RU2785661C2 - Methods for purification of adeno-associated viruses - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a method for purification of an adeno-associated virus (hereinafter – AAV). The method for purification of AAV includes loading of a solution containing AAV to affine resin; at least two washing stages; and elution of AAV from affine resin. At the same time, at least one of washing stages includes application to affine resin of a buffer containing an organic solvent or a detergent. The method additionally includes bringing into contact of the solution containing AAV with anionite, and elution of the solution containing AAV from anionite before loading of the solution containing AAV to affine resin.

EFFECT: implementation of the invention provides an increase in the efficiency of purification of AAV.

80 cl, 21 dwg, 44 tbl, 18 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 62/611,709, filed December 29, 2017, the disclosure of which is hereby incorporated by reference in its entirety.

FIELD OF TECHNOLOGY

[0002] The invention relates to materials and methods for purifying adeno-associated virus (AAV).

BACKGROUND OF THE INVENTION

[0003] Adeno-associated virus (AAV) is a small non-enveloped virus that packages a linear single-stranded DNA genome. AAV belongs to the family Parvoviridae and the genus Dependovirus because productive infection with AAV occurs only in the presence of a helper virus such as, for example, adenovirus or herpes virus. Even in the absence of a helper virus, AAV (serotype 2) can achieve latency by integrating into chromosome 19q13.4 of the human host genome. It is the only mammalian DNA virus known to be capable of site-specific integration (Daya and Berns, Clinical Microbiology Reviews, pages 583-593 (2008)).

[0004] For the safe use of AAV in the clinic, AAV has been genetically modified at several locations in its genome. For example, the Rep gene, which is required for viral replication, and an element required for site-specific integration, have been removed from the AAV genome in many viral vectors. Such recombinant AAVs (rAAVs) exist in an extrachromosomal state and have a very low efficiency of integration into genomic DNA. Thus, the likelihood of random mutagenesis occurring in the host cell by rAAV is reduced, if not completely eliminated. Because of these properties and lack of pathogenicity, rAAV has shown great potential as a gene therapy vector in many aspects of preclinical and clinical applications. The clinic is testing new serotypes and self-complementary vectors. Along with these ongoing vector developments, ongoing efforts have been focused on scalable manufacturing processes that can efficiently generate high titer quantities of rAAV vectors with high purity and potency.

[0005] Although efforts to develop efficient, large-scale methods for purifying an AAV product suitable for human administration have been significant, there remains a need for better methods for purifying AAV. There are various other proteins and materials from the host cell culture matrix that can be more efficiently removed during AAV purification. Therefore, methods for purifying AAV are needed that include the steps of removing host cell material from the final AAV product.

SUMMARY OF THE INVENTION

[0006] A feature of AAV vector generation in cell culture is the formation of a complex matrix that contains material from destroyed cells. In particular, host cell proteins, proteasomes, cell debris, and potential virus-specific receptors are often present in material from disrupted cells. Methods are disclosed that include the steps of removing host cell material from the final AAV product under conditions that result in greater purity at a physiologically acceptable pH.

[0007] In one aspect, the invention relates to a method for purifying adeno-associated virus (AAV) comprising

(a) loading the AAV-containing solution onto an AAV-targeting affinity resin under conduction conditions that allow binding between the AAV in solution and the affinity resin;

(b) carrying out at least two washing steps; and

(c) elution of the AAV from the affinity resin.

[0008] In some embodiments, the method further comprises contacting the AAV containing solution with an anion resin and eluting the AAV containing solution from the anion resin before loading the AAV containing solution onto the affinity resin.

[0009] In some embodiments, at least one of the washing steps includes applying a buffer containing an organic solvent or detergent to the affinity resin. In some embodiments, the buffer contains Tris-HCl and a salt. In some embodiments, the buffer contains 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 contains sodium acetate. In some embodiments, the buffer contains magnesium chloride. In some embodiments, the buffer contains Tris-HCl and ethylene glycol. In some embodiments, the buffer contains Arginine-HCl and one of sucrose and glycerol. In some embodiments, the implementation of the buffer contains Taurine and ethylene glycol. In some embodiments, the buffer contains Arginine-HCl, Lysine-HCl, and Histidine-HCl. In some embodiments, the buffer contains Tris-HCl and DMSO.

[0010] In some embodiments, at least three washing steps are carried out. In some embodiments, three washing steps are performed. In some embodiments, the three washing steps are performed sequentially.

[0011] 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 contains one or more of Triton X100, Polysorbate 80, and tri(n-butyl)phosphate (TNBP). In some embodiments, the implementation of the buffer contains Bis-Tris.

[0012] In some embodiments, the first wash step comprises applying to the affinity resin a first buffer containing about 50 to about 2000 mM sodium acetate and about 0.05 to about 0.2% polysorbate 80, and the first buffer has a pH of about 5.2 to about 6.8;

the second washing step includes applying to the affinity resin a second buffer containing from about 30 to about 200 mm Tris-HCl and from about 75 to about 500 mm salt, and the second buffer has a pH of from about 7.5 to about 9.2; and

the third stage of washing includes applying to the affinity resin of the third buffer containing from about 30 to about 200 mm Tris-HCl and from about 30 to about 75% vol. ethylene glycol, and the third buffer has a pH of from about 7.3 to about 8.8.

[0013] In some embodiments, the first washing step comprises applying to the affinity resin a first buffer containing from about 50 to about 500 mM 2-(N-morpholino)ethanesulfonic acid sodium salt (MES-Na), from about 3 to about 30 mM EDTA and a solvent/detergent mixture comprising polysorbate 80, DMSO and tri(n-butyl)phosphate (TNBP) and the first buffer has a pH of about 5.2 to about 6.8;

the second washing step includes applying to the affinity resin a second buffer containing about 30 to about 200 mM Tris-HCl or Arginine-HCl and about 75 to about 500 mM salt, and the second buffer has a pH of about 7.5 to about 9, 2; and

the third washing step includes applying to the affinity resin a third buffer containing about 20 to about 80 mM Arginine-HCl and about 50 to about 200 mM salt, and the third buffer has a pH of about 7.3 to about 8.8.

[0014] In some embodiments, the first wash step comprises applying to the affinity resin a first buffer containing about 50 to about 200 mM taurine and 0.2 to 1.5% PEG (e.g., PEG 6000), the first buffer having a pH of from about 5.2 to about 6.8;

the second washing step includes applying to the affinity resin a second buffer containing from about 30 to about 300 mm glycine, and the second buffer has a pH of from about 7.5 to about 9.2; and

the third washing stage includes applying to the affinity resin a third buffer containing from about 20 to about 150 mm taurine, from about 30 to about 75% vol. ethylene glycol and 0.05 to 0.2% octyl glycopyranoside, and the third buffer has a pH of about 7.3 to about 8.8.

[0015] In some embodiments, the first wash step comprises applying to the affinity resin a first buffer containing about 80 to about 400 mM Bis-Tris and about 10 to about 20 grams of a solvent/detergent mixture containing about Triton-X100, Polysorbate 80 and TNBP in a ratio of about 11:3:3 (by weight), and the first buffer has a pH of from about 5.2 to about 6.8;

the second washing step includes applying to the affinity resin a second buffer containing from about 5 to about 20 mmol sodium citrate, and wherein the second buffer has a pH of from about 7.5 to about 9.2; and

the third washing step comprises applying to the affinity resin a third buffer containing 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 from about 10% to about 40% (wt./mass.) polysorbate 80 and moreover, the third buffer has a pH of from about 7.3 to about 8.8.

[0016] In some embodiments, the first wash step comprises applying to the affinity resin a first buffer containing about 50 nM to about 200 mM sodium acetate and about 0.05 to about 0.2% polysorbate 80, the first buffer having a pH of about 5.2 to about 6.8;

the second washing step comprises applying to the affinity resin a second buffer containing about 20 nM to about 100 mM Tris-HCl and about 50 nM to about 200 nM salt, the second buffer having a pH of about 7.5 to about 8.8; and

the third washing step comprises applying to the affinity resin a third buffer containing about 20 to 100 mM Tris-HCl, about 40% to about 60% (w/w) ethylene glycol, and wherein the third buffer has a pH of about 7.5 up to about 8.8.

[0017] In some embodiments, the first wash step comprises applying to the affinity resin a first buffer containing about 50 nM to about 200 mM sodium acetate and about 0.05 to about 0.2% polysorbate 80, wherein the first buffer has a pH of about 5.2 to about 6.8;

the second washing step comprises applying to the affinity resin a second buffer containing about 20 nM to about 100 mM Tris-HCl and about 50 nM to about 200 nM salt, the second buffer having a pH of about 7.5 to about 8.8; and

the third washing step comprises applying to the affinity resin a third buffer containing about 20 to 100 mM Tris-HCl, about 40% to about 60% (w/w) ethylene glycol, and the third buffer has a pH of about 7.5 to approximately 8.8.

[0018] In some embodiments, the salt is selected from NaCl, KCl, MgCl ₂ , CaCl ₂ , sodium citrate, LiCl, CsCl, sodium acetate, and a combination of one or more of NaCl, KCl, MgCl ₂ , CaCl ₂ , sodium citrate, LiCl, CsCl and sodium acetate. In some embodiments, the salt is NaCl.

[0019] In some embodiments, the salt concentration does not exceed 500 mM. In some embodiments, the salt concentration does not exceed 200 mM. In some embodiments, the salt concentration in the third buffer does not exceed 500 mM. In some embodiments, the salt concentration in the third buffer does not exceed 200 mM.

[0020] In some embodiments, the method further includes a fourth wash step that occurs prior to the first wash step and includes applying to the affinity resin a fourth buffer containing about 10 to about 30 mM Tris-HCl and about 75 to about 250 mM NaCl and the fourth buffer has a pH of about 6.5 to about 8.0.

[0021] In some embodiments, the first buffer contains about 100 mM sodium acetate, about 0.1% polysorbate 80, and the first buffer has a pH of about 6.0. In some embodiments, the implementation of the second buffer contains about 50 mm Tris-HCl and about 125 mm NaCl, and the second buffer has a pH of about 8.5. In some embodiments, the implementation of the third buffer contains approximately 50 mm Tris-HCl and approximately 50% vol. ethylene glycol, and the third buffer has a pH of approximately 8.5.

[0022] In some embodiments, the first wash step comprises applying to the affinity resin a first buffer containing about 50 to about 200 mM sodium acetate and about 0.05 to about 0.2% polysorbate 80, and the first buffer has a pH of about 5.5 to about 6.5;

the second washing step includes applying to the affinity resin a second buffer containing from about 10 to about 70 mm Tris-HCl and from about 75 to about 250 mm NaCl, and the second buffer has a pH of from about 8.0 to about 9.0; and

the third stage of washing includes applying to the affinity resin of the third buffer containing from about 10 to about 70 mm Tris-HCl and from about 30 to about 75% vol. ethylene glycol, and the third buffer has a pH of from about 8.0 to about 9.0. In some embodiments, the method further includes a fourth wash step that occurs prior to the first wash step and includes applying to the affinity resin a fourth buffer containing about 10 to about 30 mM Tris-HCl and about 75 to about 250 mM NaCl, and a fourth the buffer has a pH of about 6.5 to about 8.0.

[0023] In some embodiments, the first buffer contains about 100 mM sodium acetate and about 0.1% polysorbate 80, and the first buffer has a pH of about 6.0. In some embodiments, the implementation of the second buffer contains about 50 mm Tris-HCl and about 125 mm NaCl, and the second buffer has a pH of about 8.5. In some embodiments, the implementation of the third buffer contains approximately 50 mm Tris-HCl and approximately 50% vol. ethylene glycol, and the third buffer has a pH of approximately 8.0.

[0024] In some embodiments, the implementation of the acid 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 below 250 pg per 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 below 250 pg per microgram of AAV antigen, as measured by ELISA.

[0025] In some embodiments, the implementation of the elution includes the use of a continuous linear increase in the conductivity of the elution buffer by gradient elution. In some embodiments, the elution involves the use of a continuous linear increase in the concentration of the organic solvent by gradient elution. In some embodiments, elution includes contacting the affinity resin with an elution buffer containing ethylene glycol, a salt such as NaCl, and a buffer such as Tris-HCl, 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 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 Tris-HCl.

[0026] In some embodiments, elution includes contacting the affinity resin with an elution buffer containing approximately 750 mM NaCl and 50-60% (w/w) ethylene glycol at a pH of at least 7.0. In some embodiments, the implementation of the elution buffer contains at least about 55% (wt./mass.) ethylene glycol. In some embodiments, elution includes contacting the affinity resin with an elution buffer containing 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 ,eight.

[0027] In some embodiments, elution includes contacting the affinity resin with an elution buffer containing about 2 mM magnesium chloride, about 50 mM Arginine HCl, about 750 mM to about 1000 mM NaCl, and at least about 55% (wt.) ./mass.) sucrose at a pH of at least about 8.0.

[0028] In some embodiments, the elution further comprises

(a) contacting the affinity resin with a fifth buffer containing about 20 to about 100 mM Arginine-HCl and about 75 to about 250 mM NaCl, and the fifth buffer has a pH of about 7.5 to about 8.5; and

(b) contacting the affinity resin with a second elution buffer containing 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 second elution buffer has a pH of from about 7.5 to about 8.5.

[0029] In some embodiments, the steps are performed sequentially.

[0030] In some embodiments, elution includes contacting the affinity resin with an elution buffer containing about 2 mM magnesium chloride, about 50 mM Arginine HCl, about 750 mM to about 1000 mM NaCl, and at least about 50% (wt.) ./mass.) glycerol at a pH of at least about 8.0.

[0031] In some embodiments, elution includes contacting the affinity resin with an elution buffer containing about 2 mM magnesium chloride, about 50 mM taurine, about 600 mM to about 1000 mM NaCl, about 0.05 to about 0.2 % octylglycopyranoside and about 60% (w/w) ethylene glycol at a pH of at least about 7.8. In some embodiments, the elution further comprises

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

(b) contacting the affinity resin with a second elution buffer containing 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.

[0032] In some embodiments, the steps are performed sequentially.

[0033] In some embodiments, elution includes contacting the affinity resin with an elution buffer containing 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 ,eight. In some embodiments, elution includes contacting the affinity resin with an elution buffer containing 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.

[0034] In some embodiments, the elution further comprises

(a) contacting the affinity resin with a fifth buffer containing about 20 to about 100 mM histidine and about 80 to about 120 mM NaCl, and the fifth buffer has a pH of about 8.0 to about 8.8; and

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

[0035] In some embodiments, the steps are performed sequentially. In some embodiments, elution includes contacting the affinity resin with an elution buffer containing approximately 100 mM glycine-HCl, approximately 200 mM NaCl, at a pH of approximately 2.5.

[0036] In some embodiments, the elution buffer has a pH of about 8.0. In some embodiments, the elution buffer has a pH of 8.0.

[0037] In some embodiments, the implementation of the elution includes a gradual increase in the concentration of the counterion. In some embodiments, the implementation of the elution includes a gradual increase in the concentration of the organic solvent. In some embodiments, the salt in the elution buffer is selected from monovalent, divalent, or polyvalent anions such as chloride, acetate, sulfate, and citrate.

[0038] In some embodiments, the first wash step comprises applying to the affinity resin a first buffer containing about 50 to about 200 mM sodium acetate, about 0.05 to about 0.2% polysorbate 80, and the first buffer has a pH of about 5.2 to about 6.8;

the second washing step includes applying to the affinity resin a second buffer containing from about 25 to about 100 mm Tris-HCl and from about 50 to about 200 mm NaCl, and the second buffer has a pH of from about 7.5 to about 9.2; and

the third washing step includes applying to the affinity resin a third buffer containing about 20 to about 100 mM Tris-HCl and about 75 to about 250 mM NaCl, and the third buffer has a pH of 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 elution using a gradient of 0 to 100% 20-50 mM hydrochloric acid/800-1200 mM NaCl in 0.5-2.0 mM hydrochloric acid.

[0039] In some embodiments, the AAV obtained from the elution step has an HC impurity level of ≤ 99.9%. In some embodiments, the AAV obtained from the elution step has an HC impurity level of ≤ 99.0%.

[0040] In some embodiments, the AAV is AAV8, the affinity resin is POROS™ CaptureSelect™ AAV8, and the elution buffer is acidic and does not contain ethylene glycol. In some embodiments, the AAV is AAV9, the affinity resin is POROS™ CaptureSelect™ AAV9, and the elution buffer is acidic and does not contain ethylene glycol.

[0041] In some embodiments, the AAV is AAV8 and the affinity resin is an immunoaffinity resin consisting of an immobilized ADK8 or ADK8/9 type anti-AAV8 monoclonal antibody immobilized on a chromatographic matrix. In some embodiments, the AAV is AAV9 and the affinity resin is an immunoaffinity resin consisting of an immobilized ADK9 or ADK8/9 anti-AAV9 monoclonal antibody immobilized on a chromatographic array.

[0042] In some embodiments, the method further comprises contacting the AAV containing solution with a filter containing positively charged groups effective to remove acidic charged contaminants from the AAV containing solution.

[0043] In some embodiments, the method further comprises nanofiltration of the AAV fraction to remove viruses larger than 35 nm.

[0044] In some embodiments, the method further includes a polishing step comprising performing AEX chromatography with a column containing a tentacle gel.

[0045] In some embodiments, the method further comprises testing the AAV fraction with an AAV-specific ELISA.

[0046] In some embodiments, the AAV-specific ELISA is an AAV-specific sandwich ELISA.

[0047] In another aspect, an AAV product obtained by the method of any one of paragraphs. 1-83.

BRIEF DESCRIPTION OF GRAPHICS

[0048] FIG. 1 is a Western blot showing a significantly lower amount of 70 kDa heat shock protein (HSP70) in accordance with the approximate purification with several washing steps described here (lane 4), in contrast to the comparative purification protocol without washing steps (lane 6) .

[0049] FIG. 2 illustrates silver staining of SDS-PAGE protein gels showing AAV8 proteins in lane 2, eluate containing AAV8 from the purification protocol with multiple washing steps described herein (lane 4) and a comparative purification protocol without a washing step (lane 6) .

[0050] FIG. 3 illustrates a Western blot showing more AAV8 present in the eluate prepared according to the purification protocol with multiple washing steps described herein (lane 4) than in the eluate from the comparative purification protocol without washing steps (lane 6). The AAV8 control is in lane 2.

[0051] Figure 4 illustrates silver staining of protein SDS-PAGE gels showing proteins present in various fractions from (L) initial loading of cell culture medium from AAV8-expressing HEK 293 cells to eluate (E) enriched in AAV8. A slight loss of AAV8 was observed during the passage through the washing steps (W1, W2 and W3) and a small amount of AAV8 was eluted from the column during stripping (S).

[0052] In FIG. 5 depicts a Western blot against AAV8 antigens. AAV8 was expressed in the cell culture bootstrap from HEK 293 cells expressing AAV8 (L). Significantly less AAV8 was present in the stream after this initial loading (FT) and subsequent wash steps (W1, W2 and W3). AAV8 was present in the eluate (E, E2). Again, significantly less AAV8 eluted from the column during stripping (S).

[0053] FIG. 6 shows the chromatogram of the separation procedure according to Example 3.

[0054] FIG. 7 shows a chromatogram from the separation procedure according to Example 11.

[0055] FIG. 8 illustrates silver staining of protein SDS-PAGE gels showing proteins present in different fractions from different washing steps and eluates according to Example 11.

[0056] FIG. 9 shows a chromatogram from the separation procedure according to Example 12.

[0057] FIG. 10 illustrates silver staining of protein SDS-PAGE gels showing proteins present in different fractions from different washing steps and eluates according to Example 12.

[0058] FIG. 11 shows a chromatogram from the separation procedure according to Example 13.

[0059] FIG. 12 illustrates silver staining of protein SDS-PAGE gels showing proteins present in various fractions from various washing steps and eluates according to Example 13.

[0060] FIG. 13 shows a chromatogram from the separation procedure according to Example 14.

[0061] FIG. 14 illustrates silver staining of protein SDS-PAGE gels showing proteins present in various fractions taken from the washing steps and eluates as described in Example 14.

[0062] In FIG. 15 depicts a Western blot against AAV antigens from various fractions taken from the washing steps and eluates as described in Example 14.

[0063] FIG. 16 shows a chromatogram from the separation procedure according to Example 15.

[0064] FIG. 17 shows a chromatogram from the separation procedure according to Example 16.

[0065] FIG. 18 depicts a Western blot against AAV antigens from various fractions taken from the washing steps and eluates as described in Example 16.

[0066] FIG. 19 shows a chromatogram from the separation procedure according to Example 17.

[0067] FIG. 20 illustrates silver staining of protein SDS-PAGE gels showing proteins present in various fractions taken from the washing steps and eluates as described in Example 17.

[0068] FIG. 21 is a chromatogram illustrating the results from the elution screen described in Example 18.

DETAILED DESCRIPTION OF THE INVENTION

[0069] This document provides methods for preparing an adeno-associated virus (AAV) product, methods for purifying AAV, and methods for purifying complete AAV capsids from a concentrated fraction of AAV containing empty AAV capsids and complete AAV capsids.

[0070] The use of terms in the singular and similar references in the context of the description of this invention (especially in the context of the following claims) should be construed for both the singular and the plural, unless otherwise specified herein or clearly contradicts the context. The terms "comprising", "having", "including", and "comprising" are to be construed as non-limiting terms (i.e., meaning "including but not limited to"), unless otherwise indicated.

[0071] The listing of ranges of values in this document is simply intended to serve as a concise way of individually referring to each individual value falling within the range and each endpoint, unless otherwise noted herein, and each individual value and endpoint is included in specification as if it were individually set forth in this document.

[0072] All methods described herein can be performed in any suitable order, unless otherwise specified in this document or otherwise explicitly contradicts the context. The use of any and all examples or exemplary language (eg, "such as") provided herein is merely for the better understanding of this document and does not limit the scope of this document unless otherwise stated. No wording in the specification should be construed as indicating any element not claimed to be essential to the practice of this invention.

[0073] Preferred embodiments of the present invention are described herein, including the best method known to the inventors for carrying out the present invention. Variations to these preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to use such variations as the case may be, and the inventors expect the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the appended claims, as permitted by applicable law. In addition, any combination of the above elements in all their possible variations is covered by this invention, unless otherwise specified in this document or otherwise clearly contradicts the context.

[0074] A feature of AAV vector generation in cell culture is the formation of a complex matrix that contains material from destroyed cells. In particular, host cell proteins, proteasomes, cell debris, and potential virus-specific receptors are often present in material from disrupted cells. Methods are disclosed that include the steps of removing host cell material from the final AAV product under conditions that result in greater purity at a physiologically acceptable pH.

[0075] In one aspect, a method for purifying adeno-associated virus (AAV) is provided. The method includes (a) loading an AAV-containing solution onto an AAV-targeting affinity resin at a conductivity that allows binding of the AAV in solution and the affinity resin; (b) performing at least two washing steps; and (c) eluting the AAV from the affinity resin.

[0076] 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 , serotype AAV12, serotype AAV13, serotype AAAV, serotype BAAV, serotype AAV (VR-195) and serotype AAV (VR-355). In some embodiments, AAV is modified by genetic engineering and/or chemically modified.

[0077] In some embodiments, the method further comprises contacting the AAV containing solution with the anion resin and eluting the AAV containing solution from the anion resin before loading the AAV containing solution onto the affinity resin. The anion exchanger can work in flow mode. In some embodiments, at least one of the washing steps includes applying a buffer containing an organic solvent or detergent to the affinity resin. In some embodiments, the buffer contains Tris-HCl and a salt, such as NaCl. In some embodiments, the buffer contains sodium acetate. In some embodiments, the buffer contains magnesium chloride. In some embodiments, the buffer contains Tris-HCl and ethylene glycol. In some embodiments, the buffer contains Arginine-HCl and one of sucrose and glycerol. In some embodiments, the implementation of the buffer contains Taurine and ethylene glycol. In some embodiments, the buffer contains Arginine-HCl, Lysine-HCl, and Histidine-HCl. In some embodiments, the buffer contains Tris-HCl and DMSO.

[0078] In some embodiments, the implementation of the buffer contains Arginine-HCl, and a salt, such as NaCl. In some embodiments, the buffer contains histidine and a salt, such as NaCl. In some embodiments, the buffer contains Tris-HCl 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 NaCl, KCl, MgCl ₂ , CaCl ₂ , sodium citrate, LiCl, CsCl, sodium acetate, and a combination of one or more of NaCl, KCl, MgCl ₂ , CaCl ₂ , sodium citrate, LiCl, CsCl and acetate sodium. In some embodiments, the salt concentration does not exceed 500 mM. In some embodiments, the salt concentration does not exceed 200 mM.

[0079] In some embodiments, at least three washing steps are performed. In some embodiments, three washing steps are performed. In some embodiments, the three washing steps are performed sequentially.

[0080] 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 contains one or more of Triton X100, Polysorbate 80, and tri(n-butyl)phosphate (TNBP). In some embodiments, the implementation of the buffer contains Bis-Tris.

[0081] In some embodiments, at least three washing steps are performed; the first washing step comprises applying to the affinity resin a first buffer containing about 50 to about 2000 mM sodium acetate and about 0.05 to about 0.2% polysorbate 80, wherein the first buffer has a pH of about 5.2 to about 6.8; the second washing step includes applying to the affinity resin a second buffer containing from about 30 to about 200 mm Tris-HCl and from about 75 to about 500 mm salt, and while the second buffer has a pH of from about 7.5 to about 9.2; and the third washing step includes applying to the affinity resin a third buffer containing from about 30 to about 200 mm Tris-HCl and from about 30 to about 75% vol. ethylene glycol, and while the third buffer has a pH of from about 7.3 to about 8.8. In some embodiments, the salt is selected from NaCl, KCl, MgCl ₂ , CaCl ₂ , sodium citrate, LiCl, CsCl, sodium acetate, and a combination of one or more of NaCl, KCl, MgCl ₂ , CaCl ₂ , sodium citrate, LiCl, CsCl and acetate sodium. In some embodiments, the salt is NaCl. In some embodiments, the salt concentration does not exceed 500 mM. In some embodiments, the salt concentration does not exceed 200 mM. In some embodiments, the salt concentration in the third buffer does not exceed 500 mM. In some embodiments, the salt concentration in the third buffer does not exceed 200 mM. In some embodiments, the method further includes a fourth wash step that occurs prior to the first wash step and includes applying to the affinity resin a fourth buffer containing about 10 to about 30 mM Tris-HCl and about 75 to about 250 mM NaCl, and at this fourth buffer has a pH of from about 6.5 to about 8.0. In some embodiments, the first buffer contains about 100 mM sodium acetate, about 0.1% polysorbate 80, and wherein the first buffer has a pH of about 6.0. In some embodiments, the implementation of the second buffer contains about 50 mm Tris-HCl and about 125 mm NaCl, and the second buffer has a pH of about 8.5. In some embodiments, the implementation of the third buffer contains approximately 50 mm Tris-HCl and approximately 50% vol. ethylene glycol, and while the third buffer has a pH of approximately 8.5.

[0082] In some embodiments, the organic solvent or detergent is polysorbate 80, ethylene glycol, sorbitol, mannitol, xylitol, sucrose, or trehalose.

[0083] In some embodiments, at least three washing steps are performed; the first washing step comprises applying to the affinity resin a first buffer containing about 50 to about 200 mM sodium acetate and about 0.05 to about 0.2% polysorbate 80, wherein the first buffer has a pH of about 5.5 to about 6.5; the second washing step comprises applying to the affinity resin a second buffer containing about 10 to about 70 mM Tris-HCl and about 75 to about 250 mM NaCl, wherein the second buffer has a pH of about 8.0 to about 9.0; and the third stage of washing includes applying to the affinity resin of the third buffer containing from about 10 to about 70 mm Tris-HCl and from about 30 to about 75% vol. ethylene glycol, and while the third buffer has a pH of from about 8.0 to about 9.0. In some embodiments, the method further includes a fourth wash step that occurs prior to the first wash step and includes applying to the affinity resin a fourth buffer containing about 10 to about 30 mM Tris-HCl and about 75 to about 250 mM NaCl, and at this fourth buffer has a pH of from about 6.5 to about 8.0. In some embodiments, the first buffer contains about 100 mM sodium acetate and about 0.1% polysorbate 80, and the first buffer has a pH of about 6.0. In some embodiments, the second buffer contains about 50 mM Tris-HCl and about 125 mM NaCl, and the second buffer has a pH of about 8.5. In some embodiments, the implementation of the third buffer contains approximately 50 mm Tris-HCl and approximately 50% vol. ethylene glycol, and while the third buffer has a pH of approximately 8.0.

[0084] In still more embodiments, at least three wash steps are performed; the first washing step includes applying to the affinity resin a first buffer containing about 50 to about 500 mM 2-(N-morpholino)ethanesulfonic acid sodium salt (MES-Na), about 3 to about 30 mM EDTA, and a solvent/detergent mixture , including polysorbate 80, DMSO and tri(n-butyl)phosphate (TNBP), and moreover, the first buffer has a pH of from about 5.2 to about 6.8; the second washing step comprises applying to the affinity resin a second buffer containing about 30 to about 200 mM Tris-HCl or Arginine-HCl and about 75 to about 500 mM salt, and wherein the second buffer has a pH of about 7.5 to about 9 ,2; and the third washing step comprises applying to the affinity resin a third buffer containing about 20 to about 80 mM Arginine-HCl and about 50 to about 60% (w/w) sucrose, and wherein the third buffer has a pH of about 7, 3 to about 8.8. In some embodiments, the salt is selected from NaCl, KCl, MgCl ₂ , CaCl ₂ , sodium citrate, LiCl, CsCl, sodium acetate, and a combination of one or more of NaCl, KCl, MgCl ₂ , CaCl ₂ , sodium citrate, LiCl, CsCl and acetate sodium. In some embodiments, the salt is NaCl. In some embodiments, the salt concentration does not exceed 500 mM. In some embodiments, the salt concentration does not exceed 200 mM. In some embodiments, the salt concentration in the third buffer does not exceed 500 mM. In some embodiments, the salt concentration in the third buffer does not exceed 200 mM. In some embodiments, the method further includes a fourth wash step that occurs prior to the first wash step and includes applying to the affinity resin a fourth buffer containing about 20 to about 100 mM Arginine-HCl and about 75 to about 250 mM NaCl, and this fourth buffer has a pH of from about 7.5 to about 8.8. In some embodiments, the first elution step comprises applying to the affinity resin a first elution buffer containing about 20 to about 100 mM Arginine-HCl and about 40 to about 60% (w/w) sucrose, and wherein the first elution buffer has pH from about 7.5 to about 8.5. In some embodiments, the method further includes a fifth washing step that takes place after the first elution step and before the second elution step, the washing step comprising applying to the affinity resin a fifth buffer containing about 20 to about 100 mM Arginine-HCl and about 75 to about 250 mm NaCl, and moreover, the fifth buffer has a pH of from about 7.5 to about 8.5. In some embodiments, the second elution step comprises applying to the affinity resin a second elution buffer containing about 20 to about 100 mM Arginine-HCl, about 40 to about 60% (w/w) glycerol, and about 500 to about 1000 mm salt (eg, NaCl) and moreover, the second elution buffer has a pH of from about 7.5 to about 8.5. The fifth wash stage can be effective in minimizing the effects of front smearing between the first and second elution steps, for example, to ensure that the elution is triggered only by the first and second elution buffers themselves, rather than the front smearing that can result from mixing the first and second elution buffers. . In some embodiments, the method further includes a sixth wash step that occurs after the fifth wash step and the second elution step, the wash step comprising applying to the affinity resin a sixth buffer containing about 20 to about 100 mM Arginine-HCl and about 75 to about 250 mm NaCl, and moreover, the fifth buffer has a pH of from about 7.5 to about 8.5.

[0085] In some embodiments, the first buffer contains about 100 mM 2-(N-morpholino)ethanesulfonic acid sodium (MES-Na), about 10 mM EDTA, and about 11 g/kg of a solvent/detergent mixture containing about 18 grams of polysorbate 80, 3.5 grams of DMSO and 3.5 grams of tri(n-butyl)phosphate (TNBP), and the first buffer has a pH of approximately 6.0. In some embodiments, the implementation of the second buffer contains about 50 mm Arginine-HCl and about 100 mm NaCl, and the second buffer has a pH of about 8.0. In some embodiments, the implementation of the third buffer contains approximately 50 mm Arginine-HCl and approximately 50% (wt./mass.) sucrose, and the third buffer has a pH of approximately 8.5. In some embodiments, the fourth buffer contains about 50 mM Arginine-HCl and about 100 mM NaCl, and the fourth buffer has a pH of about 8.0. In some embodiments, the implementation of the fifth buffer contains about 50 mm Arginine-HCl and about 100 mm NaCl, and while the fifth buffer has a pH of about 8.0. In some embodiments, the implementation of the sixth buffer contains about 50 mm Arginine-HCl and about 100 mm NaCl, and while the sixth buffer has a pH of about 8.0.

[0086] In some embodiments, at least three washing steps are performed; the first washing step includes applying to the affinity resin a first buffer containing from about 50 to about 200 mM taurine and from 0.2 to 1.5% PEG (for example, PEG 6000), and the first buffer has a pH of about 5, 2 to about 6.8; the second washing step includes applying to the affinity resin a second buffer containing from about 30 to about 300 mm glycine, and while the second buffer has a pH of from about 7.5 to about 9.2; and the third washing stage includes applying to the affinity resin a third buffer containing from about 20 to about 150 mm taurine, from about 30 to about 75% vol. ethylene glycol, and from 0.05 to 0.2% octylglycopyranoside, and while the third buffer has a pH of from about 7.3 to about 8.8. In some embodiments, the salt is selected from NaCl, KCl, MgCl ₂ , CaCl ₂ , sodium citrate, LiCl, CsCl, sodium acetate, and a combination of one or more of NaCl, KCl, MgCl ₂ , CaCl ₂ , sodium citrate, LiCl, CsCl and acetate sodium. In some embodiments, the salt is NaCl. In some embodiments, the salt concentration does not exceed 500 mM. In some embodiments, the salt concentration does not exceed 200 mM. In some embodiments, the method further includes a fourth wash step that occurs prior to the first wash step and includes applying to the affinity resin a fourth buffer containing about 20 to about 100 mM Tris-HCl and about 75 to about 250 mM NaCl, and at this fourth buffer has a pH of from about 7.5 to about 8.8.

[0087] In some embodiments, the implementation of the first stage of elution includes applying to the affinity resin the first elution buffer containing from about 30 to about 70 mm taurine, from about 50 to about 70% vol. ethylene glycol, 0.05 to 0.2% octylglycopyranoside, and about 600 to about 900 mM NaCl, wherein the first elution buffer has a pH of about 7.5 to about 8.5. In some embodiments, the method further includes a fifth washing step that takes place after the first elution step and before the second elution step, the washing step comprising applying to the affinity resin a fifth buffer containing about 20 to about 100 mM Tris-HCl and about 75 to about 250 mm NaCl, and moreover, the fifth buffer has a pH of from about 7.5 to about 8.8. The fifth washing stage can be effective in minimizing the effects of front smearing. In some embodiments, the second elution step includes applying to the affinity resin a second elution buffer containing about 30 to about 70 mM Tris-HCl, 0.05 to 0.2 M ammonium sulfate, and about 40 to about 60% vol. ethylene glycol and wherein the second elution buffer has a pH of from about 6.5 to about 7.5. The fifth wash stage can be effective in minimizing the effects of front smearing between the first and second elution steps, for example, to ensure that the elution is triggered only by the first and second elution buffers themselves, rather than the front smearing that can result from mixing the first and second elution buffers. .

[0088] In some embodiments, the method further comprises a sixth wash step that occurs after the fifth wash step and the second elution step, wherein the wash step comprises applying to the affinity resin a sixth buffer containing from about 20 to about 100 mM Tris-HCl and from about 75 to about 250 mm NaCl, and moreover, the fifth buffer has a pH of from about 7.5 to about 8.8.

[0089] In some embodiments, the implementation of the first buffer contains approximately 100 mm taurine, and approximately 0.5% PEG 6000, while the first buffer has a pH of approximately 6.0. In some embodiments, the implementation of the second buffer contains approximately 100 mm glycine, and while the second buffer has a pH of from about 7.5 to about 9.2. In some embodiments, the third buffer contains about 50 mM taurine, about 50% (w/w) ethylene glycol, and 0.1% octyl glycopyranoside, and the third buffer has a pH of about 8.5. In some embodiments, the fourth buffer contains approximately 50 mM Tris-HCl and approximately 125 mM NaCl, and wherein the fourth buffer has a pH of approximately 8.5. In some embodiments, the implementation of the fifth buffer contains about 50 mm Tris-HCl and about 125 mm NaCl, and while the fifth buffer has a pH of about 8.5. In some embodiments, the implementation of the sixth buffer contains about 50 mm Tris-HCl and about 125 mm NaCl, and while the sixth buffer has a pH of about 8.5.

[0090] In some embodiments, at least three washing steps are performed; the first washing step includes applying to the affinity resin a first buffer containing about 80 to about 400 mM Bis-Tris and about 10 to about 20 grams of a solvent/detergent mixture containing Triton-X100, Polysorbate 80 and TNBP in a ratio of about 11:3 :3 (by weight), and while the first buffer has a pH of from about 5.2 to about 6.8; the second washing step includes applying to the affinity resin a second buffer containing from about 5 to about 20 mmol sodium citrate, and while the second buffer has a pH of from about 7.5 to about 9.2; and the third washing step comprises applying to the affinity resin a third buffer containing about 50 to about 200 mM Arginine-HCl, about 50 to about 200 mM Lysine HCl, about 50 to about 200 mM Histidine-HCl, from about 1 mM to about 4 mm N-acetyl-D, L-tryptophan, and about 10% to about 40% (wt./mass.) polysorbate 80, and while the third buffer has a pH of from about 7.3 to about 8.8. In some embodiments, the salt is selected from NaCl, KCl, MgCl ₂ , CaCl ₂ , sodium citrate, LiCl, CsCl, sodium acetate, and a combination of one or more of NaCl, KCl, MgCl ₂ , CaCl ₂ , sodium citrate, LiCl, CsCl and acetate sodium. In some embodiments, the salt is NaCl. In some embodiments, the salt concentration does not exceed 500 mM. In some embodiments, the salt concentration does not exceed 200 mM. In some embodiments, the method further includes a fourth wash step that occurs prior to the first wash step and includes applying to the affinity resin a fourth buffer containing about 20 to about 100 mM Histidine and about 75 to about 250 mM NaCl, and wherein the fourth the buffer has a pH of about 7.5 to about 8.8.

[0091] In some embodiments, the first elution step comprises applying to the affinity resin a first elution buffer containing about 15% to 25% sucrose, 5% to 15% (w/w) sorbitol, 3% to 7% ( wt./mass.) mannitol, from 10% to 20% (wt./mass.) glycerol, from 40 to 60 mm histidine and from 700 to 900 mm salt (for example, NaCl), and the first elution buffer has a pH from about 7.5 to about 8.5. In some embodiments, the method further includes a fifth washing step that occurs after the first elution step and before the second elution step, the washing step comprising applying to the affinity resin a fifth buffer containing about 20 to about 100 mM Histidine and about 75 to about 250 mm NaCl, and moreover, the fifth buffer has a pH of from about 7.5 to about 8.8. In some embodiments, the second elution step comprises applying to the affinity resin a second elution buffer containing about 30 to about 70 mM Tris-HCl, about 700 to about 900 mM salt (e.g., NaCl), and 40% to 60% ( wt./mass.) DMSO and the second elution buffer has a pH of from about 7.5 to about 8.5. The fifth wash stage can be effective in minimizing the effects of front smearing between the first and second elution steps, for example, to ensure that the elution is driven only by the first and second elution buffers themselves, rather than the front smearing that can result from mixing the first and second elution buffers. . In some embodiments, the method further includes a sixth wash step that occurs after the fifth wash step and the second elution step, the wash step comprising applying to the affinity resin a sixth buffer containing about 20 to about 100 mM Histidine and about 75 to about 250 mm NaCl, and moreover, the fifth buffer has a pH of from about 7.5 to about 8.8.

[0092] In some embodiments, the first buffer contains about 200 mM Bis-Tris, about 16 to about 17 grams of a solvent/detergent mixture containing about Triton-X100, Polysorbate 80 and TNBP in a ratio of about 11:3:3 (by weight ), while the first buffer has a pH of approximately 6.0. In some embodiments, the implementation of the second buffer contains approximately 10 mmol sodium citrate, and while the second buffer has a pH of approximately 8.5. In some embodiments, the third buffer contains 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 third buffer has a pH of approximately 8.5. In some embodiments, the fourth buffer contains approximately 50 mM Histidine and approximately 100 mM NaCl, and wherein the fourth buffer has a pH of approximately 8.5. In some embodiments, the implementation of the fifth buffer contains approximately 50 mm Histidine and approximately 100 mm NaCl, and while the fifth buffer has a pH of approximately 8.5. In some embodiments, the sixth buffer contains approximately 50 mM Histidine and approximately 100 mM NaCl, and wherein the sixth buffer has a pH of approximately 8.5.

[0093] In some embodiments, at least three washing steps are performed; the first washing step comprises applying to the affinity resin a first buffer containing about 50 nM to about 200 mM Na acetate and about 0.05 to about 0.2% Polysorbate 80, wherein the first buffer has a pH of about 5.2 to about 6 ,eight; the second washing step comprises applying to the affinity resin a second buffer containing about 20 nM to about 100 mM Tris-HCl and about 50 nM to about 200 nM salt, the second buffer having a pH of about 7.5 to about 8.8 ; and the third washing step comprises applying to the affinity resin a third buffer containing about 20 mM to 100 mM Tris-HCl, about 40% to about 60% (w/w) ethylene glycol, wherein the third buffer has a pH of about 7.5 to about 8.8. In some embodiments, the salt is selected from NaCl, KCl, MgCl ₂ , CaCl ₂ , sodium citrate, LiCl, CsCl, sodium acetate, and a combination of one or more of NaCl, KCl, MgCl ₂ , CaCl ₂ , sodium citrate, LiCl, CsCl and acetate sodium. In some embodiments, the salt is NaCl. In some embodiments, the salt concentration does not exceed 500 mM. In some embodiments, the salt concentration does not exceed 200 mM. In some embodiments, the method further includes a fourth wash step that occurs prior to the first wash step and includes applying to the affinity resin a fourth buffer containing about 10 to about 50 mM Tris-HCl and about 75 to about 250 mM NaCl, and at this fourth buffer has a pH of from about 6.5 to about 8.0. In some embodiments, the method further includes a fifth wash step that occurs after the third wash step and includes applying to the affinity resin a fifth buffer containing about 20 mM to about 100 mM Tris-HCl, about 50% to about 70% (w/w) ./mass.) ethylene glycol, and from about 500 to about 900 mm NaCl, and while the fifth buffer has a pH of from about 7.5 to about 8.5. In some embodiments, the method further includes a sixth wash step, which is performed after the fifth wash step, and includes applying to the affinity resin a sixth buffer containing about 10 mM to about 50 mM Tris-HCl and about 75 to about 250 mM NaCl, and wherein the sixth buffer has a pH of about 7.0 to about 8.0.

[0094] In some embodiments, the implementation of the first buffer contains approximately 100 mm taurine, and approximately 0.5% PEG 6000, while the first buffer has a pH of approximately 6.0. In some embodiments, the implementation of the second buffer contains approximately 100 mm glycine, and while the second buffer has a pH of from about 7.5 to about 9.2. In some embodiments, the implementation of the third buffer contains approximately 50 mm taurine, approximately 50% vol. ethylene glycol, and from 0.1% octylglycopyranoside, and the third buffer has a pH of approximately 8.5. In some embodiments, the fourth buffer contains approximately 20 mM Tris-HCl and approximately 150 mM NaCl, and wherein the fourth buffer has a pH of approximately 7.4. In some embodiments, the implementation of the fifth buffer contains about 50 mm Tris-HCl, 60% (wt./mass.) ethylene glycol, and 750 mm NaCl, and while the fifth buffer has a pH of about 8.0. In some embodiments, the sixth buffer contains approximately 20 mM Tris-HCl and approximately 150 mM NaCl, and wherein the sixth buffer has a pH of approximately 7.4.

[0095] In some embodiments, at least three washing steps are performed; the first washing step comprises applying to the affinity resin a first buffer containing about 50 nM to about 200 mM Na acetate and about 0.05 to about 0.2% Polysorbate 80, wherein the first buffer has a pH of about 5.2 to about 6 ,eight; the second washing step comprises applying to the affinity resin a second buffer containing about 20 nM to about 100 mM Tris-HCl and about 50 nM to about 200 nM salt, the second buffer having a pH of about 7.5 to about 8.8 ; and the third washing step comprises applying to the affinity resin a third buffer containing about 20 mM to 100 mM Tris-HCl, about 40% to about 60% (w/w) ethylene glycol, wherein the third buffer has a pH of about 7.5 to about 8.8. In some embodiments, the salt is selected from NaCl, KCl, MgCl ₂ , CaCl ₂ , sodium citrate, LiCl, CsCl, sodium acetate, and a combination of one or more of NaCl, KCl, MgCl ₂ , CaCl ₂ , sodium citrate, LiCl, CsCl and acetate sodium. In some embodiments, the salt is NaCl. In some embodiments, the salt concentration does not exceed 500 mM. In some embodiments, the salt concentration does not exceed 200 mM. In some embodiments, the method further includes a fourth wash step that occurs prior to the first wash step and includes applying to the affinity resin a fourth buffer containing about 10 to about 50 mM Tris-HCl and about 75 to about 250 mM NaCl, and at this fourth buffer has a pH of from about 6.5 to about 8.0. In some embodiments, the method further includes a fifth wash step that occurs after the third wash step and includes applying to the affinity resin a fifth buffer containing about 20 mM to about 100 mM Tris-HCl, about 50% to about 70% (w/w) ./mass.) ethylene glycol, and from about 500 to about 900 mm NaCl, and while the fifth buffer has a pH of from about 7.5 to about 8.5. In some embodiments, the method further includes a sixth wash step, which is performed after the fifth wash step, and includes applying to the affinity resin a sixth buffer containing about 10 mM to about 50 mM Tris-HCl and about 75 to about 250 mM NaCl, and wherein the sixth buffer has a pH of about 7.0 to about 8.0.

[0096] In some embodiments, the implementation of the first buffer contains approximately 100 mm taurine, and approximately 0.5% PEG 6000, while the first buffer has a pH of approximately 6.0. In some embodiments, the implementation of the second buffer contains approximately 100 mm glycine, and while the second buffer has a pH of from about 7.5 to about 9.2. In some embodiments, the implementation of the third buffer contains approximately 50 mm taurine, approximately 50% vol. ethylene glycol, and from 0.1% octylglycopyranoside, and the third buffer has a pH of approximately 8.5. In some embodiments, the fourth buffer contains approximately 20 mM Tris-HCl and approximately 150 mM NaCl, and wherein the fourth buffer has a pH of approximately 7.4. In some embodiments, the implementation of the fifth buffer contains about 50 mm Tris-HCl, 60% (wt./mass.) ethylene glycol, and 750 mm NaCl, and while the fifth buffer has a pH of about 8.0. In some embodiments, the sixth buffer contains approximately 20 mM Tris-HCl and approximately 150 mM NaCl, and wherein the sixth buffer has a pH of approximately 7.4.

[0097] In some embodiments, at least three washing steps are performed; the first washing step comprises applying to the affinity resin a first buffer containing about 50 to about 200 mM sodium acetate and 0.05 to 0.2% Polysorbate 80, wherein the first buffer has a pH of about 5.2 to about 6 ,eight; the second washing step includes applying to the affinity resin a second buffer containing about 25 to about 100 mM Tris-HCl and about 50 to 200 mM salt, the second buffer having a pH of about 7.5 to about 9.2. In some embodiments, the salt is selected from NaCl, KCl, MgCl ₂ , CaCl ₂ , sodium citrate, LiCl, CsCl, sodium acetate, and a combination of one or more of NaCl, KCl, MgCl ₂ , CaCl ₂ , sodium citrate, LiCl, CsCl and acetate sodium. In some embodiments, the salt is NaCl. In some embodiments, the method further includes a third wash step that occurs prior to the first wash step and includes applying to the affinity resin a third buffer containing about 20 to about 100 mM Tris-HCl and about 75 to about 250 mM NaCl, and at this third buffer has a pH of from 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 containing about 25 to about 100 mM Tris-HCl, from about 500 to about 1000 mm NaCl, and from about 25% to about 75% DMSO (w/w), and the buffer has a pH of from about 7.5 to about 8.5. In some embodiments, the method further includes a fourth washing step that is performed after the first elution step and before the second elution step, the washing step comprising applying purified water to the affinity resin. In some embodiments, the implementation of the second stage of elution includes applying to the affinity resin from about 20 to about 50 mm HCl at a pH of from about 1.7 to about 2.5.

[0098] In some embodiments, the implementation of the first buffer contains approximately 100 mm sodium acetate, and approximately 0.1% Polysorbate 80, while the first buffer has a pH of approximately 6.0. In some embodiments, the second buffer contains approximately 50 mM Tris-HCl, approximately 125 mM NaCl, and wherein the second buffer has a pH of approximately 8.5. In some embodiments, the implementation of the third buffer contains about 50 mm Tris-HCl and from about 125 mm NaCl, and the third buffer has a pH of about 8.5. In some embodiments, the first elution step comprises applying purified water to the affinity resin, followed by applying 1 mM HCl at a pH of about 3.2, followed by loading the affinity resin with a buffer containing about 50 mM Tris-HCl, about 750 mM NaCl, and approximately 50% DMSO (wt./mass.), and the buffer has a pH of approximately 8.0. In some embodiments, the implementation of the second stage of elution includes applying to the affinity resin approximately 33 mm HCl at a pH of approximately 2.0.

[0099] In some embodiments, at least three washing steps are performed; the first washing step comprises applying to the affinity resin a first buffer containing about 50 to about 200 mM sodium acetate and 0.05 to 0.2% Polysorbate 80, wherein the first buffer has a pH of about 5.2 to about 6 ,eight; the second washing step includes applying to the affinity resin a second buffer containing about 25 to about 100 mM Tris-HCl and about 50 to 200 mM salt, the second buffer having a pH of about 7.5 to about 9.2. In some embodiments, the salt is selected from NaCl, KCl, MgCl ₂ , CaCl ₂ , sodium citrate, LiCl, CsCl, sodium acetate, and a combination of one or more of NaCl, KCl, MgCl ₂ , CaCl ₂ , sodium citrate, LiCl, CsCl and acetate sodium. In some embodiments, the salt is NaCl. In some embodiments, the method further includes a third wash step that occurs prior to the first wash step and includes applying to the affinity resin a third buffer containing about 20 to about 100 mM Tris-HCl and about 75 to about 250 mM NaCl, and at this third buffer has a pH of from 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 some embodiments, the gradient is 0 to 100% 33 mM hydrochloric acid/1000 mM NaCl in 1 mM hydrochloric acid. In certain embodiments, the first elution step is preceded by washing with purified water.

[00100] In some embodiments, the implementation of the first buffer contains approximately 100 mm sodium acetate, and approximately 0.1% Polysorbate 80, while the first buffer has a pH of approximately 6.0. In some embodiments, the second buffer contains approximately 50 mM Tris-HCl, approximately 125 mM NaCl, and wherein the second buffer has a pH of approximately 8.5. In some embodiments, the implementation of the third buffer contains about 50 mm Tris-HCl and from about 125 mm NaCl, and the third buffer has a pH of about 8.5. In some embodiments, the first elution step comprises applying to the affinity resin a buffer containing from about 50 mM Tris-HCl, about 750 mM NaCl, and about 50% DMSO (w/w), wherein the buffer has a pH of about 8, 0.

[00101] In some embodiments, the organic solvent or detergent is polysorbate 80, ethylene glycol, sorbitol, mannitol, xylitol, sucrose, or trehalose.

[00102] In some embodiments, the implementation of the acid component is removed. In some embodiments, the acidic component is host cell DNA, such as HEK293 DNA, and the acidic component is reduced to below 250 pg per 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 below 250 pg per microgram of AAV antigen, as measured by ELISA.

[00103] In some embodiments, elution includes contacting the affinity resin with an elution buffer containing ethylene glycol, a salt such as NaCl, and a buffer such as Tris-HCl, 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 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 Tris-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.

[00104] In some embodiments, elution includes contacting the affinity resin with an elution buffer containing 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 approximately 800 mM. In some embodiments, the buffer concentration is approximately 50 mM. In some embodiments, the sugar is sucrose and the elution buffer contains 50-60% (w/w) sucrose. In some embodiments, the implementation of the elution buffer contains 1-3 mm MgCl ₂ or approximately 2 mm MgCl ₂ . 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.

[00105] In some embodiments, elution includes contacting the affinity resin with an elution buffer containing 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 approximately 750 mM. In some embodiments, the buffer concentration is approximately 50 mM. In some embodiments, the implementation of the concentration of ethylene glycol is 50-70% (wt./mass.). In some embodiments, the concentration of ethylene glycol is at least 55% (w/w). In some embodiments, the elution buffer further comprises 0.05-0.2% octylglycopyranoside. 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.

[00106] In some embodiments, elution includes contacting the affinity resin with an elution buffer containing (i) a mixture of sucrose, sorbitol, mannitol and glycerol, (ii) a salt such as NaCl, and (iii) a buffer such as histidine, while the pH is not less than 7.8. In some embodiments, the salt concentration is approximately 800 mM. In some embodiments, the buffer concentration is approximately 50 mM. In some embodiments, the implementation of the concentration of sucrose is approximately 20% (wt./mass.). In some embodiments, the implementation of the concentration of sorbitol is approximately 10% (wt./mass.). In some embodiments, the implementation of the concentration of mannitol is approximately 5% (wt./mass.). In some embodiments, the implementation of the concentration of glycerol is approximately 15% (wt./mass.). 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.

[00107] In some embodiments, a second elution step is performed which includes contacting the affinity resin with an elution buffer containing (i) a buffer, (ii) a salt such as NaCl, and (iii) 50-60% (wt./ wt.) ethylene glycol, while the pH is not less than 8.0. In some embodiments, the salt concentration is approximately 1000 mM. In some embodiments, the buffer concentration is approximately 50 mM. In some embodiments, the concentration of ethylene glycol is at least 60% (w/w). In some embodiments, the buffer is Tris-HCl. 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.

[00108] In some embodiments, a second elution step is performed which includes contacting the affinity resin with an elution buffer containing (i) a buffer, (ii) a salt such as NaCl, and (iii) 50-60% (v/v) vol.) glycerin, while the pH is not less than 8.0. In some embodiments, the salt concentration is about 800 mM, the buffer concentration is about 50 mM, 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 step involves contacting the affinity resin with a fifth buffer containing 40-60 mM Arginine-HCl and 80-120 mM NaCl at pH 7.5 to 8.5. The fifth wash stage can be effective in minimizing the effects of front smearing between the first and second elution steps, for example, to ensure that the elution is triggered only by the first and second elution buffers themselves, rather than the front smearing that can result from mixing the first and second elution buffers. .

[00109] In some embodiments, a second elution step is performed which includes contacting the affinity resin with an elution buffer containing (i) a buffer, (ii) a salt such as (NH ₄ ) ₂ SO ₄ ), and (iii) 40 -60% (v/v) ethylene glycol, with a pH of at least 8.0. In some embodiments, the salt concentration is about 1 M, the buffer concentration is about 50 mM, and the ethylene glycol concentration is 50% (v/v). In some embodiments, the salt is (NH ₄ ) ₂ SO ₄ and the buffer is Tris-HCl. 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 step involves contacting the affinity resin with a fifth buffer containing 40-60 mM Taurine, 50-70% (w/w) ethylene glycol, 600-900 mM NaCl, and 0.05-0.2% Octylglycopyranoside, with pH 7.5 to 8.5. The fifth wash stage can be effective in minimizing the effects of front smearing between the first and second elution steps, for example, to ensure that the elution is triggered only by the first and second elution buffers themselves, rather than the front smearing that can result from mixing the first and second elution buffers. .

[00110] In some embodiments, a second elution step is carried out, which includes contacting the affinity resin with an elution buffer containing (i) a buffer, (ii) a salt such as NaCl, and (iii) 50-60% (wt./ wt.) DMSO, while the pH is not less than 8.0. In some embodiments, the salt concentration is approximately 1000 mM. In some embodiments, the buffer concentration is approximately 50 mM. In some embodiments, the implementation of the concentration of DMSO is 50% (wt./mass.). In some embodiments, the salt is NaCl and the buffer is Tris-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 DMSO-containing elution buffer is effective for eluting AAV9 but not AAV8 from the Capture Select AAV8 resin. In various embodiments, the DMSO-containing elution buffer is effective to elute AAV8 and AAV9 from the Capture Select AAVx resin. In various embodiments, the DMSO-containing elution buffer 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), but not for AAV8, from Capture Select AAV8 resin. In various embodiments, the DMSO-containing elution buffer 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) in Capture Select AAVx resin.

[00111] In various embodiments, the fifth washing step is performed after the first elution step and before the second elution step. The fifth step involves contacting the affinity resin with a fifth buffer containing 40-60 mM histidine and 70-130 mM NaCl at a pH of 8.0 to 8.8. The fifth wash stage can be effective in minimizing the effects of front smearing between the first and second elution steps, for example, to ensure that the elution is triggered only by the first and second elution buffers themselves, rather than the front smearing that can result from mixing the first and second elution buffers. .

[00112] In some embodiments, the elution includes a continuous linear increase in the conductivity of the elution buffer by gradient elution. In some embodiments, the implementation of the elution includes a continuous linear increase in the concentration of the organic solvent by gradient elution. In some embodiments, elution includes contacting the affinity resin with an elution buffer containing approximately 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 elution buffer has a pH of about 8.0. In some embodiments, the elution buffer has a pH of 8.0.

[00113] In some embodiments, the implementation of the elution includes a gradual increase in the concentration of the counterion. In some embodiments, the implementation of the elution includes a gradual increase in the concentration of the organic solvent. In some embodiments, the salt in the elution buffer is selected from monovalent, divalent, or polyvalent anions such as chloride, acetate, sulfate, and citrate. In some embodiments, the AAV obtained from the elution step has an HC impurity level of ≤ 99.9%. In some embodiments, the AAV obtained from the elution step has an HC impurity level of ≤ 99.0%.

[00114] In some embodiments, the AAV is AAV8, the affinity resin is POROS™ CaptureSelect™ AAV8, and the elution buffer is acidic and does not contain ethylene glycol. In some embodiments, the AAV is AAV9, the affinity resin is POROS™ CaptureSelect™ AAV9, and the elution buffer is acidic and does not contain ethylene glycol. In some embodiments, the AAV is AAV8 and the affinity resin is an immunoaffinity resin consisting of an immobilized ADK8 or ADK8/9 anti-AAV8 monoclonal antibody immobilized on a chromatographic array. In some embodiments, the AAV is AAV9 and the affinity resin is an immunoaffinity resin consisting of an immobilized ADK9 or ADK8/9 anti-AAV9 monoclonal antibody immobilized on a chromatographic array.

[00115] In some embodiments, the method further comprises contacting the AAV containing solution with a filter containing positively charged groups effective to remove acidic charged contaminants from the AAV containing solution. In some embodiments, the method further comprises nanofiltration of the AAV fraction to remove viruses larger than 35 nm. In some embodiments, the method further includes a polishing step comprising performing AEX chromatography on a column containing a tentacle gel. In some embodiments, the method further comprises testing the AAV fraction with an AAV-specific ELISA, such as AAV8-specific or AAV9-specific. The AAV-specific ELISA may be an AAV-specific sandwich ELISA, eg AAV8 or AAV9.

[00116] In another aspect, an AAV product made by any method described herein is provided.

[00117] AAV particles, e.g., AAV8 and AAV9 particles, are purified in the affinity step by performing many defined washing steps, e.g., applying wash buffers to the AAV-bound affinity matrix and eluting at near neutral pH conditions to maintain infectivity specified virus. In some embodiments, the anion exchange step is included before the affinity step. The anion exchanger step can be carried out in a flow mode. In some embodiments, contaminants may be depleted, which affects the cycle time of the affinity resin used in the affinity step.

[00118] The methods of this invention can result in higher purity of AAV, removal of host cell proteins, depletion of host cell DNA, removal of potential viral receptors, partial or complete inactivation of lipid-enveloped viruses where the AAV is bound to a ligand (e.g., on washing stage) and partly for the complete inactivation of lipid-coated viruses in the liquid phase (for example, in the elution stage). Without wishing to be bound by any theory, ethylene glycol alone or in combination with another additive may inactivate such lipid-enveloped viruses. Typical additives include non-ionic detergents, aliphatic agents (eg TnBP) and detergents (eg polysorbate (eg Tween), Triton X100, TnBP). For example, a solvent-detergent mixture may contain 1% Triton X100, 0.3% tri-N-butyl phosphate, and 0.3% Tween 80.

[00119] Inactivation of enveloped viruses may be of particular importance when using the Baculo transfection system. Elution according to the various embodiments described herein can prevent exposure to low pH and maintain high AAV activity. Further improvement can be seen by sequentially performing the washing steps and the elution steps in accordance with the various embodiments and examples described herein.

[00120] Inactivation of "on-column" lipid-enveloped viruses was tested in various affinity chromatographic assays in Examples 11, 12 and 13 above, as shown in Table 1 below.

Table 1: Solvent/detergent treatment used in Options A, B and C

Application step solvent/
detergent Example 11 Example 12 Example 13 Flushing 2
Possibly at the stage:
LOADING, WASHING 1,
WASH 2, WASH 3, WASH 4, ELUTE Potential processing solvent/
detergent
10 to 30 g/kg mixture
18.0g Twin 80
3.4 g DMSO,
3.6 g TnBP No Standard solvent/detergent treatment
16.6 g R/D solution
10.87 g Triton X100
3.31 g Polysorbate 80
3.01g TnBP Wash 4
Possibly at the stage:
WASH 1, WASH 2, WASH 3, WASH 4 No Elevated pH 8.5 in the presence of 50 to 60% (w/w) ethylene glycol and detergent
e.g. 0.1-10% octylglyopyranoside
Not in the eluate 1 to 20% (w/w) Polysorbate 80
Possible in eluate FLUSH x No No Polar organic solvent 50%
Dimethyl sulfoxide

[00121] Wash Buffer X containing DMSO may be effective in initiating elution of AAV9, but not AAV8, on CaptureSelect AAV8 resin at near neutral pH (eg, pH 8.0), which is an unexpected result. A DMSO-containing Wash Buffer X buffer can be effective for initiating the elution of various AAVs, 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) on CaptureSelect AAVx resin at near neutral pH (eg pH 8.0), a result that was unexpected. While not wishing to be bound by any theory, Wash X buffer is expected to have activity to wash the column and/or to inactivate or disintegrate lipid-coated viruses. WASH Buffer X was not expected to elute AAV9 differently than AAV8.

[00122] The affinity purification step includes one or more washing steps. One or more washing steps may be followed by one or more elution steps. In certain embodiments, the methods of this invention include a filtration step that occurs prior to the affinity purification steps.

[00123] After culturing host cells, such as HEK293 cells, to obtain AAV particles (eg, AAV8, AAV9, etc.) and the clarified cell-free culture supernatant is concentrated and/or filtered, the viral particles are loaded onto an affinity matrix. In certain embodiments, the viral particles are loaded into a solution having a pH in the range of about 7.4 to about 7.8. In certain embodiments, the viral particles are loaded into a solution having a pH in the range of about 8.3 to about 8.7. In certain embodiments, the viral particles are loaded into a solution having a pH of approximately 8.5. In some embodiments, the implementation of the pH is from 8.3 to 8.7, and the solution contains NaCl and Tris-HCl. In some embodiments, the viral particles are loaded into a solution containing about 20 mM Tris-HCl and about 150 mM NaCl and having a pH of about 8.5.

[00124] At least three different washing steps can be performed using the same or different buffers. In certain embodiments, the wash buffers are different.

[00125] In some embodiments, a first buffer is used in the first washing step, which may be a sodium acetate (Na acetate) buffer. In some embodiments, the first wash step uses a first buffer containing 2-(N-morpholino)ethanesulfonic acid sodium (MES-Na), EDTA, and a solvent/detergent mixture containing polysorbate 80, DMSO, and tri(n-butyl) phosphate (TNBP). In some embodiments, the first wash step uses a first buffer containing about 50 to about 200 mM taurine and 0.2 to 1.5% PEG (eg, PEG 6000). In some embodiments, the first wash step uses a first buffer containing Bis-Tris and a solvent/detergent mixture containing Triton-X100, Polysorbate 80, and TNBP. In some embodiments, the first wash step uses a first buffer containing sodium acetate and polysorbate 80.

[00126] In some embodiments, a second buffer is used in the second wash step, which can be a Tris-based buffer containing sodium chloride (NaCl), a glycine-based buffer, a sodium citrate buffer, or an Arginine-HCl-based buffer containing NaCl . In some embodiments, the third washing step uses a third buffer, which may be a Tris-based buffer containing ethylene glycol and/or NaCl, a taurine-based buffer, or an Arginine-HCl-based buffer containing NaCl. Alternatively, one or more of sorbitol, mannitol, xylitol, sucrose, or trehalose may be used in combination with or in place of ethylene glycol. In certain embodiments, an optional fourth wash step or re-equilibration step is performed prior to the three wash steps listed above. An optional fourth washing step uses a fourth buffer, which may be a Tris-based buffer containing NaCl.

[00127] In some embodiments, a pre-purification may be undertaken to remove one or more acidic protein structures and host cell DNA prior to affinity purification of the AAV-containing solution from host cell production. Pre-purification can be carried out by anion exchange in flow mode. The pre-purification step may be carried out prior to any of the affinity purification methods described herein. Prepurification of such an AAV-containing solution may remove one of the following: histones (eg, histone H2A type 1, histone H2B type 1-B, histone H4, histone H1.4), 60S ribosomal proteins (eg, 60S ribosomal protein L27, ribosomal protein 60S L6 and ribosomal protein 60S, L30) cytoplasmic actin (eg, cytoplasmic actin 1), tubulin (eg, tubulin beta-2A chain), heterogeneous nuclear ribonucleoprotein C, Rep68 protein, laminin receptor HEK293 form 37 kDa (LamR 37 kDa ) and the ATP-dependent molecular chaperone HSC82.

[00128] Washing steps can be effective in removing highly bound contaminants from the AAV and/or affinity matrix base resin. For example, the buffer may contain one or more of Tris-HCl, 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), sodium barbital (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), Trizma, HEPPSO(4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid)), POPSO (piperazine-1 ,4-bis(2-hydroxypropanesulfonic acid Dehydrate), TEA, EPPS (4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid), HEPBS (N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid) , AMPD (2-amino-2-methyl-1,3-propanediol), AMPSO (N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropa nsulfonic acid), individual amino acids, or any combination of two or more amino acids that provides the pH range and depletion rate of HEK-HCP, e.g., glycine, arginine, tryptophan, amino acid derivatives, e.g., taurine (oxidized cysteine), N-acetyl tryptophan, and glycylglycine. At the same time, the buffers used in the washing steps do not substantially elute the AAV.

[00129] In certain embodiments, the wash buffer may further contain an organic solvent or detergent. For example, the organic solvent or detergent may be Tween 80, Polysorbate 80, Triton X100, tri(n-butyl)phosphate (TNBP), ethylene glycol, sorbitol, mannitol, xylitol, sucrose, or trehalose, among others. For example, the detergent may be, but is not limited to, a non-ionic polyoxyethylene surfactant (e.g., Brij 35), 4-nonylphenyl polyethylene glycol (Arkopal N100), octylglucoside, n-dodecyl-β-D-maltoside, digitonin, 6 -cyclohexylhexyl β-D-maltoside or octylglycopyranoside. For example, ethylene glycol may be a PEG such as, but not limited to, PEG 2000, PEG4000, PEG6000 (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-butantetrol).

[00130] An organic solvent or detergent need not necessarily be present in all wash buffers used. However, an organic solvent or detergent is present in at least one of the wash buffers used. In some embodiments, the wash buffer, eg, the first wash buffer, contains both sodium acetate and Tween 80. In some embodiments, the wash buffer contains one or more of Tween 80, DMSO, and tri(n-butyl)phosphate (TNBP). In some embodiments, the wash buffer contains one or more of Triton-X100, Polysorbate 80, and TNBP. In some embodiments, the wash buffer, such as the third wash buffer, contains tris and ethylene glycol. Without wishing to be bound by any theory, the organic solvents and detergents in wash buffers are effective in removing highly bound host proteins and viral receptors, and also inactivate and/or disintegrate lipid-coated viruses.

[00131] The first buffer may contain from about 50 to about 2000 mm sodium acetate or from 50 to about 250 mm sodium acetate, and from about 0.05 to about 5% polysorbate 80, or Tween 80, or from 0.05 to about 0.2% Polysorbate 80 or Tween 80 pH 5.2 to 6.8. In some embodiments, the implementation of the first buffer may contain from about 50 to about 75 mm; from about 75 to about 100 mm; from about 90 to about 110 mm; from 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; 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; from about 950 to about 1000 mm sodium acetate; from about 1000 to about 1050 mm sodium acetate; from about 1050 to about 1100 mm sodium acetate; from about 1100 to about 1150 mm sodium acetate; from about 1150 to about 1200 mm sodium acetate; from about 1200 to about 1250 mm sodium acetate; from about 1250 to about 1300 mm sodium acetate; from about 1300 to about 1350 mm sodium acetate; from about 1350 to about 1400 mm sodium acetate; from about 1400 to about 1450 mm sodium acetate; from about 1450 to about 1500 mm sodium acetate; from about 1500 to about 1550 mm sodium acetate; from about 1550 to about 1600 mm sodium acetate; from about 1600 to about 1650 mm sodium acetate; from about 1650 to about 1700 mm sodium acetate; from about 1700 to about 1750 mm sodium acetate; from about 1750 to about 1800 mm sodium acetate; from about 1800 to about 1850 mm sodium acetate; from about 1850 to about 1900 mm sodium acetate; from about 1900 to about 1950 mm sodium acetate; or from about 1950 to about 2000 mm sodium acetate.

[00132] In some embodiments, the first buffer may contain about 50 to about 500 mM 2-(N-morpholino)ethanesulfonic acid sodium (MES-Na), about 3 to about 30 mM EDTA, and a solvent/detergent mixture, containing polysorbate 80, DMSO and tri(n-butyl)phosphate (TNBP). In some embodiments, the implementation of the first buffer may contain from about 50 to about 75 mm; from about 75 to about 100 mm; from about 90 to about 110 mm; from about 100 to about 125 mm; from about 125 to about 150 mm; from about 150 to about 175 mm; from about 175 to about 200 mm; from about 200 to about 250 mm; from about 250 to about 300 mm; from about 300 to about 350 mm; from about 350 to about 400 mm; from about 400 to about 450 mm; or about 450 to about 500 mM MES-Na sodium salt. In some embodiments, the implementation of the first buffer may contain approximately 50; approximately 75; approximately 90 mm; approximately 100 mm; approximately 125 mm; approximately 150 mm; approximately 175 mm; approximately 200 mm; approximately 250 mm; approximately 300 mm; approximately 350 mm; approximately 400 mm; approximately 450 mm; or approximately 500 mm sodium salt MES-Na.

[00133] In some embodiments, the implementation of the first buffer may contain from about 50 to about 200 mm taurine. In some embodiments, the implementation of the first buffer may contain from about 50 to about 75 mm; from about 75 to about 100 mm; from about 90 to about 110 mm; from about 100 to about 125 mm; from about 125 to about 150 mm; from about 150 to about 175 mm; about 175 to about 200 mM taurine. In some embodiments, the implementation of the first buffer may contain approximately 50; approximately 75; approximately 90 mm; approximately 100 mm; approximately 125 mm; approximately 150 mm; approximately 175 mm; approximately 200 mm taurine.

[00134] In some embodiments, the implementation of the first buffer may contain from about 80 to about 400 mm Bis-Tris. In some embodiments, the implementation of the first buffer may contain from about 80 to about 100 mm; from about 90 to about 110 mm; from about 100 to about 125 mm; from about 125 to about 150 mm; from about 150 to about 175 mm; from about 175 to about 200 mm; from about 200 to about 250 mm; from about 250 to about 300 mm; from about 300 to about 350 mm; from about 350 to about 400 mm Bis-Tris. In some embodiments, the implementation of the first buffer may contain approximately 50; approximately 75; approximately 90 mm; approximately 100 mm; approximately 125 mm; approximately 150 mm; approximately 175 mm; approximately 200 mm; approximately 250 mm; approximately 300 mm; approximately 350 mm; approximately 400 mm Bis-Tris.

[00135] In some embodiments, the implementation of the first buffer may contain from about 50 to about 200 mm sodium acetate. In some embodiments, the implementation of the first buffer may contain from about 50 to about 80 mm; from about 70 to about 100 mm; about 80 to about 110 mM; from about 90 to about 120 mm; from about 100 to about 130 mm; from about 120 to about 150 mm; from about 140 to about 170 mm; from about 170 to about 200 mm sodium acetate. In some embodiments, the implementation of the first buffer may contain approximately 60; approximately 70 mm; approximately 80 mm; approximately 90 mm; approximately 100 mm; approximately 110 mm; approximately 120 mm; approximately 130 mm; approximately 140 mm; approximately 150 mm; or approximately 160 mm sodium acetate.

[00136] In some embodiments, the first buffer may contain 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, approximately 200 mM sodium acetate, approximately 225 mM sodium acetate, approximately 250 mM sodium acetate, approximately 275 mM sodium acetate, approximately 300 mM sodium acetate, approximately 325 mM sodium acetate, approximately 350 mM sodium acetate, approximately 375 mM sodium acetate , approximately 400 mM sodium acetate, approximately 425 mM sodium acetate, approximately 450 mM sodium acetate, approximately 475 mM sodium acetate, approximately 500 mM sodium acetate, approximately 525 mM sodium acetate, approximately 550 mM sodium acetate, approximately 575 mM sodium acetate, approximately 600 mM sodium acetate, approximately 625 mM sodium acetate, approximately 650 mM sodium acetate, approximately 675 mM sodium acetate, approximately 700 mM sodium acetate, approximately 725 mM sodium acetate, approximately 750 mM sodium acetate, approximately 775 mM sodium acetate, approximately 800 mM sodium acetate, approximately 825 mM acetate sodium, approximately 850 mM sodium acetate, approximately 875 mM sodium acetate, approximately 900 mM sodium acetate, approximately 925 mM sodium acetate, approximately 950 mM sodium acetate, approximately 975 mM sodium acetate, approximately 1000 mM sodium acetate, approximately 1025 mM sodium acetate, approximately 1050 mM sodium acetate, approximately 1075 mM sodium acetate, approximately 1100 mM sodium acetate, approximately 1125 mM sodium acetate, approximately 1150 mM sodium acetate, approximately 1175 mM sodium acetate, approximately 1200 mM sodium acetate, approximately 1225 mM sodium acetate, approximately 1250 mM sodium acetate, approximately 1275 mM sodium acetate, approximately 1300 mM sodium acetate, approximately 1325 mM sodium acetate, approximately 1350 mM sodium acetate, approximately 1375 mM sodium acetate, approximately 1400 mM sodium acetate, approximately 1425 mM sodium acetate, approximately 1450 mM sodium acetate, approximately 1475 mM acetate sodium, approximately 1500 mM sodium acetate, approximately 1525 mM sodium acetate, approximately 1550 mM sodium acetate, approximately 1575 mM sodium acetate, approximately 1600 mM sodium acetate, approximately 1625 mM sodium acetate, approximately 1650 mM sodium acetate, approximately 1675 mM sodium acetate, approximately 1700 mM sodium acetate, approximately 1725 mM sodium acetate, approximately 1750 mM sodium acetate, approximately 1775 mM sodium acetate, approximately 1800 mM sodium acetate, approximately 1825 mM sodium acetate, approximately 1850 mM sodium acetate, approximately 1875 mM sodium acetate, approximately 1900 mM sodium acetate, approx. about 1925 mM sodium acetate, approximately 1950 mM sodium acetate, approximately 1975 mM sodium acetate, or approximately 2000 mM sodium acetate. In some embodiments, the implementation of the first buffer may contain approximately 100 mm or 100 mm sodium acetate.

[00137] In some embodiments, the implementation of the first buffer may contain from about 0.05 to about 0.08%; from about 0.08 to about 0.11%; from about 0.11 to about 0.14%; from about 0.14 to about 0.17%; or about 0.17 to about 0.20% polysorbate 80. In some embodiments, the first buffer may contain about 0.1% or 0.1% polysorbate 80. In some embodiments, the first buffer may contain from about 0.05 to about 0.08%; from about 0.08 to about 0.11%; from about 0.11 to about 0.14%; from about 0.14 to about 0.17%; or about 0.17 to about 0.20% Tween 80. In some embodiments, the first buffer may contain about 0.1% Tween 80. In some embodiments, the pH may be from about 5.2 to about 5.5; from about 5.5 to about 5.8; from about 5.8 to about 6.1; from about 6.1 to about 6.4; or from about 6.4 to about 6.8. In some embodiments, the first buffer has a pH of about 6.0 or 6.0.

[00138] In some embodiments, the implementation of the first buffer contains a chelating agent, such as EDTA.

[00139] The second buffer may contain from about 30 to about 200 mm Tris-HCl or from 30 to about 80 mm Tris-HCl and salt, and the second buffer has a pH of from about 7.5 to about 9.2. In some embodiments, the salt is NaCl, KCl, MgCl ₂ , CaCl ₂ , sodium citrate, LiCl, CsCl, sodium acetate, and combinations of one or more of NaCl, KCl, MgCl ₂ , CaCl ₂ , sodium citrate, LiCl, CsCl, and acetate sodium. In some embodiments, the salt is NaCl. The salt concentration, eg NaCl, may be from about 75 to about 500 mM. In some embodiments, the salt concentration, such as the NaCl concentration, is from about 75 to about 200 mM. In some embodiments, the implementation of the salt concentration, for example, the concentration of NaCl, does not exceed 500 mm. In some embodiments, the implementation of the salt concentration, for example, the concentration of NaCl, does not exceed 200 mm. Without wishing to be bound by any theory, a salt concentration not exceeding 500 mM or not exceeding 200 mM may prevent AAV from being eluted in the wash step because the conductivity of the saline solution is too low to cause elution.

[00140] The second buffer may contain from about 30 to about 35 mm; from about 35 to about 40 mm; from about 40 to about 45 mm; about 45 to about 50 mM; from about 50 to about 55 mm; from about 55 to about 60 mm; from about 60 to about 65 mm; about 65 to about 70 mM; from about 70 to about 75 mm; or from about 75 to about 80 mm Tris-HCl. In some embodiments, the implementation of the second buffer may contain approximately 50 mm or 50 mm Tris-HCl. In some embodiments, the implementation of the second buffer may contain from about 75 to about 100 mm; from about 100 to about 125 mm; from about 125 to about 150 mm; from about 150 to about 175 mm; from about 175 to about 200 mm; from about 200 to about 225 mm; or from about 225 to about 250 mm NaCl. In some embodiments, the implementation of the second buffer may contain approximately 150 mm or 150 mm NaCl. In some embodiments, the pH of the second buffer may be from about 7.5 to about 7.7; from about 7.7 to about 7.9; from about 7.9 to about 8.1; from about 8.1 to about 8.3; from about 8.3 to about 8.5; from about 8.5 to about 8.7; from 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 8.5.

[00141] The second buffer may contain from about 30 to about 35 mm; from about 35 to about 40 mm; from about 40 to about 45 mm; about 45 to about 50 mM; from about 50 to about 55 mm; from about 55 to about 60 mm; from about 60 to about 65 mm; about 65 to about 70 mM; from about 70 to about 75 mm; or from about 75 to about 80 mm Arginine-HCl. In some embodiments, the implementation of the second buffer may contain approximately 50 mm or 50 mm Arginine-HCl. In some embodiments, the implementation of the second buffer may contain from about 75 to about 100 mm; from about 100 to about 125 mm; from about 125 to about 150 mm; from about 150 to about 175 mm; from about 175 to about 200 mm; from about 200 to about 225 mm; or from about 225 to about 250 mm NaCl. In some embodiments, the implementation of the second buffer may contain approximately 150 mm or 150 mm NaCl. In some embodiments, the pH of the second buffer may be from about 7.5 to about 7.7; from about 7.7 to about 7.9; from about 7.9 to about 8.1; from about 8.1 to about 8.3; from about 8.3 to about 8.5; from about 8.5 to about 8.7; from 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 8.5.

[00142] The second buffer may contain from about 50 to about 200 mm glycine. In some embodiments, the implementation of the second buffer may contain from about 50 to about 100 mm; from about 70 to about 120 mm; from about 100 to about 150 mm; from about 120 to about 170 mm; from about 150 to about 200 mm glycine. In some embodiments, the pH of the second buffer may be from about 7.5 to about 7.7; from about 7.7 to about 7.9; from about 7.9 to about 8.1; from about 8.1 to about 8.3; from about 8.3 to about 8.5; from about 8.5 to about 8.7; from 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 8.5.

[00143] The second buffer may contain from about 50 to about 20 mm sodium citrate. In some embodiments, the implementation of the second buffer may contain from 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 some embodiments, the pH of the second buffer may be from about 7.5 to about 7.7; from about 7.7 to about 7.9; from about 7.9 to about 8.1; from about 8.1 to about 8.3; from about 8.3 to about 8.5; from about 8.5 to about 8.7; from 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 8.5.

[00144] In some embodiments, the implementation of the second buffer contains a chelating agent, such as EDTA.

[00145] The third buffer may contain from about 30 to about 200 mm Tris-HCl and from about 30 to about 75% vol. ethylene glycol, pH from about 7.5 to about 9.2. The third buffer may contain from about 20 to about 80 mm Arginine-HCl and from about 50 to about 200 mm salt, with a pH of from about 7.3 to about 8.8. The third buffer may contain approximately 50 mm Tris-HCl and approximately 50% vol. ethylene glycol, with a pH of approximately 8.5. The third buffer may contain from about 20 to about 150 mm taurine, from about 30 to about 75% vol. ethylene glycol and 0.05 to 0.2% octyl glycopyranoside, with a pH of about 7.3 to about 8.8. The third buffer may contain 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 from about 10% to about 40% (wt./mass.) polysorbate 80, with a pH of from about 7.3 to about 8.8. If a salt, such as NaCl, is present in the third buffer, in some embodiments the salt concentration does not exceed 500 mM, and in some embodiments the salt concentration does not exceed 200 mM. In some embodiments, the salt is NaCl, KCl, MgCl ₂ , CaCl ₂ , sodium citrate, LiCl, CsCl, sodium acetate, and combinations of one or more of NaCl, KCl, MgCl ₂ , CaCl ₂ , sodium citrate, LiCl, CsCl, and acetate sodium. In certain embodiments, the salt is NaCl. In some embodiments, one or more of sorbitol, mannitol, xylitol, sucrose, or trehalose may be used in combination with or in place of ethylene glycol.

[00146] Without wishing to be bound by theory, the degree of AAV elution is affected by both the amount of ethylene glycol and the conductivity of the salt in the third buffer. The amount of at least 55% (w/w) ethylene glycol in buffer can significantly increase the amount of elution compared to 50% (w/w) ethylene glycol. Accordingly, at a given concentration of ethylene glycol, an increased concentration of NaCl can increase the extent and rate of elution. At a given concentration of ethylene glycol, replacing NaCl with a polyvalent salt can also increase the extent and rate of elution.

[00147] Without wishing to be bound by theory, if the salt content is constant, eg 750 mM NaCl, then increasing the amount of ethylene glycol can increase the eluting strength of the buffer. If the ethylene glycol content is constant, eg 55%, then increasing the amount of salt can increase the eluting strength of the buffer. Thus, the eluting strength increases from 40% to 45%, from 50% to 55% to 60% (w/w) of ethylene glycol in 750 mM NaCl.

[00148] Increasing the content of ethylene glycol in a solution with a constant salt content can reduce the conductivity. An increased amount of ethylene glycol can reduce the solubility of the salt in the buffer.

[00149] In some embodiments, the implementation of the third buffer may contain from about 30 to about 35 mm; from about 35 to about 40 mm; from about 40 to about 45 mm; about 45 to about 50 mM; from about 50 to about 55 mm; from about 55 to about 60 mm; from about 60 to about 65 mm; about 65 to about 70 mM; from about 70 to about 75 mm; from about 75 to about 80 mm; about 80 to about 90 mM; about 90 to about 100 mM; from about 100 to about 110 mm; from about 110 to about 120 mm; from about 120 to about 130 mm; from about 130 to about 140 mm; from about 140 to about 150 mm; from about 150 to about 160 mm; from about 160 to about 170 mm; from about 170 to about 180 mm; from about 180 to about 190 mm; or from about 190 to about 200 mm Tris-HCl. In some embodiments, the implementation of the third buffer may contain from about 50 mm or 50 mm Tris-HCl. In some embodiments, the implementation of the third buffer may contain from about 30 to about 35% vol.; from 35 to about 40% vol.; from about 40 to about 45% vol.; from about 45 to about 50% vol.; from about 48 to about 52% vol.; from about 50 to about 55% vol.; from about 55 to about 60% vol.; from about 60 to about 65% vol.; from about 65 to about 70% vol.; or from about 70 to about 75% vol. ethylene glycol. In some embodiments, the implementation of the third buffer may contain from about 50% or 50% ethylene glycol. In some embodiments, the pH of the third buffer may be from about 7.5 to about 7.7; from about 7.7 to about 7.9; from about 7.9 to about 8.1; from about 8.1 to about 8.3; from about 8.3 to about 8.5; about 8.5 to about 8.7; from about 8.7 to about 8.9; or from about 8.9 to about 9.2. In certain embodiments, the pH of the third buffer may be approximately 8.5 or 8.5.

[00150] In some embodiments, the implementation of the third buffer contains a chelating agent, such as EDTA.

[00151] The fourth buffer may contain from about 10 to about 30 mm Tris-HCl and from about 75 to about 250 mm NaCl, with a pH of from about 6.5 to about 8.0. In some embodiments, the implementation of the fourth buffer may contain from about 10 to about 15 mm; about 15 to about 20 mM; about 20 to about 25 mM; or from about 25 to about 30 mm Tris-HCl. In some embodiments, the implementation of the fourth buffer may contain approximately 20 mm or 20 mm Tris-HCl. In some embodiments, the implementation of the fourth buffer may contain from about 75 to about 100 mm; from about 100 to about 125 mm; from about 125 to about 150 mm; from about 150 to about 175 mm; from about 175 to about 200 mm; from about 200 to about 225 mm NaCl; or from about 225 to about 250 mm NaCl. In some embodiments, the implementation of the fourth buffer may contain approximately 150 mm or 150 mm NaCl. In some embodiments, the implementation of the fourth buffer may have a pH of from about 6.5 to about 6.9; from about 6.8 to about 7.2; from about 7.1 to about 7.5; from about 7.4 to about 7.9; or from about 7.6 to about 8.0. In some embodiments, the fourth buffer may have a pH of about 7.4 or 7.4.

[00152] The fourth buffer may contain from about 20 to about 100 mm Histidine and from about 75 to about 250 mm NaCl, with a pH of from about 7.5 to about 8.8. In some embodiments, the implementation of the fourth buffer may contain from about 20 to about 40 mm; from about 40 to about 60 mm; from about 60 to about 75 mm; or from about 75 to about 100 mm Histidine. In some embodiments, the implementation of the fourth buffer may contain approximately 20 mm or 20 mm Histidine. In some embodiments, the implementation of the fourth buffer may contain from about 75 to about 100 mm; from about 100 to about 125 mm; from about 125 to about 150 mm; from about 150 to about 175 mm; from about 175 to about 200 mm; from about 200 to about 225 mm NaCl; or from about 225 to about 250 mm NaCl. In some embodiments, the implementation of the fourth buffer may contain approximately 150 mm or 150 mm NaCl. In some embodiments, the implementation of the fourth buffer may have a pH of from about 7.5 to about 7.9; from about 7.8 to about 8.2; about 8.1 to about 8.5; about 8.4 to about 8.9; or from about 8.6 to about 9.0. In some embodiments, the fourth buffer may have a pH of about 8.0 or 8.0.

[00153] In some embodiments, the implementation of the fourth buffer contains a chelating agent, such as EDTA. In some embodiments, an additional wash may be performed with a buffer containing a chelating agent, such as EDTA.

[00154] After the washing steps, the AAV particles are eluted using an elution buffer. The elution buffer may contain from about 30 to about 200 mm buffer, from about 30 to about 75% vol. ethylene glycol and from about 500 mm to about 2000 mm salt, while the elution buffer has a pH of from 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 Tris-HCl, Glycine, Citrate, Arginine, Phosphate-Glycine-HCl, Ammonium Sulfate, Magnesium Chloride, Borate, Bis-Tris, MOPS, Bicine, Tricine, TAPS, TAPSO, MES, PIPES, TES (2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid), sodium barbital (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), Trizma, HEPPSO (4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid)), POPSO (piperazine-1,4-bis(2-hydroxypropanesulfonic acid) dehydrate), TEA, EPPS (4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid), HEPBS (N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid), AMPD (2-amino-2- methyl-1,3-propanediol), AMPSO (N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid), from specific amino acids, or any combination of two or more amino acids to provide pH and elute AAV, eg, glycine, arginine, tryptophan, amino acid derivatives, eg, taurine (oxidized cysteine), N-acetyl tryptophan, and glycylglycine. In some embodiments, the buffer is Tris-HCl.

[00155] In some embodiments, the salt is NaCl, KCl, CaCl ₂ , CsCl, LiCl, CaCl ₂ , sodium citrate, LiCl, CsCl, or a combination of one or more of KCl, CaCl ₂ , CsCl, LiCl, CaCl ₂ , sodium citrate , LiCl and CsCl. In some embodiments, the salt is NaCl. The salt concentration, eg NaCl, may be from about 500 to about 2000 mM. In some embodiments, the salt concentration is from about 500 to about 900 mM, about 750 mM NaCl, or 750 mM NaCl. In some embodiments, when a NaCl concentration gradient is used, the target concentration is 2000 mM.

[00156] In some embodiments, one or more of sorbitol, mannitol, xylitol, sucrose, trehalose, glycerol (1,2,3-propanetriol), or erythritol (meso-1,2,3,4-butantetrol) may be used in combination with ethylene glycol or instead of ethylene glycol. In some embodiments, the implementation of the elution buffer may contain from about 30 to about 35 mm; from about 35 to about 40 mm; from about 40 to about 45 mm; about 45 to about 50 mM; from about 50 to about 55 mm; from about 55 to about 60 mm; from about 60 to about 65 mm; about 65 to about 70 mM; from about 70 to about 75 mm; or from about 75 to about 80 mm Tris-HCl. In some embodiments, the implementation of the elution buffer may contain approximately 50 mm or 50 mm Tris-HCl. In some embodiments, the implementation of the elution buffer may contain from about 30 to about 35% vol.; from about 35 to about 40% vol.; from about 40 to about 45% vol.; from about 45 to about 50% vol.; from about 48 to about 52% vol.; from about 50 to about 55% vol.; from about 55 to about 60% vol.; from about 60 to about 65% vol. about 65 to about 70% vol.; or from about 70 to about 75% vol. ethylene glycol. In some embodiments, the implementation of the elution buffer may contain approximately 50% or 50% ethylene glycol. In some embodiments, the concentration of ethylene glycol is at least 55% (w/w). In some embodiments, the ethylene glycol concentration is at least 56% (w/w). In some embodiments, the concentration of ethylene glycol is at least 57% (w/w). In some embodiments, the concentration of ethylene glycol is at least 58% (w/w). In some embodiments, the implementation of the elution buffer may contain from about 500 to about 700 mm; from about 550 to about 750 mm; from about 600 to about 800 mm; from about 650 to about 850 mm; from about 700 to about 900 mm; from about 750 to about 950 mm; from about 800 to about 1000 mm NaCl; from about 900 to about 1100 mm NaCl; from about 1000 to about 1200 mm NaCl; from about 1100 to about 1300 mm NaCl; from about 1200 to about 1400 mm NaCl; from about 1300 to about 1500 mm NaCl; from about 1400 to about 1600 mm NaCl; from about 1500 to about 1700 mm NaCl; from about 1600 to about 1800 mm NaCl; from about 1700 to about 1900 mm NaCl; from about 1800 to about 2000 mm NaCl. In some embodiments, the implementation of the elution buffer may contain approximately 750 mm or 750 mm NaCl. The pH may be from 7.3 to 7.6. The pH may be from 7.5 to 7.8. The pH may be from 7.7 to 8.0. The pH may be from 7.9 to 8.2. The pH may be from 8.1 to 8.4. The pH may be from 8.3 to 8.6. The pH may be from 8.5 to 8.8.

[00157] In some embodiments, the elution is carried out using an elution buffer containing an organic solvent. For example, the organic solvent may contain one or more of a polyol (eg, ethylene glycol, sorbitol, mannitol, xylitol), glycerol, sucrose, trehalose, or a combination of polyols. The buffer may have a pH range of 7.5 to 8.5. Without wishing to be bound by any theory, the organic solvent can inactivate lipid-enveloped viruses that can be produced if, for example, AAV production involves baculovirus transfection of Sf9 insect cells. The affinity eluate may contain an organic solvent (eg a polyol such as ethylene glycol) which can be used in a controlled density gradient in a subsequent ultracentrifugation step.

[00158] Any organic solvent in wash buffers or elution buffers may be able to disintegrate or inactivate lipid-enveloped viruses. Such inactivation can occur through a combination of column inactivation and liquid state inactivation. In some embodiments, the organic solvent in the elution buffer and/or wash buffer has a concentration of from about 50% (w/w) to about 80% (w/w) to disintegrate or inactivate viruses in the lipid envelope. In some embodiments, the implementation of the concentration is approximately 50% (wt./mass.). In some embodiments, the implementation of the concentration is approximately 55% (wt./mass.). In some embodiments, the implementation of the concentration of ethylene glycol is approximately 56% (wt./mass.). In some embodiments, the implementation of the concentration of ethylene glycol is approximately 57% (wt./mass.). In some embodiments, the implementation of the concentration of ethylene glycol is approximately 58% (wt./mass.). In some embodiments, the implementation of the concentration of ethylene glycol is approximately 59% (wt./mass.). In some embodiments, the implementation of the concentration is approximately 60% (wt./mass.). In some embodiments, the implementation of the concentration is approximately 65% (wt./mass.). In some embodiments, the implementation of the concentration is approximately 70% (wt./mass.). In some embodiments, the implementation of the concentration is approximately 75% (wt./mass.). In some embodiments, the organic solvent in the elution buffer and/or wash buffer has a concentration of from about 40% (w/w) to ensure disintegration or inactivation of viruses in the lipid envelope. In some embodiments, the organic solvent in the elution buffer and/or wash buffer has a concentration of from about 30% (w/w) to ensure disintegration or inactivation of viruses in the lipid envelope. In some embodiments, the organic solvent in the elution buffer or wash buffer may be inactive or destroy the baculovirus.

[00159] In some embodiments, the first buffer has a pH of about 5.8 to about 6.2 and contains about 90 to about 110 mM sodium acetate and about 0.09 to about 0.11% polysorbate 80/Tween 80, the second buffer has a pH of about 8.2 to 8.8 and contains about 45 to about 55 mM Tris-HCl and about 110 to about 135 mM NaCl, the third buffer has a pH of about 8.2 to about 8.8 and contains from about 45 to about 55 mm Tris-HCl, and from about 45 to about 55% ethylene glycol, an optional fourth buffer has a pH of from about 7.2 to about 7.6 and contains from about 15 to about 25 mm Tris-HCl , and from about 135 to about 165 mm NaCl. In some embodiments, the elution buffer has a pH of about 7.8 to about 8.2 and contains about 45 to about 55 mM Tris-HCl, about 45 to about 55% ethylene glycol, and about 650 to about 850 mM NaCl. Various volumes may be used, such as from about 2 column volumes to about 15 column volumes, from about 3 column volumes to about 7 column volumes, from about 4 column volumes to about 8 column volumes, from about 5 column volumes to about 10 column volumes. column or from about 7 column volumes to about 12 column volumes. Approximately 5 column volumes or 5 column volumes of each of the first, second, third, fourth and elution buffer may be used. Alternatively, approximately 10 column volumes or 10 column volumes of each of the first, second, third, fourth and elution buffer may be used. For example, 10 column volumes can be used when the column volume is from about 2 ml to about 3 ml. Increasing the duration of the washing steps can be further taken to improve the purity of the AAV.

[00160] In some embodiments, the first buffer has a pH of about 7.2 to about 7.6 and contains about 15 to about 25 mM Tris-HCl and about 135 to about 165 mM NaCl, the second buffer has a pH of about 5 8 to about 6.2 and contains from about 90 to about 110 mM sodium acetate and from about 0.09 to about 0.11% polysorbate 80, the third buffer has a pH of from about 8.2 to about 8.8 and contains from about 45 to about 55 mM Tris-HCl and about 110 to about 135 mM NaCl, the fourth buffer has a pH of 7.5 to 8.5 and contains 45 to 55 mM Tris-HCl and 45 to 55% ethylene glycol, and the elution buffer has a pH of 7.8 to 8.2 and contains about 45 to about 55 mM Tris-HCl, about 45 to about 55% ethylene glycol, and about 650 to about 850 mM NaCl. Approximately 10 column volumes or 10 column volumes of each wash buffer can be used.

[00161] In some embodiments, the first buffer has a pH of about 7.2 to about 7.6 and contains about 15 to about 25 mM Tris-HCl and about 135 to about 165 mM NaCl, the second buffer has a pH of about 5 .8 to about 6.2 and contains from about 90 to about 110 mM sodium acetate, and from about 0.09 to about 0.11% polysorbate 80, the third buffer has a pH of from about 8.2 to about 8.8 and contains about 45 to about 55 mM Tris-HCl, and from about 110 to about 135 mM NaCl, the fourth buffer has a pH of about 7.5 to about 8.5 and contains from about 45 to about 55 mM Tris-HCl, and from about 45 to about 55% ethylene glycol. The elution is carried out with a gradient starting with the first elution buffer at a pH of from about 7.8 to about 8.2, and includes from about 45 to about 55 mm Tris-HCl, from about 45 to about 55% ethylene glycol and from about 650 to about 850 mM NaCl and ending with a second elution buffer at a pH of about 7.8 to about 8.2 and containing about 45 to about 55 mM Tris-HCl, about 45 to about 55% ethylene glycol, and about 1900 to about 2100 mM NaCl. Approximately 10 column volumes or 10 column volumes of each wash buffer can be used. The elution can be carried out with 10 column volumes of the gradient.

[00162] In certain embodiments, the solution containing the AAV particles is subjected to ion exchange chromatography. In certain embodiments, the ion exchange is an anion exchange. In some embodiments, anion exchange chromatography can remove not only residual contaminant particles from the cell culture, but also acidic impurities and viral particles. In some embodiments, anion exchange chromatography can remove proteases and/or host cell DNA. In some embodiments, the implementation of ion exchange chromatography can be carried out with membrane separation, for example, hybrid membrane-anion exchange chromatography. In some embodiments, AEX purification chromatography is used, for example, in flow mode or in binding/elution mode. In some embodiments, anion exchange may be, but is not limited to, Mustang®Q, STREAMLINE Q XL™, POROS 50 PI™, Q SEPHAROSE™, Emphase™ AEX Hybrid Purifier, Nuvia Q, POROS 50 HQ, Capto Q, Capto Q impress, Unosphere Q, Q Ceramic HYPERD® F, TOYOPEARL® Q, TOYOPEARL® Super Q, mixed mode AEX resins (such as Capto Adhere, Capto Adhere Impress and MEP Hypercell) and any DEAE, TMAE, tertiary or quaternary amine, or resins based on PEI. In some embodiments, the anion exchange is Mustang® Q.

[00163] In some embodiments, DNA from HEK293 cells or any host cell used is not treated with benzonase and/or DNase. The anion exchange and washing steps described herein can be effective in removing such DNA so that there is no need to treat with benzonase or DNase.

[00164] AAV is typically recovered from the flow through anion exchange step (depending on pH). Alternatively, AAV can be used in a coupling/elution process. To exclude impurities in host cells that elute at higher conductivity (constant pH) or lower pH (constant conductivity) than AAV, either the flow method or the coupling/elution method should be used. When operating in the binding/elution mode, the pH and buffer composition are effective in eluting the product, but not the acidic contaminants of the host cell. Without wishing to be bound by any theory, each binding and elution step may cause forces in the microenvironment such that the flow-through method under non-binding conditions may cause less AAV damage or destruction than the binding/elution method.

[00165] In various embodiments, the yield of AAV, e.g., AAV8 and AAV9, after the purification steps described herein and measured by w/v ITR-qPCR analysis is at least 10%, 15%, 20% , 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% more than the comparative procedure in which no washing steps are performed.

[00166] In various embodiments, the yield of AAV, e.g., AAV8 and AAV9, after the purification steps described herein and measured by ITR-qPCR w/w analysis is at least 10%, 15%, 20% , 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% more than the comparative procedure in which no washing steps are performed.

[00167] In certain embodiments, the methods for producing and purifying AAV described herein reduce protein contaminants by approximately 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% , 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% more than that obtained by the comparative procedure without the same washing protocol. Surprisingly, the methods for producing and purifying AAV described herein have been found to reduce the amount of protein contaminants by at least 25%. It has surprisingly been found that the methods for preparing and purifying AAV described herein reduce the amount of protein contaminants by at least 75% when a solution of AAV particles is subjected to anion exchange chromatography prior to loading onto an affinity matrix. For example, the data in Table 7 of Example 2 shows that the amount of protein contaminants decreases from 20 to 14 (no pre-anion exchange) and from 20 to 4 (pre-anion exchange).

[00168] In certain embodiments, the methods for preparing and purifying AAV described herein provide for a purity of AAV, such as AAV8 and AAV9, that is 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%, if anion exchange and affinity purification with washing steps as provided herein. In certain embodiments, the methods for producing and purifying AAV described herein provide a purity of AAV, such as AAV8 and AAV9, that is 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 is carried out using washing steps as provided herein.

[00169] Advantageously, the methods can be scaled up to large volumes of starting material, such as cell culture. In certain embodiments, the methods provided herein are large scale methods capable of purifying AAV from volumes of at least or about 500 L, at least or about 600 L, at least or about 700 L, at least or about 800 liters, at least or about 900 liters, or at least or about 1000 liters. In some embodiments, the methods are scaled up to a minimum starting material (e.g., cell culture) volume of at least or about 1250 L, at least or about 1500 L, at least or about 2000 L, at least or about 2500 L, at at least or about 3000 liters, at least or about 4000 liters, at least or about 5000 liters, at least or about 6000 liters, at least or about 7000 liters, at least or about 8000 liters, at least or about 9000 liters, at least or about 10000 liters or more. For example, the methods are performed with a minimum volume of about 1000 L or about 10000 L or 25000 L or more of AAV producing cell culture.

[00170] The methods for producing and purifying AAV described herein are also preferred because the methods result in high titer AAV. In some embodiments, an AAV product containing at least about 10 ¹⁰ viral particles (VP) is obtained from about 1000 L of starting material (eg, cell culture). In some embodiments, an AAV product containing at least about 10 ¹¹ viral particles (VP) is obtained from about 1000 L of starting material (eg, cell culture). In some embodiments, an AAV product containing at least about 10 ¹² viral particles (VP) is obtained from about 1000 L of starting material (eg, cell culture). In some embodiments, an AAV product containing at least about 10 ¹³ viral particles (VP) is obtained from about 1000 L of starting material (eg, cell culture). In some embodiments, an AAV product containing at least about 10 ¹⁴ viral particles (VP) is obtained from about 1000 L of starting material (eg, cell culture). In some embodiments, an AAV product containing at least about 10 ¹⁵ viral particles (VP) is obtained from about 1000 L of starting material (eg, cell culture). In some embodiments, an AAV product containing at least about 10 ¹⁶ viral particles (VP) is obtained from about 1000 L of starting material (eg, cell culture). In some embodiments, an AAV product containing at least about 10 ¹⁷ viral particles (VP) is obtained from about 1000 L of starting material (eg, cell culture). In some embodiments, an AAV product containing at least about 2×10 ¹⁶ viral particles (vp) is obtained from about 1000 L of starting material (eg, cell culture). In some embodiments, an AAV product containing at least about 5×10 ¹⁷ viral particles (VP) is obtained from about 1000 L of starting material (eg, cell culture).

[00171] Another advantage of the methods described herein is that these methods produce a highly pure AAV product. In some embodiments, the AAV product produced by the methods of this invention is substantially free of one or more of the following contaminants: host cell proteins, host cell nucleic acids (e.g., free host cell DNA and free plasmid DNA), plasmid DNA, empty viral capsids, heat shock protein 70 (HSP70), lactate dehydrogenase (LDH), proteasomes, non-AAV contaminating viruses (e.g. lipid-enveloped viruses), host cell culture components (e.g. antibiotics), mycoplasmas, pyrogens, bacterial endotoxins, cellular debris (eg, debris composed of membrane lipids, proteins and other biological polymers) and introduced agents. One or more, or even all, of the following impurities may not be detected when AAV is purified according to the AAV preparation and purification methods described herein: H2A type 1 histone, H2B type 1-B histone, H4 histone, 70 kDa heat shock protein 1A , pyruvate kinase PKM, elongation factor 2, ATP-citrate synthase, histone H1.4, heavy immunoglobulin gamma-1 constant (immobilized ligand from acid elution method), ribosomal protein 60S L27, fructose bisphosphate aldolase A, heat shock protein cognate 71 kDa, cytoplasmic actin 1, S-formylglutathione hydrolase, asparagine synthetase (hydrolysable glutamine), 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, 60S L30 ribosomal protein, WD 1 repeat protein, adenosylhomocysteinase, heterogeneous nuclear ribonucleoprotein C, Rep68 protein, thymetoligopeptidase, D-3-phosphoglycerate dehydrogen for, the ATP-dependent molecular chaperone HSC82. The addition of an anion exchange step prior to the wash steps according to the AAV anion production and purification methods described herein can also render the following undetectable: histone H1.4, 60S L27 ribosomal protein, cytoplasmic actin 1, tubulin beta-2A chain, 60S ribosomal protein L6, 60S ribosomal protein L30, heterogeneous nuclear ribonucleoprotein C, protein Rep68, and the ATP-dependent molecular chaperone HSC82.

[00172] In exemplary embodiments, the methods of this invention provide a purified AAV product that removes at least or about 50% of the contaminant found in the starting material (eg, cell culture). In exemplary embodiments, the methods of this invention provide a purified AAV product that removes at least or about 60% of the contaminant found in the starting material (eg, cell culture). In exemplary embodiments, the methods of this invention provide a purified AAV product that removes at least or about 70% of the contaminant found in the starting material (eg, cell culture). In exemplary embodiments, the methods of this invention provide a purified AAV product that removes at least or about 80% of the contaminant found in the starting material (eg, cell culture). In exemplary embodiments, the methods of this invention provide a purified AAV product that removes at least or about 90% of the contaminant found in the starting material (eg, cell culture).

[00173] In certain embodiments, an AAV product made using the methods of this invention is suitable for administration to a human. In certain embodiments, the AAV is recombinant AAV (rAAV). In certain embodiments, the AAV product made using the methods of this invention is sterile and/or Good Manufacturing Practice (GMP) grade. In some embodiments, the AAV product obtained using the methods of this invention complies with the requirements set forth in USP Chapter 1046 or the European Pharmacopoeia for Gene Therapy Medicines or in accordance with the requirements of the US Food and Drug Administration (USFDA) or European Medicines Agency (EMA).

[00174] In addition, the AAV product obtained using the methods described herein is potent. The activity of an AAV product, eg an AAV8 or AAV9 product, can be described in terms of (1) in vivo bioactivity (eg, active protein production in mice), which is given in units (FIX or FVIII) per ml of mouse plasma; or (2) in vitro bioactivity. The in vitro bioactivity assay measures the potential of AAV vectors to infect cells, eg HepG2 cells, that express and secrete a protein of interest into the medium and quantifies by ELISA and/or enzyme activity. Suitable methods for measuring in vivo and in vitro bioactivity are known in the art and are also described herein.

[00175] In other embodiments, the implementation of the AAV product, obtained by the methods described in this document, shows excellent specific activity. The "specific activity" of AAV is represented by the ratio of qPCR to µg of AAV8. In exemplary embodiments, an AAV product prepared using the methods described herein exhibits an excellent ratio of vector genomes per µg of AAV, demonstrating that the AAV product has a large number of "complete" viral particles. In certain embodiments, the methods of this invention include testing the AAV fraction with an AAV-specific ELISA. In certain embodiments, the AAV-specific ELISA is sufficient to provide a representative reading of AAV fraction activity because most of the capsids in the AAV fraction are complete capsids.

[00176] Source of rAAV particles

[00177] With respect to the methods of this disclosure, AAV may be of 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 serotype (VR-195) and AAV serotype (VR-355) or AAV chimeric vectors. In some embodiments, the AAV is wild type. In some embodiments, AAV is modified by genetic engineering and/or chemically modified. In certain embodiments, the AAV contains a modified capsid, such as a genetically modified or chemically modified AAV capsid. In certain embodiments, the AAV particles purified by the methods described herein are of the AAV8 serotype.

[00178] With respect to the methods of this invention, an AAV fraction is, in exemplary aspects, a concentrated AAV fraction. In some embodiments, the AAV fraction contains at least 1×10 ¹⁰ , 1×10 ¹¹ , or 1×10 ¹² AAV AAV capsid per ml. In some embodiments, the AAV fraction contains at least 1×10 ¹² AAV AAV capsid per ml. AAV capsids may include empty AAV capsids and full AAV capsids.

[00179] In certain embodiments, the AAV fraction is the AAV fraction produced by the transfected host cells. In some embodiments, the AAV fraction is a supernatant collected from a cell culture comprising host cells transfected with a triple plasmid system, where one plasmid of the system contains a gene or cDNA of interest, one plasmid encodes a VP1 capsid protein, a VP2 capsid protein, and/or a capsid protein VP3. In some embodiments, VP1, VP2, and/or VP3 are AAV8 VP1, AAV8 VP2, and/or AAV8 VP3. In some embodiments, VP1, VP2 and/or VP3 are AAV9 VP1, AAV9 VP2 and/or AAV9 VP3. Triple plasmid transfection to produce rAAV is known in the art. See, for example, Qu et al., 2015, supra, and Mizukami et al., "A Protocol for AAV vector production and purification." PhD dissertation, Division of Genetic Therapeutics, Center for Molecular Medicine, 1998; and Kotin et al., Hum Mol Genet 20(R1): R2-R6 (2011). In some 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 cells are adherent cells. In certain embodiments, the host cells are suspension cells. In certain embodiments, the host cells are HEK293 cells or Sf9 cells. In certain embodiments, the implementation of the cell culture includes a culture medium that does not contain serum and protein. In certain embodiments, the medium is chemically defined and does not contain animal derived components, such as hydrolysates. In some embodiments, the fraction containing rAAV particles is a fraction containing HEK293 cells transfected with a triple plasmid system. In some embodiments, the fraction containing rAAV particles is described in US Provisional Application No. 62/417775 and International Application No. PCT/US2017/059967.

Additional stages

[00180] The methods of this disclosure include any combination of the steps disclosed herein and may optionally be combined with one or more additional steps. Accordingly, in exemplary aspects, the methods of this invention further comprise the steps of transfecting host cells with a triple plasmid system as described herein. In illustrative aspects, the methods of this invention include collecting supernatant from a cell culture containing host cells, eg HEK293 cells, transfected with a triple plasmid system. In exemplary aspects, the transfection and harvest step occurs prior to the ultracentrifugation step described herein.

[00181] The methods of this invention may contain still other additional steps that can further improve the purity of the AAV and remove other undesirable components, and/or concentrate the fraction and/or prepare the fraction for the next step. Additional steps may occur before or after the ultracentrifugation step described herein.

[00182] In exemplary aspects, the method includes a depth filtering step. In illustrative aspects, the method includes subjecting the supernatant fraction of the transfected HEK293 cell culture to depth filtration using a filter containing cellulose and perlite and having a minimum permeability of approximately 500 l/m ² . In illustrative aspects, the method further includes using a filter with a minimum pore size of approximately 0.2 microns. In illustrative aspects, depth filtration is followed by filtration through a filter with a minimum pore size of approximately 0.2 microns. In exemplary aspects, one or both of the porous filter and the filter having a minimum pore size of approximately 0.2 microns are washed and the washing fractions are collected. In illustrative aspects, the washes are pooled together and combined with the filtrate obtained after depth filtration and filtration with a filter having a minimum pore size of approximately 0.2 microns.

[00183] In illustrative aspects, the methods of this invention include one or more chromatography steps. In exemplary aspects, the methods include a negative chromatography step whereby unwanted components are bound to the chromatographic resin and the desired AAV is not bound to the chromatographic resin. In illustrative aspects, the methods include a negative anion exchange (AEX) chromatography step or an AEX chromatography step in "no binding mode". Example 2 describes such a stage. The advantages of "no-binding mode" include the relative simplicity of the procedure and subsequent analysis.

[00184] Accordingly, in exemplary embodiments, methods for purifying AAV particles include performing negative anion exchange (AEX) chromatography on a fraction containing AAV particles by applying the fraction to an AEX chromatography column or membrane under conditions that allow AAV to pass through the AEX chromatography column. or a membrane and collection of AAV particles. In illustrative aspects, the fraction is applied to an AEX chromatography column or membrane with a loading buffer containing from about 100 mM to about 150 mM salt, e.g., NaCl, optionally, wherein the pH of the loading buffer is from about 8 to about 9. In illustrative aspects, the loading buffer contains from about 115 mm to about 130 mm salt, for example, NaCl, optional, and the loading buffer contains from about 120 mm to about 125 mm salt, for example, NaCl. In exemplary aspects, the negative AEX step occurs prior to the ultracentrifugation step described herein.

[00185] In exemplary aspects, the methods of this disclosure include concentrating the AAV fraction using an ultra/diafiltration system. In exemplary aspects, the methods of this disclosure include another tangential flow filtration (TFF) step. In exemplary aspects, the AAV fraction is ultra-/diafiltered. In illustrative aspects, the AAV fraction is concentrated in the ultra/diafiltration system before a step including performing negative AEX chromatography, after a step including performing negative AEX chromatography, or before and after performing negative AEX chromatography. In exemplary aspects, the TFF steps occur prior to the ultracentrifugation step described herein.

[00186] In illustrative aspects, the methods of this disclosure include filtering a fraction containing rAAV particles to remove viruses larger than the rAAV particles in the fraction.

[00187] In illustrative aspects, the methods of this disclosure include one or more quality control steps, such as steps to measure the potency or specific activity of AAV fractions obtained after one or more steps (e.g., after each step) of the method. In exemplary aspects, the methods of this disclosure include an ELISA specific for AAV. In exemplary aspects, the ELISA is a sandwich ELISA. In exemplary aspects, the sandwich ELISA comprises an antibody specific for an AAV epitope. In exemplary aspects, an AAV epitope is a conformational epitope present on assembled AAV capsids. As discussed herein, ELISA can replace qPCR as a way to determine the potency of an AAV fraction. In illustrative aspects, the methods of this disclosure include testing the AAV fraction with an AAV-specific ELISA, and the methods do not include a method for measuring potency by quantitative PCR. In exemplary aspects, an AAV-specific ELISA is sufficient to provide a representative reading of AAV fraction activity because most of the capsids in the AAV fraction are complete capsids.

[00188] In exemplary aspects, the methods of this disclosure include an AAV-specific ELISA following one or more of the steps of this disclosure. In illustrative aspects, the methods of this disclosure include testing the AAV fraction obtained after depth filtration with an AAV-specific ELISA to determine the specific AAV activity in that fraction. In illustrative aspects, the methods of this disclosure include testing the AAV fraction obtained after concentrating the AAV fraction using an ultra-/diafiltration system with an AAV-specific ELISA to determine specific AAV activity in that fraction. In illustrative aspects, the methods of this disclosure include testing the AAV fraction obtained after the tangential flow filtration (TFF) step with an AAV-specific ELISA to determine the specific AAV activity in that fraction. In illustrative aspects, the methods of this invention include testing the fraction of AAV obtained after negative anion exchange chromatography (AEX) with an AAV-specific ELISA to determine the specific activity of AAV in this fraction. In illustrative aspects, the methods of this disclosure include testing the AAV fraction obtained after the polishing step with an AAV-specific ELISA to determine the specific AAV activity in that fraction.

[00189] The AAV product obtained using the method of this invention is additionally presented in this document. In illustrative aspects, an AAV product contains at least about 10 ¹² virus particles (vp) derived from about 1000 liters of starting material (e.g., cell culture), or at least about 10 ¹³ virus particles (vp) derived from approximately 1000 liters of starting material (eg cell culture). In exemplary aspects, the AAV product is an empty capsid generated by transfection of a rep-cap plasmid and an Ad helper plasmid without a transgene. Purified empty plasmids can be used to deplete or remove antibodies specific for AAV antigens from a patient's blood.

[00190] In illustrative aspects, the AAV product of this invention is highly pure, highly potent, and suitable for clinical use in humans. In illustrative aspects, the AAV product contains AAV particles of a homogeneous population and high purity. In illustrative aspects, the AAV product contains full length vector DNA. In illustrative embodiments, the AAV product is substantially free of undesirable contaminants, including, but not limited to, AAV particles containing truncated or incomplete vector DNA, AAV particles with incomplete protein composition and oligomerized structures, or contaminating viruses, such as non-AAV lipid-enveloped viruses. . In exemplary embodiments, the AAV product contains a large amount of the cDNA encoding the protein of interest. In exemplary aspects, the AAV product of this invention is suitable for administration to a human. In exemplary aspects, the AAV product is sterile and/or of Good Manufacturing Practice (GMP) purity. In illustrative aspects, the AAV product complies with the requirements set forth in USP Chapter 1046 or the European Pharmacopoeia for Gene Therapy Medicines or in accordance with the requirements of the US Food and Drug Administration (USFDA) or the European Medicines Agency (EMA). In illustrative aspects, the AAV product is a ready-to-use product for direct administration to a human with little or no processing.

[00191] The following examples are provided merely to illustrate the present invention and not to limit its scope.

EXAMPLE 1

[00192] The following example describes an illustrative method for transfecting a HEK293 cell line with a triple plasmid system to produce rAAV particles containing a nucleic acid encoding a protein of interest.

[00193] Adherent HEK293 cells were grown under suspension conditions in a commercially available culture medium that is chemically defined and free of animal components, protein and serum, for example, as described in paragraphs [00146] - [00150] of PCT/US2017/059967. Cells were transfected with three plasmids: (1) a helper plasmid that provides accessory viral functions important for productive AAV infection, (2) a repcap plasmid that carries all information regarding capsid formation, viral replication, and packaging, and (3) a plasmid containing the gene of interest (GOI), which is packaged in the resulting rAAV particle. rAAV particles containing the gene of interest are present in the HEK293 cell line for 3-5 days after transfection.

[00194] The transfected HEK293 cell culture supernatant was collected, for example, as described in paragraphs [00151] - [00155], Table 1 and Table 2 of PCT/US2017/059967. The collected supernatant was concentrated and conditioned (diafiltered), for example, as described in paragraphs [00156] - [00160], Table 3 and Table 4 of PCT/US2017/059967. Negative chromatography was performed on the diafiltered concentrate, for example, as described in paragraphs [00161] - [00165] and Table 5 PCT/US2017/059967.

EXAMPLE 2

[00195] AAV8 production was developed in the HEK293 cell line after transfection with a triple plasmid system containing the coding cDNA of the protein of interest and AAV8-. VP1. -VP2 and -VP3. The clarified cell-free culture supernatant was concentrated and diafiltered with a Pall Omega T-Series 100 kDa cassette. Virus particles were loaded onto a membrane adsorber (MustangQ; Pall Part Number XT140MSTGQP05) under non-binding conditions, ie in a solution containing 125 mM NaCl and 50 mM Tris-HCl at pH 8.5. pH-conditioned LOADING was obtained by adjusting the pH of the stream containing AAV8 to a pH range between 7.4 and 7.8 with 25% HCl.

[00196] The following test procedure was performed. First, a column containing POROS™ CaptureSelect™ AAV8 affinity matrix (p/n 195338010; Thermo Fisher) 32 mm ID, 59 mm bed height, and 47.45 mL volume was equilibrated with at least five column volumes of 20 mM Tris-HCl and 150 mM NaCl at pH 7.4. The pH-conditioned LOADING was applied to a column containing POROS™ CaptureSelect™ AAV8 affinity matrix (Cat. No. 195338010; Thermo Fisher). The column was then re-equilibrated with five column volumes of 20 mM Tris-HCl and 150 mM NaCl at pH 7.4 (optional fourth buffer).

[00197] The column was then washed with five column volumes of Wash 1 (W1): 100 mM sodium acetate and 0.1% Tween80 at pH 6.0. The column was then washed with five column volumes of Wash 2 (W2): 50 mM Tris-HCl and 125 mM NaCl at pH 8.5. The column is then washed with five column volumes of Wash 3 (W3): 50 mM Tris-HCl and 50% ethylene glycol at pH 8.5.

[00198] Elution was performed by using five column volumes of the following elution buffer: 50 mM Tris-HCl, 50% ethylene glycol and 750 mM NaCl, at pH 8.0. Five column volumes of the following secondary elution buffer were then applied to the column: 50 mM Tris-HCl, 50% ethylene glycol and 2000 mM NaCl.

[00199] The linear flow rate for the above steps was 60 cm/hr.

[00200] The following comparative procedure was carried out. A column containing POROS™ CaptureSelect™ AAV8 affinity matrix (p/n 195338010; Thermo Fisher) 10 mm ID, 2.5 mm bed height, and 1.96 mL volume was equilibrated with at least 10 column volumes of 20 mM Tris-HCl and 150 mM NaCl at pH 7.4. The pH-conditioned LOADING was applied to a column containing POROS™ CaptureSelect™ AAV8 affinity matrix (Cat. No. 195338010; Thermo Fisher). The column was then re-equilibrated with 10 column volumes of 20 mM Tris-HCl and 150 mM NaCl at pH 7.4.

[00201] The wash step was performed using the following TBS buffer: 20 mM Tris-HCl/150 mM NaCl/pH 7.4. Instead, elution was performed with 10 column volumes of 100 mM sodium citrate at pH 3.0.

[00202] The above test and comparison procedure is described in more detail in Table 2, where "CV" indicates the number of column volumes of the solution added in the step.

Table 2:

Stage TEST PROCEDURE CV COMPARATIVE PROCEDURE CV Flow rate one. 20 mM Tris-HCl
150 mM NaCl
pH 7.4 > 5 20 mM Tris-HCl
150 mM NaCl
pH 7.4 > 5 30 cm/h 2. Loading a Sample
pH 7.4 to 7.8 Loading a Sample
pH 7.4 to 7.8 --- 3. 20 mM Tris-HCl
150 mM NaCl
pH 7.4 five 20 mM Tris-HCl
150 mM NaCl
pH 7.4 10 4. Wash 1 (W1)
100 mM sodium acetate
0.1% Twin80
pH 6.0 five x x five. Wash 2 (W2)
50 mM Tris-HCl
125 mM NaCl
pH 8.5 five x x 6. Wash 3 (W3)
50 mM Tris-HCl
50% ethylene glycol
pH 8.5 five x x 30 cm/h 7. Elution
50 mM Tris
50% ethylene glycol
750 mM NaCl
pH 8.0 five Elution
100 mm
sodium citrate
pH 3.0 10

[00203] The following analyzes were performed with the data expressed in mass/volume in Table 3. The density of the elution buffer was measured on a DMA 4500M oscillating U-tube density meter (Anton Paar).

[00204] ELISA was used to measure the amount of AAV8 antigen. ELISA was performed with the AAV-8 Titration ELISA kit (No. PRAAV8; Progen (Heidelberg, Germany) on a TECAN Roboter system. Briefly, a monoclonal antibody specific for a conformational epitope on assembled AAV8 capsids (ADK8) was applied to microtiter strips and used for capture of AAV8 particles from the AAV fraction Capture of AAV8 particles was detected in two steps In the first step, a biotin-conjugated monoclonal antibody specific for the ADK8 antibody was bound to the immune complex (antibodies ADK8 and ADK8) Streptavidin peroxidase conjugates were added to the biotin-bound immune complexes α-conjugated monoclonal antibody and streptavidin peroxidase conjugates were reacted with biotin.The peroxidase substrate solution was added and a color reaction occurred which is proportional to the number of bound AAV particles.The color reaction was measured photometrically at 450 nm.

[00205] ITR-qPCR analysis was used to determine the genome copy titer by quantifying the inverted tandem repeats found in the vector encoding the gene of interest (eg, human factor VIII or human factor IX). HEK-HCP is a measure of the level of residual host cell protein by ELISA. LDH was determined by colorimetric activity assay.

[00206] AAV8 Ligand Leak ELISA (Linked Enzyme Immunosorbent Assay) is designed to detect AAV8 affinity ligand at 1 ng/mL, which may be present in a product purified with POROS™ CaptureSelect™ AAV8 Affinity Medium, which contains AAV8 affinity ligand as a capture agent. AAV8 ligand leak ELISA can be used as a tool to help optimize the purification process and perform regular quality control of both in-process streams and the final product.

[00207] The "Ligand Leak ELISA/AAV8 - Antigen" value reflects the ratio of "Ligand Leak ELISA" to "AAV8 Antigen", calculated as nanograms of ligand per microgram of AAV8.

[00208] In an in vitro biopotency assay, the AAV8 viral vector infects a target hepatic cell line, which subsequently secretes a functional, measurable encoded protein into the medium. In the first step, HepG2 target cells are transduced by infection with AAV8. During incubation, the encoded protein is released into the cell supernatant. In the second step, the activity of the encoded protein in the cell culture supernatant is directly measured by an activity assay. The AAV8 sample measurement is given as a percentage of the reference material. The method allows to quantify the biological function of the AAV8 gene therapy vector.

Table 3:

test procedure Comparative procedure DENSITY (elution buffer) 1.099 1.008 g/ml AAV8 Antigen 211.6 µg/ml 187.6 µg/ml ITR-qPCR 1.87E+13vg/ml 0.875+13vg/ml HEK-HCP <91ng/ml <99.2 ng/g LDH (lactate dehydrogenase) <24.6 <26.8 ng/g ELISA to determine ligand leakage 27.57 ng/ml 141.9 ng/ml ELISA to determine ligand leakage/
AAV8 - Antigen 0.13 ng/mcg 0.76 ng/mcg Stage exit AAV8 Antigen 88.8% 113.9% ITR-qPCR 105.3% 71.2% Biopotency in vitro 0.45BPU 0.33BPU

[00209] The yield per stage for ITR-qPCR for the test procedure was 105.3%, while for the comparative procedure it was 71.2%. The in vitro biopotency for the test procedure was 0.45, while for the comparative procedure it was 0.33.

[00210] Analyzes were also performed with data expressed in wt/wt in Table 4.

Table 4:

test procedure Comparative procedure AAV8 Antigen 232.6 µg/g 189.1 µg/g ITR-qPCR 2.06E+13vg/g 0.88E+13vg/g HEK-HCP <100 ng/g <100 ng/g LDH (lactate dehydrogenase) <27 ng/g <27 ng/g ELISA to determine ligand leakage 30.3 ng/g 143.0 ng/g ELISA to determine ligand leakage/
AAV8 - Antigen 0.13 ng/mcg 0.76 ng/mcg Stage exit AAV8 Antigen 107.4% 115.8% ITR-qPCR 127.4% 72.4%

[00211] The stage yield for ITR-qPCR for the test procedure was 127.4%, while for the comparative procedure it was 72.4%. The improvement in yield and purity, coupled with not having to use extreme conditions that degrade the infectivity and biopotency of AAV8, was unexpected. In addition, it was surprising that the test procedure removed most of the impurities without eluting the product from the affinity ligand, especially under conditions where there was significantly more non-specific binding and complexes of the product with impurities that were present prior to purification.

[00212] An SDS-PAGE analysis was performed to determine if there was a decrease in 70 kDa heat shock protein (HSP70) when using a test procedure with wash steps instead of a comparison procedure. Western blotting was performed using an Anti-Hsp70 antibody (Abcam, cat. no. ab79852) as primary antibody at a dilution of 1:2000 for two hours and goat anti-rabbit IgG (H+L) AP conjugate as secondary antibody (Sigma , Cat No. A8025) at a 1:1000 dilution for one hour. The results are shown in FIG. 1. When comparing lane 4 (test procedure) with lane 6 (comparative procedure), a significant decrease in HSP70 was observed during the test procedure. Lane 2 shows 20 ng HSP70 and lane 4 shows 4 ng HSP70.

[00213] SDS-PAGE analysis for silver staining was performed to determine the total level of impurities present. The results are shown in FIG. 2. Lane 2 is the AAV8 reference and shows three characteristic bands. The eluate (190 μg/ml) was purified according to the test procedure and comparative procedure. The results of the test procedure are shown in lane 4. The results of the comparative procedure are shown in lane 6. There are noticeably more impurities in the comparative procedure than in the test procedure.

[00214] Western blotting with 12% anti-AAV antibody was performed to determine the levels and purity of AAV8 isolated after purification, in accordance with the test and comparison procedures. Western blotting was performed with monoclonal antibodies to VP1, VP2 and VP3 AAV as primary antibodies, with goat anti-mouse ALP antibody (Sigma, cat. no. A4656) as secondary antibody. The results are shown in FIG. 3. Lane 2 shows the AAV reference (130 ng, corresponding to 60 μg/ml) with three lanes for each of 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 is higher with the test procedure compared with the comparative procedure.

[00215] LC-MS was performed (rp-HPLC-UV-ESI-MS/MS) to determine the identity and amount of various host cell contaminants. Samples were digested using the trypsin enzyme. The resulting peptide mixture was separated on an HPLC system using a reverse phase column (column ZORBAX 300SB-C18, 0.5×150 mm, 3.5 μm) and then the peptides were analyzed on a Q-Exactive HF mass spectrometer. The data were analyzed using the Proteome Discoverer software to identify proteins in the sample.

[00216] An Agilent HPLC1209 (1200 capHPLC) was used with a ChemStation for LC-3D systems (Rev. 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. Detailed HPLC information is provided in the following Table 5:

Table 5:

HPLC Skid: HPLC1209: 1200capHPLC, Agilent Software: ChemStation for 3D LC Systems Rev. B.04.03-SP2 (105) HPLC method: PEPMAP_CAP_170.M Speaker: ZORBAX 300SB-C18 0.5×150mm, 3.5 µm Part. No. 5064-8268 Lot No. USHTC01001 Lot No. WSB1432005 Eluent A: 0.1% (v/v) HCOOH in deionized water, Lot 140617/01/ DF3376/028 Eluent B: 0.08% (v/v) HCOOH in acetonitrile, Lot 250117/02 /DF3270/015 Pump: Initial conditions: 15 µl/min (microflow) 100% A Gradient: 0 min 100%A 0%B
110 min 60%A 40%B 125 min 30%A 70%B
135 min 0%A 100%B 140 min 100%A 0%B
Stop 170 min Sampler: Input volume: Various input volumes Temperature: 4°C Closing a contact Initial OPEN 0.02 min A - CLOSED 0.5 min A - OPEN Speaker compartment: Temperature: 40°C DAD Detector: Wavelengths 214, 280, 260 nm, Spectrum 190-500nm Flushing the needle Flush port 5 sec (20% isopropanol/80% H2O)

[00217] Detailed information about the MS is given in the following Table 6:

Table 6:

Execution: Q Exactive HF #2 Electrospray Ionization (HESI), S/N 05161L Thermo, Source: HESI-II Software: Thermo Xcalibur 3.1.66.10 Setting method: high_parameters.mstune Parameter
Shielding gas flow rate 7 Auxiliary gas flow rate 0
Purge gas flow rate 0 Spray voltage 3 kV
Capillary temperature 275 °C
S-lens RF 50 Instrumental method: 170406_PEPMAP_TOP10_120k_CAP_140.meth Parameter FULL MS Analysis time 140 min Internal source CID 0.0 eV
Default state of charge 2
Microscans 1
Resolution 120,000
AGC, target 3e6
Maximum IT 60
Number of scan ranges 1
Scan range 300-2000m/z
Spectrum Data Type Profile
Polarity positive dd-MS²
Microscans 1
Resolution 30,000
AGC, target 1e5
Maximum IT 100 ms
Number of cycles 10
MSX quantity 1
TopN 10
Insulation window 2.0m/z
Insulation displacement 0.0m/z
NCE/Step 27
Centroid Data Spectrum
Underfill ratio 0.6%
Intensity Threshold 1.0e3
Vertex trigger off
Charge exclusion not assigned, 1, 7, 8, >8
Peptide match is preferred
Exclude isotopes enabled
Dynamic exclusion 30s MS calibration/test resolution: Calibration solution
Pierce LTQ Velos ESI Positive Ion Calibration Solution Order No. 88323, lot RF231587 base parameter
Tune file: Calibration_pos_parameters_150818
Syringe pump flow rate: 5 µl
TIC Stability: ≤10%
IT (input time): ≤10ms
Ionic mode: positive Calibration parameter
eFT parameters (positive) ok
Analyzer accuracy (positive) approx.
Weight calibration (positive) ok Test Spectrum/Resolution
1. Calibration solution Resolution: FTMS 120000 (at m/z 200) Mass range: m/z 135-1800 AGC Target: 3e6
Number of scans: 50 scans
File name: 170621_Calmix.raw
2. m/z 524 MRFA (Komponente Kalibriermix)
Resolution: FTMS 120000 (at m/z 200) Mass range: m/z 520-530 AGC target: 1e5:
Number of scans 50 scans
File name: 170621_MRFA.raw
Resolution (m/z 524) 83281 (reference 80000)
Readouts: 1.67×10^7

[00218] Three different purification methods have been undertaken. One included the comparative procedure of this example, another included the test procedure of this example (which included MustangQ anion exchange purification before affinity purification), and the third was carried out according to the test procedure but without MustangQ anion exchange purification. Table 7 below summarizes the overall results.

Table 7:

Comparative procedure Test procedure with anion exchange pre-stage Test procedure without preliminary anion exchange step Purity
(AAV8 capsid proteins) 95.5% 99.0% 96.0% The number of identified protein impurities twenty 4 fourteen

[00219] In the table above, purity reflects the percentage area under the curve (AUC) associated with AAV8 capsids relative to the total AUC of all proteins.

[00220] Tables 8-10 below for each protein list the AUC value and number of peptides identified by LC/MS for the comparative procedure (Table 8), the pre-anion exchange test procedure (Table 9), and the test procedure without anion exchange pre-treatment (Table 10).

Table 8: Comparative procedure

Protein Square [%] No. of Peptides AAV8 capsid protein 95.5 36 Glyceraldehyde-3-phosphate dehydrogenase 1.4 eleven Histone H2A type 1 - - Histone H2B type 1-B - - Histone H4 - - Heat shock protein 70 kDa 1A 0.4 fourteen Acidic leucine-rich nuclear phosphoprotein 32 family member A/B 0.2 2 Pyruvate kinase PKM 0.3 17 40S ribosomal protein S7 - - Probable ATP-dependent RNA helicase DDX5 0.1 7 Elongation factor 2 0.2 13 ATP-citrate synthase 0.1 nine Histone H1.4 - - Immunoglobulin heavy constant gamma 1 0.1 3 60S ribosomal protein L27 - - Fructose bisphosphate aldolase A 0.1 3 Heat shock related protein 71 kDa 0.1 7 Actin, cytoplasmic 1 - - S-formylglutathione hydrolase 0.1 6 Asparagine synthetase [glutamine hydrolyzing] 0.1 6 L-lactate dehydrogenase chain B 0.1 2 Tubulin beta-2A chain - - RNA-binding motivic protein, X chromosome 0.1 2 60S ribosomal protein L6 - - Threonine - tRNA ligase, cytoplasmic 0.1 6 Immunoglobulin kappa constant 0.1 2 60S ribosomal protein L30 - - WD repeat protein 1 0.1 4 Adenosylhomocysteinase 0.1 3 Heterogeneous nuclear ribonucleoprotein C - - Rep68 protein - - Thymetoligopeptidase 0.02 2 D-3-phosphoglycerate dehydrogenase 0.02 3 ATP-dependent molecular chaperone HSC82 - - Other peptides - rating 0 0.8 -

Table 9: Test procedure with preliminary anion exchange

Protein Square [%] No. of Peptides AAV8 capsid protein 99.0 36 Glyceraldehyde-3-phosphate dehydrogenase 0.2 five Histone H2A type 1 - - Histone H2B type 1-B - - Histone H4 - - Heat shock protein 70 kDa 1A - - Acidic leucine-rich nuclear phosphoprotein 32 family member A/B 0.2 4 Pyruvate kinase PKM - - 40S ribosomal protein S7 0.3 3 Probable ATP-dependent RNA helicase DDX5 0.1 3 Elongation factor 2 - - ATP-citrate synthase - - Histone H1.4 - - Immunoglobulin heavy constant gamma 1 - - 60S ribosomal protein L27 - - Fructose bisphosphate aldolase A - - Heat shock related protein 71 kDa - - Actin, cytoplasmic 1 - - S-formylglutathione hydrolase - - Asparagine synthetase [glutamine hydrolyzing] - - L-lactate dehydrogenase chain B - - Tubulin beta-2A chain - - RNA-binding motivic protein, X chromosome - - 60S ribosomal protein L6 - - Threonine - tRNA ligase, cytoplasmic - - Immunoglobulin kappa constant - - 60S ribosomal protein L30 - - WD repeat protein 1 - - Adenosylhomocysteinase - - Heterogeneous nuclear ribonucleoprotein C - - Rep68 protein - - Thymetoligopeptidase - - D-3-phosphoglycerate dehydrogenase - - ATP-dependent molecular chaperone HSC82 - - Other peptides - rating 0 0.3 -

Table 10: Test procedure without prior anion exchange

Protein Square [%] No. of Peptides AAV8 capsid protein 96.0 39 Glyceraldehyde-3-phosphate dehydrogenase 0.1 4 Histone H2A type 1 1.3 2 Histone H2B type 1-B 0.9 3 Histone H4 0.8 4 Heat shock protein 70 kDa 1A - - Acidic leucine-rich nuclear phosphoprotein 32 family member A/B 0.2 7 Pyruvate kinase PKM - - 40S ribosomal protein S7 - - Probable ATP-dependent RNA helicase DDX5 - - Elongation factor 2 - - ATP-citrate synthase - - Histone H1.4 0.1 2 Immunoglobulin heavy constant gamma 1 - - 60S ribosomal protein L27 0.1 2 Fructose bisphosphate aldolase A - - Heat shock related protein 71 kDa - - Actin, cytoplasmic 1 0.1 five S-formylglutathione hydrolase - - Asparagine synthetase [glutamine hydrolyzing] - - L-lactate dehydrogenase chain B - - Tubulin beta-2A chain 0.1 4 RNA-binding motivic protein, X chromosome - - 60S ribosomal protein L6 0.1 2 Threonine - tRNA ligase, cytoplasmic - - Immunoglobulin kappa constant - - 60S ribosomal protein L30 0.1 2 WD repeat protein 1 - - Adenosylhomocysteinase - - Heterogeneous nuclear ribonucleoprotein C 0.03 2 Rep68 protein 0.03 3 Thymetoligopeptidase - - D-3-phosphoglycerate dehydrogenase - - ATP-dependent molecular chaperone HSC82 0.02 one Other peptides - rating 0 0.3 -

[00221] The smallest amount of proteins was found using a test procedure in combination with a preliminary anion exchange. Without anion exchange, the purity decreases and the number of proteins found increases. The lowest purity and highest amount of proteins found are observed in the comparative procedure.

EXAMPLE 3

[00222] AAV8 production was developed in the HEK293 cell line after transfection with a triple plasmid system containing the coding cDNA of the protein of interest and AAV8-. VP1. -VP2 and -VP3. The clarified cell-free culture supernatant was concentrated and diafiltered with a Pall Omega T-Series 100 kDa cassette. Virus particles were loaded onto a membrane adsorber (MustangQ. Pall part number XT140MSTGQP05) under non-binding conditions. The resulting stream containing AAV8 was not adjusted to a pH in the range of 7.4 to 7.8 with 25% HCl. Instead, a LOAD was generated by reconstituting the stream containing AAV-8 in loading buffer containing 125 mM NaCl and 50 mM TrisHCl at pH 8.5.

[00223] In addition to the advantage of not having to include a pH adjustment step, having a pH of 8.5 provides improved affinity stability and prevents non-specific binding of impurities to the product or resin.

[00224] Samples from various washing and elution steps were taken at various points to determine how much AAV8 was present in the sample. The analyzes show how much AAV8 was lost in the various washing steps. The following test procedure was carried out. First, a column containing POROS™ CaptureSelect™ AAV8 affinity matrix (p/n 195338010. Thermo Fisher) 10 mm ID, 25 mm bed height, and 1.96 mL volume was equilibrated with at least five column volumes of 20 mM Tris-HCl and 150 mM NaCl at pH 7.4. LOADING was applied to a column containing POROS™ CaptureSelect™ AAV8 Affinity Matrix (Cat. No. 195338010. Thermo Fisher). Part of the sample loaded on the column was saved and then analyzed by ITR qPCR, ELISA against AAV antigens and ELISA against HEK293 HCP antigens. The column was then re-equilibrated with 10 column volumes of 20 mM Tris-HCl and 150 mM NaCl at pH 7.4. The flow-through sample was saved and then analyzed by ITR qPCR, ELISA against AAV antigens, and ELISA against HEK293 HCP antigens.

[00225] The column was then washed with 10 column volumes Wash 1 (W1): 100 mM sodium acetate and 0.1% Tween80 at pH 6.0. The column was then washed with 10 column volumes Wash 2 (W2): 50 mM Tris-HCl and 125 mM NaCl at pH 8.5. The column is then washed with 10 column volumes Wash 3 (W3): 50 mM Tris-HCl and 50% ethylene glycol at pH 8.5. A sample from the eluate of each of W1, W2 and W3 was taken and analyzed according to ITR qPCR, ELISA against AAV antigens and ELISA against HEK293 HCP antigens.

[00226] Elution was performed by applying 10 column volumes of the following elution buffer to the column: 50 mM Tris-HCl, 50% ethylene glycol, and 750 mM NaCl, at pH 8.0.

[00227] The above test procedure is described in more detail in Table 11.

Table 11:

Stage LOADING WITH HIGH PH CV Flow rate one. 20 mM Tris-HCl
150 mM NaCl
pH 7.4 > 5 30 cm/h 2. Loading a Sample
pH 8.5 3. 20 mM Tris-HCl
150 mM NaCl
pH 7.4 10 4. 100 mM sodium acetate
0.1% Twin80
pH 6.0 10 five. 50 mM Tris-HCl
125 mM NaCl
pH 8.5 10 6. 50 mM Tris-HCl
50% ethylene glycol
pH 8.5 10 30 cm/h 7. Elution
50 mM Tris-HCl
50% ethylene glycol
750 mM NaCl
pH 8.0 10 eight. 50 mM Tris-HCl
50% ethylene glycol
2000 mM NaCl
pH 8.0 10

[00228] Selected samples were analyzed by each of ITR qPCR, ELISA against AAV antigens, and ELISA against HEK293 HCP to assess the yield and possible losses in stages. Table 12 shows the distribution of AAV8 analyzed by ITR qPCR in all fractions of the washing, elution and post-elution steps listed in Table 11.

Table 12:

Sample Volume (g) qPCR ITR (µg/ml) x 10 ¹¹ ITR qPCR Total (vg) (VG) x 10 ¹¹ ITR qPCR Yield (%) Sample Loading (See Step 2 in Table 11) 285.67 9.76 2788.14 100.00% Sample from the rebalance flow path (see Step 3 in Table 11) 316.93 0.289 91.59 3.29% Wash solution sample 1 (see step 4 in Table 11) 16.99 0.655 11.13 0.40% Wash solution sample 2 (step 5 in Table 11) 19.45 1.18 22.95 0.82% Wash solution sample 3 (step 6 in Table 11) 20.71 0.162 3.36 0.12% Sample from elution (step 7 in Table 11) 20.93 155.00 3244.15 116.36% Stripping sample (step 8 in Table 11) 23.52 13.00 305.76 10.97%

[00229] Table 13 below shows the distribution of AAV8 analyzed by ELISA in all fractions of the washing, elution and post-elution steps listed in Table 11.

Table 13:

Sample Volume (g) AAV ELISA (µg/ml) x 10 ¹¹ AAV ELISA Total (mcg) AAV ELISA Yield (%) Sample loading (stage 2 in Table 11) 285.67 28.41 8115.885 100.00% Sample from the rebalance flow path (step 3 in Table 11) 316.93 0.475 150.542 1.85% Wash solution sample 1 (step 4 in Table 11) 16.99 0.688 11.689 0.14% Wash solution sample 2 (step 5 in Table 11) 19.45 1.201 23.359 0.29% Wash solution sample 3 (step 6 in Table 11) 20.71 0.157 3.251 0.04% Sample from elution (step 7 in Table 11) 20.93 211.897 4435.004 54.65% Stripping sample (step 8 in Table 11) 23.52 15.014 353.13 4.35%

[00230] Table 14 below shows the results of the assays to determine the amount of HEK 293 HCP present according to ELISA in the LOAD and eluate steps only.

Table 14:

Sample Volume (g) HEK293 HCP ELISA (µg/ml) x 10 ¹¹ HEK293 HCP ELISA Total (mcg) HEK293 HCP ELISA Yield (%) Sample loading (stage 2 in Table 11) 285.67 191.06 54580.1 100.00% Sample from the rebalance flow path (step 3 in Table 11) 316.93 -- -- -- Wash solution sample 1 (step 4 in Table 11) 16.99 -- -- -- Wash solution sample 2 (step 5 in Table 11) 19.45 -- -- -- Wash solution sample 3 (step 6 in Table 11) 20.71 -- -- -- Sample from elution (step 7 in Table 11) 20.93 <0.10 <2.09 <0.004% Stripping sample (step 8 in Table 11) 23.52 -- -- --

[00231] The chromatogram associated with the data in Tables 12-14 is shown in Fig.6.

[00232] The above samples were also analyzed by SDS-PAGE and 12% silver staining for total protein. The results are shown in FIG. 4 with a label for each band indicated in FIG. The AAV8 bands are clearly visible on the eluate (E) and 50% dilution eluate (E2) lines. Very little AAV8 is observed in the stripping flow through the samples (W1, W2 and W3) and in the stripping flow (S) through the samples. Thus, the method effectively removes AAV8 without significant loss during washing or leaving AAV8 on the column after elution.

[00233] Samples were then analyzed by SDS-PAGE and Western blotting against AAV antigens. Primary antibodies are monoclonal antibodies to AAV VP1, VP2 and VP3, while secondary antibodies are goat anti-mouse antibodies linked to alkaline phosphatase. The results are shown in FIG. 5 with the same labels for each band as in FIG. 4. The loss of AAV8 during the washing and stripping steps is minimal.

EXAMPLE 4

[00234] AAV9 production was developed in the HEK293 cell line after transfection with a triple plasmid system containing the coding cDNA of the protein of interest and AAV9-. VP1. -VP2 and -VP3. The clarified cell-free culture supernatant was concentrated and diafiltered with a Pall Omega T-Series 100 kDa cassette. Virus particles were loaded onto a membrane adsorber (MustangQ; Pall part number XT140MSTGQP05) under non-binding conditions, ie in a solution containing 125 mM NaCl and 50 mM Tris-HCl at pH 8.5. pH dependent LOADING is achieved by adjusting the AAV9 containing stream to a pH range of 7.4 to 7.8 with 25% HCl.

[00235] The following test procedure was performed. First, a column containing POROS™ CaptureSelect™ AAV9 affinity matrix (p/n A27354; Thermo Fisher) 32 mm ID, 59 mm bed height, and 47.45 mL volume was equilibrated with at least five column volumes of 20 mM Tris-HCl and 150 mM NaCl at pH 7.4. The pH-conditioned LOAD was applied to a column containing POROS™ CaptureSelect™ AAV9 affinity matrix (cat. no. A27354; Thermo Fisher). The column was then re-equilibrated with five column volumes of 20 mM Tris-HCl and 150 mM NaCl at pH 7.4 (optional fourth buffer).

[00236] The column was then washed with five column volumes of Wash 1 (W1): 100 mM sodium acetate and 0.1% Tween 80 at pH 6.0. The column was then washed with five column volumes of Wash 2 (W2): 50 mM Tris-HCl and 125 mM NaCl at pH 8.5. The column was then washed with five column volumes of Wash 3 (W3): 50 mM Tris-HCl and 50% ethylene glycol at pH 8.5.

[00237] Elution is accomplished by applying five column volumes of the following elution buffer to the column: 50 mM Tris-HCl, 50% ethylene glycol, and 750 mM NaCl, at pH 8.0. Five column volumes of the following secondary elution buffer were then applied to the column: 50 mM Tris-HCl, 50% ethylene glycol and 2000 mM NaCl.

[00238] The linear flow rate for the above steps was 60 cm/hr.

[00239] The following comparative procedure was performed. A column containing POROS™ CaptureSelect™ AAV9 Affinity Matrix (P/N A27354; Thermo Fisher), 10 mm ID, 2.5 mm bed height, and 1.96 mL volume, was equilibrated with at least 10 column volumes of 20 mM Tris-HCl and 150 mM NaCl at pH 7.4. The pH-conditioned LOAD was applied to a column containing POROS™ CaptureSelect™ AAV9 affinity matrix (cat. no. A27354; Thermo Fisher). The column was then re-equilibrated with 10 column volumes of 20 mM Tris-HCl and 150 mM NaCl at pH 7.4.

[00240] The wash step was performed using the following TBS buffer: 20 mM Tris-HCl/150 mM NaCl/pH 7.4. Instead, elution was performed with 10 column volumes of 100 mM sodium citrate at pH 3.0.

[00241] The above test and comparison procedure is described in more detail in Table 15, where "CV" indicates the number of column volumes of solution added in the step.

Table 15:

Stage TEST PROCEDURE CV COMPARATIVE PROCEDURE CV Flow rate one. 20 mM Tris-HCl
150 mM NaCl
pH 7.4 > 5 20 mM Tris-HCl
150 mM NaCl
pH 7.4 > 5 30 cm/h 2. Loading a Sample
pH 7.4 to 7.8 Loading a Sample
pH 7.4 to 7.8 --- 3. 20 mM Tris-HCl
150 mM NaCl
pH 7.4 five 20 mM Tris-HCl
150 mM NaCl
pH 7.4 10 4. Wash 1 (W1)
100 mM sodium acetate
0.1% Twin80
pH 6.0 five x x five. Wash 2 (W2)
50 mM Tris-HCl
125 mM NaCl
pH 8.5 five x x 6. Wash 3 (W3)
50 mM Tris-HCl
50% ethylene glycol
pH 8.5 five x x 30 cm/h 7. Elution
50 mM Tris
50% ethylene glycol
750 mM NaCl
pH 8.0 five Elution
100 mm
sodium citrate
pH 3.0 10

50% ethylene glycol w/w in the entire buffer (mass/mass)

[00242] The density of the elution buffer was measured on a DMA 4500M oscillating U-tube density meter (Anton Paar).

[00243] ELISA was used to measure the amount of AAV9 antigen. ELISA was performed with an AAV-9 ELISA titration kit (No. PRAAV9; Progen (Heidelberg, Germany) on a TECAN Roboter system. Briefly, a monoclonal antibody specific for a conformational epitope on assembled AAV9 capsids (ADK9) was applied to microtiter strips and used for capture of AAV9 particles from the AAV fraction Captured AAV9 particles were detected in two steps In the first step, a biotin-conjugated monoclonal antibody specific for the ADK9 antibody binds to an immune complex (antibodies ADK9 and ADK9) Streptavidin peroxidase conjugates were added to the biotin-bound immune complexes α-conjugated monoclonal antibody and streptavidin peroxidase conjugates were reacted with biotin A peroxidase substrate solution was added and a color reaction occurred in which the response is proportional to the number of bound AAV particles The color reaction was measured photometrically at 450 nm.

[00244] The ITR-qPCR assay is used to determine the titer of a genome copy by quantifying the inverted tandem repeats found in a vector encoding a gene of interest (eg, human factor VIII or human factor IX). HEK-HCP is a measure of the level of residual host cell protein by ELISA. LDH is determined by colorimetric activity assay.

[00245] AAV9 Ligand Leak ELISA (Linked Enzyme Immunosorbent Assay) can detect 1 ng/mL of AAV9 affinity ligand that may be present in a product purified with a POROS™ CaptureSelect™ AAV9 affinity matrix that contains AAV9 affinity ligand as a capturing agent. AAV9 ligand leak ELISA can be used as a tool to help optimize the purification process and perform regular quality control of both in-process streams and the final product. The "Ligand Leak ELISA/AAV9 - Antigen" value reflects the ratio of "Ligand Leak ELISA" to "AAV9 Antigen", calculated as nanograms of ligand per microgram of AAV9.

[00246] In an in vitro biopotency assay, the AAV9 viral vector infects a target hepatic cell line, which subsequently secretes a functional, measurable encoded protein into the medium. In the first step, HepG2 target cells are transduced by infection with AAV9. During incubation, the encoded protein is released into the cell supernatant. In the second step, the activity of the encoded protein in the cell culture supernatant is directly measured by an activity assay. The AAV9 sample measurement is given as a percentage of the reference material. The method allows to quantify the biological function of the AAV9 gene therapy vector.

[00247] SDS-PAGE analysis was performed to determine if there was a decrease in the level of heat shock protein 70 kDa (HSP70) when using a test procedure with washing steps instead of a comparative procedure. Western blotting was performed using an anti-Hsp70 antibody (Abcam, cat. no. ab79852) as the primary antibody at a dilution of 1:2000 for two hours and goat anti-rabbit IgG (H+L) AP conjugate as the secondary antibody (Sigma, No. A8025) at a 1:1000 dilution for one hour.

[00248] SDS-PAGE analysis for silver staining was performed to determine the total level of impurities present.

[00249] Western blot with 12% anti-AAV antibodies was performed to determine the levels and purity of AAV9 isolated after purification, in accordance with the test and comparative procedures. Western blotting was performed with monoclonal antibodies to VP1, VP2 and VP3 AAV9 as primary antibodies, with goat anti-mouse ALP antibody (Sigma, cat. no. A4656) as secondary antibody.

[00250] LC-MS was performed (rp-HPLC-UV-ESI-MS/MS) to determine the identity and amount of various host cell contaminants. Samples were digested using the trypsin enzyme. The resulting mixture of peptides was separated on an HPLC system using an RP column (column ZORBAX 300SB-C18, 0.5×150 mm, 3.5 μm), and then the peptides were analyzed on a Q-Exactive HF mass spectrometer. Data was analyzed using Proteome Discoverer software to identify proteins in the sample.

[00251] An Agilent HPLC1209 (1200 capHPLC) was used with a ChemStation for 3D LC systems (Rev. B.04.03-SP2 (105)). HPLC method - PEPMAP_CAP_170.M. Eluent A is 0.1% (v/v) HCOOH in deionized water and eluent B is 0.08% (v/v) HCOOH in acetonitrile. Detailed HPLC information is provided in the following Table 16:

Table 16:

HPLC Skid: HPLC1209: 1200capHPLC, Agilent Software: ChemStation for 3D LC Systems Rev. B.04.03-SP2 (105) HPLC method: PEPMAP_CAP_170.M Speaker: ZORBAX 300SB-C18 0.5×150mm, 3.5 µm Part. No. 5064-8268 Lot No. USHTC01001 Lot No. WSB1432005 Eluent A: 0.1% (v/v) HCOOH in deionized water, Lot 140617/01/ DF3376/028 Eluent B: 0.08% (v/v) HCOOH in acetonitrile, Lot 250117/02 /DF3270/015 Pump: Initial conditions: 15 µl/min (microflow) 100% A Gradient: 0 min 100%A 0%B
110 min 60%A 40%B 125 min 30%A 70%B
135 min 0%A 100%B 140 min 100%A 0%B
Stop 170 min Sampler: Input volume: Various input volumes Temperature: 4°C Closing a contact Initial OPEN 0.02 min A - CLOSED 0.5 min A - OPEN Speaker compartment: Temperature: 40°C DAD Detector: Wavelengths 214, 280, 260 nm, Spectrum 190-500nm Flushing the needle Flush port 5 sec (20% isopropanol/80% H2O)

[00252] Detailed information about the MS is given in the following Table 17:

Table 17:

Execution: Q Exactive HF #2 Electrospray Ionization (HESI), S/N 05161L Thermo, Source: HESI-II Software: Thermo Xcalibur 3.1.66.10 Setting method: high_parameters.mstune Parameter
Shielding gas flow rate 7 Auxiliary gas flow rate 0
Purge gas flow rate 0 Spray voltage 3 kV
Capillary temperature 275 °C
S-lens RF 50 Instrumental method: 170406_PEPMAP_TOP10_120k_CAP_140.meth Parameter FULL MS Analysis time 140 min Internal source CID 0.0 eV
Default state of charge 2
Microscans 1
Resolution 120,000
AGC, target 3e6
Maximum IT 60
Number of scan ranges 1
Scan range 300-2000m/z
Spectrum Data Type Profile
Polarity positive dd-MS²
Microscans 1
Resolution 30,000
AGC, target 1e5
Maximum IT 100 ms
Number of cycles 10
MSX quantity 1
TopN 10
Insulation window 2.0m/z
Insulation displacement 0.0m/z
NCE/Step 27
Centroid Data Spectrum
Underfill ratio 0.6%
Intensity Threshold 1.0e3
Vertex trigger off
Charge exclusion not assigned, 1, 7, 8, >8
Peptide match is preferred
Exclude isotopes enabled
Dynamic exclusion 30s MS calibration/test resolution: Calibration solution
Pierce LTQ Velos ESI Positive Ion Calibration Solution Order No. 88323, lot RF231587 base parameter
Tune file: Calibration_pos_parameters_150818
Syringe pump flow rate: 5 µl
TIC Stability: ≤10%
IT (input time): ≤10ms
Ionic mode: positive Calibration parameter
eFT parameters (positive) ok
Analyzer accuracy (positive) approx.
Weight calibration (positive) ok Test Spectrum/Resolution
1. Calibration solution Resolution: FTMS 120000 (at m/z 200) Mass range: m/z 135-1800 AGC Target: 3e6
Number of scans: 50 scans
File name: 170621_Calmix.raw
2. m/z 524 MRFA (Komponente Kalibriermix)
Resolution: FTMS 120000 (at m/z 200) Mass range: m/z 520-530 AGC target: 1e5:
Number of scans 50 scans
File name: 170621_MRFA.raw
Resolution (m/z 524) 83281 (reference 80000)
Readouts: 1.67×10^7

[00253] Three different cleaning modes were carried out. One included a comparative procedure for this example, another included a test procedure for testing this example (which includes MustangQ anion exchange purification prior to affinity purification), and a third was performed according to the test procedure but without MustangQ anion exchange purification.

EXAMPLE 5

[00254] AAV9 production was developed in the HEK293 cell line after transfection with a triple plasmid system containing the coding cDNA of the protein of interest and AAV9-. VP1. -VP2 and -VP3. The clarified cell-free culture supernatant was concentrated and diafiltered with a Pall Omega T-Series 100 kDa cassette. Virus particles were loaded onto a membrane adsorber (MustangQ. Pall part number XT140MSTGQP05) under non-binding conditions. The resulting stream containing AAV9 was not adjusted to a pH in the range of 7.4 to 7.8 with 25% HCl. Instead, LOADING was generated by reconstituting a stream containing AAV-8 in loading buffer containing 125 mM NaCl and 50 mM Tris-HCl at pH 8.5.

[00255] In addition to the advantage of not having to include a pH adjustment step, having a pH of 8.5 can provide improved affinity stability and prevent non-specific binding of impurities to the product or resin.

[00256] Samples from various washing and elution steps were taken at various points to determine how much AAV9 was present in the sample. The analyzes showed how much AAV9 was lost during the various washing steps. The following test procedure was carried out. First, a column containing POROS™ CaptureSelect™ AAV9 affinity matrix (p/n A27354; Thermo Fisher) 10 mm ID, 25 mm bed height, and 1.96 ml volume was equilibrated with at least five column volumes of 20 mM Tris-HCl and 150 mM NaCl at pH 7.4. LOAD was applied to a column containing POROS™ CaptureSelect™ AAV9 affinity matrix (cat. A27354; Thermo Fisher). A portion of the sample loaded onto the column was saved and then analyzed by ITR qPCR, ELISA against AAV antigens, and ELISA against HEK293 HCP antigens. The column was then re-equilibrated with 10 column volumes of 20 mM Tris-HCl and 150 mM NaCl at pH 7.4. The flow-through sample was saved and then analyzed by ITR qPCR, ELISA against AAV antigens, and ELISA against HEK293 HCP antigens.

[00257] The column was then washed with 10 column volumes of Wash 1 (W1): 100 mM sodium acetate and 0.1% Tween 80 at pH 6.0. The column was then washed with 10 column volumes of Wash 2 (W2): 50 mM Tris-HCl and 125 mM NaCl at pH 8.5. The column was then washed with 10 column volumes of Wash 3 (W3): 50 mM Tris-HCl and 50% ethylene glycol at pH 8.5. A sample from the eluate of each of W1, W2 and W3 was taken and analyzed according to ITR qPCR, ELISA against AAV antigens and ELISA against HEK293 HCP antigens.

[00258] Elution was performed by applying 10 column volumes of the following elution buffer to the column: 50 mM Tris-HCl, 50% ethylene glycol and 750 mM NaCl, at pH 8.0.

[00259] The above test procedure is described in more detail in Table 18.

Table 18:

Stage LOADING WITH HIGH PH CV Flow rate one. 20 mM Tris-HCl
150 mM NaCl
pH 7.4 > 5 30 cm/h 2. Loading a Sample
pH 8.5 3. 20 mM Tris-HCl
150 mM NaCl
pH 7.4 10 4. 100 mM sodium acetate
0.1% Twin80
pH 6.0 10 five. 50 mM Tris-HCl
125 mM NaCl
pH 8.5 10 6. 50 mM Tris-HCl
50% ethylene glycol
pH 8.5 10 30 cm/h 7. Elution
50 mM Tris-HCl
50% ethylene glycol
750 mM NaCl
pH 8.0 10 eight. 50 mM Tris-HCl
50% ethylene glycol
2000 mM NaCl
pH 8.0 10

[00260] Selected samples were analyzed by each of ITR qPCR, ELISA against AAV antigens, and ELISA against HEK293 HCP to assess the yield and possible losses in stages.

[00261] The above samples were also analyzed using SDS-PAGE and 12% silver staining to determine the level of total protein.

[00262] Samples were then analyzed by SDS-PAGE and Western blotting against AAV9 antigens. The primary antibodies were monoclonal antibodies against VP1, VP2 and VP3 AAV9, while the secondary antibodies were a goat anti-mouse phosphatase-linked antibody.

EXAMPLE 6

[00263] AAV9 production was developed in the HEK293 cell line after transfection with a triple plasmid system containing the coding cDNA of the protein of interest and AAV9-. VP1. -VP2 and -VP3. The clarified cell-free culture supernatant was concentrated and diafiltered with a Pall Omega T-Series 100 kDa cassette. Virus particles were loaded onto a membrane adsorber (MustangQ; Pall part number XT140MSTGQP05) under non-binding conditions, ie in a solution containing 125 mM NaCl and 50 mM Tris-HCl at pH 8.5. pH dependent LOADING is achieved by adjusting the AAV9 containing stream to a pH range of 7.4 to 7.8 with 25% HCl.

[00264] The following test procedure was carried out. First, a column containing AAV8/9 anti-intact antibody (p/n 03-651161, American Research Products, Inc., Waltham, MA) immobilized on resin (“ADK8/9 affinity resin”) 32 mm ID, 59 mm bed height and volume of 47.45 ml was balanced with at least five column volumes of 20 mm Tris-HCl and 150 mm NaCl at pH 7.4. The pH conditioned LOADING was applied to a column containing ADK8/9 affinity resin. The column was then re-equilibrated with five column volumes of 20 mM Tris-HCl and 150 mM NaCl at pH 7.4 (optional fourth buffer).

[00265] The column was then washed with five column volumes of Wash 1 (W1): 100 mM sodium acetate and 0.1% Tween 80 at pH 6.0. The column was then washed with five column volumes of Wash 2 (W2): 50 mM Tris-HCl and 125 mM NaCl at pH 8.5. The column was then washed with five column volumes of Wash 3 (W3): 50 mM Tris-HCl and 50% ethylene glycol at pH 8.5.

[00266] Elution is accomplished by applying five column volumes of the following elution buffer to the column: 50 mM Tris-HCl, 50% ethylene glycol, and 750 mM NaCl, at pH 8.0. Five column volumes of the following secondary elution buffer were then applied to the column: 50 mM Tris-HCl, 50% ethylene glycol and 2000 mM NaCl.

[00267] The linear flow rate for the above steps was 60 cm/hr.

[00268] The following comparative procedure was carried out. A column containing ADK8/9 affinity resin, ID 10 mm, bed height 2.5 mm and volume 1.96 ml, was equilibrated with at least 10 column volumes of 20 mM Tris-HCl and 150 mM NaCl at pH 7.4. The pH conditioned LOADING was applied to a column containing ADK8/9 affinity resin. The column was then re-equilibrated with 10 column volumes of 20 mM Tris-HCl and 150 mM NaCl at pH 7.4.

[00269] The wash step was performed using the following TBS buffer: 20 mM Tris-HCl/150 mM NaCl/pH 7.4. Instead, elution was performed with 10 column volumes of 100 mM sodium citrate at pH 3.0.

[00270] The above test and comparison procedure is described in more detail in Table 1, where "CV" indicates the number of column volumes of the solution added in the step.

[00271] The density of the elution buffer was measured on a density meter with an oscillating U-tube DMA 4500M (Anton Paar).

[00272] ELISA was used to measure the amount of AAV9 antigen. ELISA was performed with an AAV-9 titration kit ELISA (No. PRAAV9; Progen (Heidelberg, Germany) on a TECAN Roboter system. Briefly, a monoclonal antibody specific for AAV9 capsids (antibody AAV8/9 (“antibody ADK8/9”, cat. 03 -651161, American Research Products, Inc., Waltham, MA) was applied to microtiter strips and used to capture AAV9 particles from the AAV fraction. Captured AAV9 particles were detected in two steps. /9, binds to immune complex (antibodies ADK8/9 and ADK8/9) Streptavidin peroxidase conjugates were added to immune complexes bound to biotin-conjugated monoclonal antibody and streptavidin peroxidase conjugates reacted with biotin Peroxidase substrate solution was added and color reaction occurred, response in which is proportional to the number of bound AAV particles The color reaction was measured photometrically at 450 nm.

[00273] The ITR-qPCR assay is used to determine the titer of a genome copy by quantifying the inverted tandem repeats found in a vector encoding a gene of interest (eg, human factor VIII or human factor IX). HEK-HCP is a measure of the level of residual host cell protein by ELISA. LDH is determined by colorimetric activity assay.

[00274] AAV8/9 ligand leak ELISA (linked enzyme immunosorbent assay) can detect an affinity ligand that may be present in a product purified with ADK8/9 affinity resin.

[00275] In an in vitro biopotency assay, the AAV9 viral vector infects a target hepatic cell line, which subsequently secretes a functional, measurable encoded protein into the medium. In the first step, HepG2 target cells are transduced by infection with AAV9. During incubation, the encoded protein is released into the cell supernatant. In the second step, the activity of the encoded protein in the cell culture supernatant is directly measured by the activity assay. The AAV9 sample measurement is given as a percentage of the reference material. The method allows to quantify the biological function of the AAV9 gene therapy vector.

[00276] SDS-PAGE analysis was performed to determine if a decrease in 70 kDa heat shock protein (HSP70) occurred when using a test procedure with washing steps instead of a comparative procedure. Western blotting was performed using an anti-Hsp70 antibody (Abcam, cat. no. ab79852) as the primary antibody at a dilution of 1:2000 for two hours and goat anti-rabbit IgG (H+L) AP conjugate as the secondary antibody (Sigma, No. A8025) at a 1:1000 dilution for one hour.

[00277] SDS-PAGE analysis for silver staining was performed to determine the total level of impurities present.

[00278] Western blot with 12% anti-AAV antibodies was performed to determine the levels and purity of AAV9 isolated after purification, in accordance with the test and comparison procedures. Western blotting was performed with monoclonal antibodies to VP1, VP2 and VP3 AAV9 as primary antibodies, with goat anti-mouse ALP antibody (Sigma, cat. no. A4656) as secondary antibody.

[00279] LC-MS was performed (rp-HPLC-UV-ESI-MS/MS) to determine the identity and amount of various host cell contaminants. Samples were digested using the trypsin enzyme. The resulting mixture of peptides was separated on an HPLC system using an RP column (column ZORBAX 300SB-C18, 0.5×150 mm, 3.5 μm), and then the peptides were analyzed on a Q-Exactive HF mass spectrometer. Data was analyzed using Proteome Discoverer software to identify proteins in the sample.

[00280] An Agilent HPLC1209 (1200 capHPLC) was used with a ChemStation for 3D LC systems (Rev. B.04.03-SP2 (105)). HPLC method - PEPMAP_CAP_170.M. Eluent A is 0.1% (v/v) HCOOH in deionized water and eluent B is 0.08% (v/v) HCOOH in acetonitrile. Detailed HPLC information is provided in the following Table 19:

Table 19:

HPLC Skid: HPLC1209: 1200capHPLC, Agilent Software: ChemStation for 3D LC Systems Rev. B.04.03-SP2 (105) HPLC method: PEPMAP_CAP_170.M Speaker: ZORBAX 300SB-C18 0.5×150mm, 3.5 µm Part. No. 5064-8268 Lot No. USHTC01001 Lot No. WSB1432005 Eluent A: 0.1% (v/v) HCOOH in deionized water, Lot 140617/01/ DF3376/028 Eluent B: 0.08% (v/v) HCOOH in acetonitrile, Lot 250117/02 /DF3270/015 Pump: Initial conditions: 15 µl/min (microflow) 100% A Gradient: 0 min 100%A 0%B
110 min 60%A 40%B 125 min 30%A 70%B
135 min 0%A 100%B 140 min 100%A 0%B
Stop 170 min Sampler: Input volume: Various input volumes Temperature: 4°C Closing a contact Initial OPEN 0.02 min A - CLOSED 0.5 min A - OPEN Speaker compartment: Temperature: 40°C DAD Detector: Wavelengths 214, 280, 260 nm, Spectrum 190-500nm Flushing the needle Flush port 5 sec (20% isopropanol/80% H2O)

[00281] Detailed information about the MS is given in the following Table 20:

Table 20:

Execution: Q Exactive HF #2 Electrospray Ionization (HESI), S/N 05161L Thermo, Source: HESI-II Software: Thermo Xcalibur 3.1.66.10 Setting method: high_parameters.mstune Parameter
Shielding gas flow rate 7 Auxiliary gas flow rate 0
Purge gas flow rate 0 Spray voltage 3 kV
Capillary temperature 275 °C
S-lens RF 50 Instrumental method: 170406_PEPMAP_TOP10_120k_CAP_140.meth Parameter FULL MS Analysis time 140 min Internal source CID 0.0 eV
Default state of charge 2
Microscans 1
Resolution 120,000
AGC, target 3e6
Maximum IT 60
Number of scan ranges 1
Scan range 300-2000m/z
Spectrum Data Type Profile
Polarity positive dd-MS²
Microscans 1
Resolution 30,000
AGC, target 1e5
Maximum IT 100 ms
Number of cycles 10
MSX quantity 1
TopN 10
Insulation window 2.0m/z
Insulation displacement 0.0m/z
NCE/Step 27
Centroid Data Spectrum
Underfill ratio 0.6%
Intensity Threshold 1.0e3
Vertex trigger off
Charge exclusion not assigned, 1, 7, 8, >8
Peptide match is preferred
Exclude isotopes enabled
Dynamic exclusion 30s MS calibration/test resolution: Calibration solution
Pierce LTQ Velos ESI Positive Ion Calibration Solution Order No. 88323, lot RF231587 base parameter
Tune file: Calibration_pos_parameters_150818
Syringe pump flow rate: 5 µl
TIC Stability: ≤10%
IT (input time): ≤10ms
Ionic mode: positive Calibration parameter
eFT parameters (positive) ok
Analyzer accuracy (positive) approx.
Weight calibration (positive) ok Test Spectrum/Resolution
1. Calibration solution Resolution: FTMS 120000 (at m/z 200) Mass range: m/z 135-1800 AGC Target: 3e6
Number of scans: 50 scans
File name: 170621_Calmix.raw
2. m/z 524 MRFA (Komponente Kalibriermix)
Resolution: FTMS 120000 (at m/z 200) Mass range: m/z 520-530 AGC target: 1e5:
Number of scans 50 scans
File name: 170621_MRFA.raw
Resolution (m/z 524) 83281 (reference 80000)
Readouts: 1.67×10^7

[00282] Three different cleaning modes were carried out. One included a comparative procedure for this example, another included a test procedure for testing this example (which includes MustangQ anion exchange purification prior to affinity purification), and a third was performed according to the test procedure but without MustangQ anion exchange purification.

EXAMPLE 7

[00283] AAV9 production was developed in the HEK293 cell line after transfection with a triple plasmid system containing the coding cDNA of the protein of interest and AAV9-. VP1. -VP2 and -VP3. The clarified cell-free culture supernatant was concentrated and diafiltered with a Pall Omega T-Series 100 kDa cassette. Virus particles were loaded onto a membrane adsorber (MustangQ. Pall part number XT140MSTGQP05) under non-binding conditions. The resulting stream containing AAV9 was not adjusted to a pH in the range of 7.4 to 7.8 with 25% HCl. Instead, the LOAD was formed by reconstituting the stream containing AAV9 in a loading buffer containing 125 mM NaCl and 50 mM Tris-HCl at pH 8.5.

[00284] In addition to the advantage of not having to include a pH adjustment step, having a pH of 8.5 can provide improved affinity stability and prevent non-specific binding of impurities to the product or resin.

[00285] Samples from various washing and elution steps were taken at various points to determine how much AAV9 was present in the sample. The analyzes showed how much AAV9 was lost during the various washing steps. The following test procedure was carried out. First, a column containing ADK8/9 affinity resin 10 mm ID, 25 mm bed height and 1.96 ml volume was equilibrated with at least five column volumes of 20 mM Tris-HCl and 150 mM NaCl at pH 7.4. LOAD was applied to a column containing ADK8/9 affinity resin. A portion of the sample loaded onto the column was saved and then analyzed by ITR qPCR, ELISA against AAV antigens, and ELISA against HEK293 HCP antigens. The column was then re-equilibrated with 10 column volumes of 20 mM Tris-HCl and 150 mM NaCl at pH 7.4. The flow-through sample was saved and then analyzed by ITR qPCR, ELISA against AAV antigens, and ELISA against HEK293 HCP antigens.

[00286] The column was then washed with 10 column volumes of Wash 1 (W1): 100 mM sodium acetate and 0.1% Tween 80 at pH 6.0. The column was then washed with 10 column volumes of Wash 2 (W2): 50 mM Tris-HCl and 125 mM NaCl at pH 8.5. The column was then washed with 10 column volumes of Wash 3 (W3): 50 mM Tris-HCl and 50% ethylene glycol at pH 8.5. A sample from the eluate of each of W1, W2 and W3 was taken and analyzed according to ITR qPCR, ELISA against AAV antigens and ELISA against HEK293 HCP antigens.

[00287] Elution was performed by applying 10 column volumes of the following elution buffer to the column: 50 mM Tris-HCl, 50% ethylene glycol and 750 mM NaCl, at pH 8.0.

[00288] The above test procedure is described in more detail in Table 21.

Table 21:

Stage LOADING WITH HIGH PH CV Flow rate one. 20 mM Tris-HCl
150 mM NaCl
pH 7.4 > 5 30 cm/h 2. Loading a Sample
pH 8.5 3. 20 mM Tris-HCl
150 mM NaCl
pH 7.4 10 4. 100 mM sodium acetate
0.1% Twin80
pH 6.0 10 five. 50 mM Tris-HCl
125 mM NaCl
pH 8.5 10 6. 50 mM Tris-HCl
50% ethylene glycol
pH 8.5 10 30 cm/h 7. Elution
50 mM Tris-HCl
50% ethylene glycol
750 mM NaCl
pH 8.0 10 eight. 50 mM Tris-HCl
50% ethylene glycol
2000 mM NaCl
pH 8.0 10

[00289] Selected samples were analyzed by each of ITR qPCR, ELISA against AAV antigens, and ELISA against HEK293 HCP to assess the yield and possible losses in stages.

[00290] The above samples were also analyzed by SDS-PAGE and 12% silver staining to determine the level of total protein.

[00291] Samples were then analyzed by SDS-PAGE and Western blotting against AAV9 antigens. The primary antibodies were monoclonal antibodies against VP1, VP2 and VP3 AAV9, while the secondary antibodies were a goat anti-mouse phosphatase-linked antibody.

EXAMPLE 8

[00292] The AAV8 ELISA was performed with the AAV-8 Titration ELISA kit (No. PRAAV8; Progen (Heidelberg, Germany) on a TECAN Roboter system. A monoclonal antibody specific for the conformational epitope on the assembled AAV8 capsids (ADK8) was applied to microtiter strips and was used to capture AAV8 particles from the AAV fraction Capture of AAV8 particles was detected in two steps In the first step, a biotin-conjugated monoclonal antibody specific for the ADK8 antibody was bound to the immune complex Streptavidin peroxidase conjugates were added to the immune complexes associated with the biotin-conjugated monoclonal antibody , and streptavidin peroxidase conjugates were reacted with biotin.The peroxidase substrate solution was added and a color reaction occurred which is proportional to the number of bound AAV particles.The color reaction was measured photometrically at 450 nm.

EXAMPLE 9

[00293] For the ITR-qPCR assays performed in the examples, the following procedure was undertaken. The titer of the vector genome (vg) per milliliter (ml) was determined using TaqMan-based qPCR with primers and a fluorescently labeled probe detecting the sequence in the ITR sequences of the vector genome. To detect the ITR-specific sequence in the AAV particle, the samples were subjected to various treatments. Samples were treated with DNase I followed by proteinase K so that the scAAV genome was released from the capsid. Restriction enzyme digestion was then performed to resolve the T-shaped structures of the AAV ITR.

[00294] The plasmid used as a reference material was linearized with a single cleavage restriction enzyme and then purified from the agarose gel. The UV A260 absorbance of the gel-extracted DNA was measured three times on a UV spectrophotometer, with the average DNA concentration calculated in μg per ml.

[00295] To determine the number of copies per ml for a linearized standard plasmid, the molecular weight of dsDNA was calculated in grams per mol, taking into account the exact mass for each individual nucleotide of the main sequence.

[00296] The titer of the vector genome in the test article (in vector genomes per ml) was calculated by fitting a standard plasmid curve with linear regression.

EXAMPLE 10

[00297] AAV8 production was developed in the HEK293 cell line after transfection with a triple plasmid system containing the coding cDNA of the protein of interest and AAV8-. VP1. -VP2 and -VP3. The clarified cell-free culture supernatant was concentrated and diafiltered with a Pall Omega T-Series 300 kDa cassette. Virus particles were loaded onto a membrane adsorber (MustangQ. Pall part number XT140MSTGQP05) under non-binding conditions for AAV8. The resulting stream containing AAV8 was diluted with dilution buffer containing 100 mM arginine, 200 mM NaCl at pH 8.0 to prepare the AAV8 affinity matrix load. The arginine-containing loading was applied to a column containing POROS ^™ CaptureSelect ^™ AAV8 affinity matrix (Thermo Fisher, cat. no. A30793).

[00298] Samples from various washing and elution steps were taken at various points to determine how much AAV8 was present in the sample. The analyzes show how much AAV8 was lost in the various washing steps. The following test procedure was carried out. First, a column containing ADK8/9 affinity resin 10 mm ID, 25 mm bed height and 2.04 ml volume was equilibrated with at least ten column volumes of 20 mM Tris-HCl and 125 mM NaCl at pH 8.5. The loading was applied to a column containing ADK8/9 affinity resin. Part of the sample loaded on the column was saved and then analyzed by ITR qPCR, ELISA against AAV antigens and ELISA against HEK293 HCP antigens. The column was then re-equilibrated (Wash 1, W1) with 10 column volumes of 50 mM Tris-HCl and 125 mM NaCl at pH 8.5. The flow-through sample was saved and then analyzed by ITR qPCR, ELISA against AAV antigens, and ELISA against HEK293 HCP antigens.

[00299] The column was then washed with 10 column volumes Wash 2 (W2): 100 mM sodium acetate and 0.1% Tween80 at pH 6.0. The column was then washed with 10 column volumes Wash 3 (W3): 50 mM Tris-HCl and 125 mM NaCl at pH 8.5. The column is then washed with 10 column volumes Wash 4 (W4): 50 mM Tris-HCl and 50% ethylene glycol at pH 8.5. A sample from the eluate of each of W2, W3 and W4 was taken and analyzed according to ITR qPCR, ELISA against AAV antigens and ELISA against HEK293 HCP antigens.

[00300] Elution was performed by injecting 10 column volumes of the following elution buffer per column: 50 mM Tris-HCl, 60% (w/w) ethylene glycol, and 750 mM NaCl, at pH 8.0. The washing step was subsequently omitted. Elution was performed by introducing 10 column volumes of the following elution buffer per column: 50 mM Tris-HCl, 60% (w/w) ethylene glycol and 1000 mM NaCl, at pH 8.0. The column was then washed with 20 mM Tris-HCl at pH 7.4. The column was then regenerated by applying to the column 10 column volumes of a solution containing 100 mM glycine and 200 mM NaCl at pH 2.5.

[00301] The above test procedure is described in more detail in Table 22.

Table 22:

STAGE Balancing 50 mM Tris-HCl
125 mM NaCl
pH 8.5 LOADING MUQ-FT Wash 1
(Rebalancing) 50 mM Tris-HCl
125 mM NaCl
pH 8.5 Flushing 2 100 mM sodium acetate
0.1% Twin 80
pH 6.0 Flushing 3 50 mM Tris-HCl
125 mM NaCl
pH 8.5 Wash 4 50 mM Tris-HCl
50% (w/w) ethylene glycol
pH 8.5 ELUTION 1 50 mM Tris-HCl
60% (w/w) ethylene glycol
750 mM NaCl
pH 8.0 ELUTION 2 50 mM Tris-HCl
60% (w/w) ethylene glycol
1000 mM NaCl
pH 8.0 Flushing 6 20 mM Tris-HCl
pH 7.4 Regeneration 100 mM glycine
200 mM NaCl
pH 2.5

EXAMPLE 11

[00302] AAV8 production was developed in the HEK293 cell line after transfection with a triple plasmid system containing the coding cDNA of the protein of interest and AAV8-. VP1. -VP2 and -VP3. From a collected 30 L aliquot, cells were disrupted using a Megatron MT3000 (Pall) followed by filtration of the solution containing AAV8 through: a) PDP8 depth filter, area 0.5m ² , b) V100 depth filter, area: 0.5m ² and c ) Kleenpak Capsule 0.2 µm, area 0.15 m ² . The clarified cell-free culture supernatant was concentrated and diafiltered with a Pall Omega T-Series 300 kDa cassette. Virus particles were loaded onto a membrane adsorber (MustangQ. Pall part number XT140MSTGQP05) under non-binding conditions for AAV8. The resulting stream containing AAV8 was diluted 1:2 with a dilution buffer containing 100 mM arginine, 200 mM NaCl at pH 8.0 to prepare the loading for the AAV8 affinity matrix. The arginine-containing loading was applied to a column containing POROS ^™ CaptureSelect ^™ AAV8 affinity matrix (Thermo Fisher, cat. no. A30793; ID 10 mm, layer height 26 mm, volume 2.04 ml).

[00303] Samples from various washing and elution steps were taken at various points to determine how much AAV8 was present in the sample. The analyzes show how much AAV8 was lost in the various washing steps. The following test procedure was carried out. First, a column containing ADK8/9 affinity resin 10 mm ID, 25 mm bed height and 2.04 ml volume was equilibrated with at least ten column volumes of 50 mM Arginine-HCl and 100 mM NaCl at pH 8.0. The loading was applied to a column containing ADK8/9 affinity resin. Part of the sample loaded on the column was saved and then analyzed by ITR qPCR, ELISA against AAV antigens and ELISA against HEK293 HCP antigens. The column was then re-equilibrated (Wash 1, W1) with 10 column volumes of 50 mM Arginine-HCl and 100 mM NaCl at pH 8.0. The flow-through sample was saved and then analyzed by ITR qPCR, ELISA against AAV antigens, and ELISA against HEK293 HCP antigens.

[00304] 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/D II solution (18.0 g Tween 80, 3.4 g DMSO and 3.6 g TnBP) at pH 6.0. The column was then washed with 10 column volumes of Wash 3 (W3): 50 mM Tris-HCl and 100 mM NaCl at pH 8.0. The column was then washed with 10 column volumes of Wash 4 (W4): 50 mM Tris-HCl and 50% sucrose at pH 8.5. A sample from the eluate of each of W2, W3 and W4 was taken and analyzed according to ITR qPCR, ELISA against AAV antigens and ELISA against HEK293 HCP antigens.

[00305] Elution was performed by injecting 10 column volumes of the following elution buffer per column: 50 mM Arginine-HCl, 55% (w/w) sucrose, 2 mM MgCl ₂ and 800 mM NaCl at pH 8.0. The column was then washed with 10 column volumes of Wash 5 (W5): 50 mM Arginine-HCl and 100 mM NaCl at pH 8.0. Elution was performed by injecting 10 column volumes of the following elution buffer per column: 50 mM Arginine-HCl, 50% (v/v) glycerol and 800 mM NaCl at pH 8.0. The column was then washed with 50 mM Arginine-HCl and 100 mM NaCl at pH 8.0. The column was then regenerated by applying to the column 10 column volumes of a solution containing 100 mM glycine and 200 mM NaCl at pH 2.7.

[00306] The above test procedure is described in more detail in Table 23. A linear flow rate of 39 cm/h was used for all stages.

Table 23:

STAGE Balancing 50 mM Arginine-HCl
100 mM NaCl
pH 8.0 LOADING MUQ-FT razb. 1:2 Wash 1
(Rebalancing) 50 mM Arginine-HCl
100 mM NaCl
pH 8.0 Flushing 2 100 mM MES sodium
10 mM EDTA
pH 6.0
11.2 g/kg S/D II solution
(18.0 g Tween 80, 3.4 g DMSO, 3.6 g TnBP) Flushing 3 50 mM Arginine-HCl
100 mM NaCl
pH 8.0 Wash 4 50 mM Arginine-HCl
50% (w/w) sucrose
pH 8.5 ELUTION 1 50 mM Arginine-HCl
55 (w/w)% sucrose
2 mM MgCl2
+ 800 mM NaCl
pH 8.0 Wash 5 50 mM Arginine-HCl
100 mM NaCl
pH 8.0 ELUTION 2 50 mM Arginine-HCl
50% (v/v) glycerin
+ 800 mM NaCl
pH 8.0 Flushing 6 50 mM Arginine-HCl
100 mM NaCl
pH 8.0 Regeneration 100 mM glycine
200 mM NaCl
pH 2.7

[00307] In the loading step above, a 1:2 dilution was made in the Mustang Q column to adjust the loading to conditions close to the equilibration buffer matrix. This step was done to investigate the effect of buffers in terms of AAV8 binding to the ligand. Any new compound introduced may have potential competitive properties and/or may potentially cause elution.

[00308] Selected samples were analyzed by each of ITR qPCR, ELISA against AAV antigens, and ELISA against HEK293 HCP to assess the yield and possible losses in stages. The chromatogram associated with the above example is shown in FIG. 7. The following Table 24 shows the samples taken from each of the above steps and the yield of each component is shown in Table 25.

Table 24:

Stage Buffer Flow rate CV Fraction Resin activation 100 mM glycine, pH 2.7 39 cm/h 10 Waste Balancing 50 mM arginine, 100 mM NaCl, pH 8.0 10 Waste Sample application MUQ_FT diluted 1:2 X FT/Flush 1 Wash 1 Rebalance 50 mM arginine, 100 mM NaCl, pH 8.0 10 FT/Flush 1 Flushing 2 100 mM MES sodium, 10 mM EDTA, pH 6.0+
9.09 g/kg polysorbate 80, 1.53 g/kg DMSO, 1.62 g/kg TnBP 10 Flushing 2 Flushing 3 50 mM arginine, 100 mM NaCl, pH 8.0 10 Flushing 3 Wash 4 50 mM Arginine-HCl, 50% (w/w) sucrose, pH 8.5 10 Wash 4 Elution 1 50 mM Arginine-HCl, 55 (w/w)% sucrose,
2 mM MgCl2, 800 mM NaCl, pH 8.0 10 Elution 1 Wash 5 50 mM arginine, 100 mM NaCl, pH 8.0 10 Wash 5 Elution 2 50 mM Arginine-HCl, 50% (w/w) glycerol
800 mM NaCl, pH 8.0 10 Elution 1 Flushing 6 50 mM arginine, 100 mM NaCl, pH 8.0 10 Flushing 6 Strip 100 mM glycine, pH 2.7 10 Str/Ntr

[00309] The "Wash 5" step reduced the elution frontal effects.

Table 25:

Stage Quantity AAV8
Antigen Total AAV8
Antigen % AAV8 antigen ITR-
qPCR Total
ITR-qPCR %
ITR-qPCR LOADING 273.18 14.1 3851.838 100.00% 18.2 4970 100.0% FT/Flush 1 279.53 0.1 27.953 0.73% on. on. on. Flushing 2 20.56 0.062 1.275 0.03% on. on. on. Flushing 3 20.55 0.065 1.336 0.03% on. on. on. Wash 4 24.33 6.35 154.496 4.01% on. on. on. Elution 1 25.76 50.92 1311.699 34.05% 29.8 768 15.44% Wash 5 21.44 10.53 225.763 5.86% on. on. on. Elution 2 23.07 19.79 456.555 11.85% 14.9 344E 6.91% Flushing 6 21.31 3.84 81.830 2.12% on. on. on. Strip 22.68 57.2 1297.296 33.68% 70.6 1600 32.21%

[00310] The above samples were also analyzed by SDS-PAGE and 12% silver staining for total protein. The results of the SDS-PAGE analysis are shown in FIG. eight.

EXAMPLE 12

[00311] AAV8 production was developed in the HEK293 cell line after transfection with a triple plasmid system containing the coding cDNA of the protein of interest and AAV8-. VP1. -VP2 and -VP3. From a collected 30 L aliquot, cells were disrupted using a Megatron MT3000 (Pall) followed by filtration of the solution containing AAV8 through: a) PDP8 depth filter, area 0.5m ² , b) V100 depth filter, area: 0.5m ² and c ) Kleenpak Capsule 0.2 µm, area 0.15 m ² . The clarified cell-free culture supernatant was concentrated and diafiltered with a Pall Omega T-Series 300 kDa cassette. Virus particles were loaded onto a membrane adsorber (MustangQ. Pall part number XT140MSTGQP05) under non-binding conditions for AAV8. The loading was loaded onto a column containing AAV8 POROS ^™ CaptureSelect ^™ affinity matrix (Thermo Fisher, cat. no. A30793; ID 10 mm, bed height 26 mm, volume 2.04 ml).

[00312] Samples from various washing and elution steps were taken at various points to determine how much AAV8 was present in the sample. The analyzes show how much AAV8 was lost in the various washing steps. The following test procedure was carried out. First, a column containing ADK8/9 affinity resin 10 mm ID, 25 mm bed height and 2.04 ml volume was equilibrated with at least ten column volumes of 50 mM Tris-HCl and 125 mM NaCl at pH 8.5. The loading was applied to a column containing ADK8/9 affinity resin. Part of the sample loaded on the column was saved and then analyzed by ITR qPCR, ELISA against AAV antigens and ELISA against HEK293 HCP antigens. The column was then re-equilibrated (Wash 1, W1) with 10 column volumes of 50 mM Tris-HCl and 125 mM NaCl at pH 8.5. The flow-through sample was saved and then analyzed by ITR qPCR, ELISA against AAV antigens, and ELISA against HEK293 HCP antigens.

[00313] The column was then washed with 10 column volumes Washes 2 (W2): 100 mM Taurine, 125 mM NaCl, at pH 8.5. The column was then washed with 10 column volumes Washes 3 (W3): 100 mM glycine at 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% octyl glycopyranoside, at pH 8.5. A sample from the eluate of each of W2, W3 and W4 was taken and analyzed according to ITR qPCR, ELISA against AAV antigens and ELISA against HEK293 HCP antigens.

[00314] Elution was performed by injecting 10 column volumes of the following elution buffer per column: 50 mM taurine, 60% (w/w) ethylene glycol, 750 mM NaCl, 0.1% octyl glycopyranoside at pH 8.0. The column was then washed with 10 column volumes of Wash 5 (W5): 50 mM Tris-HCl and 125 mM NaCl at pH 8.5. Elution was performed by injecting 10 column volumes of the following elution buffer per column: 1 M (NH ₄ ) ₂ SO ₄ , 50 mM Tris-HCl, 50% (v/v) ethylene glycol at pH 7.0. The column was then washed with 50 mM Tris-HCl and 125 mM NaCl at pH 8.5. The column was then regenerated by applying to the column 10 column volumes of a solution containing 100 mM glycine and 200 mM NaCl at pH 2.7.

[00315] The above test procedure is described in more detail in Table 26. A linear flow rate of 39 cm/h was used for all stages.

Table 26:

STAGE Balancing 100 mM glycine
200 mM NaCl
pH 2.7 LOADING MUQ-FT Wash 1
(Rebalancing) 50 mM Tris-HCl
125 mM NaCl
pH 8.5 Flushing 2 100 mM taurine
0.5% PEG 6000
pH 6.0 Flushing 3 100 mM glycine
pH 8.5 Wash 4 50 mM Taurine
50% (w/w) ethylene glycol
0.1% octylglycopyranoside
pH 8.5 ELUTION 1 50 mM Taurine
60% (w/w) ethylene glycol
750 mM NaCl
0.1% octylglycopyranoside
pH 8.0 Wash 5 50 mM Tris-HCl
125 mM NaCl
pH 8.5 ELUTION 2 1M(NH4)2SO4
50 mM Tris-HCl
50% (v/v) ethylene glycol
pH 7.0 Flushing 6 50 mM Tris-HCl
125 mM NaCl
pH 8.5 Regeneration 100 mM glycine
200 mM NaCl
pH 2.7

[00316] Selected samples were analyzed by each of ITR qPCR and ELISA (as described in Example 8 above) against AAV antigens and ELISA against HEK293 HCP to assess the yield and whether losses could occur at the stages. The chromatogram associated with the above example is shown in FIG. 9. The following Table 27 shows the samples taken from each of the above steps and the yield of each component is shown in Table 28.

Table 27:

Stage Buffer Flow rate CV Fraction Resin activation 100 mM glycine, pH 2.7 39cm/h 10 Waste Balancing 50 mM Tris-HCl, 125 mM NaCl, pH 8.5 10 Waste Sample application MUQ_FT (undiluted) X FT/Flush 1 Wash 1 Rebalance 50 mM Tris-HCl, 125 mM NaCl, pH 8.5 10 FT/Flush 1 Flushing 2 100 mM Taurine, 0.5% PEG 6000, pH 6.0 10 Flushing 2 Flushing 3 100 mM Glycine, pH 8.5 10 Flushing 3 Wash 4 50% ethylene glycol (w/w), 50 mM taurine,
0.1% octylglycopyranoside, pH 8.5 10 Wash 4 Elution 1 50% ethylene glycol (w/w), 50 mM taurine,
0.1% octylglycopyranoside, pH 8.0 + 750 mM NaCl 10 Elution 1 Wash 5 50 mM Tris-HCl, 125 mM NaCl, pH 8.5 10 Wash 5 Elution 2 1M ammonium sulfate, 50% ethylene glycol,
50 mM Tris-HCl, pH 7.0 10 Elution 1 Flushing 6 50 mM Tris-HCl, 125 mM NaCl, pH 8.5 10 Flushing 6 Strip 100 mM glycine, pH 2.7 10 Str/Ntr

[00317] The "Wash 5" step reduced the frontal elution effects.

Table 28:

Stage Quantity AAV8
Antigen Total AAV8
Antigen % AAV8 antigen ITR-
qPCR Total
ITR-qPCR %
ITR-qPCR LOADING 118.33 34.8 4111.968 100.00% 30.4 4111.97 100.0 FT/Flush 1 142.21 0.045 6.399 0.16% on. on. on. Flushing 2 20.47 0.0325 0.665 0.02% on. on. on. Flushing 3 20.44 0.0319 0.652 0.02% on. on. on. Wash 4 21.45 0.0159 0.341 0.01% on. on. on. Elution 1 22.14 177.95 3939.813 95.81% 177.95 3939.81 95.81 Wash 5 20.82 1.02 21.236 0.52% on. on. on. Elution 2 22.31 0.55 12.271 0.30% on. on. on. Flushing 6 21.11 0.0778 1.642 0.04% on. on. on. Strip 22.69 6.349 144.059 3.50% on. on. on.

[00318] The above samples were also analyzed by SDS-PAGE and 12% silver staining to detect total protein. The results of the SDS-PAGE analysis are shown in FIG. 10.

EXAMPLE 13

[00319] AAV8 production was developed in the HEK293 cell line after transfection with a triple plasmid system containing the coding cDNA of the protein of interest and AAV8-. VP1. -VP2 and -VP3. From a collected 30 L aliquot, cells were disrupted using a Megatron MT3000 (Pall) followed by filtration of the solution containing AAV8 through: a) PDP8 depth filter, area 0.5m ² , b) V100 depth filter, area: 0.5m ² and c ) Kleenpak Capsule 0.2 µm, area 0.15 m ² . The clarified cell-free culture supernatant was concentrated and diafiltered with a Pall Omega T-Series 300 kDa cassette. Virus particles were loaded onto a membrane adsorber (MustangQ. Pall part number XT140MSTGQP05) under non-binding conditions for AAV8. The resulting stream containing AAV8 was diluted 1:2 with dilution buffer [100 mM histidine, 200 mM NaCl, pH 8.5], an additional 4 g of polysorbate 80/kg was added to prepare a load on the AAV8 affinity resin. The histidine-containing load was applied to a column containing POROS™ CaptureSelect™ AAV8 affinity matrix (p/n A30793, Thermo Fisher; id 10 mm, layer height 26 mm, volume 2.04 ml).

[00320] Samples from various washing and elution steps were taken at various points to determine how much AAV8 was present in the sample. The analyzes show how much AAV8 was lost in the various washing steps. The following test procedure was carried out. First, a column containing ADK8/9 affinity resin 10 mm ID, 26 mm bed height and 2.04 ml volume was equilibrated with at least ten column volumes of 50 mM Tris-HCl and 125 mM NaCl at pH 8.5. The loading was applied to a column containing ADK8/9 affinity resin. A portion of the sample loaded on the column was saved and then analyzed by ITR qPCR and ELISA against AAV antigens (as described in Example 9 above) and ELISA against HEK293 HCP antigens (as described in Example 8 above). The column was then re-equilibrated (Wash 1, W1) with 10 column volumes of 50 mM histidine and 100 mM NaCl at pH 8.5. The flow-through sample was saved and then analyzed by ITR qPCR and ELISA (as described in Examples 8 and 9 above) against AAV antigens and ELISA against HEK293 HCP antigens.

[00321] The column was washed with 10 column volumes 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 (w/w), at pH 6.0 The column was then washed with 10 column volumes Wash 3 (W3): 10 mM sodium citrate at pH 8.5 The column was then washed with 10 column volumes Wash 4 (W4): 100 mM Arginine-HCl , 100 mM Lysine-HCl, 100 mM histidine-HCl, 2 mM N-acetyl-D, L-tryptophan, at pH 8.5, with 20% (w/w) polysorbate 80. Sample from eluate of each of W2 , W3 and W4 were selected and analyzed according to ITR qPCR, ELISA against AAV antigens and ELISA against HEK293 HCP antigens (as described in Examples 8 and 9 above).

[00322] Elution was performed by injecting 10 column volumes of the following elution buffer per column: 20% (w/w) sucrose, 10% (w/w) sorbitol, 5% (w/w) mannitol ( sucrose), 15% (w/w) glycerol, 50 mM histidine, 800 mM NaCl, at pH 8.0. The column was then washed with 10 column volumes Wash 5 (W5): 50 mM Histidine and 100 mM NaCl at pH 8.5. Elution was performed by injecting 10 column volumes of the following elution buffer per column: 50 mM Tris-HCl, 750 mM NaCl and 50% (w/w) DMSO at pH 8.0. The column was then washed with 50 mM histidine and 100 mM NaCl at pH 8.5. The column was then regenerated by applying to the column 10 column volumes of a solution containing 100 mM glycine and 200 mM NaCl at pH 2.7.

[00323] The above test procedure is described in more detail in Table 29. A linear flow rate of 39 cm/h was used for all stages.

TABLE 29:

STAGE Balancing 100 mM glycine
200 mM NaCl
pH 2.7 LOADING MUQ-FT razb. 1:2 Wash 1
(Rebalancing) 50 mM histidine
100 mM NaCl
pH 8.5 Flushing 2 200 mM Bis-Tris
16.6 g S/D solution
(Triton X100 : Polysorbate 80 : TNBP=10.87 : 3.31 : 3.01 (by mass)
pH 6.0 Flushing 3 10 mmol sodium citrate
pH 8.5 Wash 4 100 mM Arginine-HCl
100 mM Lysine-HCl
100 mM Histidine-HCl
2 mM N-Acetyl-D, L-Tryptophan
pH 8.5
20% (w/w) Polysorbate 80
(Elution/Wash+SD) ELUTION 1 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 Wash 5 50 mM histidine
100 mM NaCl
pH 8.5 ELUTION 2 50 mM Tris-HCl
750 mM NaCl
50% (w/w) DMSO
pH 8.0 Flushing 6 50 mM histidine
100 mM NaCl
pH 8.5 Regeneration 100 mM glycine
200 mM NaCl
pH 2.7

[00324] The "Wash 5" step reduced the elution frontal effects. No elution of AAV8 was detected in the second elution step (elution 2). For AAV8, DMSO may still be suitable as a wash buffer and/or as a buffer for potential inactivation or disintegration of lipid-enveloped viruses.

[00325] Selected samples were analyzed by each of ITR qPCR, ELISA against AAV antigens and ELISA against HEK293 HCP (as described in Examples 8 and 9 above) to assess the yield and possible losses in stages. The chromatogram associated with the above example is shown in FIG. 11. Table 30 below shows the samples taken from each of the above steps and the yield of each component is shown in Table 31.

[00326] Unexpectedly, elution of AAV8 from Capture Select AAV8 resin occurs under near neutral conditions in the presence of polyols and a certain amount of salt. The resin manufacturer suggested that the elution required acidic conditions below pH 3.5. It was also surprising that the elution can be initiated by sugars, sugar alcohols and/or a mixture of sugars and sugar alcohols. In addition, it was unexpected that DMSO as a polar solvent could not elute AAV8 from the Capture Select AAV8 resin.

Table 30:

Stage Buffer Flow rate CV Fraction Resin activation 100 mM glycine, pH 2.7 39cm/h 10 Waste Balancing 50 mM Histidine, 100 mM NaCl, pH 8.5 10 Waste Sample application MUQ_FT 1:2 X FT/Flush 1 Wash 1 Rebalance 50 mM Histidine, 100 mM NaCl, pH 8.5 10 FT/Flush 1 Flushing 2 200 mM Bis-Tris+16.6 g/kg S/D solution 10 Flushing 2 Flushing 3 10 mmol sodium citrate, pH 8.5 10 Flushing 3 Wash 4 100 mM Arginine, 100 mM Lysine,
100 mM Histidine
2 mM N-Acetyl-D, L-Tryptophan,
20% (w/w) Polysorbate 80 pH 8.5 10 Wash 4 Elution 1 20% Sucrose, 10% (w/w) Sorbitol,
5% (w/w) Mannitol, 15% (w/w) Glycerin
50 mM Histidine, 800 mM NaCl, pH 8.0 10 Elution 1 Wash 5 50 mM Histidine, 100 mM NaCl, pH 8.5 10 Wash 5 Elution 2 50 mM Tris-HCl, 750 mM NaCl,
50% (w/w) DMSO, pH 8.0 10 Elution 2 Flushing 6 50 mM Histidine, 100 mM NaCl, pH 8.5 10 Flushing 6 Strip 100 mM glycine, pH 2.7 10 Str/Ntr

[00327] The "Wash 5" step reduced the elution frontal effects. No elution of AAV8 was detected in the second elution step (elution 2).

Table 31:

Stage Quantity AAV8
Antigen Total AAV8
Antigen % AAV8 antigen ITR-
qPCR Total
ITR-qPCR %
ITR-qPCR LOADING 244.41 17.2 4191.632 100.00% 3.63 4191.6 100 FT/Flush 1 368.03 0.037 13.617 0.32% on. on. on. Flushing 2 21.24 0.0755 1.604 0.04% on. on. on. Flushing 3 20.61 0.031 0.639 0.02% on. on. on. Wash 4 21.09 0.016 0.337 0.01% on. on. on. Elution 1 23.68 47.35 1121.248 26.75% 126 2980 33.63 Wash 5 21.45 6.1 130.845 3.12% on. on. on. Elution 2 22.22 0.016 0.356 0.01% on. on. on. Flushing 6 21.72 0.031 0.673 0.02% on. on. on. Strip 23.47 113.4 2661.5 63.5% 8.27 194 2.19

[00328] The above samples were also analyzed by SDS-PAGE and 12% silver staining for total protein. The results of the SDS-PAGE analysis are shown in FIG. 12.

EXAMPLE 14

[00329] AAV2 production was developed in the HEK293 cell line after transfection with a triple plasmid system containing the coding cDNA of the protein of interest and AAV2-. VP1. -VP2 and -VP3. AAV2 sample was diluted with 150 mM NaCl, 20 mM Tris-HCl, pH 7.4 to ensure loading. Loaded on a column containing POROS™ CaptureSelect™ AAVX affinity matrix (Thermo Fisher, cat. no.: A36739, Thermo Fisher; id 10 mm, layer height 25 mm, volume 1.96 ml).

[00330] Samples from various washing and elution steps were taken at various points to determine how much AAV2 was present in the sample. The analyzes show how much AAV2 was lost in the various washing steps. The following test procedure was carried out. First, a column containing ADK8/9 affinity resin 10 mm ID, 25 mm bed height and 1.96 ml volume was equilibrated with at least ten column volumes of 20 mM Tris-HCl, 150 mM NaCl at pH 7.4. The loading was applied to a column containing ADK8/9 affinity resin. A portion of the sample loaded on the column was saved and then analyzed by ITR qPCR, ELISA against AAV antigens and ELISA against HEK293 HCP antigens (as described in Examples 8 and 9 above). The column was then re-equilibrated (Wash 1, W1) with 10 column volumes of 20 mM Tris-HCl, 150 mM NaCl at pH 7.4. The flow-through sample was saved and then analyzed by ITR qPCR, ELISA against AAV antigens, and ELISA against HEK293 HCP antigens (as described in Examples 8 and 9 above).

[00331] The column was then washed with 10 column volumes Wash 2 (W2): 100 mM sodium acetate, 0.1% Polysorbate 80 at pH 6.0. The column was then washed with 10 column volumes Wash 3 (W3): 50 mM Tris-HCl and 125 mM NaCl at pH 8.5. The column was then washed with 10 column volumes Wash 4 (W4): 50 mM Tris-HCl, 50% (w/w) ethylene glycol at pH 8.5. The column was then washed with 10 column volumes Wash 5 (W5): 50 mM Tris-HCl, 60% (w/w) ethylene glycol+750 mM NaCl, pH 8.0. The column was then washed with 10 column volumes Wash 6 (W6): 20 mM Tris-HCl and 150 mM NaCl at pH 7.4. A sample from the eluate of each of W2, W3 and W4 was taken and analyzed according to ITR qPCR, ELISA against AAV antigens and ELISA against HEK293 HCP antigens (as described in Examples 8 and 9 above).

[00332] Elution was performed by introducing 10 column volumes of the following elution buffer per column: 100 mM Glycine-HCl, 200 mM NaCl, pH 2.5. The column was then washed with 10 column volumes Wash 7 (W7): 20 mM Tris-HCl and 150 mM NaCl at pH 7.4. The column was stripped with 10 column volumes of 50 mM phosphoric acid at pH 2.0, washed with 10 column volumes of Wash 8 (W8): 20 mM Tris-HCl, 150 mM NaCl at pH 7.4, and then stripped with 10 column volumes of 50 mM phosphoric acid at pH 2.0.

[00333] The above test procedure is described in more detail in Table 32. A linear flow rate of 39 cm/h was used for all stages.

Table 32:

STAGE Balancing 20 mM Tris-HCl, 150 mM NaCl, pH 7.4 Wash 1
(Rebalancing) 20 mM Tris-HCl, 150 mM NaCl, pH 7.4 Flushing 2 100 mM sodium acetate, 0.1% polysorbate 80, pH 6.0 Flushing 3 50 mM Tris-HCl, 125 mM NaCl, pH 8.5 Wash 4 50 mM Tris-HCl, 50% (w/w) ethylene glycol, pH 8.5 Wash 5 50 mM Tris-HCl, 60% (w/w) ethylene glycol+750 mM NaCl
pH 8.0 Flushing 6 20 mM Tris-HCl, 150 mM NaCl, pH 7.4 ELUTION 1 100 mM Glycine HCl, 200 mM NaCl, pH 2.5 Wash 7 20 mM Tris-HCl, 150 mM NaCl, pH 7.4 Stripping - first 50 mM phosphoric acid pH 2.0 Flushing 8 20 mM Tris-HCl, 150 mM NaCl, pH 7.4 Stripping - second 50 mM phosphoric acid pH 2.0

[00334] Selected samples were analyzed by each of ITR qPCR, ELISA against AAV antigens and ELISA against HEK293 HCP (as described in Examples 8 and 9 above) to assess the yield and possible losses in stages. The chromatogram associated with the above example is shown in FIG. 13. Table 33 below shows the samples taken from each of the above steps and the yield of each component is shown in Table 34.

Table 33:

Stage Buffer Flow rate CV Fraction Resin activation --- 39cm/h 10 Waste Balancing 20 mM Tris-HCl, 150 mM NaCl, pH 7.4 10 Waste Sample application 2x flow via TMAE X FT/Flush 1 Wash 1 Rebalance 20 mM Tris-HCl, 150 mM NaCl, pH 7.4 10 FT/Flush 1 Flushing 2 100 mM sodium acetate, 0.1% polysorbate 80, pH 6.0 10 Flushing 2 Flushing 3 50 mM Tris-HCl, 125 mM NaCl, pH 8.5 10 Flushing 3 Wash 4 50 mM Tris-HCl, 50% (w/w) ethylene glycol, pH 8.5 10 Wash 4 Wash 5 50 mM Tris-HCl, 60% (w/w) ethylene glycol+750 mM NaCl
pH 8.0 10 Wash 5 Flushing 6 20 mM Tris-HCl, 150 mM NaCl, pH 7.4 10 Flushing 6 ELUTION 1 100 mM Glycine HCl, 200 mM NaCl, pH 2.5 10 elution Wash 7 20 mM Tris-HCl, 150 mM NaCl, pH 7.4 Wash 7 Strip 50 mM phosphoric acid pH 2.0 10 Strip 1 Flushing 8 20 mM Tris-HCl, 150 mM NaCl, pH 7.4 10 Flushing 8 Strip 2 50 mM phosphoric acid pH 2.0 10 Strip 2

Table 34:

Stage Quantity
[ML] ITR-
qPCR
E+11
vg/ml Total
ITR-qPCR
E+11
vg %
ITR-qPCR LOADING 22.57 12.6 284.38 100.0% Elution 1 19.97 14.0 279.58 98.3%

[00335] The above samples were also analyzed by SDS-PAGE and 12% silver staining to detect total protein. The results of the SDS-PAGE analysis are shown in FIG. 14 (see lanes 2-11).

[00336] Samples were then analyzed by SDS-PAGE and Western blotting against AAV antigens. Primary antibodies are monoclonal antibodies to AAV VP1, VP2 and VP3, while secondary antibodies are goat anti-mouse antibodies linked to alkaline phosphatase. The results of the Western blot analysis are shown in FIG. 15 (see lanes 2-11).

EXAMPLE 15

[00337] AAV9 production was developed in the HEK293 cell line after transfection with a triple plasmid system containing the coding cDNA of the protein of interest and AAV9-, VP1, -VP2 and -VP3. AAV9 sample was diluted with 150 mM NaCl, 20 mM Tris-HCl, pH 7.4 to ensure loading. Loaded on a column containing POROS™ CaptureSelect™ AAVX affinity matrix (Thermo Fisher, cat. no.: A36739, Thermo Fisher; id 10 mm, layer height 25 mm, volume 1.96 ml).

[00338] Samples from various washing and elution steps were taken at various points to determine how much AAV9 was present in the sample. The analyzes show how much AAV9 was lost in the various washing steps. The following test procedure was carried out. First, a column containing ADK8/9 affinity resin 10 mm ID, 25 mm bed height and 1.96 ml volume was equilibrated with at least ten column volumes of 20 mM Tris-HCl, 150 mM NaCl at pH 7.4. The loading was applied to a column containing ADK8/9 affinity resin. A portion of the sample loaded onto the column was saved and then analyzed by ITR qPCR, ELISA against AAV antigens, and ELISA against HEK293 HCP antigens (as described in Examples 8 and 9 above). The column was then re-equilibrated (Wash 1, W1) with 10 column volumes of 20 mM Tris-HCl, 150 mM NaCl at pH 7.4. The flow-through sample was saved and then analyzed by ITR qPCR, ELISA against AAV antigens, and ELISA against HEK293 HCP antigens (as described in Examples 8 and 9 above).

[00339] The column was then washed with 10 column volumes Wash 2 (W2): 100 mM sodium acetate, 0.1% Polysorbate 80 at pH 6.0. The column was then washed with 10 column volumes Wash 3 (W3): 50 mM Tris-HCl and 125 mM NaCl at pH 8.5. The column was then washed with 10 column volumes Wash 4 (W4): 50 mM Tris-HCl, 50% (w/w) ethylene glycol at pH 8.5. The column was then washed with 10 column volumes Wash 5 (W5): 50 mM Tris-HCl, 60% (w/w) ethylene glycol+750 mM NaCl, pH 8.0. The column was then washed with 10 column volumes Wash 6 (W6): 20 mM Tris-HCl and 150 mM NaCl at pH 7.4. A sample from the eluate of each of W2, W3 and W4 was taken and analyzed according to ITR qPCR, ELISA against AAV antigens and ELISA against HEK293 HCP antigens (as described in Examples 8 and 9 above).

[00340] Elution was performed by introducing 10 column volumes of the following elution buffer per column: 100 mM Glycine HCl, 200 mM NaCl, pH 2.5. The column was then washed with 10 column volumes Wash 7 (W7): 20 mM Tris-HCl and 150 mM NaCl at pH 7.4. The column was stripped with 10 column volumes of 50 mM phosphoric acid at pH 2.0, washed with 10 column volumes of Wash 8 (W8): 20 mM Tris-HCl, 150 mM NaCl at pH 7.4, and then stripped with 10 column volumes of 50 mM phosphoric acid at pH 2.0.

[00341] The above test procedure is described in more detail in Table 35. A linear flow rate of 39 cm/h was used for all stages.

Table 35:

STAGE Balancing 20 mM Tris-HCl, 150 mM NaCl, pH 7.4 Sample application 2x flow via TMAE Wash 1
(Rebalancing) 20 mM Tris-HCl, 150 mM NaCl, pH 7.4 Flushing 2 100 mM sodium acetate, 0.1% polysorbate 80, pH 6.0 Flushing 3 50 mM Tris-HCl, 125 mM NaCl, pH 8.5 Wash 4 50 mM Tris-HCl, 50% (w/w) ethylene glycol, pH 8.5 Wash 5 50 mM Tris-HCl, 60% (w/w) ethylene glycol+750 mM NaCl
pH 8.0 Flushing 6 20 mM Tris-HCl, 150 mM NaCl, pH 7.4 ELUTION 1 100 mM Glycine HCl, 200 mM NaCl, pH 2.5 Wash 7 20 mM Tris-HCl, 150 mM NaCl, pH 7.4 Stripping - first 50 mM phosphoric acid pH 2.0 Flushing 8 20 mM Tris-HCl, 150 mM NaCl, pH 7.4 Stripping - second 50 mM phosphoric acid pH 2.0

[00342] Selected samples were analyzed by each of ITR qPCR, ELISA against AAV antigens and ELISA against HEK293 HCP (as described in Examples 8 and 9 above) to assess the yield and possible losses in stages. The chromatogram associated with the above example is shown in FIG. 16. Table 36 below shows the samples taken from each of the above steps and the yield of each component is shown in Table 37.

Table 36:

Stage Buffer Flow rate CV Fraction Resin activation --- 39cm/h 10 Waste Balancing 20 mM Tris-HCl, 150 mM NaCl, pH 7.4 10 Waste Sample application 2x flow via TMAE X FT/Flush 1 Wash 1 Rebalance 20 mM Tris-HCl, 150 mM NaCl, pH 7.4 10 FT/Flush 1 Flushing 2 100 mM sodium acetate, 0.1% polysorbate 80, pH 6.0 10 Flushing 2 Flushing 3 50 mM Tris-HCl, 125 mM NaCl, pH 8.5 10 Flushing 3 Wash 4 50 mM Tris-HCl, 50% (w/w) ethylene glycol, pH 8.5 10 Wash 4 Wash 5 50 mM Tris-HCl, 60% (w/w) ethylene glycol+750 mM NaCl
pH 8.0 10 Wash 5 Flushing 6 20 mM Tris-HCl, 150 mM NaCl, pH 7.4 10 Flushing 6 ELUTION 1 100 mM Glycine HCl, 200 mM NaCl, pH 2.5 10 elution Wash 7 20 mM Tris-HCl, 150 mM NaCl, pH 7.4 Wash 7 Strip 50 mM phosphoric acid pH 2.0 10 Strip 1 Flushing 8 20 mM Tris-HCl, 150 mM NaCl, pH 7.4 10 Flushing 8 Strip 2 50 mM phosphoric acid pH 2.0 10 Strip 2

Table 37:

Stage Quantity
[ML] ITR-
qPCR
E+11
vg/ml Total
ITR-qPCR
E+11
vg %
ITR-qPCR LOADING 22.46 41.1 923.11 100.0% Elution 1 19.97 53.10 1060.41 114.9%

[00343] The above samples were also analyzed by SDS-PAGE and 12% silver staining for total protein. The results of the SDS-PAGE analysis are shown in FIG. 14 (see lanes 12-20).

[00344] Samples were then analyzed by SDS-PAGE and Western blotting against AAV antigens. Primary antibodies are monoclonal antibodies to AAV VP1, VP2 and VP3, while secondary antibodies are goat anti-mouse antibodies linked to alkaline phosphatase. The results of the Western blot analysis are shown in FIG. 15 (see lanes 12-20).

EXAMPLE 16

[00345] This example demonstrates elution conditions improved for AAV9 with respect to the use of acid glycine or phosphoric acid.

[00346] AAV9 production was developed in the HEK293 cell line after transfection with a triple plasmid system containing the coding cDNA of the protein of interest and AAV9-. VP1. -VP2 and -VP3. From a collected 30 L aliquot, cells were disrupted using a Megatron MT3000 (Pall) followed by filtration of the solution containing AAV9 through: a) PDP8 depth filter, area 0.5m ² , b) V100 depth filter, area: 0.5m ² and c ) Kleenpak Capsule 0.2 µm, area 0.15 m ² . The clarified cell-free culture supernatant was concentrated and diafiltered with a Pall Omega T-Series 300 kDa cassette. Virus particles were loaded onto a membrane adsorber (MustangQ. Pall part number XT140MSTGQP05) under non-binding conditions for AAV9. The loading was applied to a column containing POROS™ CaptureSelect™ AAVX affinity matrix (Thermo Fisher, cat. no. A36739, Thermo Fisher), ID 16 mm, bed height 50 mm, volume 10.0 ml, followed by buffer steps with column volume , as stated.

[00347] Samples from various washing and elution steps were taken at various points to determine how much AAV9 was present in the sample. The analyzes show how much AAV9 was lost in the various washing steps. The following test procedure was carried out. First, the column was equilibrated with at least five column volumes of 50 mM Tris-HCl, 125 mM NaCl at pH 8.5. The loading was applied to a column containing an affinity resin. Part of the sample loaded on the column was saved and then analyzed by ITR qPCR, ELISA against AAV antigens and ELISA against HEK293 HCP antigens. The column was then re-equilibrated (Wash 1, W1) with 5 column volumes of 50 mM Tris-HCl, 125 mM NaCl at pH 8.5. The flow-through sample was saved and then analyzed by ITR qPCR, ELISA against AAV antigens, and ELISA against HEK293 HCP antigens.

[00348] The column was then washed with 5 column volumes Wash 2 (W2): 100 mM sodium acetate, 0.1% Polysorbate 80 at pH 6.0. The column was then washed with 10 column volumes Wash 3 (W3): 50 mM Tris-HCl and 125 mM NaCl at pH 8.5. A sample from the eluate of each of W2 and W3 was taken and analyzed according to ITR rPCR, ELISA against AAV antigens and ELISA against HEK293 HCP antigens.

[00349] Elution was performed by first using 20 column volumes of purified water, then 20 column volumes of 1 mM HCl at pH 3.2, and then 20 column volumes of 50 mM Tris-HCl, 750 mM NaCl, 50% DMSO (w/w .) at pH 8.0. The column was washed with 20 volumes of purified water followed by 10 volumes of 33 mM HCl at pH 2.0.

[00350] The above test procedure is described in more detail in Table 38. A linear flow rate of 39 cm/h was used for all stages.

Table 38:

STAGE Balancing 50 mM Tris-HCl
125 mM NaCl
pH 8.5 LOADING AAV9 PPHT2_1813MUQ_FT , pH8.5 Wash 1
(Rebalancing) 50 mM Tris-HCl
125 mM NaCl
pH 8.5 Flushing 2 100 mM sodium acetate
0.1% Polysorbate 80
pH 6.0 Flushing 3 50 mM Tris-HCl
125 mM NaCl
pH 8.5 ELUTION 1 Purified water ELUTION 2 1 mM HCl, pH 3.2 ELUTION 3 50 mM Tris-HCl
750 mM NaCl
50% DMSO (w/w)
pH 8.0 Wash 4 Purified water ELUTION 4 33 mM HCl, pH 2.0

[00351] Selected samples were analyzed by each of the ITR qPCR and ELISA (as described in Example 8 above) against AAV antigens and ELISA against HEK293 HCP to assess the yield and whether losses could occur at the stages. The chromatogram associated with the above example is shown in FIG. 17. Table 39 below shows the samples taken from each of the above steps and the yield of some components is shown in Table 40.

Table 39:

Stage Buffer Flow rate CV Fraction Balancing 50 mM Tris-HCl, 125 mM NaCl, pH 8.5 39cm/h five Waste Sample application AAV9 PPHT2_1813MUQ_FT , pH8.5 X FT/Flush 1 Wash 1 Rebalance 50 mM Tris-HCl, 125 mM NaCl, pH 8.5 five FT/Flush 1 Flushing 2 100 mM sodium acetate, 0.1% polysorbate 80, pH 6.0 five Flushing 2 Flushing 3 50 mM Tris-HCl, 125 mM NaCl, pH 8.5 five Flushing 3 Elution 1 Purified water twenty Wash 5 Elution 2 1 mM Hydrochloric acid, pH 3.2 twenty Flushing 6 Elution 3 50 mM Tris-HCl, 750 mM NaCl, 50% DMSO (w/w), pH 8.0 10 elution flushing Purified water twenty Wash 7 Elution 4 33 mM hydrochloric acid, pH 2.0 10 Strip 1 Other stages Regeneration procedure x X

Table 40:

Stage Quantity
[ML] AAV9
Antigen
cp/ml
[10+11] Total
AAV9:AG
cp
[10+11] % LOADING 1011.85 7.44 7528.16 100.0 Elution 2 1 mM HCl 40.06 117.0 4687.02 62.3 Elution with 3 DMSO 6.19 61 377.6 5.0 Strip 33 mM HCl 9.31 54.5 507.4 6.7

[00352] The above samples were also analyzed by SDS-PAGE and Western blot with the results shown in FIG. eighteen.

[00353] AAV9 can be eluted from Capture select AAVx with 1 mM hydrochloric acid, which has the advantage that only a very small amount of free acid is introduced into the aqueous system. A further near neutral pH elution procedure can be carried out using DMSO containing an aqueous buffer solution (750 mM NaCl, 50 mM Tris-HCl, 50% w/w dimethyl sulfoxide). DMSO may be useful if the eluate containing DMSO is used for ultracentrifugation using a sucrose gradient. Both elution strategies potentially have improved properties for maintaining biopotency during elution from the AAVX resin for all AAV subtypes compared to stringent elution conditions at 100 mM below pH 3 and/or in the presence of large amounts of sodium or magnesium chloride.

EXAMPLE 17

[00354] This example demonstrates elution conditions improved for AAV9 with respect to the use of acid glycine or phosphoric acid.

[00355] AAV9 production was developed in the HEK293 cell line after transfection with a triple plasmid system containing the cDNA coding for the protein of interest (FIX-padua, double stranded) and AAV9-. VP1. -VP2 and -VP3. From a collected 30 L aliquot, cells were disrupted using a Megatron MT3000 (Pall) followed by filtration of the solution containing AAV9 through: a) PDP8 depth filter, area 0.5m ² , b) V100 depth filter, area: 0.5m ² and c ) Kleenpak Capsule 0.2 µm, area 0.15 m ² . The clarified cell-free culture supernatant was concentrated and diafiltered with a Pall Omega T-Series 300 kDa cassette. Virus particles were loaded onto a membrane adsorber (MustangQ. Pall part number XT140MSTGQP05) under non-binding conditions for AAV9. The loading was applied to a column containing POROS™ CaptureSelect™ AAVX affinity matrix (Thermo Fisher, cat. no. A36739; 10 mm, layer height 28 mm, volume 2.2 ml).

[00356] Samples from various washing and elution steps were taken at various points to determine how much AAV9 was present in the sample. The analyzes show how much AAV9 was lost in the various washing steps. The following test procedure was carried out. First, a column containing 10 mm ID affinity resin with a bed height of 28 mm and a volume of 2.2 ml was equilibrated with at least ten column volumes of 50 mM Tris-HCl, 125 mM NaCl at pH 8.5. The loading was applied to a column containing an affinity resin. Part of the sample loaded on the column was saved and then analyzed by ITR qPCR, ELISA against AAV antigens and ELISA against HEK293 HCP antigens. The column was then re-equilibrated (Wash 1, W1) with 10 column volumes of 50 mM Tris-HCl and 125 mM NaCl at pH 8.5. The flow-through sample was saved and then analyzed by ITR qPCR, ELISA against AAV antigens, and ELISA against HEK293 HCP antigens.

[00357] The column was then washed with 10 column volumes Wash 2 (W2): 100 mM sodium acetate, 0.1% Polysorbate 80 at pH 6.0. The column was then washed with 10 column volumes Wash 3 (W3): 50 mM Tris-HCl and 125 mM NaCl at pH 8.5. The column was then washed with 30 volumes of purified water (Wash 4 (W4)). A sample from the eluate of each of W2, W3 and W4 was taken and analyzed according to ITR qPCR, ELISA against AAV antigens and ELISA against HEK293 HCP antigens.

[00358] Elution was performed by first using 20 column volumes with a gradient of 0 to 100% 33 mM hydrochloric acid/1000 mM NaCl in 1 mM hydrochloric acid.

[00359] The above test procedure is described in more detail in Table 41. A linear flow rate of 39 cm/h was used for all stages.

Table 41:

STAGE Balancing 50 mM Tris-HCl
125 mM NaCl
pH 8.5 LOADING AAV9 EHT2_1831_MUQ_FT Wash 1
(Rebalancing) 50 mM Tris-HCl
125 mM NaCl
pH 8.5 Flushing 2 100 mM sodium acetate
0.1% Polysorbate80
pH 6.0 Flushing 3 50 mM Tris-HCl
125 mM NaCl
pH 8.5 Wash 4 Purified water ELUTION Gradient from 0 to 100% 33 mM hydrochloric acid/
1000 mM NaCl in 1 mM hydrochloric acid Regeneration

[00360] Selected samples were analyzed by each of ITR qPCR and ELISA (as described in Example 8 above) against AAV antigens and ELISA against HEK293 HCP to assess the yield and whether losses could occur in stages. The chromatogram associated with the above example is shown in FIG. 19. The silver spot is shown in FIG. twenty.

[00361] The biopotency of eluted AAV9 was 0.51 BPU.

EXAMPLE 18

[00362] The elution properties of different types of resins were evaluated against different elution buffers. A polyol elution buffer was prepared which contains 50% ethylene glycol (w/w), 50 mM Tris-HCl, 750-2000 mM NaCl, pH 8.0. An acidic elution buffer was prepared which contains 100 mM Glycine pH 2.5, 100 mM sodium citrate pH 3.0 and 50 mM phosphoric acid pH 2.0. Prepared MgCl2-containing elution buffer, which contains 2M MgCl ₂ and 50 mm Tris-HCl at pH 7.4.

[00363] Several resins were tested. The results are shown in Table 42 below. In the table, "Yes" indicates that AAV8 can be eluted from the resin with a potential "elution buffer" in excess of 10% of the load, while "No" indicates that AAV can be eluted from the resin with a "potential elution buffer" less than 1% of the load.

Table 42:

resin type Polyol buffer acid buffer MgCl2 Capture Select AAV8 ¹ Yes Yes Not verified Capture Select AAV9 ¹ No Yes Yes Capture SelectAAVX ¹ No Yes Yes ADK8-Sepharose ² Yes Yes Not verified AVB Sepharose ³ Not verified Yes Not verified

¹ Thermo Fisher Scientific

² Resin was prepared by immobilizing a monoclonal antibody type ADK8 from Progen AG on CNBr-SepharoseFastFlow affinity resin at a resin density of 0.94 mg/mL.

^3GE Healthcare

[00364] Various buffers were tested for their ability to elute AAV8 from the above Capture Select AAV8 resin. The results are shown in Table 43 below.

Table 43:

resin type
AAV8 DMSO
organic solvent
50 mM Tris-HCl
750 mM NaCl
50% (mass/
wt.) DMSO
pH 8.0 Carbohydrate
Sucrose, mannose, sorbitol
(Polyol)
50 mM Arginine Arginine-HCl, 55% (w/w) sucrose,
2 mM MgCl2, 800 mM NaCl, pH 8.0
or
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 Glycerol
(Polyol)
50 mM Arginine Arginine-HCl, 50% (w/w) glycerol
800 mM NaCl, pH 8.0 ethylene glycol
(Polyol)
50 mM Taurine
60% (w/w) ethylene glycol
750 mM NaCl
0.1% octylglycopyranoside
pH 8.0
or
50 mM Tris-HCl
55% (w/w) ethylene glycol
750 mM NaCl
pH 8.0
or
50 mM Tris-HCl, 1 M ammonium sulfate,
50% Ethylene glycol
pH 7.0 Polyol/organic solvent content ≥ 50% ≥ 50% ≥ 50% ≥ 50% Elution of AAV8 No Yes Yes Yes

[00365] The elution buffers listed in Table 44 were also tested to determine if they were satisfactory for eluting AAV from Capture Select® AAV9 available from Thermo Fisher Scientific.

Table 44

Buffer pH Composition Glycine, alkaline pH 8.0 ±0.2 20 mM Glycine 2M NaCl/Tris-HCl pH 7.4 ±0.2 50 mM Tris, 2M NaCl sodium citrate pH 3.0 ±0.2 100 mM sodium citrate Magnesium chloride/Tris-HCl pH 7.4 ±0.2 50 mM Tris, 2M MgCl2 Regeneration Buffer NaP/EG pH 2.5 ±0.2 50 mM sodium phosphate, 50% ethylene glycol Regeneration Buffer Arginine/EDTA/
Urea pH 8.5 ±0.2 20 mM Arginine, 2 mM EDTA. 4M Urea

[00366] Each of the potential elution buffers, as described in Table 44, was applied sequentially as part of the elution selection to determine which elution buffers could be used in the AAVx Capture select program. The selection started with the elution buffer with the potentially lowest elution strength and ended with the buffer with the potentially highest elution strength. The chromatogram is shown in Fig. 21. This is an example of what elution options can be applied to Capture select AAVx.

[00367] All references, including publications, patent applications, and patents cited herein, are incorporated herein by reference to the same extent as if each reference were separately and specifically indicated to be incorporated by reference and set forth throughout its entirety in this document.

Claims (84)

1. A method for purifying an adeno-associated virus (AAV), including: (a) loading the AAV-containing solution onto an AAV-targeting affinity resin under conditions that allow binding between the AAV in solution and the affinity resin; (b) carrying out at least two washing steps; and (c) eluting the AAV from the affinity resin, wherein at least one of the washing steps includes applying a buffer containing an organic solvent or detergent to the affinity resin, and further comprising contacting the solution containing AAV with an anion exchanger and eluting the solution containing AAV from the anion exchanger before loading the solution containing AAV, for affinity resin. 2. The method of claim 1 wherein the organic solvent or detergent is polysorbate 80, ethylene glycol, sorbitol, mannitol, xylitol, DMSO, sucrose, trehalose, or a combination thereof. 3. The method of claim 1 or 2, wherein the detergent contains one or more of Triton X100, polysorbate 80, and tri(n-butyl)phosphate (TNBP). 4. The method according to any one of paragraphs. 1-3, in which at least one of the buffers contains Tris-HCl and salt. 5. The method according to any one of paragraphs. 1-4, in which at least one of the buffers contains one or more of the following components: Histidine, Histidine-HCl, Arginine-HCl, Lysine-HCl, Glycine, Taurine, MES-Na, Bis-Tris and N-acetyl- D, L-tryptophan. 6. The method according to any one of paragraphs. 1-5, in which at least one of the buffers contains sodium acetate. 7. The method according to any one of paragraphs. 1-6, in which at least one of the buffers contains magnesium chloride. 8. The method according to any one of paragraphs. 1-7, in which at least one of the buffers contains Tris-HCl and ethylene glycol. 9. The method according to any one of paragraphs. 1-8, in which at least one of the buffers contains Arginine-HCl and one component of sucrose and glycerol. 10. The method according to any one of paragraphs. 1-9, in which at least one of the buffers contains Taurine and ethylene glycol. 11. The method according to any one of paragraphs. 1-10, in which at least one of the buffers contains Arginine-HCl, Lysine-HCl and Histidine-HCl. 12. The method according to any one of paragraphs. 1-11, in which at least one of the buffers contains Tris-HCl and DMSO. 13. The method according to any one of paragraphs. 1-12, in which at least three washing steps are performed. 14. The method of claim 13 wherein three washing steps are performed. 15. The method of claim 14, wherein the three washing steps are performed sequentially. 16. The method according to any one of paragraphs. 1-3, wherein the first washing step comprises applying to the affinity resin a first buffer containing 50 to 2000 mM sodium acetate and 0.05 to 0.2% polysorbate 80, and wherein the first buffer has a pH of 5.2 to 6.8; wherein the second washing step comprises applying to the affinity resin a second buffer containing 30 to 200 mM Tris-HCl and 75 to 500 mM salt, and wherein the second buffer has a pH of 7.5 to 9.2. 17. The method according to claim 16, in which the third stage of washing includes applying to the affinity resin a third buffer containing from 30 to 200 mm Tris-HCl and from 30 to 75% vol. ethylene glycol, and in which the third buffer has a pH of 7.3 to 8.8. 18. The method of claim 16 wherein the third washing step comprises applying purified water to the affinity resin. 19. The method according to any one of paragraphs. 1-3, in which at least three washing steps are carried out, and in which the first washing step includes applying to the affinity resin a first buffer containing from 50 to 500 mm sodium salt of 2-(N-morpholino)ethanesulfonic acid (MES-Na) , from 3 to 30 mm EDTA and a mixture of solvent/detergent containing polysorbate 80, DMSO and tri(n-butyl)phosphate (TNBP), and in which the first buffer has a pH of from 5.2 to 6.8; wherein the second washing step comprises applying to the affinity resin a second buffer containing 30 to 200 mM Tris-HCl or Arginine-HCl and 75 to 500 mM salt, and wherein the second buffer has a pH of 7.5 to 9.2; and wherein the third washing step comprises applying to the affinity resin a third buffer containing 20 to 80 mM Arginine-HCl and 50 to 200 mM salt, and wherein the third buffer has a pH of 7.3 to 8.8. 20. The method according to any one of paragraphs. 1-3, in which at least three washing stages are carried out, and in which the first washing stage includes applying to the affinity resin a first buffer containing from 50 to 200 mm taurine and from 0.2 to 1.5% PEG, in which the first the buffer has a pH of 5.2 to 6.8; wherein the second washing step comprises applying to the affinity resin a second buffer containing 30 to 300 mM glycine, and wherein the second buffer has a pH of 7.5 to 9.2; and in which the third stage of washing includes applying to the affinity resin of the third buffer containing from 20 to 150 mm taurine, from 30 to 75% wt./mass. ethylene glycol and 0.05 to 0.2% octyl glycopyranoside, and in which the third buffer has a pH of 7.3 to 8.8. 21. The method of claim 20 wherein the PEG is PEG 6000. 22. The method according to any one of paragraphs. 1-3, in which at least three washing steps are carried out, and in which the first washing step comprises applying to the affinity resin a first buffer containing 80 to 400 mM Bis-Tris and 10 to 20 g of a solvent/detergent mixture containing Triton -X100, polysorbate 80 and TNBP in a ratio of 11:3:3 wt./mass., in which the first buffer has a pH of from 5.2 to 6.8; wherein the second washing step comprises applying to the affinity resin a second buffer containing 5 to 20 mM sodium citrate, and wherein the second buffer has a pH of 7.5 to 9.2; and wherein the third washing step comprises applying to the affinity resin a third buffer containing 50 to 200 mM Arginine-HCl, 50 to 200 mM Lysine-HCl, 50 to 200 mM Histidine-HCl, and 1 to 4 mM N-acetyl -D, L-tryptophan, and from 10 to 40% wt./mass. polysorbate 80, and in which the third buffer has a pH of 7.3 to 8.8. 23. The method according to any one of paragraphs. 1-3, in which the first stage of washing includes applying to the affinity resin the first buffer containing from 50 nm to 200 mm sodium acetate and from 0.05 to 0.2% wt./mass. polysorbate 80, in which the first buffer has a pH of 5.2 to 6.8; wherein the second washing step comprises applying to the affinity resin a second buffer containing 20 nM to 100 mM Tris-HCl and 50 to 200 nM salt, wherein the second buffer has a pH of 7.5 to 8.8. 24. The method according to claim 23, in which the third stage of washing includes applying to the affinity resin a third buffer containing from 20 to 100 mm Tris-HCl, from 40 to 60% wt./mass. ethylene glycol, and in which the third buffer has a pH of 7.5 to 8.8. 25. The method of claim 23 wherein the third washing step comprises applying purified water to the affinity resin. 26. The method according to any one of paragraphs. 1-3, in which at least three washing stages are carried out and in which the first washing stage includes applying to the affinity resin a first buffer containing from 50 nm to 200 mm sodium acetate and from 0.05 to 0.2% wt./mass . polysorbate 80, in which the first buffer has a pH of 5.2 to 6.8; wherein the second washing step comprises applying to the affinity resin a second buffer containing 20 to 100 nM Tris-HCl and 50 to 200 nM salt, wherein the second buffer has a pH of 7.5 to 8.8; and in which the third stage of washing includes applying to the affinity resin of the third buffer containing from 20 to 100 mm Tris-HCl, from 40 to 60% wt./mass. ethylene glycol, and in which the third buffer has a pH of 7.5 to 8.8. 27. The method according to any one of paragraphs. 4-19 or 23-26 wherein the salt is selected from NaCl, KCl, MgCl ₂ , CaCl ₂ , sodium citrate, LiCl, CsCl, sodium acetate and a combination of one or more of NaCl, KCl, MgCl ₂ , CaCl ₂ , sodium citrate , LiCl, CsCl and sodium acetate. 28. The method of claim 27 wherein the salt is NaCl. 29. The method according to any one of paragraphs. 4-15, 27 or 28, in which the salt concentration does not exceed 500 mm. 30. The method according to any one of paragraphs. 4-15, 27 or 28, in which the salt concentration does not exceed 200 mm. 31. The method according to any one of paragraphs. 17-22 or 24-30, further comprising a fourth washing step that occurs prior to the first washing step and comprises applying to the affinity resin a buffer containing 10 to 30 mM Tris-HCl and 75 to 250 mM NaCl, and wherein the fourth the buffer has a pH of 6.5 to 8.0. 32. The method according to any one of paragraphs. 23-25, in which the first buffer contains 100 mM sodium acetate, 0.1% polysorbate 80, and in which the first buffer has a pH of 6.0. 33. The method according to any one of paragraphs. 23-25, in which the second buffer contains 50 mM Tris-HCl and 125 mM NaCl, and in which the second buffer has a pH of 8.5. 34. The method according to claim 24, in which the third buffer contains 50 mm Tris-HCl and 50% wt./mass. ethylene glycol and in which the third buffer has a pH of 8.5. 35. The method according to any one of paragraphs. 1-3, in which the first stage of washing includes applying to the affinity resin the first buffer containing from 50 to 200 mm sodium acetate and from 0.05 to 0.2% wt./mass. polysorbate 80, and in which the first buffer has a pH of 5.5 to 6.5; wherein the second washing step comprises applying to the affinity resin a second buffer containing 10 to 70 mM Tris-HCl and 75 to 250 mM NaCl, and wherein the second buffer has a pH of 8.0 to 9.0. 36. The method according to item 35, in which the third stage of washing includes applying to the affinity resin a third buffer containing from 10 to 70 mm Tris-HCl and from 30 to 75% wt./mass. ethylene glycol, and in which the third buffer has a pH of 8.0 to 9.0. 37. The method of claim 35, wherein the third washing step comprises applying purified water to the affinity resin. 38. The method according to any one of paragraphs. 35-37, further comprising a washing step that occurs before the first washing step and includes applying to the affinity resin a buffer containing 10 to 30 mM Tris-HCl and 75 to 250 mM NaCl, and in which the fourth buffer has a pH of 6, 5 to 8.0. 39. The method according to any one of paragraphs. 35-38, in which the first buffer contains 100 mm sodium acetate and 0.1% polysorbate 80 and in which the first buffer has a pH of 6.0. 40. The method according to any one of paragraphs. 35-39, in which the second buffer contains 50 mM Tris-HCl and 125 mM NaCl, and in which the second buffer has a pH of 8.5. 41. The method according to item 36, in which the third buffer contains 50 mm Tris-HCl and approximately 50% wt./mass. ethylene glycol and in which the third buffer has a pH of 8.0. 42. The method according to any one of paragraphs. 1-41, wherein the elution comprises applying a continuous linear increase in the conductivity of the elution buffer by gradient elution. 43. The method according to any one of paragraphs. 1-42, in which the elution involves the application of a continuous linear increase in the concentration of the organic solvent by gradient elution. 44. The method according to any one of paragraphs. 1-43, wherein the elution comprises contacting the affinity resin with an elution buffer containing ethylene glycol, a salt, and a buffer in which the pH is at least 7.0. 45. The method of claim 44 wherein the salt is NaCl. 46. The method according to claim 44 or 45, wherein the buffer is Tris-HCl. 47. The method according to any one of paragraphs. 44-46, in which the salt concentration is 750 mm, the buffer concentration is 50 mm and the concentration of ethylene glycol is from 50 to 60% wt./mass. 48. The method according to any one of paragraphs. 44-47, in which the pH is 8.0. 49. The method according to any one of paragraphs. 44-48 in which the salt is NaCl and the buffer is Tris-HCl. 50. The method according to any one of paragraphs. 1-47 or 49, in which the elution includes bringing into contact the affinity resin with an elution buffer containing 750 mm NaCl and 50-60% wt./mass. ethylene glycol at a pH of at least 7.0. 51. The method according to item 50, in which the elution buffer contains at least 55% wt./mass. ethylene glycol. 52. The method according to any one of paragraphs. 1-41, in which the elution includes bringing into contact the affinity resin with an elution buffer containing 50 mm Tris-HCl, from 750 to 1250 mm NaCl and 60% wt./mass. ethylene glycol at a pH of at least 7.8. 53. The method according to any one of paragraphs. 1-41, in which the elution includes bringing into contact the affinity resin with an elution buffer containing 2 mm magnesium chloride, 50 mm Arginine-HCl, from 750 to 1000 mm NaCl and at least 55% wt./mass. sucrose at a pH of at least 8.0. 54. The method according to any one of paragraphs. 1-41, in which the elution includes bringing into contact the affinity resin with an elution buffer containing 2 mm magnesium chloride, 50 mm Arginine-HCl, from 750 to 1000 mm NaCl and at least 50% wt./mass. glycerol at a pH of at least 8.0. 55. The method according to any one of paragraphs. 1-41, in which the elution includes bringing into contact the affinity resin with an elution buffer containing 2 mm magnesium chloride, 50 mm Taurine, from 600 to 1000 mm NaCl, from 0.05 to 0.2% octylglycopyranoside and 60% wt./ mass. ethylene glycol at a pH of at least 7.8. 56. The method according to any one of paragraphs. 1-41, in which the elution includes bringing into contact the affinity resin with an elution buffer containing 1 M ammonium sulfate, 50 mm Tris-HCl and 50% wt./mass. ethylene glycol at a pH of at least 6.8. 57. The method according to any one of paragraphs. 1-41, in which the elution includes bringing into contact the affinity resin with an elution buffer containing 20% wt./mass. sucrose, 10% wt./mass. sorbitol, 5% wt./mass. mannitol or 5% wt./mass. sucrose, 15% wt./mass. glycerol, 50 mM Histidine, and 750 mM to 1000 mM NaCl at a pH of at least 7.8. 58. The method according to any one of paragraphs. 1-41, wherein the elution comprises contacting the affinity resin with an elution buffer containing 100 mM Glycine-HCl, 200 mM NaCl at pH 2.5. 59. The method according to any one of paragraphs. 50-57, in which the elution buffer has a pH of 8.0. 60. The method according to any one of paragraphs. 50, 54 or 56, in which the elution buffer has a pH of 7.4. 61. The method according to any one of paragraphs. 1-41, in which the elution includes a stepwise increase in the concentration of the counterion. 62. The method according to any one of paragraphs. 1-41, in which the elution includes a stepwise increase in the concentration of the organic solvent. 63. The method according to any one of paragraphs. 1-41, wherein the salt in the elution buffer is selected from monovalent, divalent, or polyvalent anions such as chloride, acetate, sulfate, and citrate. 64. The method according to any one of paragraphs. 1-3, wherein the first washing step comprises applying to the affinity resin a first buffer containing 50 to 200 mM sodium acetate, 0.05 to 0.2% polysorbate 80, and wherein the first buffer has a pH of 5.2 to 6.8; wherein the second washing step comprises applying to the affinity resin a second buffer containing 25 to 100 mM Tris-HCl and 50 to 200 mM NaCl, and wherein the second buffer has a pH of 7.5 to 9.2; and wherein the third washing step comprises applying to the affinity resin a third buffer containing 20 to 100 mM Tris-HCl and 75 to 250 mM NaCl, and wherein the third buffer has a pH of 7.5 to 8.8. 65. The method of claim 64, further comprising eluting by applying purified water to the affinity resin, followed by applying 20 to 50 mM HCl at a pH of 1.7 to 2.5 to the affinity resin. 66. The method according to any one of paragraphs. 1-41, 64 or 65, wherein the elution comprises applying a gradient from 0 to 100% 20-50 mM HCl to 800-1200 mM NaCl in 0.5-2.0 mM HCl solution. 67. The method according to any one of paragraphs. 1-66, in which the AAV obtained in the elution step has an HC impurity level of ≤ 99.9%. 68. The method according to any one of paragraphs. 1-67, in which the AAV obtained in the elution step has an HC impurity level of ≤ 99.0%. 69. The method according to any one of paragraphs. 1-68, in which the AAV is AAV8, the affinity resin is POROS™ CaptureSelect™ AAV8, and in which the elution buffer is acidic and does not contain ethylene glycol. 70. The method according to any one of paragraphs. 1-68, in which the AAV is AAV9, the affinity resin is POROS™ CaptureSelect™ AAV9, and in which the elution buffer is acidic and does not contain ethylene glycol. 71. The method according to any one of paragraphs. 1-68, wherein the AAV is AAV8 and wherein the affinity resin is an immune affinity resin consisting of an immobilized anti-AAV8 monoclonal antibody of the type ADK8 or ADK8/9 immobilized on a chromatographic matrix. 72. The method according to any one of paragraphs. 1-68, wherein the AAV is AAV9 and wherein the affinity resin is an immune affinity resin consisting of an immobilized anti-AAV9 monoclonal antibody of the type ADK9 or ADK8/9 immobilized on a chromatographic matrix. 73. The method according to any one of paragraphs. 1-72, the method further comprising contacting the AAV containing solution with a filter containing positively charged groups effective to remove acidic charged contaminants from the AAV containing solution. 74. The method according to any one of paragraphs. 1-73, further comprising nanofiltration of the AAV fraction to remove viruses larger than 35 nm. 75. The method according to any one of paragraphs. 1-74, further comprising an additional purification step comprising performing AEX chromatography on a column containing a tentacle gel. 76. The method according to any one of paragraphs. 1-75, further comprising testing the AAV fraction with an AAV-specific ELISA. 77. The method of claim 76, wherein the AAV-specific ELISA is an AAV-specific sandwich ELISA. 78. The method according to any one of paragraphs. 1-41, wherein the elution comprises contacting the affinity resin with an elution buffer containing 1 mM HCl in purified water at pH 3.2. 79. The method according to any one of paragraphs. 1-41, in which the elution includes bringing the affinity resin into contact with an elution buffer containing from 30 to 70 mm Tris-HCl, from 700 to 900 mm NaCl and from 40 to 60% wt./mass. DMSO at pH 7.5 to 8.5. 80. The method according to any one of paragraphs. 1-41, wherein the elution comprises contacting the affinity resin with an elution buffer containing 2 M MgCl ₂ and 50 mM Tris-HCl at pH 7.4.

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