8, AAV9, etc.), the cell-free culture supernatant of the clarification is concentrated and / or filtered, and the viral particles are loaded onto an affinity matrix. In certain embodiments, the viral particles are loaded into a solution having a pH range of about 7.4 to about 7.8. In certain embodiments, the viral particles are loaded into a solution having a pH range of about 8.3 to about 8.7. In certain embodiments, the viral particles are loaded into a solution having a pH of about 8.5. In certain embodiments, the pH is 8.3 to 8.7, and the solution comprises NaCl and TrisHCl. In certain embodiments, the viral particles are loaded into a solution having a pH of about 8.5 comprising about 20mM TrisHCl and about 150mM NaCl.

[0153] At least 3 different washing steps may be performed, each step involving the same or different buffers. In certain embodiments, the washing buffers are different.

[0154] In certain embodiments, the first washing step uses the first buffer, which can be a buffer based on sodium acetate (NaAcetate).In certain embodiments, the first washing step uses the first buffer comprising the sodium salt (MES-Na) of 2-(N-morpholino) ethanesulfonic acid, EDTA and the solvent / detergent mixture comprising polysorbate 80, DMSO and tri(n-butyl) phosphate (TNBP).In certain embodiments, the first washing step uses the first buffer comprising the taurine of about 50 to about 200mM and 0.2% to 1.5% PEG (for example, PEG 6000).In certain embodiments, the first washing step uses the first buffer comprising Bis-Tris and the solvent / detergent mixture comprising Triton-X100, polysorbate 80 and TNBP.In certain embodiments, the first washing step uses the first buffer comprising sodium acetate and polysorbate 80.

[0155] In certain embodiments, the second washing step uses the second buffer, which can be a buffer based on Tris, a buffer based on glycine, a buffer based on sodium citrate or a buffer based on arginine-HCl comprising sodium chloride (NaCl). In certain embodiments, the third washing step uses the third buffer, which can be a buffer based on Tris, a buffer based on taurine or a buffer based on arginine-HCl comprising ethylene glycol and / or NaCl. Alternatively, one or more of sorbitol, mannitol, xylitol, sucrose or trehalose can be used in combination with ethylene glycol or replace ethylene glycol. In certain embodiments, the optional fourth washing step or rebalancing step are carried out before the three washing steps listed above. In the optional fourth washing step, the fourth buffer is used, which can be a buffer based on Tris comprising NaCl.

[0156] In certain embodiments, before affinity purification of a solution containing AAV from host cell production, pre-purification can be performed to remove one or more complex acidic protein structures and host cell DNA. Pre-purification can be performed by anion exchange in flow-through mode. The pre-purification step can be performed before any affinity purification method described herein. One or more of the following substances can be removed by pre-purifying this solution containing AAV: histones (e.g., histone H2A type 1, histone H2B type 1-B, histone H4, histone H1.4), 60S ribosomal proteins (e.g., 60S ribosomal protein L27, 60S ribosomal protein L6 and 60S ribosomal protein L30), cytoplasmic actin (e.g., cytoplasmic actin 1), tubulin (e.g., tubulin β-2A chain), heterogeneous nuclear ribonucleoprotein C, Rep68 protein, HEK293 laminin receptor 37kDa form (LamR 37kDa) and ATP-dependent molecular chaperone HSC82.

[0157] Washing steps can effectively remove strongly bound contaminants from the AAV and / or the base resin of the affinity matrix. For example, the buffer may comprise one or more of the following: TrisHCl, acetate, phosphate, histidine, imidazole, lysine, arginine, glycine, taurine, citrate, HEPES, MES, MES-Na, borate, Bis-Tris, MOPS, bicine, tricine, TAPS, TAPSO, MES, PIPES, TES (2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid), Veronal, ADA (N-(2-acetamido)iminodiacetic acid), ACES (N-(2-acetamido)-2-aminoethanesulfonic acid), Bis-Tris propane, BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), DIPSO (3-(N,N-bis[2-hydroxyethyl] The invention also provides a method for preparing a HEK-HCP-containing aqueous solution of at least one amino acid, wherein the aqueous solution comprises at least one amino acid, such as 1,4-diamino-2-hydroxypropanesulfonic acid, 1,4-bis(2-hydroxypropanesulfonic acid) dihydrate, 1,4-diamino-2-hydroxypropanesulfonic acid ...

[0158] In certain embodiments, the washing buffer may also include an organic solvent or a detergent. For example, the organic solvent or detergent may be, but is not limited to, Tween 80, polysorbate 80, Triton X100, tri(n-butyl) phosphate (TNBP), ethylene glycol, sorbitol, mannitol, xylitol, sucrose or trehalose. For example, the detergent may be, but is not limited to, a nonionic polyoxyethylene surfactant (e.g., Brij 35), 4-nonylphenyl-polyethylene glycol (Arkopal N100), octyl glycoside, n-dodecyl β-D-maltoside, digitonin, 6-cyclohexylhexyl β-D-maltoside or octyl pyranoglucoside. For example, ethylene glycol may be PEG, such as, but not limited to, PEG 2000, PEG 4000, PEG 6000 (Macrogol). For example, the organic solvent may be, but is not limited to, glycerol (1,2,3-propanetriol) and erythritol (meso-1,2,3,4-butanetetrol).

[0159] Organic solvent or detergent need not be present in all wash buffers used. However, organic solvent or detergent is present in at least one wash buffer used. In some embodiments, wash buffer, such as the first wash buffer, comprises sodium acetate and Tween 80. In some embodiments, wash buffer comprises one or more of Tween 80, DMSO and tri(n-butyl) phosphate (TNBP). In some embodiments, wash buffer comprises one or more of Triton-X100, polysorbate 80 and TNBP. In some embodiments, wash buffer, such as the third wash buffer, comprises Tris and ethylene glycol. Without wishing to be bound by theory, organic solvent and detergent in wash buffer can effectively remove strongly bound host proteins and viral receptors, while also inactivating and / or decomposing lipid enveloped viruses.

[0160] The first buffer may comprise about 50 to about 2000 mM sodium acetate, or 50 to about 250 mM sodium acetate, and about 0.05% to about 5% polysorbate 80 or Tween 80, or 0.05% to about 0.2% polysorbate 80 or Tween 80, wherein the pH is 5.2 to 6.8. In certain embodiments, the first buffer may comprise about 50 to about 75 mM, about 75 to about 100 mM, about 90 to about 110 mM, about 100 to about 125 mM, about 125 to about 150 mM sodium acetate, about 150 to about 175 mM sodium acetate, about 175 to about 200 mM sodium acetate, about 200 to about 250 mM sodium acetate, about 250 to about 300 mM sodium acetate, about 300 to about 350 mM sodium acetate, about 350 to about 400 mM sodium acetate, about 400 to about 450 mM sodium acetate, or about 500 to about 500 mM sodium acetate. 0 mM sodium acetate, about 450 to about 500 mM sodium acetate, about 500 to about 550 mM sodium acetate, about 550 to about 600 mM sodium acetate, about 600 to about 650 mM sodium acetate, about 650 to about 700 mM sodium acetate, about 700 to about 750 mM sodium acetate, about 750 to about 800 mM sodium acetate, about 800 to about 850 mM sodium acetate, about 850 to about 900 mM sodium acetate, about 900 to about 950 mM sodium acetate, about 950 to about 1000 mM sodium acetate, about 1000 to about 15 ... about 1000 to about 1050 mM sodium acetate, about 1050 to about 1100 mM sodium acetate, about 1100 to about 1150 mM sodium acetate, about 1150 to about 1200 mM sodium acetate, about 1200 to about 1250 mM sodium acetate, about 1250 to about 1300 mM sodium acetate, about 1300 to about 1350 mM sodium acetate, about 1350 to about 1400 mM sodium acetate, about 1400 to about 1450 mM sodium acetate, about 1450 to about 1500 mM acetic acid In some embodiments, the present invention relates to sodium acetate at about 1500 to about 1550 mM sodium acetate, about 1550 to about 1600 mM sodium acetate, about 1600 to about 1650 mM sodium acetate, about 1650 to about 1700 mM sodium acetate, about 1700 to about 1750 mM sodium acetate, about 1750 to about 1800 mM sodium acetate, about 1800 to about 1850 mM sodium acetate, about 1850 to about 1900 mM sodium acetate, about 1900 to about 1950 mM sodium acetate, or about 1950 to about 2000 mM sodium acetate.

[0161] In certain embodiments, the first buffer may comprise about 50 to about 500 mM of the sodium salt of 2-(N-morpholino)ethanesulfonic acid (MES-Na), about 3 to about 30 mM EDTA, and a solvent / detergent mixture comprising polysorbate 80, DMSO, and tri(n-butyl) phosphate (TNBP). In certain embodiments, the first buffer may comprise about 50 to about 75 mM, about 75 to about 100 mM, about 90 to about 110 mM, about 100 to about 125 mM, about 125 to about 150 mM, about 150 to about 175 mM, about 175 to about 200 mM, about 200 to about 250 mM, about 250 to about 300 mM, about 300 to about 350 mM, about 350 to about 400 mM, about 400 to about 450 mM, or about 450 to about 500 mM of the sodium salt of MES-Na. In certain embodiments, the first buffer may include about 50, about 75, about 90 mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, or about 500 mM of the sodium salt of MES-Na.

[0162] In certain embodiments, the first buffer may include about 50 to about 200 mM taurine. In certain embodiments, the first buffer may include about 50 to about 75 mM, about 75 to about 100 mM, about 90 to about 110 mM, about 100 to about 125 mM, about 125 to about 150 mM, about 150 to about 175 mM, about 175 to about 200 mM taurine. In certain embodiments, the first buffer may include about 50, about 75, about 90 mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM taurine.

[0163] In certain embodiments, the first buffer may include about 80 to about 400mM Bis-Tris. In certain embodiments, the first buffer may include about 80 to about 100mM, about 90 to about 110mM, about 100 to about 125mM, about 125 to about 150mM, about 150 to about 175mM, about 175 to about 200mM, about 200 to about 250mM, about 250 to about 300mM, about 300 to about 350mM, about 350 to about 400mM Bis-Tris. In certain embodiments, the first buffer may include about 50, about 75, about 90mM, about 100mM, about 125mM, about 150mM, about 175mM, about 200mM, about 250mM, about 300mM, about 350mM, about 400mM Bis-Tris.

[0164] In certain embodiments, the first buffer may include sodium acetate of about 50 to about 200 mM. In certain embodiments, the first buffer may include sodium acetate of about 50 to about 80 mM, about 70 to about 100 mM, about 80 to about 110 mM, about 90 to about 120 mM, about 100 to about 130 mM, about 120 to about 150 mM, about 140 to about 170 mM, about 170 to about 200 mM. In certain embodiments, the first buffer may include sodium acetate of about 60, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, or about 160 mM.

[0165] In certain embodiments, the first buffer may comprise about 25 mM sodium acetate, about 50 mM sodium acetate, about 75 mM sodium acetate, about 100 mM sodium acetate, about 125 mM sodium acetate, about 150 mM sodium acetate, about 175 mM sodium acetate, about 200 mM sodium acetate, about 225 mM sodium acetate, about 250 mM sodium acetate, about 275 mM sodium acetate, about 300 mM sodium acetate, about 325 mM sodium acetate, about 350 mM sodium acetate, about 375 mM sodium acetate, about 400 mM sodium acetate, about 425 mM sodium acetate, about 450 mM sodium acetate, about 475 mM sodium acetate, about 500 mM sodium acetate, or about 600 mM sodium acetate. , about 525mM sodium acetate, about 550mM sodium acetate, about 575mM sodium acetate, about 600mM sodium acetate, about 625mM sodium acetate, about 650mM sodium acetate, about 675mM sodium acetate, about 700mM sodium acetate, about 725mM sodium acetate, about 750mM sodium acetate, about 775mM sodium acetate, about 800mM sodium acetate, about 825mM sodium acetate, about 850mM sodium acetate, about 875mM sodium acetate, about 900mM sodium acetate, about 925mM sodium acetate, about 950mM sodium acetate, about 975mM sodium acetate, about 1000mM sodium acetate, about 1025mM sodium acetate, about 1050 mM sodium acetate, about 1075 mM sodium acetate, about 1100 mM sodium acetate, about 1125 mM sodium acetate, about 1150 mM sodium acetate, about 1175 mM sodium acetate, about 1200 mM sodium acetate, about 1225 mM sodium acetate, about 1250 mM sodium acetate, about 1275 mM sodium acetate, about 1300 mM sodium acetate, about 1325 mM sodium acetate, about 1350 mM sodium acetate, about 1375 mM sodium acetate, about 1400 mM sodium acetate, about 1425 mM sodium acetate, about 1450 mM sodium acetate, about 1475 mM sodium acetate, about 1500 mM sodium acetate, about 1525 mM sodium acetate, about In some embodiments, the first buffer may comprise about 100 mM sodium acetate or 100 mM sodium acetate. In some embodiments, the first buffer may comprise about 100 mM sodium acetate or 100 mM sodium acetate.

[0166] In certain embodiments, the first buffer may comprise about 0.05% to about 0.08%, about 0.08% to about 0.11%, about 0.11% to about 0.14%, about 0.14% to about 0.17%, or about 0.17% to about 0.20% polysorbate 80. In certain embodiments, the first buffer may comprise about 0.1% polysorbate 80 or 0.1% polysorbate 80. In certain embodiments, the first buffer may comprise about 0.05% to about 0.08%, about 0.08% to about 0.11%, about 0.11% to about 0.14%, about 0.14% to about 0.17%, or about 0.17% to about 0.20% Tween 80. In certain embodiments, the first buffer may comprise about 0.1% Tween 80. In certain embodiments, the pH can be about 5.2 to about 5.5, about 5.5 to about 5.8, about 5.8 to about 6.1, about 6.1 to about 6.4, or about 6.4 to about 6.8. In certain embodiments, the pH of the first buffer is about 6.0 or is 6.0.

[0167] In certain embodiments, the first buffer comprises a chelating agent, such as EDTA.

[0168] The second buffer may include about 30 to about 200mM TrisHCl or 30 to about 80mM TrisHCl and salt, wherein the pH of the second buffer is about 7.5 to about 9.2. In some embodiments, the salt is a combination of one or more of NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl, sodium acetate, and NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl, and sodium acetate. In some embodiments, the salt is NaCl. The concentration of salt such as NaCl may be about 75 to about 500mM. In some embodiments, the salt concentration, such as NaCl concentration, is about 75 to about 200mM. In some embodiments, the salt concentration, such as NaCl concentration, is no more than 500mM. In some embodiments, the salt concentration, such as NaCl concentration, is no more than 200mM. Without wishing to be bound by theory, salt concentrations of no more than 500 mM or no more than 200 mM can prevent elution of AAV during the wash steps because the conductivity of the salt solution is too low to facilitate elution.

[0169] The second buffer may include about 30 to about 35mM, about 35 to about 40mM, about 40 to about 45mM, about 45 to about 50mM, about 50 to about 55mM, about 55 to about 60mM, about 60 to about 65mM, about 65 to about 70mM, about 70 to about 75mM or about 75 to about 80mM TrisHCl. In certain embodiments, the second buffer may include about 50mM TrisHCl or 50mM TrisHCl. In certain embodiments, the second buffer may include about 75 to about 100mM, about 100 to about 125mM, about 125 to about 150mM, about 150 to about 175mM, about 175 to about 200mM, about 200 to about 225mM or about 225 to about 250mM NaCl. In certain embodiments, the second buffer may include about 150mM NaCl or 150mM NaCl. In certain embodiments, the pH of the second buffer may be about 7.5 to about 7.7, about 7.7 to about 7.9, about 7.9 to about 8.1, about 8.1 to about 8.3, about 8.3 to about 8.5, about 8.5 to about 8.7, about 8.7 to about 8.9, or about 8.9 to about 9.2. In certain embodiments, the pH of the second buffer may be about 8.5 or may be 8.5.

[0170] The second buffer may include about 30 to about 35mM, about 35 to about 40mM, about 40 to about 45mM, about 45 to about 50mM, about 50 to about 55mM, about 55 to about 60mM, about 60 to about 65mM, about 65 to about 70mM, about 70 to about 75mM or about 75 to about 80mM arginine-HCl. In certain embodiments, the second buffer may include about 50mM arginine-HCl or 50mM arginine-HCl. In certain embodiments, the second buffer may include about 75 to about 100mM, about 100 to about 125mM, about 125 to about 150mM, about 150 to about 175mM, about 175 to about 200mM, about 200 to about 225mM or about 225 to about 250mM NaCl. In certain embodiments, the second buffer may include about 150mM NaCl or 150mM NaCl. In certain embodiments, the pH of the second buffer may be about 7.5 to about 7.7, about 7.7 to about 7.9, about 7.9 to about 8.1, about 8.1 to about 8.3, about 8.3 to about 8.5, about 8.5 to about 8.7, about 8.7 to about 8.9, or about 8.9 to about 9.2. In certain embodiments, the pH of the second buffer may be about 8.5 or may be 8.5.

[0171] The second buffer may include about 50 to about 200 mM of glycine. In certain embodiments, the second buffer may include about 50 to about 100 mM, about 70 to about 120 mM, about 100 to about 150 mM, about 120 to about 170 mM, about 150 to about 200 mM of glycine. In certain embodiments, the pH of the second buffer may be about 7.5 to about 7.7, about 7.7 to about 7.9, about 7.9 to about 8.1, about 8.1 to about 8.3, about 8.3 to about 8.5, about 8.5 to about 8.7, about 8.7 to about 8.9, or about 8.9 to about 9.2. In certain embodiments, the pH of the second buffer may be about 8.5 or may be 8.5.

[0172] The second buffer may include about 50 to about 20 mM sodium citrate. In certain embodiments, the second buffer may include about 5 to about 10 mM, about 7 to about 12 mM, about 10 to about 15 mM, about 12 to about 17 mM, about 15 to about 20 mM sodium citrate. In certain embodiments, the pH of the second buffer may be about 7.5 to about 7.7, about 7.7 to about 7.9, about 7.9 to about 8.1, about 8.1 to about 8.3, about 8.3 to about 8.5, about 8.5 to about 8.7, about 8.7 to about 8.9, or about 8.9 to about 9.2. In certain embodiments, the pH of the second buffer may be about 8.5 or may be 8.5.

[0173] In certain embodiments, the second buffer comprises a chelating agent, such as EDTA.

[0174] The third buffer may comprise about 30 to about 200 mM TrisHCl and about 30% to about 75% by volume ethylene glycol, wherein the pH is about 7.5 to about 9.2. The third buffer may comprise about 20 to about 80 mM arginine-HCl and about 50 to about 200 mM salt, wherein the pH is about 7.3 to about 8.8. The third buffer may comprise about 50 mM TrisHCl and about 50% by volume ethylene glycol, wherein the pH is about 8.5. The third buffer may comprise about 20 to about 150 mM taurine, about 30% to about 75% by volume ethylene glycol, and 0.05% to 0.2% octylpyranoglucoside, wherein the pH is about 7.3 to about 8.8. The third buffer may include about 50 to about 200 mM arginine-HCl, about 50 to about 200 mM lysine-HCl, about 50 to about 200 mM histidine-HCl, and about 1 mM to about 4 mM N-acetyl-D, L-tryptophan and about 10% (w / w) to about 40% (w / w) polysorbate 80, wherein the pH is about 7.3 to about 8.8. If a salt, such as NaCl, is present in the third buffer, in certain embodiments, the concentration of the salt does not exceed 500 mM, and in certain embodiments, the concentration of the salt does not exceed 200 mM. In certain embodiments, the salt is a combination of one or more of NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl, sodium acetate, and NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl, and sodium acetate. In certain embodiments, the salt is NaCl. In certain embodiments, one or more of sorbitol, mannitol, xylitol, sucrose, or trehalose may be used in combination with or in place of ethylene glycol.

[0175] Without wishing to be bound by theory, the extent of elution of AAV is affected by the amount of ethylene glycol in the third buffer and the conductivity of the salt. An amount of ethylene glycol of at least 55% (w / w) in the buffer can significantly increase the amount of elution compared to 50% (w / w) ethylene glycol. Thus, at a given ethylene glycol concentration, an increased NaCl concentration can increase the extent and rate of elution. At a given ethylene glycol concentration, replacing NaCl with a multivalent salt can also increase the extent and rate of elution.

[0176] Without wishing to be bound by theory, if the salt is constant, e.g., 750 mM NaCl, then increasing the amount of ethylene glycol can increase the elution strength of the buffer. If the ethylene glycol content is constant, e.g., 55%, then increasing the amount of salt can increase the elution strength of the buffer. Thus, in 750 mM NaCl, as the ethylene glycol increases from 40% (w / w) to 45% (w / w) to 50% (w / w) to 55% (w / w) to 60 (w / w), the elution strength increases.

[0177] Increasing the amount of ethylene glycol in the solution will decrease the conductivity while keeping the salt content constant. Increasing the amount of ethylene glycol decreases the amount of salt solubility in the buffer.

[0178] In certain embodiments, the third buffer may comprise about 30 to about 35 mM, about 35 to about 40 mM, about 40 to about 45 mM, about 45 to about 50 mM, about 50 to about 55 mM, about 55 to about 60 mM, about 60 to about 65 mM, about 65 to about 70 mM, about 70 to about 75 mM, about 75 to about 80 mM, about 80 to about 90 mM, about 90 to about 100 mM, about 100 to about 110 mM, about 110 to about 120 mM, about 120 to about 130 mM, about 130 to about 140 mM, about 140 to about 150 mM, about 150 to about 160 mM, about 160 to about 170 mM, about 170 to about 180 mM, about 180 to about 190 mM, or about 190 to about 200 mM TrisHCl. In certain embodiments, the third buffer may include about 50mM or 50mM TrisHCl.In certain embodiments, the third buffer may include about 30% by volume to about 35% by volume, 35% by volume to about 40% by volume, about 40% by volume to about 45% by volume, about 45% by volume to about 50% by volume, about 48% by volume to about 52% by volume, about 50% by volume to about 55% by volume, about 55% by volume to about 60% by volume, about 60% by volume to about 65% by volume, about 65% by volume to about 70% by volume or about 70% by volume to about 75% by volume of ethylene glycol.In certain embodiments, the third buffer may include about 50% ethylene glycol or 50% ethylene glycol. In certain embodiments, the pH of the third buffer may be about 7.5 to about 7.7, about 7.7 to about 7.9, about 7.9 to about 8.1, about 8.1 to about 8.3, about 8.3 to about 8.5, about 8.5 to about 8.7, about 8.7 to about 8.9, or about 8.9 to about 9.2. In certain embodiments, the pH of the third buffer may be about 8.5 or may be 8.5.

[0179] In certain embodiments, the third buffer comprises a chelating agent, such as EDTA.

[0180] The 4th buffer may include about 10 to about 30mM TrisHCl and about 75 to about 250mM NaCl, wherein pH is about 6.5 to about 8.0. In certain embodiments, the 4th buffer may include about 10 to about 15mM, about 15 to about 20mM, about 20 to about 25mM or about 25 to about 30mM TrisHCl. In certain embodiments, the 4th buffer may include about 20mM or 20mMTrisHCl. In certain embodiments, the 4th buffer may include about 75 to about 100mM, about 100 to about 125mM, about 125 to about 150mM, about 150 to about 175mM, about 175 to about 200mM, about 200 to about 225mM NaCl or about 225 to about 250mMNaCl. In certain embodiments, the second buffer may include about 150mM NaCl or 150mM NaCl. In certain embodiments, the pH of the fourth buffer may be about 6.5 to about 6.9, about 6.8 to about 7.2, about 7.1 to about 7.5, about 7.4 to about 7.9, or about 7.6 to about 8.0. In certain embodiments, the pH of the fourth buffer may be about 7.4 or may be 7.4.

[0181] The 4th buffer may include about 20 to about 100mM of histidine and about 75 to about 250mM NaCl, wherein pH is about 7.5 to about 8.8. In certain embodiments, the 4th buffer may include about 20 to about 40mM, about 40 to about 60mM, about 60 to about 75mM or about 75 to about 100mM of histidine. In certain embodiments, the 4th buffer may include about 20mM of histidine or 20mM of histidine. In certain embodiments, the 4th buffer may include about 75 to about 100mM, about 100 to about 125mM, about 125 to about 150mM, about 150 to about 175mM, about 175 to about 200mM, about 200 to about 225mM NaCl or about 225 to about 250mM NaCl. In certain embodiments, the second buffer may include about 150mM NaCl or 150mM NaCl. In certain embodiments, the pH of the fourth buffer may be about 7.5 to about 7.9, about 7.8 to about 8.2, about 8.1 to about 8.5, about 8.4 to about 8.9, or about 8.6 to about 9.0. In certain embodiments, the pH of the fourth buffer may be about 8.0 or may be 8.0.

[0182] In certain embodiments, the fourth buffer comprises a chelating agent, such as EDTA. In certain embodiments, additional washes may be performed with a buffer comprising a chelating agent, such as EDTA.

[0183] After the washing step, the AAV particles are eluted using an elution buffer. The elution buffer may comprise about 30 to about 200 mM of a buffer, about 30% to about 75% by volume of ethylene glycol, and about 500 mM to about 2000 mM of a salt, wherein the pH of the elution buffer is about 7.3 to about 8.8. In some embodiments, the concentration of ethylene glycol is at least 50% (w / w). In some embodiments, the concentration of ethylene glycol is at least 55% (w / w). In some embodiments, the concentration of ethylene glycol is at least 60% (w / w). In some embodiments, the buffer is TrisHCl, glycine, citrate, arginine, phosphoglycine-HCl, ammonium sulfate, magnesium chloride, borate, bis-Tris, MOPS, N,N-bicine, N-tricine, TAPS, TAPSO, MES, PIPES, TES (2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid), barbital sodium (Veronal), ADA (N-(2-acetamido)iminodiacetic acid), ACES (N-(2-acetamido)-2-aminoethanesulfonic acid), Bis-Tris propane, BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), DIPSO (3-(N,N-bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid), Triz In some embodiments, the buffer is TrisHCl.

[0184] In some embodiments, the salt is NaCl, KCl, CaCl2, CsCl, LiCl, CaCl2, sodium citrate, LiCl, CsCl or a combination of one or more of KCl, CaCl2, CsCl, LiCl, CaCl2, sodium citrate, LiCl and CsCl. In some embodiments, the salt is NaCl. The concentration of salt such as NaCl can be about 500 to about 2000mM. In some embodiments, the salt concentration is about 500 to about 900mM, about 750mM NaCl or 750mM NaCl. In some embodiments, when using a concentration gradient of NaCl, the target concentration is 2000mM.

[0185] In certain embodiments, sorbitol, mannitol, xylitol, sucrose, trehalose, glycerol (1,2,3-propanetriol) or erythritol (meso-1,2,3,4-butanetetrol) can be used in combination with ethylene glycol or replace ethylene glycol. In certain embodiments, elution buffer can include about 30 to about 35mM, about 35 to about 40mM, about 40 to about 45mM, about 45 to about 50mM, about 50 to about 55mM, about 55 to about 60mM, about 60 to about 65mM, about 65 to about 70mM, about 70 to about 75mM or about 75 to about 80mM TrisHCl. In certain embodiments, elution buffer can include about 50mM TrisHCl or 50mMTrisHCl. In certain embodiments, elution buffer can include about 30% by volume to about 35% by volume, about 35% by volume to about 40% by volume, about 40% by volume to about 45% by volume, about 45% by volume to about 50% by volume, about 48% by volume to about 52% by volume, about 50% by volume to about 55% by volume, about 55% by volume to about 60% by volume, about 60% by volume to about 65% by volume, about 65% by volume to about 70% by volume or about 70% by volume to about 75% by volume of ethylene glycol. In certain embodiments, elution buffer can include about 50% ethylene glycol or 50% ethylene glycol. In certain embodiments, the concentration of ethylene glycol is at least 55% (w / w). In certain embodiments, the concentration of ethylene glycol is at least 56% (w / w). In certain embodiments, the concentration of ethylene glycol is at least 57% (w / w). In certain embodiments, the concentration of ethylene glycol is at least 58% (w / w). In certain embodiments, the elution buffer may comprise about 500 to about 700 mM, about 550 to about 750 mM, about 600 to about 800 mM, about 650 to about 850 mM, about 700 to about 900 mM, about 750 to about 950 mM, about 800 to about 1000 mM NaCl, about 900 to about 1100 mM NaCl, about 1000 to about 1200 mM NaCl, about 1100 to about 1300 mM NaCl, about 1200 to about 1400 mM NaCl, about 1300 to about 1500 mM NaCl, about 1400 to about 1600 mM NaCl, about 1500 to about 1700 mM NaCl, about 1600 to about 1800 mM NaCl, about 1700 to about 1900 mM NaCl, about 1800 to about 2000 mM NaCl. In certain embodiments, the elution buffer may comprise about 750 mM NaCl or 750 mM NaCl. The pH may be between 7.3 and 7.6. The pH may be between 7.5 and 7.8. The pH may be between 7.7 and 8.0. The pH may be between 7.9 and 8.2. The pH may be between 8.1 and 8.4. The pH may be between 8.3 and 8.6. The pH may be between 8.5 and 8.8.

[0186] In certain embodiments, elution is performed using an elution buffer comprising an organic solvent. For example, the organic solvent may include one or more of the following: a polyol (e.g., ethylene glycol, sorbitol, mannitol, xylitol), a combination of glycerol, sucrose, trehalose or a polyol. The pH range of the buffer may be between 7.5 and 8.5. Without wishing to be bound by theory, if, for example, the production of AAV involves the transfection of baculovirus to Sf9 insect cells, the organic solvent may inactivate the lipid enveloped virus that can be produced. The affinity eluate may include an organic solvent (e.g., a polyol such as ethylene glycol), which may be used in a subsequent ultracentrifugation step with an adjustable density gradient.

[0187] Any organic solvent in the washing buffer or elution buffer can decompose or inactivate lipid envelope viruses. This inactivation can be carried out by a combination of on-column inactivation and liquid inactivation. In some embodiments, the concentration of the organic solvent in the elution buffer and / or the washing buffer is about 50% (w / w) to about 80% (w / w), to ensure the decomposition or inactivation of lipid envelope viruses. In some embodiments, the concentration is about 50% (w / w). In some embodiments, the concentration is about 55% (w / w). In certain embodiments, the concentration of ethylene glycol is about 56% (w / w). In certain embodiments, the concentration of ethylene glycol is about 57% (w / w). In certain embodiments, the concentration of ethylene glycol is about 58% (w / w). In certain embodiments, the concentration of ethylene glycol is about 59% (w / w). In some embodiments, the concentration is about 60% (w / w). In some embodiments, the concentration is about 65% (w / w). In some embodiments, the concentration is about 70% (w / w). In some embodiments, the concentration is about 75% (w / w). In some embodiments, the concentration of the organic solvent in the elution buffer and / or the wash buffer is about 40% (w / w) to ensure the decomposition or inactivation of lipid enveloped viruses. In some embodiments, the concentration of the organic solvent in the elution buffer and / or the wash buffer is about 30% (w / w) to ensure the decomposition or inactivation of lipid enveloped viruses. In some embodiments, the organic solvent in the elution buffer or the wash buffer may inactivate or decompose the baculovirus.

[0188] In some embodiments, the pH of the first buffer is about 5.8 to about 6.2, and comprises about 90 to about 110 mM sodium acetate and about 0.09% to about 0.11% polysorbate 80 / Tween 80, the pH of the second buffer is about 8.2 to about 8.8, and comprises about 45 to about 55 mM TrisHCl and about 110 to about 135 mM NaCl, the pH of the third buffer is about 8.2 to about 8.8, and comprises about 45 to about 55 mM TrisHCl and about 45% to about 55% ethylene glycol, and the pH of the optional fourth buffer is about 7.2 to about 7.6, and comprises about 15 to about 25 mM TrisHCl and about 135 to about 165 mM NaCl. In certain embodiments, the pH of the elution buffer is about 7.8 to about 8.2, and comprises about 45 to about 55 mM TrisHCl, about 45% to about 55% ethylene glycol, and about 650 to about 850 mM NaCl. Various volumes can be used, such as about 2 column volumes to about 15 column volumes, about 3 column volumes to about 7 column volumes, about 4 column volumes to about 8 column volumes, about 5 column volumes to about 10 column volumes, or about 7 column volumes to about 12 column volumes. About 5 column volumes or 5 column volumes of each of the first buffer, the second buffer, the third buffer, the fourth buffer, and the elution buffer can be used. Alternatively, about 10 column volumes or 10 column volumes of each of the first buffer, the second buffer, the third buffer, the fourth buffer, and the elution buffer can be used. For example, when the column volume is about 2 ml to about 3 ml, 10 column volumes can be used. The time of the washing step can be further extended to improve the purity of the AAV.

[0189] In some embodiments, the first buffer has a pH of about 7.2 to about 7.6 and comprises about 15 to about 25 mM TrisHCl and about 135 to about 165 mM NaCl, the second buffer has a pH of about 5.8 to about 6.2 and comprises about 90 to about 110 mM sodium acetate and about 0.09% to about 0.11% polysorbate 80, the third buffer has a pH of about 8.2 to about 8.8 and comprises about 45 to about 55 mM TrisHCl and about 110 to about 135 mM NaCl, the fourth buffer has a pH of about 7.5 to about 8.5 and comprises about 45 to about 55 mM TrisHCl and about 45% to about 55% ethylene glycol, and the elution buffer has a pH of about 7.8 to about 8.2 and comprises about 45 to about 55 mM TrisHCl, about 45% to about 55% ethylene glycol, and about 650 to about 850 mM NaCl. About 10 column volumes or 10 column volumes of each wash buffer may be used.

[0190] In some embodiments, the first buffer has a pH of about 7.2 to about 7.6 and comprises about 15 to about 25 mM TrisHCl and about 135 to about 165 mM NaCl, the second buffer has a pH of about 5.8 to about 6.2 and comprises about 90 to about 110 mM sodium acetate and about 0.09% to about 0.11% polysorbate 80, the third buffer has a pH of about 8.2 to about 8.8 and comprises about 45 to about 55 mM TrisHCl and about 110 to about 135 mM NaCl, and the fourth buffer has a pH of about 7.5 to about 8.5 and comprises about 45 to about 55 mM TrisHCl and about 45% to about 55% ethylene glycol. Elution is performed with a gradient starting with a first elution buffer having a pH of about 7.8 to about 8.2 and comprising about 45 to about 55 mM TrisHCl, about 45% to about 55% ethylene glycol, and about 650 to about 850 mM NaCl and ending with a second elution buffer having a pH of about 7.8 to about 8.2 and comprising about 45 to about 55 mM TrisHCl, about 45% to about 55% ethylene glycol, and about 1900 to about 2100 mM NaCl. About 10 column volumes or 10 column volumes of each wash buffer may be used. Elution may be performed with a gradient of 10 column volumes.

[0191] In certain embodiments, a solution comprising AAV particles is subjected to ion exchange chromatography. In certain embodiments, the ion exchange is an anion exchange. In certain embodiments, the anion exchange chromatography carrier can not only remove residual particle contaminants from the cell culture, but also remove acidic impurities and viral particles. In certain embodiments, anion exchange chromatography is capable of removing proteases and / or host cell DNA. In certain embodiments, ion exchange chromatography can be performed using membrane-based separations (e.g., hybrid membrane-anion exchange chromatography). In certain embodiments, pure AEX chromatography is used, for example, in a flow-through mode or a bind / elute mode. In certain embodiments, the anion exchange carrier can be, but is not limited to, STREAMLINE Q XL TM 、POROS 50PI TM 、Q SEPHAROSE TM 、Emphase TM AEX HybridPurifier, Nuvia Q, POROS 50HQ, Capto Q, Capto Q impress, Unosphere Q, Q Ceramic F. Q. Super Q, mixed mode AEX resins (e.g., Capto Adhere, Capto adhere impress, and MEP Hypercell), and any DEAE, TMAE, tertiary or quaternary amine, or PEI-based resins. In certain embodiments, the anion exchange support is Q.

[0192] In certain embodiments, DNA from HEK293 cells or any host cells used is not treated with Benzonase and / or DNase. The anion exchanger and washing steps described herein can effectively remove such DNA, making treatment with Benzonase or DNase unnecessary.

[0193] AAV is typically recovered from the flow-through fraction by an anion exchange step (depending on the pH). Alternatively, AAV can be used in a bind / elute method. A flow-through method or a bind / elute method should be performed to exclude host cell impurities, which elute at a higher conductivity (at a constant pH) or lower pH (at a constant conductivity) than AAV. When operating in a bind / elute mode, the pH and composition of the buffer effectively trigger the elution of the product, but do not trigger the elution of acidic host cell impurities. Without wishing to be bound by theory, each binding and elution step can generate forces in the microenvironment, so that a flow-through method under non-binding conditions can cause less AAV damage or decomposition than a bind / elute method.

[0194] In various embodiments, the yield of AAV (e.g., AAV8 and AAV9), following the purification steps described herein and as measured by weight / volume by ITR-qPCR assay, is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% higher than the yield obtained by a comparable procedure without a wash step.

[0195] In various embodiments, the yield of AAV (e.g., AAV8 and AAV9), after the purification steps described herein and as measured by ITR-qPCR assay on a weight / weight basis, is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% higher than the yield obtained by a comparable procedure without a wash step.

[0196] In certain embodiments, the methods of producing and purifying AAV described herein reduce the number of protein impurities by more than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% compared to the yield obtained by a comparative procedure without the same washing scheme. Surprisingly, it was found that the methods of producing and purifying AAV described herein reduce the number of protein impurities by at least 25%. Surprisingly, it was found that when a solution of AAV particles is exposed to anion exchange chromatography before loading on an affinity matrix, the methods of producing and purifying AAV described herein reduce the number of protein impurities by at least 75%. For example, the data in Table 7 of Example 2 show that the number of protein impurities is reduced from 20 to 14 (without prior anion exchange) and from 20 to 4 (with prior anion exchange).

[0197] In certain embodiments, the methods of producing and purifying AAV described herein provide a purity of AAV (e.g., AAV8 and AAV9) of at least 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1% or 99.2% when both anion exchange and affinity purification with wash steps are performed as provided herein. In certain embodiments, the methods of producing and purifying AAV described herein provide a purity of AAV (e.g., AAV8 and AAV9) of at least 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9% or 98.0% when affinity purification with a wash step is performed as provided herein.

[0198] Advantageously, the methods are scalable to large volumes of starting material, such as cell cultures. In certain embodiments, the methods provided herein are large-scale methods capable of purifying AAV from a volume of at least 500 L or about 500 L, at least 600 L or about 600 L, at least 700 L or about 700 L, at least 800 L or about 800 L, at least 900 L or about 900 L, or at least 1000 L or about 1000 L. In certain embodiments, the methods can be expanded to a minimum volume of starting material (e.g., cell culture) of at least 1250 L or about 1250 L, at least 1500 L or about 1500 L, at least 2000 L or about 2000 L, at least 2500 L or about 2500 L, at least 3000 L or about 3000 L, at least 4000 L or about 4000 L, at least 5000 L or about 5000 L, at least 6000 L or about 6000 L, at least 7000 L or about 7000 L, at least 8000 L or about 8000 L, at least 9000 L or about 9000 L, at least 10,000 L or about 10,000 L, or more. For example, the methods are performed using a minimum volume of AAV-producing cell culture of about 1000 L or about 10,000 L or 25,000 L or more.

[0199] The methods of producing and purifying AAV described herein are also advantageous because they result in the production of high titers of AAV. 10 An AAV product containing at least about 10 virus particles (vp) is produced from about 1000 L of starting material (e.g., cell culture). 11 An AAV product containing at least about 10 virus particles (vp) is produced from about 1000 L of starting material (e.g., cell culture). 12 An AAV product containing at least about 10 virus particles (vp) is produced from about 1000 L of starting material (e.g., cell culture). 13 An AAV product containing at least about 10 virus particles (vp) is produced from about 1000 L of starting material (e.g., cell culture). 14 An AAV product containing at least about 10 virus particles (vp) is produced from about 1000 L of starting material (e.g., cell culture). 15 An AAV product containing at least about 10 virus particles (vp) is produced from about 1000 L of starting material (e.g., cell culture). 16 An AAV product containing at least about 10 virus particles (vp) is produced from about 1000 L of starting material (e.g., cell culture). 17An AAV product containing at least about 2 x 10 virus particles (vp) is produced from about 1000 L of starting material (e.g., cell culture). 16 An AAV product containing at least about 5 x 10 virus particles (vp) is produced from about 1000 L of starting material (e.g., cell culture). 17 AAV product of 10 viral particles (vp) is produced from approximately 1000 L of starting material (e.g., cell culture).

[0200] Another advantageous aspect of the methods described herein is that the methods produce highly pure AAV products. In certain embodiments, the AAV products produced by the methods disclosed herein are substantially free of one or more contaminants: host cell proteins, cellular nucleic acids of host cells (e.g., free host cell DNA and free plasmid DNA), plasmid DNA, empty viral capsids, heat shock protein 70 (HSP70), lactate dehydrogenase (LDH), proteasomes, contaminant non-AAV viruses (e.g., lipid enveloped viruses), host cell culture components (e.g., antibiotics), mycoplasmas, pyrogens, bacterial endotoxins, cell debris (e.g., fragments composed of membrane lipids, proteins, and other biopolymers), and adventitious agents. When AAV is purified according to the methods for producing and purifying AAV described herein, one or more or even all of the following impurities may not be detected: histone H2A type 1, histone H2B type 1-B, histone H4, heat shock protein 70 kDa, pyrogens, endotoxins ... 1A, pyruvate kinase PKM, elongation factor 2, ATP-citrate synthase, histone H1.4, immunoglobulin heavy chain constant region gamma 1 (from immobilized ligand by acid elution method), 60S ribosomal protein L27, fructose bisphosphate aldolase A, 71 kDa heat shock homologous protein, cytoplasmic actin 1, S-formylglutathione hydrolase, asparagine synthetase (glutamine hydrolysis), L-lactate dehydrogenase B chain, tubulin beta-2A chain, X chromosome RNA binding motif protein , 60S ribosomal protein L6, cytoplasmic threonine tRNA ligase, immunoglobulin kappa constant region, 60S ribosomal protein L30, WD repeat-containing protein 1, adenosylhomocysteinase, heterogeneous nuclear ribonucleoprotein C, protein Rep68, thimetoligopeptidase, D-3-phosphoglycerate dehydrogenase, ATP-dependent molecular chaperone HSC82. According to the method for producing and purifying anionic AAV described herein, adding an anion exchange step before the washing step can also result in the following substances being undetectable: histone H1.4, 60S ribosomal protein L27, cytoplasmic actin 1, tubulin beta-2A chain, 60S ribosomal protein L6, 60S ribosomal protein L30, heterogeneous nuclear ribonucleoprotein C, protein Rep68 and ATP-dependent molecular chaperone HSC82.

[0201] In an exemplary embodiment, the method of the present disclosure provides a purified AAV product wherein at least 50% or about 50% of the contaminants found in the starting material (e.g., cell culture) have been removed. In an exemplary embodiment, the method of the present disclosure provides a purified AAV product wherein at least 60% or about 60% of the contaminants found in the starting material (e.g., cell culture) have been removed. In an exemplary embodiment, the method of the present disclosure provides a purified AAV product wherein at least 70% or about 70% of the contaminants found in the starting material (e.g., cell culture) have been removed. In an exemplary embodiment, the method of the present disclosure provides a purified AAV product wherein at least 80% or about 80% of the contaminants found in the starting material (e.g., cell culture) have been removed. In an exemplary embodiment, the method of the present disclosure provides a purified AAV product wherein at least 90% or about 90% of the contaminants found in the starting material (e.g., cell culture) have been removed.

[0202] In certain embodiments, the AAV product produced by the methods of the present disclosure is suitable for administration to humans. In certain embodiments, the AAV is a recombinant AAV (rAAV). In certain embodiments, the AAV product produced by the methods of the present disclosure is sterile and / or has a good manufacturing practice (GMP) grade. In certain embodiments, the AAV product produced by the methods of the present disclosure complies with the requirements of Chapter 1046 of the United States Pharmacopoeia or the European Pharmacopoeia for gene therapy drug products, or complies with the requirements of the U.S. Food and Drug Administration (USFDA) or the European Medicines Agency (EMA).

[0203] In addition, the AAV products produced by the methods described herein have strong efficacy. The efficacy of AAV products (e.g., AAV8 or AAV9 products) can be described in the following terms: (1) in vivo bioefficacy (e.g., production of active protein in mice), which is given in units (FIX or FVIII) per mL of mouse plasma; or (2) in vitro bioefficacy. In vitro bioefficacy tests measure the potential of AAV vectors to infect cells (e.g., HepG2 cells) expressing target proteins and secreting them into culture media, and measure the amount by ELISA technology and / or enzyme activity. Suitable methods for measuring in vivo and in vitro bioefficacy are known in the art and are also described herein.

[0204] In another embodiment, the AAV product produced by the methods described herein shows excellent specific activity. The "specific activity" of AAV is expressed by the ratio of qPCR to μg AAV8. In an exemplary embodiment, the AAV product produced by the methods described herein shows an excellent ratio of vector genomes per μg AAV, proving that the AAV product has a large number of "complete" virus particles. In certain embodiments, the methods of the present disclosure include testing the AAV fractions by an AAV-specific ELISA. In certain embodiments, the AAV-specific ELISA is sufficient to provide a representative reading of the potency of the AAV fractions because most of the capsids in the AAV fractions are complete capsids.

[0205] Source of rAAV particles

[0206] With regard to the methods disclosed herein, AAV can be any AAV serotype. In some embodiments, the AAV purified by the methods described herein is AAV1 serotype, AAV2 serotype, AAV3 serotype, AAV4 serotype, AAV5 serotype, AAV6 serotype, AAV7 serotype, AAV8 serotype, AAV9 serotype, AAV10 serotype, AAV11 serotype, AAV12 serotype, AAV13 serotype, AAAV serotype, BAAV serotype, AAV (VR-195) serotype and AAV (VR-355) serotype. In certain embodiments, AAV is wild type. In certain embodiments, AAV is genetically engineered and / or chemically modified. In certain embodiments, AAV comprises a modified capsid, such as a genetically engineered or chemically modified AAV capsid. In certain embodiments, the AAV particles purified by the methods described herein are AAV8 serotype.

[0207] With respect to the methods of the invention, in exemplary aspects, the AAV fraction is a concentrated AAV fraction. In certain embodiments, the AAV fraction comprises at least 1 x 10 10 AAV capsids / mL, 1x 10 11 AAV capsids / mL or 1x 10 12 AAV capsids / mL. In certain embodiments, the AAV fraction comprises at least 1 x 10 12 AAV capsids / mL. AAV capsids may include empty AAV capsids and complete AAV capsids.

[0208] In certain embodiments, the AAV fraction represents the AAV fraction produced by the transfected host cells. In certain embodiments, the AAV fraction represents the supernatant harvested from a cell culture comprising a host cell transfected with a triple plasmid system, wherein a plasmid of the system comprises a target gene or cDNA, and a plasmid encodes capsid protein VP1, capsid protein VP2 and / or capsid protein VP3. In certain embodiments, VP1, VP2 and / or VP3 are AAV8VP1, AAV8 VP2 and / or AAV8 VP3. In certain embodiments, VP1, VP2 and / or VP3 are AAV9 VP1, AAV9 VP2 and / or AAV9 VP3. Triple plasmid transfection for rAAV production purposes is known in the art. See, e.g., Qu et al., 2015, supra, and Mizukami et al., "A Protocol for AAV vector production and purification." PhD thesis, Division of Genetic Therapeutics, Center for Molecular Medicine, 1998; and Kotin et al., Hum Mol Genet 20(R1):R2-R6(2011). In certain embodiments, transfection can be performed using inorganic compounds, such as calcium phosphate, or organic compounds, polyethyleneimine (PEI), or non-chemical means, such as electroporation. In certain embodiments, the host cell is an adherent cell. In certain embodiments, the host cell is a suspension cell. In certain embodiments, the host cell is a HEK293 cell or a Sf9 cell. In certain embodiments, the cell culture comprises a culture medium free of serum and protein. In certain embodiments, the culture medium is chemically defined and free of animal-derived components, such as hydrolysates. In certain embodiments, the fraction comprising rAAV particles represents a fraction comprising HEK293 cells transfected with a triple plasmid system. In certain embodiments, fractions comprising rAAV particles are described in U.S. Provisional Application No. 62 / 417,775 and International Application No. PCT / US2017 / 059967.

[0209] Additional Steps

[0210] The method of the present disclosure includes any combination of the steps disclosed herein, and can be optionally combined with one or more additional steps. Therefore, in exemplary aspects, the method of the present disclosure also includes the step of transfecting host cells with the triple plasmid system described herein. In exemplary aspects, the method of the present disclosure includes harvesting supernatant from a cell culture, wherein the cell culture comprises host cells, such as HEK293 cells, transfected with the triple plasmid system. In exemplary aspects, the transfection and harvesting steps are performed before the ultracentrifugation step described herein.

[0211] The methods of the present disclosure may include additional additional steps that can further improve the purity of the AAV and remove other unwanted components and / or concentrate the fractions and / or adjust the fractions for subsequent steps. The additional steps can be performed before or after the ultracentrifugation step described above.

[0212] In an exemplary aspect, the method includes a deep filtration step. In an exemplary aspect, the method includes using a filter (which comprises cellulose and diatomaceous earth and has a flow rate of about 500 L / m 2 In an exemplary aspect, the method further comprises depth filtering a fraction of the transfected HEK293 cell culture supernatant using a filter having a minimum pore size of about 0.2 μm. In an exemplary aspect, the depth filtration is followed by filtration through a filter having a minimum pore size of about 0.2 μm. In an exemplary aspect, one or both of the depth filter and the filter having a minimum pore size of about 0.2 μm are washed and the washings are collected. In an exemplary aspect, the washings are combined and combined with the filtrate obtained after the depth filtration and filtration using a filter having a minimum pore size of about 0.2 μm.

[0213] In exemplary aspects, the methods of the present disclosure include one or more chromatography steps. In exemplary aspects, the methods include a negative chromatography step, whereby unwanted components bind to the chromatography resin and the desired AAV does not bind to the chromatography resin. In exemplary aspects, the methods include a negative anion exchange (AEX) chromatography step, or an AEX chromatography step performed in a "non-binding mode". Example 2 describes such a step. Advantages of the "non-binding mode" include the relative ease of performing the procedure and subsequent assays.

[0214] Thus, in an exemplary embodiment, a method for purifying AAV particles comprises subjecting the fraction comprising the AAV particles to negative anion exchange (AEX) chromatography by applying the fraction to an AEX column or membrane under conditions that allow AAV to flow through the AEX column or membrane, and collecting the AAV particles. In an exemplary aspect, the fraction is applied to an AEX column or membrane having a loading buffer comprising about 100 mM to about 150 mM of a salt, such as NaCl, optionally wherein the loading buffer has a pH of about 8 to about 9. In an exemplary aspect, the loading buffer comprises about 115 mM to about 130 mM of a salt, such as NaCl, optionally wherein the loading buffer comprises about 120 mM to about 125 mM of a salt, such as NaCl. In an exemplary aspect, the negative AEX step is performed prior to the ultracentrifugation step described herein.

[0215] In exemplary aspects, the methods of the present disclosure include concentrating the AAV fraction using an ultrafiltration / diafiltration system. In exemplary aspects, the methods of the present disclosure include one or more tangential flow filtration (TFF) steps. In exemplary aspects, the AAV fraction undergoes ultrafiltration / diafiltration. In exemplary aspects, the AAV fraction is concentrated using an ultrafiltration / diafiltration system before a step including performing negative AEX chromatography, after a step including performing negative AEX chromatography, or before and after a step including performing negative AEX chromatography. In exemplary aspects, the TFF step is performed before the ultracentrifugation step described herein.

[0216] In exemplary aspects, the methods of the present disclosure include filtering a fraction comprising rAAV particles to remove viruses in the fraction that have a size larger than the rAAV particles.

[0217] In exemplary aspects, the methods of the present disclosure include one or more quality control steps, for example, a step of measuring the potency or specific activity of the AAV fraction obtained after one or more steps of the process (e.g., after each step). In exemplary aspects, the methods of the present disclosure include an ELISA specific for AAV. In exemplary aspects, the ELISA is a sandwich ELISA. In exemplary aspects, the sandwich ELISA comprises antibodies specific for an AAV epitope. In exemplary aspects, the AAV epitope is a conformational epitope present on the assembled AAV capsid. As discussed herein, ELISA can replace qPCR as a method for determining the potency of the AAV fraction. In exemplary aspects, the methods of the present disclosure include testing the AAV fraction by an AAV-specific ELISA, and the method does not include a method for measuring potency by quantitative PCR. In exemplary aspects, the AAV-specific ELISA is sufficient to provide a representative reading of the potency of the AAV fraction because most of the capsids in the AAV fraction are intact capsids.

[0218] In exemplary aspects, after one or more steps of the present disclosure, the method of the present disclosure includes an ELISA specific for AAV. In exemplary aspects, the method of the present disclosure includes testing the AAV fraction obtained after depth filtration by an AAV-specific ELISA to determine the specific activity of AAV in the fraction. In exemplary aspects, the method of the present disclosure includes testing the AAV fraction obtained after concentrating the AAV fraction using an ultrafiltration / diafiltration system by an AAV-specific ELISA to determine the specific activity of AAV in the fraction. In exemplary aspects, the method of the present disclosure includes testing the AAV fraction obtained after the tangential flow filtration (TFF) step by an AAV-specific ELISA to determine the specific activity of AAV in the fraction. In exemplary aspects, the method of the present disclosure includes testing the AAV fraction obtained after the negative anion exchange (AEX) chromatography by an AAV-specific ELISA to determine the specific activity of AAV in the fraction. In exemplary aspects, the method of the present disclosure includes testing the AAV fraction obtained after the polish step by an AAV-specific ELISA to determine the specific activity of AAV in the fraction.

[0219] Also provided herein are AAV products produced by the methods of the present disclosure. In exemplary aspects, the AAV product comprises at least about 10 μL of starting material (e.g., cell culture) produced. 12 virus particles (vp), or at least about 10 produced from about 1000 L of starting material (e.g., cell culture). 13 Virus particles (vp). In exemplary aspects, the AAV product is an empty capsid produced by transfection of rep-cap and Ad helper plasmids in the absence of a transgenic plasmid. The purified empty plasmid can be used to deplete or remove antibodies specific for AAV antigens from the patient's blood.

[0220] In exemplary aspects, the AAV products of the present disclosure are highly purified, highly effective and suitable for human clinical use. In exemplary aspects, the AAV products include homogenous populations and highly purified AAV particles. In exemplary aspects, the AAV products include full-length vector DNA. In exemplary embodiments, the AAV products are substantially free of unwanted contaminants, including but not limited to AAV particles containing truncated or incomplete vector DNA, AAV particles with incomplete protein composition and oligomerization structure, or contaminating viruses, such as non-AAV viruses, lipid envelope viruses. In exemplary embodiments, the AAV products contain a large amount of coding cDNAs of related proteins. In exemplary aspects, the AAV products of the present disclosure are suitable for administration to humans. In exemplary aspects, the AAV products are sterile and / or have a good manufacturing practice (GMP) level. In exemplary aspects, the AAV products meet the requirements of Chapter 1046 of the United States Pharmacopoeia or the European Pharmacopoeia for gene therapy drug products, or meet the requirements of the U.S. Food and Drug Administration (USFDA) or the European Medicines Agency (EMA). In exemplary aspects, the AAV product is a ready-to-use product for direct administration to humans with little or no processing or handling.

[0221] The following examples are given merely to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0222] Example 1

[0223] The following example describes an exemplary method for transfecting a HEK293 cell line using a triple plasmid system to produce rAAV particles containing nucleic acids encoding a protein of interest.

[0224] Adherent HEK293 cells are grown under suspension conditions in a commercially available culture medium that is chemically defined and free of animal-derived components, proteins, and serum, such as described in paragraphs

[00146] –

[00150] of PCT / US2017 / 059967. The cells are transfected with three plasmids: (1) a helper plasmid that provides the helper virus functions necessary for productive AAV infection, (2) a repcap-plasmid that carries all the information for viral capsid production, replication, and packaging, and (3) a plasmid containing the gene of interest (GOI), which is packaged into the resulting rAAV particles. Within a period of 3-5 days after transfection, rAAV particles carrying the gene of interest are present in the HEK293 cell line.

[0225] For example, the supernatant of the transfected HEK293 cell culture is harvested as described in paragraphs

[00151] -

[00155] , Tables 1 and 2 of PCT / US2017 / 059967. The harvested supernatant is concentrated and conditioned (diafiltered), for example, as described in paragraphs

[00156] -

[00160] , Tables 3 and 4 of PCT / US2017 / 059967. The diafiltered concentrate is subjected to negative chromatography, for example, as described in paragraphs

[00161] -

[00165] and Table 5 of PCT / US2017 / 059967.

[0226] Example 2

[0227] AAV8 was produced in the HEK293 cell line after transfection with a triple plasmid system containing the relevant proteins and the coding cDNAs of AAV8-.VP1, -VP2 and -VP3. The clarified cell-free culture supernatant was concentrated and diafiltered using a Pall Omega T-series cassette 100 kDa. The viral particles were loaded onto a membrane adsorber (MustangQ; Pall part number XT140MSTGQP05) under non-binding conditions (i.e., in a solution containing 125 mM NaCl and 50 mM TrisHCl, pH 8.5). The pH-adjusted load was obtained by adjusting the AAV8-containing effluent to a pH range between 7.4 and 7.8 with 25% HCl.

[0228] The following test procedure was performed. First, the POROS TM CaptureSelect TM AAV8 affinity matrix (Cat. No. 195338010; Thermo Fisher) with an ID of 32 mm, a bed height of 59 mm, and a volume of 47.45 ml was equilibrated with at least 5 column volumes of 20 mM TrisHCl and 150 mM NaCl, pH 7.4. The pH adjusted load was applied to the POROS TM CaptureSelect TM The column was then re-equilibrated with five column volumes of 20 mM TrisHCl and 150 mM NaCl, pH 7.4 (optional fourth buffer).

[0229] The column was then washed with five column volumes of wash 1 (W1): 100 mM sodium acetate and 0.1% Tween 80, pH 6.0. The column was then washed with five column volumes of wash 2 (W2): 50 mM TrisHCl and 125 mM NaCl, pH 8.5. The column was then washed with five column volumes of wash 3 (W3): 50 mM TrisHCl and 50% ethylene glycol, pH 8.5.

[0230] Elution was performed by applying five column volumes of the following elution buffer to the column: 50 mM TrisHCl, 50% ethylene glycol and 750 mM NaCl, pH 8.0. Five column volumes of the following second elution buffer were then applied to the column: 50 mM TrisHCl, 50% ethylene glycol and 2000 mM NaCl.

[0231] The linear flow rate in the above steps is 60 cm / h.

[0232] The following comparison procedure was performed. TM CaptureSelect TM AAV8 affinity matrix (Cat. No. 195338010; Thermo Fisher) with an ID of 10 mm and a bed height of 2.5 mm was equilibrated in a 1.96 ml column with at least 10 column volumes of 20 mM TrisHCl and 150 mM NaCl, pH 7.4. The pH adjusted load was applied to the column containing POROS TM CaptureSelect TM The column was then re-equilibrated with 10 column volumes of 20 mM TrisHCl and 150 mM NaCl, pH 7.4.

[0233] The washing steps were performed using the following TBS buffer: 20 mM TrisHCl / 150 mM NaCl / pH 7.4. Alternatively, elution was performed with 10 column volumes of 100 mM sodium citrate, pH 3.0.

[0234] The above test and comparison procedures are described in more detail in Table 2, where "CV" represents the number of column volumes of solution added during that step.

[0235] Table 2:

[0236]

[0237]

[0238] The following determinations were performed using the data shown in weight / volume in Table 3. The density of the elution buffer was measured on an oscillating U-tube densitometer DMA 4500M (Anton Paar).

[0239] The amount of AAV8 antigen was measured by ELISA. The ELISA was performed on a TECAN robotic system using an AAV-8 titration ELISA kit (Article No. PRAAV8; Progen (Heidelberg, Germany). Briefly, a monoclonal antibody specific for a conformational epitope on the assembled AAV8 capsid (ADK8) was coated onto a microtiter strip and used to capture AAV8 particles from the AAV fraction. The captured AAV8 particles were detected in two steps. In the first step, a biotin-conjugated monoclonal antibody specific for the ADK8 antibody was bound to the immune complex (of ADK8 and ADK8 antibody). A streptavidin peroxidase conjugate was added to the immune complex bound to the biotin-conjugated monoclonal antibody, which reacts with biotin. A peroxidase substrate solution was added, and a color reaction occurred that was proportional to the amount of bound AAV particles. The color reaction was measured photometrically at 450 nm.

[0240] Using ITR-qPCR assay, genomic copy titer is determined by quantifying the inverted tandem repeats found in vectors encoding the gene of interest (e.g., human factor VIII or human factor IX). HEK-HCP is a measurement of residual host cell protein by ELISA. LDH is determined by colorimetric activity assay.

[0241] The AAV8 ligand leakage ELISA (enzyme-linked immunosorbent assay) was designed to detect 1 ng / mL AAV8 affinity ligand that may be present in POROS TM CaptureSelect TM The AAV8 affinity matrix contains the AAV8 affinity ligand as the capture agent in the purified product. The AAV8 ligand leakage ELISA can be used as a tool to aid in the development of optimal purification processes and for routine quality control of in-process streams and final products.

[0242] The amount of "Ligand leakage ELISA / AAV8-antigen" reflects the ratio of "Ligand leakage ELISA" to "AAV8 antigen" calculated as nanograms of ligand / micrograms of AAV8.

[0243] In an in vitro biopotency assay, the viral vector AAV8 infects a liver target cell line, which then secretes a functional, measurable encoded protein into the culture medium. In a first step, HepG2 target cells are transduced by AAV8 infection. During incubation, the encoded protein is released into the cell supernatant. In a second step, the activity of the encoded protein entering the cell culture supernatant is measured directly by an activity assay. The measurements of the AAV8 samples are given as a percentage relative to the reference material. The method allows for quantitative assessment of the biological function of the AAV8 gene therapy vector.

[0244] Table 3:

[0245]

[0246] The step yield of ITR-qPCR for the test procedure was 105.3%, while the step yield for the comparative procedure was 71.2%. The in vitro biopotency for the test procedure was 0.45, while the in vitro biopotency for the comparative procedure was 0.33.

[0247] The determination was also performed using the data shown in Table 4 on a weight / weight basis.

[0248] Table 4.

[0249]

[0250] The step yield of ITR-qPCR for the test procedure was 127.4%, while the step yield of the comparative procedure was 72.4%. The improvement in yield and purity, coupled with not having to use extreme conditions that would reduce AAV8 infectivity and biopotency, was surprising. Likewise, it was surprising that the test procedure removed a large portion of the impurities without eluting the product from the affinity ligand, especially under conditions in which there was significantly greater nonspecific binding and complexing of the product to the impurities (present prior to purification).

[0251] SDS-PAGE analysis was performed to determine whether heat shock protein 70 kDa (HSP70) was reduced when the test procedure with a wash step was used instead of the comparative procedure. Western blotting was performed using an anti-Hsp70 antibody (Abcam, catalog number ab79852) as the primary antibody at a dilution of 1:2000 (for 2 hours) and a goat anti-rabbit IgG (H+L) AP conjugate (Sigma, catalog number A8025) as the secondary antibody at a dilution of 1:1000 (for 1 hour). The results are shown in Figure 2. Figure 1 When comparing lane 4 (test procedure) to lane 6 (comparative procedure), a significant reduction in HSP70 was observed using the test procedure. Lane 2 shows 20 ng of HSP70 and lane 4 shows 4 ng of HSP70.

[0252] SDS-PAGE silver staining assay was performed to determine the overall level of impurities present. Figure 2 Lane 2 represents the AAV8 reference and shows 3 characteristic bands. The eluate (190 μg / ml) was purified according to the test procedure and the comparative procedure. The results of the test procedure are shown in lane 4. The results of the comparative procedure are shown in lane 6. The impurities observed for the comparative procedure were significantly more than those observed for the test procedure.

[0253] Western blotting was performed with 12% anti-AAV antibody to determine the level and purity of AAV8 recovered after purification according to the test and comparison procedures. Western blotting was performed with monoclonal antibodies against VP1, VP2, and VP3 of AAV as primary antibodies and goat anti-mouse ALP antibody (Sigma, catalog number A4656) as secondary antibody. The results are shown in Figure 3 Lane 2 shows the AAV reference (130 ng, corresponding to 60 μg / ml), with 3 bands each for VP1, VP2 and VP3. Lane 4 shows the eluate according to the test procedure (190 μg / ml AAV8 antigen), and lane 6 shows the eluate according to the comparative procedure. According to Western blotting, the yield of the test procedure is higher compared to the comparative procedure.

[0254] LC-MS (rp-HPLC-UV-ESI-MS / MS) was performed to determine the identity and amount of various host cell impurities. The samples were digested with trypsin. The resulting peptide mixture was separated on an HPLC system using a RP column (ZORBAX 300SB-C18 column, 0.5x150mm, 3.5 μm), and subsequently, peptides were analyzed on a Q-Exactive HF mass spectrometer. The data were analyzed using software Proteome Discoverer to identify proteins in the samples.

[0255] Agilent HPLC1209 (1200capHPLC) was used with the ChemStation of LC 3D system (Revision B.04.03-SP2 (105)). The HPLC method was PEPMAP_CAP_170.M. Eluent A was 0.1% (v / v) HCOOH in deionized water and eluent B was 0.08% (v / v) HCOOH in acetonitrile. Details on the HPLC are provided in Table 5 below:

[0256] Table 5:

[0257]

[0258]

[0259] Detailed information on MS is provided in Table 6 below:

[0260] Table 6:

[0261]

[0262]

[0263]

[0264] Three different purification modes were performed. One included the comparative procedure of this example, another included the test procedure of this example (which included anion exchange purification by MustangQ before affinity purification), and the third was performed according to the test procedure but without anion exchange purification by MustangQ. The overall results are summarized in Table 7 below.

[0265] Table 7:

[0266]

[0267] In the table above, purity reflects the percentage of the area under the curve (AUC) associated with AAV8 capsid relative to the total AUC of all proteins.

[0268] Tables 8-10 below list the AUC values ​​for each protein and the number of peptides identified by LC / MS for the comparative procedure (Table 8), the test procedure with prior anion exchange (Table 9), and the test procedure without prior anion exchange (Table 10).

[0269] Table 8: Comparison procedures

[0270]

[0271]

[0272]

[0273] Table 9: Test procedure with prior anion exchange

[0274]

[0275]

[0276] Table 10: Test procedure without prior anion exchange

[0277]

[0278] The lowest amount of protein was detected using the test procedure coupled with prior anion exchange. Without anion exchange, the purity decreased and the amount of protein detected increased. The lowest purity and the highest amount of protein detected were observed for the comparative procedure.

[0279] Example 3

[0280] AAV8 was produced in the HEK293 cell line after transfection with a triple plasmid system containing the relevant proteins and the coding cDNAs of AAV8-.VP1, -VP2 and -VP3. The clarified cell-free culture supernatant was concentrated and diafiltered with a Pall Omega T-series box 100 kDa. The viral particles were loaded onto a membrane adsorber (MustangQ.Pall part number XT140MSTGQP05) under non-binding conditions. The pH of the AAV8-containing effluent obtained was not adjusted to a pH range between 7.4 and 7.8 with 25% HCl. Instead, the load was formed by reconstitution of the AAV-8-containing effluent in a loading buffer comprising 125 mM NaCl and 50 mM TrisHCl, pH 8.5.

[0281] Besides the inherent advantage of not having to include a pH adjustment step, having a pH of 8.5 allows for increased robustness of affinity performance and prevents non-specific binding of impurities to the product or resin.

[0282] At various points, samples were taken from each wash and elution step to determine how much AAV8 was present in the sample. The determination indicated how much AAV8 was lost in each wash step. The following test procedure was performed. First, a sample containing POROS TM CaptureSelect TM AAV8 affinity matrix (Cat. No. 195338010, Thermo Fisher) with an ID of 10 mm, a bed height of 25 mm, and a volume of 1.96 ml was equilibrated with at least 5 column volumes of 20 mM TrisHCl and 150 mM NaCl, pH 7.4. The load was applied to the column containing POROS TM CaptureSelect TMAAV8 affinity matrix (Cat. No. 195338010, Thermo Fisher) column. A portion of the sample loaded on the column was saved and subsequently assayed by ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP antigens. The column was then rebalanced with 10 column volumes of 20 mM TrisHCl and 150 mM NaCl, pH 7.4. A portion of the sample loaded on the column was saved and subsequently assayed by ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP antigens.

[0283] The column was then washed with 10 column volumes of wash 1 (W1): 100 mM sodium acetate and 0.1% Tween 80, pH 6.0. The column was then washed with 10 column volumes of wash 2 (W2): 50 mM TrisHCl and 125 mM NaCl, pH 8.5. The column was then washed with 10 column volumes of wash 3 (W3): 50 mM TrisHCl and 50% ethylene glycol, pH 8.5. Samples were taken from each of the eluates of W1, W2, and W3 and assayed by ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP antigens.

[0284] Elution was performed by applying 10 column volumes of the following elution buffer to the column: 50 mM TrisHCl, 50% ethylene glycol and 750 mM NaCl, pH 8.0.

[0285] The above test procedure is described in more detail in Table 11.

[0286] Table 11:

[0287]

[0288] The samples obtained were assayed by each of ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP to assess yield and whether losses may have occurred during the steps. Table 12 shows the distribution of AAV8 in all fractions of the wash, elution, and post-elution steps listed in Table 11 as determined by ITR qPCR.

[0289] Table 12:

[0290]

[0291] Table 13 below shows the distribution of AAV8 as determined by ELISA in all fractions of the wash, elution and post-elution steps listed in Table 11.

[0292] Table 13:

[0293]

[0294] Table 14 below shows the results of a determination of the amount of HEK 293 HCP present by ELISA during the loading and elution steps only.

[0295] Table 14:

[0296]

[0297] Figure 6 Chromatograms associated with the data in Tables 12-14 are shown in FIG.

[0298] The above samples were also assayed by SDS-PAGE and 12% silver staining to detect total protein. Figure 4 , wherein the label of each lane is shown in the figure. The AAV8 band is clearly visible in the eluate (E) and 50% eluate dilution (E2) lanes. Almost no AAV8 is visible in the wash band effluent samples (W1, W2 and W3) and the back extraction (S) effluent sample. Therefore, the method efficiently removes AAV8 without significant loss during the washing process or AAV8 remaining on the column after elution.

[0299] The samples were then assayed by SDS-PAGE and Western blotting against AAV antigens. The primary antibodies were monoclonal antibodies against VP1, VP2, and VP3 of AAV, while the secondary antibodies were goat anti-mouse antibodies conjugated to alkaline phosphatase. Figure 5 As shown, each lane uses Figure 4 Same tag. AAV8 shows minimal loss during wash and back extraction steps.

[0300] Example 4

[0301] AAV9 was produced in the HEK293 cell line after transfection with a triple plasmid system containing the relevant proteins and the coding cDNAs of AAV9-.VP1, -VP2 and -VP3. The clarified cell-free culture supernatant was concentrated and diafiltered using a Pall Omega T-series cassette 100 kDa. The viral particles were loaded onto a membrane adsorber (MustangQ; Pall part number XT140MSTGQP05) under non-binding conditions (i.e., in a solution containing 125 mM NaCl and 50 mM TrisHCl, pH 8.5). The pH-adjusted load was obtained by adjusting the AAV9-containing effluent to a pH range between 7.4 and 7.8 with 25% HCl.

[0302] The following test procedure was performed. First, the POROSTM CaptureSelect TM AAV9 affinity matrix (Cat. No. A27354; Thermo Fisher) with an ID of 32 mm, a bed height of 59 mm, and a volume of 47.45 ml was equilibrated with at least five column volumes of 20 mM TrisHCl and 150 mM NaCl, pH 7.4. The pH adjusted load was applied to the POROS TM CaptureSelect TM AAV9 affinity resin (Cat. No. A27354; Thermo Fisher) was loaded onto a column. The column was then re-equilibrated with five column volumes of 20 mM TrisHCl and 150 mM NaCl, pH 7.4 (optional fourth buffer).

[0303] The column was then washed with five column volumes of wash 1 (W1): 100 mM sodium acetate and 0.1% Tween 80, pH 6.0. The column was then washed with five column volumes of wash 2 (W2): 50 mM TrisHCl and 125 mM NaCl, pH 8.5. The column was then washed with five column volumes of wash 3 (W3): 50 mM TrisHCl and 50% ethylene glycol, pH 8.5.

[0304] Elution was performed by applying five column volumes of the following elution buffer to the column: 50 mM TrisHCl, 50% ethylene glycol and 750 mM NaCl, pH 8.0. Five column volumes of the following second elution buffer were then added to the column: 50 mM TrisHCl, 50% ethylene glycol and 2000 mM NaCl.

[0305] The linear flow rate in the above steps is 60 cm / h.

[0306] The following comparison procedure was performed. TM CaptureSelect TM AAV9 affinity resin (Cat. No. A27354; Thermo Fisher) with an ID of 10 mm and a bed height of 2.5 mm was equilibrated in a 1.96 ml column with at least 10 column volumes of 20 mM TrisHCl and 150 mM NaCl, pH 7.4. The pH adjusted load was applied to the column containing POROS TM CaptureSelect TMThe column was then re-equilibrated with 10 column volumes of 20 mM TrisHCl and 150 mM NaCl, pH 7.4.

[0307] The washing steps were performed using the following TBS buffer: 20 mM TrisHCl / 150 mM NaCl / pH 7.4. Alternatively, elution was performed with 10 column volumes of 100 mM sodium citrate, pH 3.0.

[0308] The above test and comparison procedures are described in more detail in Table 15, where "CV" indicates the number of column volumes of solution added in that step.

[0309] Table 15:

[0310]

[0311] In all buffers, 50% ethylene glycol weight / weight (w / w)

[0312] The density of the elution buffer was measured on an oscillating U-tube densitometer DMA 4500M (Anton Paar).

[0313] The amount of AAV9 antigen was measured by ELISA. The ELISA was performed on a TECAN robotic system using an AAV-9 titration ELISA kit (Article No. PRAAV9; Progen (Heidelberg, Germany). Briefly, monoclonal antibodies specific for conformational epitopes on assembled AAV9 capsids (ADK9) were coated onto microtiter strips and used to capture AAV9 particles from the AAV fraction. The captured AAV9 particles were detected in two steps. In the first step, a biotin-conjugated monoclonal antibody specific for the ADK9 antibody was bound to the immune complex (of ADK9 and ADK9 antibody). A streptavidin peroxidase conjugate was added to the immune complex bound to the biotin-conjugated monoclonal antibody, which reacts with biotin. A peroxidase substrate solution was added, and a color reaction occurred that was proportional to the amount of bound AAV particles. The color reaction was measured photometrically at 450 nm.

[0314] Using ITR-qPCR assay, genomic copy titer is determined by quantifying the inverted tandem repeats found in vectors encoding the gene of interest (e.g., human factor VIII or human factor IX). HEK-HCP is a measurement of residual host cell protein by ELISA. LDH is determined by colorimetric activity assay.

[0315] The AAV9 ligand leakage ELISA (enzyme-linked immunosorbent assay) detects 1 ng / mL AAV9 affinity ligands that may be present in POROS TM CaptureSelect TM The product of AAV9 affinity medium purification contains AAV9 affinity ligand as capture agent. The AAV9 ligand leakage ELISA can be used as a tool to aid in the development of optimal purification processes and routine quality control of process flows and final products. The amount of "Ligand leakage ELISA / AAV9-antigen" reflects the ratio of "Ligand leakage ELISA" to "AAV9 antigen" calculated as nanograms of ligand / microgram of AAV9.

[0316] In an in vitro biopotency assay, the viral vector AAV9 infects a liver target cell line, which then secretes a functional, measurable encoded protein into the culture medium. In a first step, HepG2 target cells are transduced by AAV9 infection. During incubation, the encoded protein is released into the cell supernatant. In a second step, the activity of the encoded protein entering the cell culture supernatant is measured directly by an activity assay. The measurements of the AAV9 samples are given as a percentage relative to the reference material. The method allows for quantitative assessment of the biological function of the AAV9 gene therapy vector.

[0317] When the test procedure with a washing step is used instead of the comparative procedure, SDS-PAGE analysis is performed to determine whether heat shock protein 70 kDa (HSP70) is reduced. Western blotting is performed using an anti-Hsp70 antibody (Abcam, catalog number ab79852) as the primary antibody at a dilution of 1:2000 (for 2 hours) and a goat anti-rabbit IgG (H+L) AP conjugate (Sigma, catalog number A8025) as the secondary antibody at a dilution of 1:1000 (for 1 hour).

[0318] SDS-PAGE silver staining assay was performed to determine the overall level of impurities present.

[0319] Western blots were performed with 12% anti-AAV antibody to determine the level and purity of AAV9 recovered after purification according to the test and comparative procedures. Western blots were performed with monoclonal antibodies against VP1, VP2, and VP3 of AAV9 as primary antibodies and goat anti-mouse ALP antibody (Sigma, catalog number A4656) as secondary antibodies.

[0320] LC-MS (rp-HPLC-UV-ESI-MS / MS) was performed to determine the identity and amount of various host cell impurities. The samples were digested with trypsin. The resulting peptide mixture was separated on an HPLC system using a RP column (ZORBAX 300SB-C18 column, 0.5x150mm, 3.5 μm), and subsequently, peptides were analyzed on a Q-Exactive HF mass spectrometer. The data were analyzed using software Proteome Discoverer to identify proteins in the samples.

[0321] Agilent HPLC1209 (1200capHPLC) was used with ChemStation of LC 3D system (Revision B.04.03-SP2 (105)). The HPLC was PEPMAP_CAP_170.M. Eluent A was 0.1% (v / v) HCOOH in deionized water and eluent B was 0.08% (v / v) HCOOH in acetonitrile. Details on the HPLC are provided in Table 16 below:

[0322] Table 16:

[0323]

[0324]

[0325] Detailed information on MS is provided in Table 17 below:

[0326] Table 17:

[0327]

[0328]

[0329]

[0330] Three different purification modes were performed. One mode included the comparative procedure of this example, another mode included the test procedure of this example (which included anion exchange purification by MustangQ before affinity purification), and the third mode was performed according to the test procedure but without anion exchange purification by MustangQ.

[0331] Example 5

[0332] AAV9 was produced in the HEK293 cell line after transfection with a triple plasmid system containing the relevant proteins and the coding cDNAs of AAV9-.VP1, -VP2 and -VP3. The clarified cell-free culture supernatant was concentrated and diafiltrated with a Pall Omega T-series box 100 kDa. The viral particles were loaded onto a membrane adsorber (Mustang Q.Pall part number XT140MSTGQP05) under non-binding conditions. The pH of the AAV9-containing effluent obtained was not adjusted to a pH range between 7.4 and 7.8 with 25% HCl. Instead, the load was formed by reconstructing the AAV-8-containing effluent in a loading buffer containing 125 mM NaCl and 50 mM TrisHCl at pH 8.5.

[0333] In addition to the inherent advantage of not having to include a pH adjustment step, having a pH of 8.5 can allow for increased robustness of affinity performance and prevent non-specific binding of impurities to the product or resin.

[0334] Samples were taken at different points from the different wash and elution steps to determine how much AAV9 was present in the sample. This assay indicates how much AAV9 was lost during the different wash steps. The following test procedure was performed. First, the POROS TM CaptureSelect TM AAV9 affinity matrix (Cat. No. A27354; Thermo Fisher) with an ID of 10 mm, a bed height of 25 mm, and a volume of 1.96 ml was equilibrated with at least five column volumes of 20 mM TrisHCl and 150 mM NaCl, pH 7.4. The load was applied to the column containing POROS TM CaptureSelect TM AAV9 affinity matrix (Cat. No. A27354; ThermoFisher) was loaded onto a column. A portion of the sample loaded onto the column was saved and subsequently assayed by ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP antigens. The column was then re-equilibrated with 10 column volumes of 20 mM TrisHCl and 150 mM NaCl, pH 7.4. A sample of the effluent was saved and subsequently assayed by ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP antigens.

[0335] The column was then washed with 10 column volumes of wash 1 (W1): 100 mM sodium acetate and 0.1% Tween 80, pH 6.0. The column was then washed with 10 column volumes of wash 2 (W2): 50 mM TrisHCl and 125 mM NaCl, pH 8.5. The column was then washed with 10 column volumes of wash 3 (W3): 50 mM TrisHCl and 50% ethylene glycol, pH 8.5. Samples were taken from each of the eluates of W1, W2, and W3 and assayed by ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP antigens.

[0336] Elution was performed by applying 10 column volumes of the following elution buffer to the column: 50 mM TrisHCl, 50% ethylene glycol and 750 mM NaCl, pH 8.0.

[0337] The above test procedure is described in more detail in Table 18.

[0338] Table 18:

[0339]

[0340]

[0341] The obtained samples were assayed by each of ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCPs to assess yield and whether losses may have occurred during the steps.

[0342] The above samples were also assayed by SDS-PAGE and 12% silver staining to detect total protein.

[0343] The samples were then assayed by SDS-PAGE and Western blotting against AAV9 antigens. The primary antibodies were monoclonal antibodies against VP1, VP2, and VP3 of AAV9, while the secondary antibody was a goat anti-mouse antibody conjugated to alkaline phosphatase.

[0344] Example 6

[0345] AAV9 was produced in the HEK293 cell line after transfection with a triple plasmid system containing the relevant proteins and the coding cDNAs of AAV9-.VP1, -VP2 and -VP3. The clarified cell-free culture supernatant was concentrated and diafiltered using a Pall Omega T-series cassette 100 kDa. The viral particles were loaded onto a membrane adsorber (MustangQ; Pall part number XT140MSTGQP05) under non-binding conditions (i.e., in a solution containing 125 mM NaCl and 50 mM TrisHCl, pH 8.5). The pH-adjusted load was obtained by adjusting the AAV9-containing effluent to a pH range between 7.4 and 7.8 with 25% HCl.

[0346] The following test procedure was performed. First, a column with an ID of 32 mm, a bed height of 59 mm, and a volume of 47.45 ml containing anti-whole AAV8 / 9 antibodies (Catalog No. 03-651161, American Research Products, Inc., Waltham, MA) immobilized on a resin ("ADK8 / 9 affinity resin") was equilibrated with at least 5 column volumes of 20 mM TrisHCl and 150 mM NaCl, pH 7.4. The pH-adjusted load was applied to the column containing the ADK8 / 9 affinity resin. The column was then re-equilibrated with five column volumes of 20 mM TrisHCl and 150 mM NaCl, pH 7.4 (optional fourth buffer).

[0347] The column was then washed with five column volumes of wash 1 (W1): 100 mM sodium acetate and 0.1% Tween 80, pH 6.0. The column was then washed with five column volumes of wash 2 (W2): 50 mM TrisHCl and 125 mM NaCl, pH 8.5. The column was then washed with five column volumes of wash 3 (W3): 50 mM TrisHCl and 50% ethylene glycol, pH 8.5.

[0348] Elution was performed by applying five column volumes of the following elution buffer to the column: 50 mM TrisHCl, 50% ethylene glycol and 750 mM NaCl, pH 8.0. Five column volumes of the following second elution buffer were then added to the column: 50 mM TrisHCl, 50% ethylene glycol and 2000 mM NaCl.

[0349] The linear flow rate in the above steps is 60 cm / h.

[0350] The following comparative procedure was performed. A column containing ADK8 / 9 affinity resin with an ID of 10 mm, a bed height of 2.5 mm, and a volume of 1.96 ml was equilibrated with at least 10 column volumes of 20 mM TrisHCl and 150 mM NaCl, pH 7.4. The pH adjusted load was applied to the column containing the ADK8 / 9 affinity resin. The column was then re-equilibrated with 10 column volumes of 20 mM TrisHCl and 150 mM NaCl, pH 7.4.

[0351] The washing steps were performed using the following TBS buffer: 20 mM TrisHCl / 150 mM NaCl / pH 7.4. Alternatively, elution was performed with 10 column volumes of 100 mM sodium citrate, pH 3.0.

[0352] The above test and comparison procedures are described in more detail in Table 1, where "CV" indicates the number of column volumes of solution added in that step.

[0353] The density of the elution buffer was measured on an oscillating U-tube densitometer DMA 4500M (Anton Paar).

[0354] The amount of AAV9 antigen was measured by ELISA. The ELISA was performed on a TECAN robotic system using an AAV-9 titration ELISA kit (Article No. PRAAV9; Progen (Heidelberg, Germany). Briefly, a monoclonal antibody specific for the AAV9 capsid (AAV8 / 9 antibody (“ADK8 / 9 antibody”, catalog No. 03-651161, American Research Products, Inc., Waltham, MA)) is coated onto a microtiter strip and used to capture AAV9 particles from the AAV fraction. The captured AAV9 particles are detected in two steps. In the first step, a monoclonal antibody conjugated with biotin specific for the ADK8 / 9 antibody is bound to the immune complex (of ADK8 / 9 and ADK8 / 9 antibodies). A streptavidin peroxidase conjugate is added to the immune complex bound to the monoclonal antibody conjugated with biotin, and the streptavidin peroxidase conjugate reacts with biotin. A peroxidase substrate solution is added, and a color reaction proportional to the amount of bound AAV particles occurs. The color reaction is measured photometrically at 450 nm.

[0355] Using ITR-qPCR assay, genomic copy titer is determined by quantifying the inverted tandem repeats found in vectors encoding the gene of interest (e.g., human factor VIII or human factor IX). HEK-HCP is a measurement of residual host cell protein by ELISA. LDH is determined by colorimetric activity assay.

[0356] The AAV8 / 9 ligand leakage ELISA (enzyme-linked immunosorbent assay) detects affinity ligands that may be present in products purified using ADK8 / 9 affinity resin.

[0357] In an in vitro biopotency assay, the viral vector AAV9 infects a liver target cell line, which then secretes a functional, measurable encoded protein into the culture medium. In a first step, HepG2 target cells are transduced by AAV9 infection. During incubation, the encoded protein is released into the cell supernatant. In a second step, the activity of the encoded protein entering the cell culture supernatant is measured directly by an activity assay. The measurements of the AAV9 samples are given as a percentage relative to the reference material. The method allows for quantitative assessment of the biological function of the AAV9 gene therapy vector.

[0358] When the test procedure with a washing step is used instead of the comparative procedure, SDS-PAGE analysis is performed to determine whether heat shock protein 70 kDa (HSP70) is reduced. Western blotting is performed using an anti-Hsp70 antibody (Abcam, catalog number ab79852) as the primary antibody at a dilution of 1:2000 (for 2 hours) and a goat anti-rabbit IgG (H+L) AP conjugate (Sigma, catalog number A8025) as the secondary antibody at a dilution of 1:1000 (for 1 hour).

[0359] SDS-PAGE silver staining assay was performed to determine the overall level of impurities present.

[0360] Western blots were performed with 12% anti-AAV antibody to determine the level and purity of AAV9 recovered after purification according to the test and comparative procedures. Western blots were performed with monoclonal antibodies against VP1, VP2, and VP3 of AAV9 as primary antibodies and goat anti-mouse ALP antibody (Sigma, catalog number A4656) as secondary antibodies.

[0361] LC-MS (rp-HPLC-UV-ESI-MS / MS) was performed to determine the identity and amount of various host cell impurities. The samples were digested with trypsin. The resulting peptide mixture was separated on an HPLC system using a RP column (ZORBAX 300SB-C18 column, 0.5x150mm, 3.5 μm), and subsequently, peptides were analyzed on a Q-Exactive HF mass spectrometer. The data were analyzed using software Proteome Discoverer to identify proteins in the samples.

[0362] Agilent HPLC1209 (1200capHPLC) was used with the ChemStation of LC 3D system (Revision B.04.03-SP2 (105)). The HPLC was PEPMAP_CAP_170.M. Eluent A was 0.1% (v / v) HCOOH in deionized water and eluent B was 0.08% (v / v) HCOOH in acetonitrile. Details on the HPLC are provided in Table 19 below:

[0363] Table 19:

[0364]

[0365] Detailed information on MS is provided in Table 20 below:

[0366] Table 20:

[0367]

[0368]

[0369]

[0370] Three different purification modes were performed. One mode included the comparative procedure of this example, another mode included the test procedure of this example (which included anion exchange purification by MustangQ before affinity purification), and the third mode was performed according to the test procedure but without anion exchange purification by MustangQ.

[0371] Example 7

[0372] AAV9 was produced in the HEK293 cell line after transfection with a triple plasmid system containing the relevant proteins and the coding cDNAs of AAV9-.VP1, -VP2 and -VP3. The clarified cell-free culture supernatant was concentrated and diafiltered with a Pall Omega T-series box 100 kDa. The viral particles were loaded onto a membrane adsorber (Mustang Q.Pall part number XT140MSTGQP05) under non-binding conditions. The pH of the AAV9-containing effluent obtained was not adjusted to a pH range between 7.4 and 7.8 with 25% HCl. Instead, the load was formed by reconstructing the AAV9-containing effluent in a loading buffer comprising 125 mM NaCl and 50 mM TrisHCl, pH 8.5.

[0373] In addition to the inherent advantage of not having to include a pH adjustment step, having a pH of 8.5 can allow for increased robustness of affinity performance and prevent non-specific binding of impurities to the product or resin.

[0374] Samples were taken from different washing and elution steps at different points to determine how much AAV9 was present in the sample. The determination showed how much AAV9 was lost in different washing steps. The following test procedure was performed. First, a column containing an ADK8 / 9 affinity resin with an ID of 10 mm, a bed height of 25 mm, and a volume of 1.96 ml was balanced with at least 5 column volumes of 20 mM TrisHCl and 150 mM NaCl, pH 7.4. The pH-adjusted load was applied to the column containing the ADK8 / 9 affinity resin. A portion of the sample loaded on the column was saved and subsequently measured by ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP antigens. The column was then rebalanced with 10 column volumes of 20 mM TrisHCl and 150 mM NaCl, pH 7.4. A sample of the effluent was saved and subsequently measured by ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP antigens.

[0375] The column was then washed with 10 column volumes of wash 1 (W1): 100 mM sodium acetate and 0.1% Tween 80, pH 6.0. The column was then washed with 10 column volumes of wash 2 (W2): 50 mM TrisHCl and 125 mM NaCl, pH 8.5. The column was then washed with 10 column volumes of wash 3 (W3): 50 mM TrisHCl and 50% ethylene glycol, pH 8.5. Samples were taken from each of the eluates of W1, W2, and W3 and assayed by ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP antigens.

[0376] Elution was performed by applying 10 column volumes of the following elution buffer to the column: 50 mM TrisHCl, 50% ethylene glycol and 750 mM NaCl, pH 8.0.

[0377] The above test procedure is described in more detail in Table 21.

[0378] Table 21:

[0379]

[0380]

[0381] The obtained samples were assayed by each of ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCPs to assess yield and whether losses may have occurred during the steps.

[0382] The above samples were also assayed by SDS-PAGE and 12% silver staining to detect total protein.

[0383] The samples were then assayed by SDS-PAGE and Western blotting against AAV9 antigens. The primary antibody was a monoclonal antibody against VP1, VP2, and VP3 of AAV9, while the secondary antibody was a goat anti-mouse antibody conjugated to alkaline phosphatase.

[0384] Example 8

[0385] The AAV8 ELISA was performed on a TECAN robotic system using an AAV-8 titration ELISA kit (Article No. PRAAV8; Progen (Heidelberg, Germany). Monoclonal antibodies specific for a conformational epitope on the assembled AAV8 capsid (ADK8) were coated onto microtiter strips and used to capture AAV8 particles from the AAV fraction. The captured AAV8 particles were detected in two steps. In the first step, a biotin-conjugated monoclonal antibody specific for the ADK8 antibody was bound to the immune complexes. A streptavidin peroxidase conjugate was added to the immune complexes bound to the biotin-conjugated monoclonal antibody, which reacted with biotin. A peroxidase substrate solution was added, and a color reaction occurred that was proportional to the amount of bound AAV particles. The color reaction was measured photometrically at 450 nm.

[0386] Example 9

[0387] For the ITR-qPCR assay performed in the examples, the following procedures were performed. Using TaqMan-based qPCR, the vector genome titer (vg) / ml (ml) was determined using primers and fluorescently labeled probes that detect sequences within the ITR sequence of the vector genome. In order to detect ITR-specific sequences in AAV particles, the samples were treated differently. The samples were treated with DNase I and then with proteinase K to release the scAAV genome from the capsid. Then, restriction enzyme digestion was performed to resolve the AAV ITR T-shaped structure.

[0388] Plasmids used as reference materials were linearized with a single-cutting restriction enzyme and then further purified from agarose gels. The UV A260 absorbance of gel-extracted DNA was measured three times in a UV spectrophotometer and the average DNA concentration was calculated in μg / ml.

[0389] To determine the copy number / ml of the linearized standard plasmid, the molecular weight of the ds DNA was calculated in grams / mol, taking into account the exact mass of each individual nucleotide in the underlying sequence.

[0390] The vector genome titer (in vector genomes / mL) in the test article was calculated by fitting the plasmid standard curve with linear regression.

[0391] Example 10

[0392] AAV8 was produced in the HEK293 cell line after transfection with a triple plasmid system containing the relevant proteins and the coding cDNAs of AAV8-.VP1, -VP2 and -VP3. The clarified cell-free culture supernatant was concentrated and diafiltered with a Pall Omega T-series cassette 300 kDa. The viral particles were loaded onto a membrane adsorber (Mustang Q. Pall part number XT140MSTGQP05) under non-binding conditions for AAV8. The AAV8-containing effluent obtained was diluted with a dilution buffer containing 100 mM arginine, 200 mM NaCl, pH 8.0 to prepare a load for the AAV8-affinity matrix. The arginine-containing load was applied to a POROS TM CaptureSelect TM AAV8 affinity matrix (Thermo Fisher, catalog number A30793) was used as the column.

[0393] Samples were taken from each washing and elution step at each point to determine how much AAV8 was present in the sample. The determination showed how much AAV8 was lost in each washing step. The following test procedure was performed. First, a column containing an ADK8 / 9 affinity resin with an ID of 10 mm, a bed height of 25 mm, and a volume of 2.04 ml was balanced with at least 10 column volumes of 20 mM TrisHCl and 125 mM NaCl, pH 8.5. The load was applied to the column containing the ADK8 / 9 affinity resin. A portion of the sample loaded on the column was saved and subsequently measured by ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP antigens. The column was then rebalanced (wash 1, W1) with 10 column volumes of 50 mM TrisHCl and 125 mM NaCl, pH 8.5. Samples of the effluent were saved and subsequently assayed by ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP antigens.

[0394] The column was then washed with 10 column volumes of wash 2 (W2): 100 mM sodium acetate and 0.1% Tween 80, pH 6.0. The column was then washed with 10 column volumes of wash 3 (W3): 50 mM TrisHCl and 125 mM NaCl, pH 8.5. The column was then washed with 10 column volumes of wash 4 (W4): 50 mM TrisHCl and 50% ethylene glycol, pH 8.5. Samples were taken from each of the eluates of W2, W3, and W4 and assayed according to ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP antigens.

[0395] Elution was performed by first applying 10 column volumes of the following elution buffer to the column: 50 mM TrisHCl, 60% (w / w) ethylene glycol and 750 mM NaCl, pH 8.0. No subsequent washing step was performed. Elution was continued by then applying 10 column volumes of the following elution buffer to the column: 50 mM TrisHCl, 60% (w / w) ethylene glycol and 1000 mM NaCl, pH 8.0. The column was then washed with 20 mM Tris HCl, pH 7.4. The column was then regenerated by applying 10 column volumes of a solution comprising 100 mM glycine and 200 mM NaCl, pH 2.5 to the column.

[0396] The above test procedure is described in more detail in Table 22.

[0397] Table 22:

[0398]

[0399] Embodiment 11

[0400] AAV8 was produced in the HEK293 cell line after transfection with a triple plasmid system containing the coding cDNAs for the proteins of interest and AAV8-.VP1, -VP2 and -VP3. Cells were disrupted from a 30 L harvest aliquot using a Megatron MT3000 (Pall) and subsequently filtered through a) depth filter PDP8 region (0.5 m 2 ), b) Depth filter V100 area (0.5m 2 ) and c) Kleenpak capsule 0.2 μm area (0.15 μm 2 ) was used to filter the AAV8-containing solution. The clarified cell-free culture supernatant was concentrated and diafiltered using a Pall Omega T-series cassette 300 kDa. The viral particles were loaded onto a membrane adsorber (Mustang Q. Pall part number XT140MSTGQP05) under non-binding conditions for AAV8. The AAV8-containing effluent obtained was diluted 1:2 with a dilution buffer containing 100 mM arginine, 200 mM NaCl, pH 8.0 to prepare a load for the AAV8-affinity matrix. The arginine-containing load was applied to a POROS TM CaptureSelect TM The cells were loaded onto a column of AAV8 affinity matrix (Thermo Fisher, catalog number A30793; ID 10 mm, bed height 26 mm, volume 2.04 ml).

[0401] Samples were taken from each washing and elution step at each point to determine how much AAV8 was present in the sample. The determination showed how much AAV8 was lost in each washing step. The following test procedure was performed. First, a column containing an ADK8 / 9 affinity resin with an ID of 10 mm, a bed height of 25 mm, and a volume of 2.04 ml was balanced with at least 10 column volumes of 50 mM arginine-HCl and 100 mM NaCl, pH 8.0. The load was applied to the column containing the ADK8 / 9 affinity resin. A portion of the sample loaded on the column was saved and subsequently measured by ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP antigens. The column was then rebalanced (wash 1, W1) with 10 column volumes of 50 mM arginine-HCl and 100 mM NaCl, pH 8.0. Samples of the effluent were saved and subsequently assayed by ITR qPCR, ELISA against AAV antigens, and ELISA against HEK293 HCP antigens.

[0402] The column was then washed with 10 column volumes of wash 2 (W2): 100 mM MES-sodium, 10 mM EDTA, and 11.2 g / kg S / DII solution (18.0 g Tween 80, 3.4 g DMSO, and 3.6 g TnBP), pH 6.0. The column was then washed with 10 column volumes of wash 3 (W3): 50 mM arginine-HCl and 100 mM NaCl, pH 8.0. The column was then washed with 10 column volumes of wash 4 (W4): 50 mM arginine-HCl and 50% sucrose, pH 8.5. Samples were taken from each of the eluates of W2, W3, and W4 and assayed by ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP antigens.

[0403] Elution was performed by first applying 10 column volumes of the following elution buffer to the post: 50 mM arginine-HCl, 55% (w / w) sucrose, 2 mM MgCl2 and 800 mM NaCl, pH 8.0. The post was then washed with 10 column volumes of wash solution 5 (W5): 50 mM arginine-HCl and 100 mM NaCl, pH 8.0. Elution was continued by then applying 10 column volumes of the following elution buffer to the post: 50 mM arginine-HCl, 50% (v / v) glycerol and 800 mM NaCl, pH 8.0. The post was then washed with 50 mM arginine-HCl and 100 mM NaCl, pH 8.0. The post was then regenerated by applying 10 column volumes of a solution comprising 100 mM glycine and 200 mM NaCl, pH 2.7 to the post.

[0404] The above test procedure is described in more detail in Table 23. A linear flow rate of 39 cm / h was applied in all steps.

[0405] Table 23:

[0406]

[0407]

[0408] In the loading step described above, a 1:2 dilution was performed in the Mustang Q column to adjust the load to conditions close to the matrix of the equilibration buffer. This step was performed to investigate the effect of the buffer substance on AAV8 binding to the ligand. Any newly introduced compound could have potential competitive properties and / or it could potentially trigger elution.

[0409] Samples obtained were assayed by each of ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCPs to assess yield and whether losses may have occurred during the steps. Figure 7 Table 24 below shows the samples obtained in each of the above steps, wherein the yield of each component is shown in Table 25.

[0410] Table 24:

[0411]

[0412]

[0413] The "Wash 5" step reduces the front effect in the elution.

[0414] Table 25:

[0415]

[0416] The above samples were also assayed by SDS-PAGE and 12% silver staining to detect total protein. Figure 8 shown.

[0417] Example 12

[0418] AAV8 was produced in the HEK293 cell line after transfection with a triple plasmid system containing the coding cDNAs for the proteins of interest and AAV8-.VP1, -VP2 and -VP3. Cells were disrupted from a 30 L harvest aliquot using a Megatron MT3000 (Pall) and subsequently filtered through a) depth filter PDP8 region (0.5 m 2), b) Depth filter V100 area (0.5m 2 ) and c) Kleenpak capsule 0.2 μm area (0.15 μm 2 ) was used to filter the AAV8-containing solution. The clarified cell-free culture supernatant was concentrated and diafiltered using a Pall Omega T-series cassette 300 kDa. The viral particles were loaded onto a membrane adsorber (Mustang Q. Pall part number XT140MSTGQP05) under non-binding conditions for AAV8. The load was applied to a POROS TM CaptureSelect TM The AAV8 affinity matrix was loaded onto a column (Thermo Fisher, catalog number A30793; ID 10 mm, bed height 26 mm, volume 2.04 ml).

[0419] Samples were taken from each washing and elution step at each point to determine how much AAV8 was present in the sample. The determination showed how much AAV8 was lost in each washing step. The following test procedure was performed. First, a column containing an ADK8 / 9 affinity resin with an ID of 10 mm, a bed height of 25 mm, and a volume of 2.04 ml was balanced with at least 10 column volumes of 50 mM TrisHCl and 125 mM NaCl, pH 8.5. The load was applied to the column containing the ADK8 / 9 affinity resin. A portion of the sample loaded on the column was saved and subsequently measured by ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP antigens. The column was then rebalanced (wash 1, W1) with 10 column volumes of 50 mM TrisHCl and 125 mM NaCl, pH 8.5. Samples of the effluent were saved and subsequently assayed by ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP antigens.

[0420] The column was then washed with 10 column volumes of wash 2 (W2): 100 mM taurine, 125 mM NaCl, pH 8.5. The column was then washed with 10 column volumes of wash 3 (W3): 100 mM glycine, pH 8.5. The column was then washed with 10 column volumes of wash 4 (W4): 50 mM taurine, 50% (w / w) ethylene glycol, 0.1% octylpyranoglucoside, pH 8.5. Samples were taken from each of the eluates of W2, W3, and W4 and assayed by ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP antigens.

[0421] Elution was performed by first applying 10 column volumes of the following elution buffer to the column: 50 mM taurine, 60% (w / w) ethylene glycol, 750 mM NaCl, 0.1% octyl glucopyranoside, pH 8.0. The column was then washed with 10 column volumes of wash 5 (W5): 50 mM TrisHCl and 125 mM NaCl, pH 8.5. Elution was continued by then applying 10 column volumes of the following elution buffer to the column: 1 M (NH4)2SO4, 50 mM TrisHCl and 50% (v / v) ethylene glycol, pH 7.0. The column was then washed with 50 mM TrisHCl and 125 mM NaCl, pH 8.5. The column was then regenerated by applying 10 column volumes of a solution comprising 100 mM glycine and 200 mM NaCl, pH 2.7 to the column.

[0422] The above test procedure is described in more detail in Table 26. A linear flow rate of 39 cm / h was applied in all steps.

[0423] Table 26:

[0424]

[0425] The obtained samples were assayed by ITR qPCR and each of ELISA for AAV antigens (as described in Example 8 above) and ELISA for HEK293 HCP to assess the yield and whether losses may have occurred during the steps. The chromatograms associated with the above examples are shown in Fig. 9 Table 27 below shows the samples obtained in each of the above steps, wherein the yield of each component is shown in Table 28.

[0426] Table 27:

[0427]

[0428] The "Wash 5" step reduces the front effect in the elution.

[0429] Table 28:

[0430]

[0431] The above samples were also assayed by SDS-PAGE and 12% silver staining to detect total protein. Fig.10 shown.

[0432] Example 13

[0433] AAV8 was produced in the HEK293 cell line after transfection with a triple plasmid system containing the coding cDNAs for the proteins of interest and AAV8-.VP1, -VP2 and -VP3. Cells were disrupted from a 30 L harvest aliquot using a Megatron MT3000 (Pall) and subsequently filtered through a) depth filter PDP8 region (0.5 m 2 ), b) Depth filter V100 area (0.5m 2 ) and c) Kleenpak capsule 0.2 μm area (0.15 μm 2 ) was used to filter the AAV8-containing solution. The clarified cell-free culture supernatant was concentrated and diafiltered using a Pall Omega T-series cassette 300 kDa. The viral particles were loaded onto a membrane adsorber (Mustang Q. Pall part number XT140MSTGQP05) under non-binding conditions for AAV8. The AAV8-containing effluent obtained was diluted 1:2 with dilution buffer [100 mM histidine, 200 mM NaCl, pH 8.5] and an additional 4 g of polysorbate 80 / kg was added to prepare a load for AAV8-affinity. The histidine-containing load was applied to a POROS TM CaptureSelect TM The PCR products were loaded onto a column of AAV8 affinity matrix (Catalog No. A30793, Thermo Fisher; ID: 10 mm, bed height: 26 mm, volume: 2.04 ml).

[0434] Samples were taken from each washing and elution step at each point to determine how much AAV8 was present in the sample. The determination showed how much AAV8 was lost in each washing step. The following test procedure was performed. First, a column containing an ADK8 / 9 affinity resin with an ID of 10 mm, a bed height of 26 mm, and a volume of 2.04 ml was balanced with at least 10 column volumes of 50 mM TrisHCl and 125 mM NaCl, pH 8.5. The load was applied to the column containing the ADK8 / 9 affinity resin. A portion of the sample loaded on the column was saved and subsequently measured by ITR qPCR, ELISA for AAV antigens (as described in Example 9 above), and ELISA for HEK293 HCP antigens (as described in Example 8 above). The column was then rebalanced (wash 1, W1) with 10 column volumes of 50 mM histidine and 100 mM NaCl, pH 8.5. Samples of the effluent were saved and subsequently assayed by ITR qPCR and ELISA for AAV antigens (as described in Examples 8 and 9 above) and ELISA for HEK293 HCP antigens.

[0435] The column was then washed with 10 column volumes of wash 2 (W2): 200 mM Bis-Tris, 16.6 g S / D solution (Triton X-100; polysorbate 80; TNBP=10.87; 3.31:3.01 (by weight), pH 6.0. The column was then washed with 10 column volumes of wash 3 (W3): 10 mM sodium citrate, pH 8.5. The column was then washed with 10 column volumes of wash 4 (W4): 100 mM arginine-HCl, 100 mM lysine-HCl, 100 mM histidine-HCl, 2 mM N-acetyl-D,L-tryptophan, pH 8.5, and 20% (w / w) polysorbate 80. Samples were taken from each of the eluates of W2, W3, and W4 and eluted according to the ITR. The assays were performed by qPCR, ELISA against AAV antigens, and ELISA against HEK293 HCP antigens (as described in Examples 8 and 9 above).

[0436] Elution was performed by first applying 10 column volumes of the following elution buffer to the column: 20% (w / w) sucrose, 10% (w / w) sorbitol, 5% (w / w) mannitol (sucrose), 15% (w / w) glycerol, 50 mM histidine, 800 mM NaCl, pH 8.0. The column was then washed with 10 column volumes of wash 5 (W5): 50 mM histidine and 100 mM NaCl, pH 8.5. Elution was continued by then applying 10 column volumes of the following elution buffer to the column: 50 mM TrisHCl, 750 mM NaCl and 50% (w / w) DMSO, pH 8.0. The column was then washed with 50 mM histidine and 100 mM NaCl, pH 8.5. Then, the column was regenerated by applying 10 column volumes of a solution containing 100 mM glycine and 200 mM NaCl, pH 2.7 to the column.

[0437] The above test procedure is described in more detail in Table 29. A linear flow rate of 39 cm / h was applied in all steps.

[0438] Table 29:

[0439]

[0440] The "Wash 5" step reduced the front effect in the elution. No elution of AAV8 was observed in the second elution step (Elution 2). For AAV8, DMSO may still be suitable as a wash buffer and / or buffer to potentially inactivate or decompose lipid enveloped viruses.

[0441] The obtained samples were assayed by each of ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 (as described in Examples 8 and 9 above) to assess yield and whether losses may have occurred during the steps. The chromatograms associated with the above examples are shown in Fig.11 Table 30 below shows the samples obtained in each of the above steps, wherein the yield of each component is shown in Table 31.

[0442] Surprisingly, AAV8 was eluted from CaptureSelect AAV8 resin under near neutral conditions in the presence of polyols and a certain amount of salt. The resin manufacturer suggested that elution would require acidic conditions below pH 3.5. Also surprisingly, elution could be triggered by sugars, sugar alcohols, and / or mixtures of sugars and sugar alcohols. Likewise, unexpectedly, DMSO as a polar solvent did not elute AAV8 from Capture Select AAV8 resin.

[0443] Table 30:

[0444]

[0445] The "Wash 5" step reduced the front effect in the elution. No elution of AAV8 was observed in the second elution step (Elu 2).

[0446] Table 31:

[0447]

[0448] The above samples were also assayed by SDS-PAGE and 12% silver staining to detect total protein. Fig.12 shown.

[0449] Embodiment 14

[0450] AAV2 was produced in the HEK293 cell line after transfection with a triple plasmid system containing the relevant proteins and the coding cDNAs for AAV2-.VP1, -VP2, and -VP3. The AAV2 samples were diluted with 150 mM NaCl, 20 mM TrisHCl, pH 7.4 to provide the loading. The loading was applied to the POROS TM CaptureSelect TM The PCR products were loaded onto a column of AAVX affinity matrix (Catalog No.: A36739, Thermo Fisher; ID: 10 mm, bed height: 25 mm, volume: 1.96 ml).

[0451] Samples were taken from each washing and elution step at each point to determine how much AAV2 was present in the sample. The assay showed how much AAV2 was lost in the different washing steps. The following test procedure was performed. First, a column containing an ADK8 / 9 affinity resin with an ID of 10 mm, a bed height of 25 mm, and a volume of 1.96 ml was balanced with at least 10 column volumes of 20 mM TrisHCl, 150 mM NaCl, pH 7.4. The load was applied to the column containing the ADK8 / 9 affinity resin. A portion of the sample loaded onto the column was saved and subsequently assayed by ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP antigens (as described in Examples 8 and 9 above). The column was then rebalanced (wash 1, W1) with 10 column volumes of 20 mM TrisHCl, 150 mM NaCl, pH 7.4. A sample of the effluent was saved and subsequently assayed by ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP antigens (as described in Examples 8 and 9 above).

[0452] The column was then washed with 10 column volumes of wash 2 (W2): 100 mM sodium acetate, 0.1% polysorbate 80, pH 6.0. The column was then washed with 10 column volumes of wash 3 (W3): 50 mM TrisHCl, 125 mM NaCl, pH 8.5. The column was then washed with 10 column volumes of wash 4 (W4): 50 mM TrisHCl, 50% (w / w) ethylene glycol, pH 8.5. The column was then washed with 10 column volumes of wash 5 (W5): 50 mM TrisHCl, 60% (w / w) ethylene glycol + 750 mM NaCl pH 8.0. The column was then washed with 10 column volumes of wash 6 (W6): 20 mM TrisHCl, 150 mM NaCl, pH 7.4. Samples were taken from the eluate of each of W2, W3 and W4 and assayed by ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP antigens (as described in Examples 8 and 9 above).

[0453] Elution was performed by first applying 10 column volumes of the following elution buffer to the column: 100 mM glycine-HCl, 200 mM NaCl, pH 2.5. The column was then washed with 10 column volumes of wash 7 (W7): 20 mM TrisHCl, 150 mM NaCl, pH 7.4. The column was back-extracted with 10 column volumes of 50 mM phosphoric acid, pH 2.0, washed with 10 column volumes of wash 8 (W8): 20 mM TrisHCl, 150 mM NaCl, pH 7.4, and then back-extracted with 10 column volumes of 50 mM phosphoric acid, pH 2.0.

[0454] The above test procedure is described in more detail in Table 32. A linear flow rate of 39 cm / h was applied in all steps.

[0455] Table 32:

[0456]

[0457] The obtained samples were assayed by each of ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP (as described in Examples 8 and 9 above) to assess yield and whether losses may have occurred during the steps. The chromatograms associated with the above examples are shown in Fig.13 Table 33 below shows the samples obtained in each of the above steps, wherein the yield of each component is shown in Table 34.

[0458] Table 33:

[0459]

[0460] Table 34:

[0461]

[0462] The above samples were also assayed by SDS-PAGE and 12% silver staining to detect total protein. The results of SDS-PAGE assay are shown in Fig.14 As shown (see lanes 2-11).

[0463] The samples were then assayed by SDS-PAGE and Western blotting against AAV antigens. The primary antibodies were monoclonal antibodies against VP1, VP2, and VP3 of AAV, while the secondary antibodies were goat anti-mouse antibodies conjugated to alkaline phosphatase. The results of the Western blotting assay are shown in Figure 2. Fig.15 As shown (see lanes 2-11).

[0464] Embodiment 15

[0465] AAV9 was produced in the HEK293 cell line after transfection with a triple plasmid system containing the relevant proteins and the coding cDNAs of AAV9-.VP1, -VP2 and -VP3. The AAV9 samples were diluted with 150 mM NaCl, 20 mM TrisHCl, pH 7.4 to provide the loading material. The loading material was applied to the POROS TM CaptureSelect TM The PCR products were loaded onto a column of AAVX affinity matrix (Catalog No.: A36739, Thermo Fisher; ID: 10 mm, bed height: 25 mm, volume: 1.96 ml).

[0466] Samples were obtained from each washing and elution step at each point to determine how much AAV9 was present in the sample. The determination showed how much AAV9 was lost in each washing step. The following test procedure was performed. First, a column containing an ADK8 / 9 affinity resin with an ID of 10 mm, a bed height of 25 mm, and a volume of 1.96 ml was balanced with at least 10 column volumes of 20 mM TrisHCl, 150 mM NaCl, pH 7.4. The load was applied to the column containing the ADK8 / 9 affinity resin. A portion of the sample loaded on the column was saved and subsequently measured by ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP antigens (as described in Examples 8 and 9 above). The column was then rebalanced (wash 1, W1) with 10 column volumes of 20 mM TrisHCl, 150 mM NaCl, pH 7.4. A sample of the effluent was saved and subsequently assayed by ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP antigens (as described in Examples 8 and 9 above).

[0467] The column was then washed with 10 column volumes of wash 2 (W2): 100 mM sodium acetate, 0.1% polysorbate 80, pH 6.0. The column was then washed with 10 column volumes of wash 3 (W3): 50 mM TrisHCl, 125 mM NaCl, pH 8.5. The column was then washed with 10 column volumes of wash 4 (W4): 50 mM TrisHCl, 50% (w / w) ethylene glycol, pH 8.5. The column was then washed with 10 column volumes of wash 5 (W5): 50 mM TrisHCl, 60% (w / w) ethylene glycol + 750 mM NaCl pH 8.0. The column was then washed with 10 column volumes of wash 6 (W6): 20 mM TrisHCl, 150 mM NaCl, pH 7.4. Samples were taken from the eluate of each of W2, W3 and W4 and assayed by ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP antigens (as described in Examples 8 and 9 above).

[0468] Elution was performed by first applying 10 column volumes of the following elution buffer to the column: 100 mM glycine HCl, 200 mM NaCl, pH 2.5. The column was then washed with 10 column volumes of wash 7 (W7): 20 mM TrisHCl, 150 mM NaCl, pH 7.4. The column was back-extracted with 10 column volumes of 50 mM phosphoric acid, pH 2.0, washed with 10 column volumes of wash 8 (W8): 20 mM TrisHCl, 150 mM NaCl, pH 7.4, and then back-extracted with 10 column volumes of 50 mM phosphoric acid, pH 2.0.

[0469] The above test procedure is described in more detail in Table 35. A linear flow rate of 39 cm / h was applied in all steps.

[0470] Table 35:

[0471]

[0472]

[0473] The obtained samples were assayed by each of ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP (as described in Examples 8 and 9 above) to assess yield and whether losses may have occurred during the steps. The chromatograms associated with the above examples are shown in Fig.16 Table 36 below shows the samples obtained in each of the above steps, wherein the yield of each component is shown in Table 37.

[0474] Table 36:

[0475]

[0476] Table 37:

[0477]

[0478] The above samples were also assayed by SDS-PAGE and 12% silver staining to detect total protein. The results of SDS-PAGE assay are shown in Fig.14 As shown (see lanes 12-20).

[0479] The samples were then assayed by SDS-PAGE and Western blotting against AAV antigens. The primary antibodies were monoclonal antibodies against VP1, VP2, and VP3 of AAV, while the secondary antibodies were goat anti-mouse antibodies conjugated to alkaline phosphatase. The results of the Western blotting assay are shown in Figure 2. Fig.15 As shown (see lanes 12-20).

[0480] Example 16

[0481] This example demonstrates that elution conditions for AAV9 are enhanced relative to the use of acidic glycine or phosphate.

[0482] AAV9 was produced in the HEK293 cell line after transfection with a triple plasmid system containing the coding cDNAs for the proteins of interest and AAV9-.VP1, -VP2 and -VP3. Cells were disrupted from a 30 L harvest aliquot using a Megatron MT3000 (Pall) and subsequently filtered through a) depth filter PDP8 region (0.5 m 2 ), b) Depth filter V100 area (0.5m 2 ) and c) Kleenpak capsule 0.2 μm area (0.15 μm 2 ) was used to filter the AAV9-containing solution. The clarified cell-free culture supernatant was concentrated and diafiltered using a Pall Omega T-series cassette 300 kDa. The viral particles were loaded onto a membrane adsorber (Mustang Q. Pall part number XT140MSTGQP05) under non-binding conditions for AAV9. The load was applied to a POROS TM CaptureSelect TM AAVX affinity matrix (Thermo Fisher, Cat. No. A36739, Thermo Fisher) was loaded onto a column with an ID of 16 mm, a bed height of 50 mm, and a volume of 10.0 ml, followed by a buffer step using the column volumes indicated.

[0483] Samples were obtained from each washing and elution step at each point to determine how much AAV9 was present in the sample. The determination showed how much AAV9 was lost in each washing step. The following test procedure was performed. First, the column was balanced with at least 5 column volumes of 50mM Tris HCl, 125mM NaCl, pH 8.5. The load was applied to a column containing affinity resin. A portion of the sample loaded on the column was saved and then measured by ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP antigens. The column was then rebalanced (washing 1, W1) with 5 column volumes of 50mM TrisHCl, 125mM NaCl, pH 8.5. The sample of the effluent was saved and then measured by ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP antigens.

[0484] The column was then washed with 5 column volumes of wash 2 (W2): 100 mM sodium acetate, 0.1% polysorbate 80, pH 6.0. The column was then washed with 10 column volumes of wash 3 (W3): 50 mM TrisHCl, 125 mM NaCl, pH 8.5. Samples were taken from each of the eluates of W2 and W3 and assayed by ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP antigens.

[0485] Elution was performed by first applying 20 column volumes of purified water, then 20 column volumes of 1 mM HCl, pH 3.2, then 20 column volumes of 50 mM TrisHCl, 750 mM NaCl, 50% DMSO (w / w), pH 8.0. The column was washed with 20 column volumes of purified water, then 10 column volumes of 33 mM HCl, pH 2.0.

[0486] The above test procedure is described in more detail in Table 38. A linear flow rate of 39 cm / h was applied in all steps.

[0487] Table 38:

[0488]

[0489]

[0490] The obtained samples were assayed by ITR qPCR and each of ELISA for AAV antigens (as described in Example 8 above) and ELISA for HEK293 HCP to assess the yield and whether losses may have occurred during the steps. The chromatograms associated with the above examples are shown in Fig.17 Table 39 below shows the samples obtained in each of the above steps, wherein the yields of certain components are shown in Table 40.

[0491] Table 39:

[0492]

[0493] Table 40:

[0494]

[0495] The above samples were also assayed by SDS-PAGE and Western blotting, and the results were as follows Fig.18 shown.

[0496] AAV9 can be eluted from Capture select AAVx using 1 mM hydrochloric acid, which has the benefit of introducing only very small amounts of free acid into the aqueous system. Further elution procedures at near neutral pH can be performed with a buffered aqueous solution containing DMSO (750 mM NaCl, 50 mM TrisHCl, 50% w / w dimethyl sulfoxide). DMSO may be beneficial if the eluate containing DMSO is applied to ultracentrifugation using a sucrose gradient. Both elution strategies may have improved properties compared to harsh elution conditions (100 mM (pH below 3) and / or large amounts of sodium chloride or magnesium chloride) to maintain the biopotency of all AAV subtypes during elution from the AAVX resin.

[0497] Embodiment 17

[0498] This example demonstrates that elution conditions for AAV9 are enhanced relative to the use of acidic glycine or phosphate.

[0499] AAV9 was produced in the HEK293 cell line after transfection with a triple plasmid system containing the protein of interest (FIX-padua, double-stranded) and the coding cDNAs for AAV9-.VP1, -VP2 and -VP3. Cells were disrupted from a 30 L harvest aliquot using a Megatron MT3000 (Pall) and subsequently filtered through a) depth filter PDP8 region (0.5 m 2 ), b) Depth filter V100 area (0.5m 2 ) and c) Kleenpak capsule 0.2 μm area (0.15 μm 2) was used to filter the AAV9-containing solution. The clarified cell-free culture supernatant was concentrated and diafiltered using a Pall Omega T-series cassette 300 kDa. The viral particles were loaded onto a membrane adsorber (Mustang Q. Pall part number XT140MSTGQP05) under non-binding conditions for AAV9. The load was applied to a POROS TM CaptureSelect TM The PCR products were loaded onto a column of AAVX affinity matrix (Thermo Fisher, catalog number A36739; 10 mm, bed height 28 mm, volume 2.2 ml).

[0500] Samples were obtained from each washing and elution step at each point to determine how many AAV9s were present in the sample. The determination showed how many AAV9s were lost in each washing step. The following test procedure was performed. First, a column containing an affinity resin with an ID of 10 mm, a column height of 28 mm, and a volume of 2.2 ml was balanced with at least 10 column volumes of 50 mM TrisHCl, 125 mM NaCl, pH 8.5. The load was applied to the column containing the affinity resin. A portion of the sample loaded on the column was saved and then measured by ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP antigens. The column was then rebalanced (washing 1, W1) with 10 column volumes of 50 mM TrisHCl and 125 mM NaCl, pH 8.5. The sample of the effluent was saved and then measured by ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP antigens.

[0501] The column was then washed with 10 column volumes of wash 2 (W2): 100 mM sodium acetate and 0.1% polysorbate 80, pH 6.0. The column was then washed with 10 column volumes of wash 3 (W3): 50 mM TrisHCl and 125 mM NaCl, pH 8.5. The column was then washed with 30 column volumes of purified water (wash 4 (W4)). Samples were taken from each of the eluates of W2, W3, and W4 and assayed according to ITR qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP antigens.

[0502] Elution was performed by first applying a gradient of 0% to 100% 33 mM HCl / 1000 mM NaCl in 1 mM HCl over 20 column volumes.

[0503] The above test procedure is described in more detail in Table 41. A linear flow rate of 39 cm / h was applied in all steps.

[0504] Table 41:

[0505]

[0506] The obtained samples were assayed by ITR qPCR and each of ELISA for AAV antigens (as described in Example 8 above) and ELISA for HEK293 HCP to assess the yield and whether losses may have occurred during the steps. The chromatograms associated with the above examples are shown in Fig.19 Silver staining as shown. Fig. 20 shown.

[0507] The biological potency of the eluted AAV9 was 0.51 BPU.

[0508] Embodiment 18

[0509] The elution properties of various resin types were evaluated for different elution buffers. A polyol elution buffer was prepared containing 50% ethylene glycol (w / w), 50 mM TrisHCl, 750-2000 mM NaCl, pH 8.0. An acidic elution buffer was prepared containing 100 mM glycine (pH 2.5), 100 mM sodium citrate (pH 3.0), and 50 mM phosphoric acid (pH 2.0). An elution buffer containing MgCl2 was prepared containing 2M MgCl2 and 50 mM TrisHCl, pH 7.4.

[0510] Several resins were tested. The results are shown in Table 42 below. In this table, "yes" indicates that AAV8 can be eluted from the resin with a potential "elution buffer" at an amount greater than 10% of the load, while "no" indicates that AAV can be eluted from the resin with a potential "elution buffer" at an amount less than 1% of the load.

[0511] Table 42:

[0512]

[0513] 1 Thermo Fisher Scientific

[0514] 2 The resin was prepared by immobilizing the monoclonal antibody type ADK8 from Progen AG onto CNBr-Sepharose Fast Flow affinity resin at a density of 0.94 mg / ml resin.

[0515] 3 GE Healthcare

[0516] Various buffers were tested for their ability to elute AAV8 from the Capture Select AAV8 resin described above. The results are shown in Table 43 below.

[0517] Table 43:

[0518]

[0519]

[0520] The elution buffers listed in Table 44 were also tested to determine if they would satisfactorily elute from Capture ELISA Kit available from ThermoFisher Scientific. AAV was eluted from AAV9.

[0521] Table 44.

[0522]

[0523] Each potential elution buffer described in Table 44 was applied sequentially as part of an elution screen to determine which elution buffers could be used on Capture select AAVx. The screen started with the elution buffer with the lowest potential elution strength and ended with the buffer with the highest potential elution strength. Fig.21 This is an example of how the Elution option can be applied on Capture select AAVx.

[0524] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

Claims

1. A method for purifying adeno-associated virus (AAV), the method comprising (a) loading the solution onto an affinity resin for AAV under conditions that allow binding between AAV in the solution and the affinity resin; (b) performing at least three washing steps; and (c) eluting the AAV from the affinity resin, wherein at least one of the washing steps comprises applying a buffer comprising an organic solvent or a detergent to the affinity resin, and The method further comprises contacting the AAV-containing solution with an anion exchanger, and eluting the AAV-containing solution from the anion exchanger before loading the AAV-containing solution onto the affinity resin, wherein the first washing step comprises applying a first buffer to the affinity resin, the first buffer comprising 50 mM to 200 mM sodium acetate and 0.05% (w / w) to 0.2% (w / w) polysorbate 80, wherein the pH of the first buffer is 5.2 to 6.8; wherein the second washing step comprises applying a second buffer to the affinity resin, the second buffer comprising 30 mM to 200 mM TrisHCl and 75 mM to 500 mM salt, wherein the pH of the second buffer is 7.5 to 8.8, wherein the salt is selected from the group consisting of NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl, sodium acetate, and combinations thereof; The third washing step comprises applying a third buffer to the affinity resin, wherein the third buffer comprises 20 mM to 100 mM TrisHCl, 40% (w / w) to 60% (w / w) ethylene glycol, and wherein the pH of the third buffer is 7.5 to 8.

8.

2. The method of claim 1, wherein the salt is NaCl.

3. The method of claim 1, further comprising a washing step, which is performed before the first washing step and comprises applying a buffer comprising 10 mM to 30 mM TrisHCl and 75 mM to 250 mM NaCl to the affinity resin, and wherein the pH of the buffer is 6.5 to 8.

0.

4. The method of claim 1, wherein the first washing step comprises applying a first buffer to the affinity resin, the first buffer comprising 50 mM to 200 mM sodium acetate and 0.05% (w / w) to 0.2% (w / w) polysorbate 80, and wherein the pH of the first buffer is 5.5 to 6.5; and The second washing step comprises applying a second buffer to the affinity resin, the second buffer comprising 30 mM to 70 mM TrisHCl and 75 mM to 250 mM NaCl, and wherein the pH of the second buffer is 8.0 to 8.

8.

5. The method of claim 4, wherein the third washing step comprises applying a third buffer to the affinity resin, the third buffer comprising 20 mM to 70 mM TrisHCl and 40% (w / w) to 60% (w / w) ethylene glycol, and wherein the pH of the third buffer is 8.0 to 8.

8.

6. The method of claim 4, further comprising a washing step, which is performed before the first washing step and comprises applying a buffer comprising 10 mM to 30 mM TrisHCl and 75 mM to 250 mM NaCl to the affinity resin, and wherein the pH of the buffer is 6.5 to 8.

0.

7. The method of claim 4, wherein the first buffer comprises 100 mM sodium acetate and 0.1% polysorbate 80, and wherein the pH of the first buffer is 6.

0.

8. The method of claim 4, wherein the second buffer comprises 50 mM TrisHCl and 125 mM NaCl, and wherein the pH of the second buffer is 8.

5.

9. The method of claim 5, wherein the third buffer comprises 50 mM TrisHCl and 50% (w / w) ethylene glycol, and wherein the pH of the third buffer is 8.

0.

10. The method of any one of claims 1 to 9, wherein acidic components are removed.

11. The method of claim 10, wherein the acidic component is host cell DNA, and wherein the acidic component is reduced to a value below 250 pg / microgram AAV antigen as measured by qPCR.

12. The method of claim 10, wherein the acidic component is host cell DNA, and wherein the acidic component is reduced to a value below 250 pg / microgram AAV antigen as measured by ELISA.

13. The method of claim 11 or 12, wherein the acidic component is HEK293 DNA.

14. The method of any one of claims 1 to 9, wherein eluting comprises applying an elution buffer having a continuously linearly increasing conductivity by gradient elution.

15. The method of any one of claims 1 to 9, wherein eluting comprises applying a continuously linearly increasing concentration of an organic solvent by gradient elution.

16. The method of any one of claims 1 to 9, wherein eluting comprises contacting the affinity resin with an elution buffer comprising ethylene glycol, a salt, and a buffer, wherein the salt is NaCl, the buffer is TrisHCl, and the pH of the elution buffer is at least 7.

0.

17. The method of claim 16, wherein the concentration of the salt is 750 mM, the concentration of the buffer is 50 mM, and the concentration of the ethylene glycol is 50% (w / w) to 60% (w / w).

18. The method of claim 16, wherein the pH of the elution buffer is 8.

0.

19. The method of any one of claims 1 to 9, wherein eluting comprises contacting the affinity resin with an elution buffer comprising 750 mM NaCl and 50-60% (w / w) ethylene glycol at a pH of at least 7.

0.

20. The method of claim 19, wherein the elution buffer comprises at least 55% (w / w) ethylene glycol.

21. The method of any one of claims 1 to 9, wherein eluting comprises contacting the affinity resin with an elution buffer comprising 50 mM Tris HCl, 750 mM to 1250 mM NaCl, and 60% (w / w) ethylene glycol at a pH of at least 7.

8.

22. The method of any one of claims 1 to 9, wherein eluting comprises contacting the affinity resin with an elution buffer comprising 2 mM magnesium chloride, 50 mM arginine-HCl, 750 mM to 1000 mM NaCl, and at least 55% (w / w) sucrose, at a pH of at least 8.

0.

23. The method of claim 22, wherein eluting further comprises (a) contacting the affinity resin with a fifth buffer, the fifth buffer comprising 20 mM to 100 mM arginine-HCl and 75 mM to 250 mM NaCl, and wherein the pH of the fifth buffer is 7.5 to 8.5; and (b) contacting the affinity resin with a second elution buffer comprising 20 mM to 100 mM arginine-HCl, 40% (w / w) to 60% (w / w) glycerol, and 500 mM to 1000 mM salt, and wherein the pH of the second elution buffer is 7.5 to 8.

5.

24. The method of claim 23, wherein the steps are performed continuously.

25. The method of any one of claims 1 to 9, wherein eluting comprises contacting the affinity resin with an elution buffer comprising 2 mM magnesium chloride, 50 mM arginine-HCl, 750 mM to 1000 mM NaCl, and at least 50% (w / w) glycerol, at a pH of at least 8.

0.

26. The method of any one of claims 1 to 9, wherein eluting comprises contacting the affinity resin with an elution buffer comprising 2 mM magnesium chloride, 50 mM taurine, 600 mM to 1000 mM NaCl, 0.05% to 0.2% octylpyranoglucoside, and 60% (w / w) ethylene glycol at a pH of at least 7.

8.

27. The method of claim 26, wherein eluting further comprises (a) contacting the affinity resin with a fifth buffer, the fifth buffer comprising 20 mM to 100 mM Tris-HCl and 75 mM to 250 mM NaCl, and wherein the pH of the fifth buffer is 8.0 to 8.8; and (b) contacting the affinity resin with a second elution buffer comprising 1 M ammonium sulfate, 50 mM Tris HCl and 50% (w / w) ethylene glycol at a pH of at least 6.

8.

28. The method of claim 27, wherein the steps are performed continuously.

29. The method of any one of claims 1 to 9, wherein eluting comprises contacting the affinity resin with an elution buffer comprising 1 M ammonium sulfate, 50 mM Tris HCl, and 50% (w / w) ethylene glycol at a pH of at least 6.

8.

30. The method of any one of claims 1 to 9, wherein eluting comprises contacting the affinity resin with an elution buffer comprising 20% ​​(w / w) sucrose, 10% (w / w) sorbitol, 5% (w / w) mannitol or 5% (w / w) sucrose, 15% (w / w) glycerol, 50 mM histidine, and 750 mM to 1000 mM NaCl, at a pH of at least 7.

8.

31. The method of claim 30, wherein eluting further comprises (a) contacting the affinity resin with a fifth buffer, the fifth buffer comprising 20 mM to 100 mM histidine, 80 mM to 120 mM NaCl, and wherein the pH of the fifth buffer is 8.0 to 8.8; and (b) contacting the affinity resin with a second elution buffer comprising 20 mM to 100 mM histidine, 600 mM to 900 mM NaCl and 5% (w / w) to 60% (w / w) DMSO, and wherein the pH of the fifth buffer is 6.5 to 8.

5.

32. The method of claim 31 , wherein the steps are performed continuously.

33. The method of any one of claims 1 to 9, wherein eluting comprises contacting the affinity resin with an elution buffer comprising 100 mM glycine-HCl, 200 mM NaCl, pH 2.

5.

34. The method of any one of claims 1 to 9, wherein the pH of the elution buffer is 8.

0.

35. The method of any one of claims 1 to 9, wherein the pH of the elution buffer is 7.

4.

36. The method of any one of claims 1 to 9, wherein elution comprises a stepwise increase in counterion concentration.

37. The method of any one of claims 1 to 9, wherein elution comprises a stepwise increase in the concentration of the organic solvent.

38. The method of any one of claims 1 to 9, wherein the salt in the elution buffer is a monovalent or multivalent anion.

39. The method of any one of claims 1 to 9, wherein the salt in the elution buffer is a divalent anion.

40. The method of any one of claims 1 to 9, wherein the salt in the elution buffer is selected from chloride, acetate, sulfate and citrate.

41. The method of claim 1, wherein the first wash step comprises applying a first buffer to the affinity resin, the first buffer comprising 50 mM to 200 mM sodium acetate, 0.05% to 0.2% polysorbate 80, and wherein the pH of the first buffer is 5.2 to 6.8; wherein the second washing step comprises applying a second buffer to the affinity resin, the second buffer comprising 30 mM to 100 mM TrisHCl and 75 mM to 200 mM NaCl, and wherein the pH of the second buffer is 7.5 to 8.8; and The third washing step comprises applying a third buffer to the affinity resin, the third buffer comprising 20 mM to 100 mM TrisHCl and 75 mM to 250 mM NaCl, and wherein the pH of the third buffer is 7.5 to 8.

8.

42. The method of claim 41, further comprising eluting by applying purified water to the affinity resin followed by applying 20 mM to 50 mM HCl at a pH of 1.7 to 2.5 to the affinity resin.

43. The method of claim 41, further comprising eluting by applying a gradient of 0% to 100% of 20-50 mM HCl to 800-1200 mM NaCl in 0.5-2.0 mM HCl.

44. The method of any one of claims 1 to 9 and 41, wherein the HC impurity level of the AAV obtained from the elution step is ≤ 99.9%.

45. The method of any one of claims 1 to 9 and 41, wherein the HC impurity level of the AAV obtained from the elution step is ≤ 99.0%.

46. ​​The method of any one of claims 1 to 9 and 41, wherein the AAV is AAV8 and the affinity resin is POROS TM CaptureSelect TM AAV8, and wherein the elution buffer is acidic and does not contain ethylene glycol.

47. The method of any one of claims 1 to 9 and 41, wherein the AAV is AAV9 and the affinity resin is POROS TM CaptureSelect TM AAV9, and wherein the elution buffer is acidic and does not contain ethylene glycol.

48. The method of any one of claims 1 to 9 and 41, wherein the AAV is AAV8, and wherein the affinity resin is an immunoaffinity resin consisting of an immobilized monoclonal antibody against AAV8 from ADK8 or ADK8 / 9 immobilized on a chromatography matrix.

49. The method of any one of claims 1 to 9 and 41, wherein the AAV is AAV9, and wherein the affinity resin is an immunoaffinity resin consisting of an immobilized monoclonal antibody against AAV9 from the ADK9 type or the ADK8 / 9 type immobilized on a chromatography matrix.

50. The method of any one of claims 1 to 9 and 41, wherein the method further comprises contacting the AAV-containing solution with a filter comprising positively charged groups that effectively deplete the AAV-containing solution of acidic charged contaminants.

51. The method of any one of claims 1 to 9 and 41, further comprising nanofiltration of the AAV fraction to remove viruses larger than 35 nm.

52. The method of any one of claims 1 to 9 and 41, further comprising a polishing step comprising AEX chromatography using a column comprising a whisker gel.

53. The method of any one of claims 1 to 9 and 41, further comprising testing the AAV fraction by an AAV-specific ELISA.

54. The method of claim 53, wherein the AAV-specific ELISA is a sandwich ELISA specific for AAV.

55. The method of any one of claims 1 to 9 and 41, wherein eluting comprises a) applying purified water to the affinity resin followed by 1 mM HCl at a pH of 3.2; and / or b) contacting the affinity resin with an elution buffer comprising 30 mM to 70 mM TrisHCl, 700 mM to 900 mM NaCl, and 40% (w / w) to 60% (w / w) DMSO at a pH of 7.5 to 8.5.

Images