Compositions and methods for reducing host cell proteins in recombinant adeno-associated virus preparations

Abstract

Provided are methods and compositions for removal of host cell protein contaminants from recombinant adeno-associated viral vector preparations.

Description

COMPOSITIONS AND METHODS FOR REDUCING HOST CELL PROTEINS IN RECOMBINANT ADENO-ASSOCIATED VIRUS PREPARATIONSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 509,873, filed on June 23, 2023, the entire disclosure of which is incorporated by reference herein in its entirety for all purposes.BACKGROUND

[0002] Recombinant adeno-associated virus (rAAV) production has seen significant process improvement in the past decade. However, challenges in impurity clearance remain. Host cell protein (HCP) impurity levels are considered as one of the important quality attributes in biologies therapeutic drugs, including rAAV. In the downstream purification process, the HCPs are generally reduced in multiple unit operations, including affinity chromatography (AC), anion exchange chromatography (AEX). tangential flow filtration (TFF) steps or combinations thereof. High levels of HCP impurities in purified rAAV preparations significantly dampen efficacy of the rAAV product, mainly by increasing immunogenicity in the host and reducing stability and transduction efficiency of the rAAV particles.

[0003] Commonly used affinity stationary’ phases for purifying rAAVs utilize a proteinbased ligand to provide an affinity interaction with rAAV capsids. Modification of specific binding sites on the rAAV capsid to increase affinity for ligands bound to chromatographic resins and modification of pH conditions for reducing HCP levels while maintaining biologies product quality have been investigated w ith limited success.SUMMARY

[0004] The present disclosure is based in part on the insight that certain additives, such as sodium octanoate and L-arginine, when added at different steps of chromatographic purification of rAAV can reduce HCP impurity levels while maintaining the stability and integrity of purified rAAV product. The present disclosure provides compositions and methods utilizing additives in rAAV chromatographic purification with improved HCP reduction without negatively affecting other product qualities of rAAV, including percentage of full capsids and potency.

[0005] The present disclosure provides improved compositions and methods for reducing the level of HCP impurities or contaminants in drug products prepared by rAAV-specific chromatographic purification.

[0006] In one aspect, the present disclosure provides a method of reducing host cell protein (HCP) contaminants in a recombinant adeno-associated virus (rAAV) preparation, the method comprising the steps of: a) providing a cell lysate and / or a cell culture supernatant comprising a rAAV; b) applying the cell lysate and / or the cell culture supernatant to a chromatography resin; c) passing at least one wash solution comprising an effective amount of an additive selected from L-arginine or a derivative thereof, an octanoate salt, and combinations thereof, through the chromatography resin; d) passing an elution solution through the washed chromatography resin to elute the rAAV to generate an eluent flowthrough comprising the rAAV; and e) collecting one or more fractions of the eluent flowthrough comprising said rAAV, wherein said rAAV comprises an AAV capsid and a vector genome packaged therein.

[0007] In one aspect, the present disclosure also provides an elution method for separating host cell protein (HCP) from a recombinant adeno-associated virus (rAAV) preparation, the method comprising the steps of: a) contacting a chromatography resin with the rAAV preparation; b) applying at least one wash solution to the chromatography resin and allowing the wash solution to flow through, wherein the wash solution comprises an additive selected from L-arginine or a derivative thereof, an octanoate salt, and combinations thereof; c) applying an elution solution to the washed chromatography resin to elute the rAAV to generate an eluent flow through comprising the rAAV; and d) collecting one or more fractions of the eluent flowthrough comprising said rAAV, wherein said rAAV comprises an AAV capsid and a vector genome packaged therein.

[0008] In some embodiments, the one or more fractions of the eluent flowthrough are pooled to form a combined eluent fraction. In some embodiments, the methods disclosed herein comprises determining the amount of HCP in the combined eluent fraction. In some embodiments, the combined eluent fraction comprises an HCP amount that is 4 log reduction value (LRV) or more relative to the HCP amount of the rAAV preparation. In some embodiments, the combined eluent fraction comprises HCP at a concentration of 0 ng / ml to 5 ng / ml. In some embodiments, the combined eluent fraction comprises HCP at a concentration of 0 ng / ml to 4 ng / ml. In some embodiments, the combined eluent fraction comprises HCP at a concentration of 1 ng / ml to 2 ng / ml.

[0009] In some embodiments, the method disclosed herein further comprises determining the recovery of rAAV in the combined eluent fraction. In some embodiments, the combined eluent fraction comprises at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the vector genome present in the rAAV preparation. In some embodiments, the at least one wash solution comprises L-arginine or a derivative thereof. In some embodiments, the at leastone wash solution comprises L-arginine or a derivative thereof at a concentration of about 50 mM to about 200 mM.

[0010] In some embodiments, the at least one wash solution comprises an octanoate salt. In some embodiments, the at least one wash solution comprises an octanoate salt at a concentration of about 10 mM to about 50 mM.

[0011] In some embodiments, the at least one wash solution comprises L-arginine or a derivative thereof and an octanoate salt. In some embodiments, the at least one wash solution comprises L-arginine or a derivative thereof at a concentration of about 50 mM to about 200 mM and an octanoate salt at a concentration of about 10 mM to about 50 mM.

[0012] In some embodiments, passing at least one wash solution comprises passing a first wash solution through the chromatography resin, followed by passing a second wash solution through the chromatography resin. In some embodiments, the first and the second wash solutions comprise the same additive(s). In some embodiments, the concentration of the additive(s) in the first and the second wash solutions is the same. In some embodiments, the concentration of the additive(s) in the first and the second wash solutions is different. In some embodiments, the concentration of the additive(s) in the first wash solution is less than the concentration of the additive(s) in the second wash solution. In some embodiments, the concentration of the additive(s) in the first wash solution is more than the concentration of the additive(s) in the second wash solution.

[0013] In some embodiments, the first and second wash solutions comprise L-arginine or a derivative thereof.

[0014] In some embodiments, the first and second wash solutions comprise an octanoate salt.

[0015] In some embodiments, the first and second wash solutions comprise L-arginine or a derivative thereof and an octanoate salt.

[0016] In some embodiments, the first and the second wash solutions comprise different additives.

[0017] In some embodiments, the first wash solution comprises L-arginine or a derivative thereof, and the second wash solution comprises an octanoate salt.

[0018] In some embodiments, the first wash solution comprises octanoate salt, and the second wash solution comprises L-arginine or a derivative thereof.

[0019] In some embodiments, the first wash solution comprises L-arginine or a derivative thereof, and the second wash solution comprises a combination of L-arginine or a derivative thereof and an octanoate salt.

[0020] In some embodiments, the first wash solution comprises a combination of L- arginine or a derivative thereof and an octanoate salt, and the second wash solution comprises L-arginine or a derivative thereof.

[0021] In some embodiments, the first wash solution comprises octanoate salt, and the second wash solution comprises a combination of L-arginine or a derivative thereof and an octanoate salt.

[0022] In some embodiments, the first wash solution comprises a combination of L- arginine or a derivative thereof and an octanoate salt, and the second wash solution comprises an octanoate salt.

[0023] In some embodiments, the methods disclosed herein comprises passing at least three wash solutions sequentially through the chromatography resin. In some embodiments, each of the at least three wash solutions comprise the same additive(s).

[0024] In some embodiments, each of the at least three wash solutions comprise L-arginine or a derivative thereof. In some embodiments, each of the at least three wash solutions comprise L-arginine or a derivative thereof at a concentration of about 50 mM to about 200 mM.

[0025] In some embodiments, each of the at least three wash solutions comprise an octanoate salt. In some embodiments, each of the at least three wash solutions comprise an octanoate salt at a concentration of about 10 mM to about 50 mM.

[0026] In some embodiments, each of the at least three wash solutions comprise L-arginine or a derivative thereof and an octanoate salt. In some embodiments, each of the at least three wash solutions comprise L-arginine or a derivative thereof at a concentration of about 50 mM to about 200 mM and an octanoate salt at a concentration of about 10 mM to about 50 mM.

[0027] In some embodiments, the concentrations of the same additive in each of the at least three wash solutions are different.

[0028] In some embodiments, the concentration of the additive increases in each sequentially used wash solution.

[0029] In some embodiments, the concentration of the additive increases linearly in each sequentially used wash solution.

[0030] In some embodiments, the concentration of the additive increases stepwise in each sequentially used wash solution.

[0031] In some embodiments, the concentration of the additive decreases in each sequentially used wash solution.

[0032] In some embodiments, the concentration of the additive decreases linearly in each sequentially used wash solution.

[0033] In some embodiments, the concentration of the additive decreases stepwise sequentially used wash solution.

[0034] In some embodiments, each of the at least three wash solutions comprise an additive that is different from an additive in any of the previously or subsequently used wash solutions or both.

[0035] In some embodiments, the first wash solution comprises L-arginine or a derivative thereof, the second wash solution comprises an octanoate salt, and the third wash solution comprises L-arginine or a derivative thereof and an octanoate salt.

[0036] In some embodiments, the first wash solution comprises an octanoate salt, the second wash solution comprises L-arginine or a derivative thereof, and the third wash solution comprises L-arginine or a derivative thereof and an octanoate salt.

[0037] In some embodiments, the first wash solution comprises L-arginine or a derivative thereof and octanoate salt, the second wash solution comprises L-arginine or a derivative thereof, and the third wash solution comprises an octanoate salt.

[0038] In some embodiments, the first wash solution comprises L-arginine or a derivative thereof and an octanoate salt, the second wash solution comprises an octanoate salt, and the third wash solution comprises L-arginine or a derivative thereof.

[0039] In some embodiments, the first wash solution comprises an octanoate salt, the second wash solution comprises L-arginine or a derivative thereof and an octanoate salt, and the third wash solution comprises L-arginine or a derivative thereof.

[0040] In some embodiments, the first wash solution comprises L-arginine or a derivative thereof, the second wash solution comprises L-arginine or a derivative thereof and an octanoate salt, and the third wash solution comprises an octanoate salt.

[0041] In some embodiments, the first and the second wash solutions comprise L-arginine or a derivative thereof, and the third wash solution comprises an octanoate salt.

[0042] In some embodiments, the first and the second wash solutions comprise L-arginine or a derivative thereof, and the third wash solution comprises L-arginine or a derivative thereof and an octanoate salt.

[0043] In some embodiments, the first and the second wash solutions comprise an octanoate salt, and the third wash solution comprises L-arginine or a derivative thereof.

[0044] In some embodiments, the first and the second wash solutions comprise an octanoate salt, and the third wash solution comprises L-arginine or a derivative thereof and an octanoate salt.

[0045] In some embodiments, the first and the second wash solutions comprise L-arginine or a derivative thereof and an octanoate salt and the third wash solution comprises L-arginine or a derivative thereof.

[0046] In some embodiments, the first wash solution comprises L-argimne or a derivative thereof, and the second and the third wash solutions comprise an octanoate salt.

[0047] In some embodiments, the first wash solution comprises L-arginine or a derivative thereof, and the second and the third wash solutions comprise L-arginine or a derivative thereof and an octanoate salt.

[0048] In some embodiments, the first wash solution comprises an octanoate salt, and the second and the third wash solutions comprise L-arginine or a derivative thereof.

[0049] In some embodiments, the first wash solution comprises an octanoate salt, and the second and the third wash solutions comprise L-arginine or a derivative thereof and an octanoate salt.

[0050] In some embodiments, the first wash solution comprises L-arginine or a derivative thereof and an octanoate salt, and the second and the third wash solutions comprise L-arginine or a derivative thereof.

[0051] In some embodiments, the first wash solution comprises L-arginine or a derivative thereof and an octanoate salt, and the second and the third wash solutions comprise an octanoate salt.

[0052] In some embodiments, the L-arginine derivative is selected from the group consisting of L-arginine hydrochloride. L-arginine dihydrochloride, L-arginine methyl ester dihydrochloride, L-arginine ethyl ester dihydrochloride, acetyl L-arginine, N-alpha-butyroyl- L-arginine, agmatine, L-arginic acid, and N-alpha-pyvaloyl L-arginine.

[0053] In some embodiments, the octanoate salt is selected from the group consisting of sodium octanoate, magnesium octanoate, potassium octanoate, and calcium octanoate.

[0054] In some embodiments, the chromatography resin comprises an affinity chromatography medium, an anion exchange (AEX) chromatography medium, a hydrophobic interaction chromatography (HIC) medium, a mix-mode chromatography medium, a tangential flow (TFF) chromatography medium, or a combination thereof.

[0055] In some embodiments, the affinity chromatography medium comprises a protein or ligand that binds to the AAV capsid.

[0056] In some embodiments, the affinity chromatography medium comprises an AAV- specific antibody or a fragment thereof.

[0057] In some embodiments, the at least one wash solution comprises an organic solvent or a detergent.

[0058] In some embodiments, the at least one wash solution comprises an organic solvent selected from any one of polysorbate 80, ethylene glycol, t-butanol, sorbitol, mannitol, xylitol, DMSO, sucrose trehalose and polyethyleneimine (PEI).

[0059] In some embodiments, the at least one wash solution comprises a detergent selected from the group consisting of Nonidet P-40, 3-[(3-cholamidopropyl)dimethylammonio]-l- propanesulfonate (CHAPS), triton x-100, and tri (n-but l) phosphate (TNBP).

[0060] In some embodiments, the at least one wash solution comprises Tris-HCl, Bis-Tris or MES-Na.

[0061] In some embodiments, the at least one wash solution has a pH of between about 6.0 to about 9.0.

[0062] In some embodiments, the elution solution comprises glycine-HCl or citric acid.

[0063] In some embodiments, the elution solution further comprises an ionic strength modifier selected from the group consisting of sodium chloride, sodium iodide, sodium thiocyanate, calcium chloride, magnesium chloride, and a combination thereof.

[0064] In some embodiments, the elution solution has a pH of about 2.5 to about 3.0.

[0065] In some embodiments, the AEX chromatography medium is a weak ionic exchanger.

[0066] In some embodiments, the AEX chromatography medium is a strong ionic exchanger.

[0067] In some embodiments, the AEX chromatography medium is in the form of a packed bed.

[0068] In some embodiments, the AEX chromatography medium is a chromatographic monolithic column.

[0069] In some embodiments, the AEX chromatography medium comprises a combined eluent fraction CIMmultus® QA column. CIMac® QA column. NuviaQ® column, POROS HQ1Mcolumn, Eshmuno® Q column, POROS XQ1Mcolumn, FractoGel® TMAE column, or a CaptoQ® column.

[0070] In some embodiments, the at least one wash solution comprises a quaternary ammonium salt.

[0071] In some embodiments, the quaternary ammonium salt comprises a tetraalkylammonium salt, a choline chloride, or a divalent salt.

[0072] In some embodiments, the tetraalkylammonium salt is a tetraalkylammonium chloride or a tetraalkyl ammonium acetate.

[0073] In some embodiments, the tetraalkylammonium chloride is selected from the group consisting of tetramethylammonium chloride (TMAC), tetraethylammonium chloride (TEAC), tetrapropylammonium chloride (TPAC). tetrabutylammonium chloride (TBAC). benzyhributylammonium chloride (BTBAC), and any combination(s) thereof.

[0074] In some embodiments, the tetraalkylammonium acetate is selected from the group consisting of tetramethylammonium acetate, tetraethylammonium acetate (TEA-Ac), tetrapropylammonium acetate, tetrabutylammonium acetate, and any combination(s) thereof.

[0075] In some embodiments, the wash solution has a pH of between about 6.0 to about9.0.

[0076] In some embodiments, the elution solution comprises a salt in the form of any one of an isocratic salt solution or a gradient salt solution.

[0077] In some embodiments, the concentration of the salt within the elution solution increases continuously over time throughout the elution step.

[0078] In some embodiments, the concentration of the salt within the elution solution increases linearly over time throughout the elution step.

[0079] In some embodiments, the concentration of the salt within the elution solution increases stepwise throughout the elution step.

[0080] In some embodiments, the concentration of the salt within the elution solution decreases continuously over time throughout the elution step.

[0081] In some embodiments, the concentration of the salt within the elution solution decreases linearly over time throughout the elution step.

[0082] In some embodiments, the concentration of the salt within the elution solution decreases stepwise throughout the elution step.

[0083] In some embodiments, the salt is any one of sodium chloride, sodium sulfate, magnesium chloride, magnesium sulfate, or any combination thereof.

[0084] In some embodiments, the elution solution further comprises a bis-Tris Propanebased buffer or a potassium phosphate-based buffer.

[0085] In some embodiments, the elution solution has a pH of about 6.0 to about 10.0.

[0086] In some embodiments, the HIC medium is selected from the group consisting of methyl HIC, Source ETH, Toyopearl Ether-650, Toyopearl PPG-600, Source Iso, Source Phe,Phenyl Sepharose, Toyopearl Phenyl-600, Toyopearl Phenyl-650. Butyl Sepharose, Toyopearl Butyl-600, Toyopearl Butyl-650, Toyopearl SuperButyl-550. t-Butyl HIC. Toyopearl Hexyl- 650, and Octyl Sepharose.

[0087] In some embodiments, the wash solution comprises a wash buffer comprising a salt selected from the group consisting of a sulfate salt, a citrate salt, ammonium sulfate, sodium sulfate, sodium phosphate, sodium chloride, ammonium chloride, sodium bromide, potassium phosphate, and a combination thereof.

[0088] In some embodiments, the wash solution has a pH of about 5.0 to about 7.0.

[0089] In some embodiments, the elution solution comprises the same salt as in the wash buffer, at a lower concentration as compared to a corresponding wash buffer.

[0090] In some embodiments, the elution solution has a pH of about 5.0 to about 7.0.

[0091] In some embodiments, the AAV capsid is from an AAV of serotype 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, rhlO, hu37, or a variant thereof.

[0092] In one aspect, the present disclosure also provides a composition comprising an effective amount of an additive selected from L-arginine or a derivative thereof, an octanoate salt, and combinations thereof, for use in reducing host cell protein (HCP) contaminants during chromatographic purification of rAAV from a rAAV preparation.

[0093] In some embodiments, the additive is L-arginine or a derivative thereof at a concentration of about 50 mM to about 200 mM.

[0094] In some embodiments, the additive is an octanoate salt at a concentration of about 10 mM to about 50 mM.

[0095] In some embodiments, the additive comprises L-arginine or a derivative thereof at a concentration of about 50 mM to about 200 mM and an octanoate salt at a concentration of about 10 mM to about 50 mM.

[0096] In some embodiments, the L-arginine derivative is selected from the group consisting of L-arginine hydrochloride, L-arginine dihydrochloride, L-arginine methyl ester dihydrochloride, L-arginine ethyl ester dihydrochloride, acetyl L-arginine, N-alpha-butyroyl- L-arginine. agmatine, L-arginic acid, and N-alpha-py valoyl L-arginine.

[0097] In some embodiments, the octanoate salt is selected from the group consisting of sodium octanoate, magnesium octanoate, potassium octanoate, and calcium octanoate.

[0098] In some embodiments, the composition disclosed herein is a wash solution in a chromatographic medium post-application of a rAAV preparation to the medium.

[0099] In some embodiments, the chromatographic medium is any one of an affinity chromatography medium, an anion exchange (AEX) chromatography medium, a hydrophobicinteraction chromatography (HIC) medium, a mix-mode chromatography medium, a tangential flow (TFF) chromatography medium, or a combination thereof.

[0100] In some embodiments, the affinity chromatography medium comprises a protein or ligand that binds to AAV capsid protein.

[0101] In some embodiments, the affinity chromatography medium comprises an AAV- specific antibody or a fragment thereof.

[0102] In some embodiments, the compositions disclosed herein further comprises an organic solvent or a detergent.

[0103] In some embodiments, the at least one wash solution comprises an organic solvent selected from any one of polysorbate 80, ethylene glycol, t-butanol, sorbitol, mannitol, xylitol, DMSO, sucrose trehalose and polyethyleneimine (PEI).

[0104] In some embodiments, the at least one wash solution comprises a detergent selected from the group consisting of Nonidet P-40, 3-[(3-cholamidopropyl)dimethylammonio]-l- propanesulfonate (CHAPS), triton x-100, and tri (n-butyl) phosphate (TNBP).

[0105] In some embodiments, the at least one wash solution comprises Tris-HCl, Bis-Tris or MES-Na.

[0106] In some embodiments, the composition has a pH of between about 6.0. to about 9.0.

[0107] In some embodiments, the AEX chromatography medium is a weak ionic exchanger.

[0108] In some embodiments, the AEX chromatography medium is a strong ionic exchanger.

[0109] In some embodiments, the AEX chromatography medium is in the form of a packed bed.

[0110] In some embodiments, the AEX chromatography medium is a chromatographic monolithic column.

[0111] In some embodiments, the AEX chromatography medium comprises a CIMmultus® QA column, CIMac® QA column, NuviaQ® column, POROS HQ CIMac® QAY™ column, Eshmuno® Q column, POROS® XQ column, FractoGel® TMAE column, or a CaptoQ® column.

[0112] In some embodiments, the compositions disclosed herein further comprises a quaternary ammonium salt.

[0113] In some embodiments, the quaternary ammonium salt comprises a tetraalkylammonium salt, a choline chloride, or a divalent salt.

[0114] In some embodiments, the tetraalkylammonium salt is a tetraalkylammonium chloride or a tetraalkylammonium acetate.

[0115] In some embodiments, the tetraalkylammonium chloride is selected from the group consisting of tetramethyl ammonium chloride (TMAC), tetraethylammonium chloride (TEAC), tetrapropylammonium chloride (TPAC), tetrabutylammonium chloride (TBAC), benzyltributylammonium chloride (BTBAC), and any combination(s) thereof.

[0116] In some embodiments, the tetraalkylammonium acetate is selected from the group consisting of tetramethylammonium acetate, tetraethylammonium acetate (TEA-Ac), tetrapropylammonium acetate, tetrabuty lammonium acetate, and any combination(s) thereof.

[0117] In some embodiments, the composition has a pH of between about 6.0. to about 9.0.

[0118] In some embodiments, the HIC medium is selected from the group consisting of methyl HIC, Source ETH, Toy opearl Ether-650, Toy opearl PPG-600, Source Iso, Source Phe, Phenyl Sepharose, Toyopearl Phenyl-600, Toyopearl Phenyl-650, Butyl Sepharose, Toyopearl Butyl-600, Toyopearl Butyl-650, Toy opearl SuperButyl-550, t-Butyl HIC, Toyopearl Hexyl- 650, and Octyl Sepharose.

[0119] In some embodiments, the wash solution comprises sodium phosphate or potassium phosphate.

[0120] In some embodiments, the composition has a pH of about 5.0 to about 7.0.

[0121] In some embodiments, the AAV capsid is from an AAV of serotype 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, rhlO, hu37 or a variant thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0122] FIG. 1. Initial Additive Screen in Affinity Chromatography. FIG. 1 is a graph depicting the effect of five additives, sodium octanoate, urea, isopropyl alcohol, Pluronic® F68, and L-arginine ("Arginine” in FIG. 1) on HCP levels and rAAV genome recovery percentage, when spiked into affinity load material and affinity wash buffers in affinity chromatography. The x-axis depicts the different additives, as indicated. The left and right y-axes depict the percent rAAV genome recovery (measured as elution peak area ratio relative to control), and elution HCP concentration (ng / ml), respectively. The HCP concentration in eluent for each additive is shown in filled shapes, with the filled triangle representing BLOQ (<2 ng / mL) while the filled circle represents a detectable level reading (7 ng / mL). The normalized affinity elution peak area (percentage to negative control) of A254 nm trace and A280 nm trace are indicated by black (denoted as “UV 254nm Area Ratio'’) and white bars (denoted as "UV 280nm Area Ratio”), respectively.

[0123] FIG. 2. Effect of sodium octanoate and L-arginine on HCP reduction and rAAV stability in affinity chromatography runs. FIG. 2 is a graph plot of affinity chromatography GOI genome recovery as a function of HCP LRV, for negative control, 20 mM sodium octanoate, and 0.1 M L-arginine runs, as indicated. The x-axis depicts the HCP log-reduction value as determined by ELISA. The y-axis depicts percent GOI genome recovery as determined by qPCR.

[0124] FIG. 3. The impact of additive spiking location in affinity chromatography. FIG. 3 is a graph plotting the GOI genome recovery percentage as a function of HCP mass log reduction value for affinity runs with 20 mM sodium octanoate spiked in load material, wash buffer, and both load material and wash buffer are represented by open circle, open triangle, and open square, respectively. The affinity runs with 0. 1 M arginine spiked in load material, wash buffer, and both load material and wash buffer are represented by filled circle, filled triangle, and filled square, respectively. The x-axis depicts the HCP log-reduction value as determined by ELISA. The y-axis depicts percent GOI genome recovery as determined by qPCR.DETAILED DESCRIPTION OF THE DISCLOSURE

[0125] Use of additives (e.g., arginine, sodium octanoate) for reducing impurities in purification of biologies (e.g., antibodies) using chromatographic resins is in practice. However, use of additives (e.g., arginine, sodium octanoate) in buffers for purification of viral vectors, such as recombinant AAV based vectors, is particularly difficult due to potential loss of viral vector yield (e.g, induced disintegration of the purified viral particles caused by L- arginine and sodium octanoate). The present disclosure provides improved compositions and methods for reducing the level of HCP impurities or contaminants in drug products prepared by rAAV specific chromatographic purification.Methods for reducing HCP during rAA V chromatographic puri fication

[0126] In one aspect, the present disclosure provides a method of reducing host cell protein (HCP) contaminants in a recombinant adeno-associated virus (rAAV) preparation, wherein the method comprises: a) providing a cell lysate and / or a cell culture supernatant comprising the rAAV; b) applying the cell lysate and / or a cell culture supernatant to a chromatography resin; c) passing at least one wash solution comprising an effective amount of an additive selected from L-arginine or a derivative thereof, an octanoate salt, and combinations thereof, through the chromatography resin; d) passing an elution solution through the chromatography resin to elute the rAAV to generate an eluent flowthrough comprising the rAAV; and e) collecting oneor more fractions of the eluent flowthrough comprising said rAAV, wherein said rAAV comprises an AAV capsid and a vector genome packaged therein.

[0127] In one aspect the present disclosure also provides an elution method for separating host cell protein (HCP) from a recombinant adeno-associated virus (rAAV) preparation, wherein the method comprises: a) contacting a chromatography resin with the rAAV preparation; b) applying at least one wash solution to the chromatography resin and allowing the wash solution to flow through, wherein the wash solution comprises an additive selected from L-arginine or a derivative thereof, an octanoate salt, and combinations thereof; c) applying an elution solution to the chromatography resin to elute the rAAV to generate an eluent flowthrough comprising the rAAV; and d) collecting one or more fractions of the eluent flowthrough comprising said rAAV, wherein said rAAV comprises an AAV capsid and a vector genome packaged therein.

[0128] In some embodiments of the method of the present disclosure, the one or more fractions of the eluent flowthrough are pooled to form a combined eluent fraction. In some embodiments, the method of the present disclosure further comprises determining the amount of HCP in the combined eluent fraction. In some embodiments, the method of the present disclosure further comprises determining the recovery of rAAV in the combined eluent fraction.

[0129] In some embodiments of the method of the present disclosure, the combined eluent fraction comprises an HCP amount that is at least 4 log reduction value (LRV) (e.g.. at least 4 LRV, at least 5 LRV, at least 6 LRV, at least 7 LRV, at least 8 LRV, at least 9 LRV or at least 10 LRV) relative to the HCP amount of the rAAV preparation. In some embodiments of the method of the present disclosure, the combined eluent fraction comprises HCP at a concentration of 0 to 5 ng / ml (0 to 1 ng / ml, 1 to 2 ng / ml, 2 to 3 ng / ml. 3 to 4 ng / ml, 4 to 5 ng / ml, 0 to 4 ng / ml, 1 to 5 ng / ml, 1 to 3 ng / ml, 2 to 4 ng / ml, 3 to 5 ng / ml and all integers including and in between 0 to 5 ng / ml). In some embodiments of the method of the present disclosure, the combined eluent fraction comprises HCP at a concentration of 0 to 4 ng / ml. In some embodiments of the method of the present disclosure, the combined eluent fraction comprises HCP at a concentration of 1 to 2 ng / ml.

[0130] In some embodiments of the method of the present disclosure, the combined eluent fraction comprises at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the vector genome present in the rAAV preparation. In some embodiments of the method of the present disclosure, the combined eluent fraction comprises at least 80% of the vector genome present in the rAAV preparation. In some embodiments of the method of the present disclosure,the combined eluent fraction comprises at least 90% of the vector genome present in the rAAV preparation.

[0131] In some embodiments of the method of the present disclosure, the at least one wash solution comprises L-arginine or a derivative thereof. In some embodiments of the method of the present disclosure, the at least one wash solution comprises L-arginine or a derivative thereof at a concentration of about 50 mM to about 200 mM (e.g., about 50 mM to about 75 mM, about 75 mM to about 100 mM, about 100 mM to about 125 mM, about 125 mM to about 150 mM, 150 mM to 175 mM, 175 mM to 200 mM, about 75 mM to about 175 mM, about 100 mM to about 150 mM, about 125 mM to about 175 mM, about 75 mM to about 125 mM, and all integers including and in between about 50 mM and about 200 mM). In some embodiments of the method of the present disclosure, the at least one wash solution comprises L-arginine or a derivative thereof at a concentration of about 100 mM.

[0132] In some embodiments of the method of the present disclosure, the at least one wash solution comprises an octanoate salt. In some embodiments of the method of the present disclosure, the at least one wash solution comprises an octanoate salt at a concentration of about 10 mM to about 50 mM (e.g.. about 10 mM to about 20 mM, about 20 mM to about 30 mM. about 30 mM to about 40 mM, about 40 mM to about 50 mM, about 20 mM to about 40 mM, about 30 mM to 50 mM, about 10 mM to about 30 mM, about 20 mM to about 50 mM, and all integers including and in between about 10 mM and about 50 mM). In some embodiments of the method of the present disclosure, the at least one wash solution comprises an octanoate salt at a concentration of about 20 mM.

[0133] In some embodiments of the method of the present disclosure, the at least one wash solution comprises L-arginine or a derivative thereof and an octanoate salt. In some embodiments of the method of the present disclosure, the at least one wash solution comprises L-arginine or a derivative thereof at a concentration of about 50 mM to about 200 mM (e.g., about 50 mM to about 75 mM, about 75 mM to about 100 mM, about 100 mM to about 125 mM, about 125 mM to about 150 mM, 150 mM to 175 mM, 175 mM to 200 mM, about 75 mM to about 175 mM, about 100 mM to about 150 mM, 125 mM to about 175 mM, about 150 mM to 200 mM. and all integers including and in between about 50 mM and about 200 mM) and an octanoate salt at a concentration of about 10 mM to about 50 mM (e.g., about 10 mM to about 20 mM, about 20 mM to about 30 mM, about 30 mM to about 40 mM, about 40 mM to about 50 mM, about 20 mM to 40 mM, about 30 mM to about 50 mM, about 10 mM to about 30 mM, and all integers including and in between about 10 mM and about 50 mM).

[0134] In some embodiments of the method of the present disclosure, passing at least one wash solution comprises passing a first wash solution through the chromatography resin, followed by passing a second wash solution through the chromatography resin. In some embodiments of the method of the present disclosure, the first and the second wash solutions comprise the same additive(s). In some embodiments of the method of the present disclosure, the concentration of the additive(s) in the first and the second wash solutions is the same. In some embodiments of the method of the present disclosure, the concentration of the additive(s) in the first and the second wash solutions is different. In some embodiments of the method of the present disclosure, the concentration of the additive(s) in the first wash solution is less than the concentration of the additive(s) in the second wash solution. In some embodiments of the method of the present disclosure, the concentration of the additive(s) in the first wash solution is more than the concentration of the additive(s) in the second wash solution.

[0135] In some embodiments of the method of the present disclosure, the first and second wash solutions comprise L-arginine or a derivative thereof. In some embodiments of the method of the present disclosure, the first and second wash solutions comprise an octanoate salt. In some embodiments of the method of the present disclosure, the first and second wash solutions comprise L-arginine or a derivative thereof and an octanoate salt.

[0136] In some embodiments of the method of the present disclosure, the first and the second wash solutions comprise different additives. In some embodiments of the method of the present disclosure, the first wash solution comprises L-arginine or a derivative thereof, and the second wash solution comprises an octanoate salt. In some embodiments of the method of the present disclosure, the first wash solution comprises an octanoate salt, and the second wash solution comprises L-arginine or a derivative thereof.

[0137] In some embodiments of the method of the present disclosure, the first wash solution comprises L-arginine or a derivative thereof, and the second wash solution comprises a combination of L-arginine or a derivative thereof and an octanoate salt. In some embodiments of the method of the present disclosure, the first wash solution comprises a combination of L- arginine or a derivative thereof and an octanoate salt, and the second wash solution comprises L-arginine or a derivative thereof.

[0138] In some embodiments of the method of the present disclosure, the first wash solution comprises an octanoate salt, and the second wash solution comprises a combination of L-arginine or a derivative thereof and an octanoate salt. In some embodiments of the method of the present disclosure, the first wash solution comprises a combination of L-arginine or aderivative thereof and an octanoate salt, and the second wash solution comprises an octanoate salt.

[0139] In some embodiments, the method of the present disclosure comprises passing at least three wash solutions sequentially through the chromatography resin.

[0140] In some embodiments of the method of the present disclosure, each of the at least three wash solutions comprises the same additive. In some embodiments of the method of the present disclosure, each of the at least three wash solutions comprises L-arginine or a derivative thereof. In some embodiments of the method of the present disclosure, each of the at least three wash solutions comprises L-arginine or a derivative thereof at a concentration of about 50 mM to about 200 mM.

[0141] In some embodiments of the method of the present disclosure, each of the at least three wash solutions comprises an octanoate salt. In some embodiments of the method of the present disclosure, each of the at least three wash solutions comprises an octanoate salt at a concentration of about 10 mM to about 50 mM.

[0142] In some embodiments of the method of the present disclosure, the concentration of the same additive in each of the at least three wash solutions are different. In some embodiments of the method of the present disclosure, the concentration of the additive increases in each sequentially used wash solution. In some embodiments of the method of the present disclosure, the concentration of the additive increases linearly in each sequentially used wash solution. In some embodiments of the method of the present disclosure, the concentration of the additive increases linearly in each stepwise wash solution. In some embodiments of the method of the present disclosure, the concentration of the additive decreases in each sequentially used wash solution. In some embodiments of the method of the present disclosure, the concentration of the additive decreases linearly in each sequentially used wash solution. In some embodiments of the method of the present disclosure, the concentration of the additive decreases stepwise in each sequentially used wash solution.

[0143] In some embodiments of the method of the present disclosure, each of the at least three wash solutions comprises an additive that is different from an additive in any of the previously or subsequently used wash solutions. In some embodiments of the method of the present disclosure, the first wash solution comprises L-arginine or a derivative thereof, the second wash solution comprises an octanoate salt, and the third wash solution comprises L- arginine or a derivative thereof and an octanoate salt. In some embodiments of the method of the present disclosure, the first wash solution comprises an octanoate salt, the second wash solution comprises L-arginine or a derivative thereof, and the third wash solution comprises L-arginine or a derivative thereof and an octanoate salt. In some embodiments of the method of the present disclosure, the first wash solution comprises L-arginine or a derivative thereof, the second wash solution comprises an octanoate salt, and the third wash solution comprises L- arginine or a derivative thereof and an octanoate salt. In some embodiments of the method of the present disclosure, the first wash solution comprises L-arginine or a derivative thereof and an octanoate salt, the second wash solution comprises L-arginine or a derivative thereof, and the third wash solution comprises an octanoate salt. In some embodiments of the method of the present disclosure, the first wash solution comprises L-arginine or a derivative thereof and an octanoate salt, the second wash solution comprises an octanoate salt, and the third wash solution comprises L-arginine or a derivative thereof. In some embodiments of the method of the present disclosure, the first wash solution comprises an octanoate salt, the second wash solution comprises L-arginine or a derivative thereof and an octanoate salt, and the third wash solution comprises L-arginine or a derivative thereof.

[0144] In some embodiments of the method of the present disclosure, the first wash solution comprises L-arginine or a derivative thereof, the second wash solution comprises L- arginine or a derivative thereof and an octanoate salt, and the third wash solution comprises an octanoate salt.

[0145] In some embodiments of the method of the present disclosure, the first and the second wash solutions comprise L-arginine or a derivative thereof, and the third wash solution comprises an octanoate salt. In some embodiments of the method of the present disclosure, the first and the second wash solutions comprise L-arginine or a derivative thereof, and the third wash solution comprises L-arginine or a derivative thereof and an octanoate salt.

[0146] In some embodiments of the method of the present disclosure, the first and the second wash solutions comprise an octanoate salt, and the third wash solution comprises L- arginine or a derivative thereof. In some embodiments of the method of the present disclosure, the first and the second wash solutions comprise an octanoate salt, and the third wash solution comprises L-arginine or a derivative thereof and an octanoate salt.

[0147] In some embodiments of the method of the present disclosure, the first and the second wash solutions comprise L-arginine or a derivative thereof and an octanoate salt and the third wash solution comprises L-arginine or a derivative thereof.

[0148] In some embodiments of the method of the present disclosure, the first wash solution comprises L-arginine or a derivative thereof, and the second and the third wash solution comprise an octanoate salt. In some embodiments of the method of the presentdisclosure, the first wash solution comprises L-arginine or a derivative thereof, and the second and the third wash solution comprise L-arginine or a derivative thereof and an octanoate salt.

[0149] In some embodiments of the method of the present disclosure, the first wash solution comprises an octanoate salt, and the second and the third wash solution comprise L- arginine or a derivative thereof. In some embodiments of the method of the present disclosure, the first wash solution comprises an octanoate salt, and the second and the third wash solution comprise L-arginine or a derivative thereof and an octanoate salt.

[0150] In some embodiments of the method of the present disclosure, the first wash solution comprises L-arginine or a derivative thereof and an octanoate salt, and the second and the third wash solution comprise L-arginine or a derivative thereof. In some embodiments of the method of the present disclosure, the first wash solution comprises L-arginine or a derivative thereof and an octanoate salt, and the second and the third wash solution comprise an octanoate salt.

[0151] In some embodiments of the method of the present disclosure, the L-arginine derivative is selected from the group consisting of L-arginine hydrochloride, L-arginine dihydrochloride. L-arginine methyl ester dihydrochloride, L-arginine ethyl ester dihydrochloride, acetyl L-arginine, N-alpha-butyroyl-L-arginine, agmatine, L-arginic acid and N-alpha-py valoyl L-arginine. In exemplary7embodiments, the L-arginine or arginine derivative comprises L-arginine hydrochloride. In some embodiments of the method of the present disclosure, the octanoate salt is selected from the group consisting of sodium octanoate, magnesium octanoate, potassium octanoate and calcium octanoate.

[0152] In some embodiments of the method of the present disclosure, the cell lysate and / or a cell culture supernatant comprising the rAAV or the rAAV preparation comprises an effective amount of an additive that is same as the additive in the wash buffer for passing through or applying to the resin. In some embodiments, the cell lysate and / or a cell culture supernatant comprising the rAAV or the rAAV preparation comprises an additive at the same concentration as compared to the concentration of the same additive in the wash buffer for passing through or applying to the resin. In some embodiments, the cell lysate and / or a cell culture supernatant comprising the rAAV or the rAAV preparation comprises the additive at a concentration that is between at least 2x to at least lOx (e.g., at least 2x to at least 3x, at least 3x to at least 4x, at least 4x to at least 5x, at least 5x to at least 6x, at least 6x to at least 7x, at least 7x to at least 8x, at least 8x to at least 9x, at least 9x to at least lOx, at least 3x to at least 9x, at least 4x to at least 8x, at least 5x to at least 7x, and all integers including and in between about 2x to aboutlOx) as compared to the concentration of the same additive in the wash buffer for passing through or applying to the resin.

[0153] In some embodiments of the method of the present disclosure, the chromatography resin is an affinity chromatography medium, an anion exchange (AEX) chromatography medium, a hydrophobic interaction chromatography (HIC) medium, a mix-mode chromatography medium, a tangential flow (TFF) chromatography medium, or a combination thereof.Method ofHCP reduction using r AAV affinity chromatographic purification

[0154] In some embodiments of the method of the present disclosure, the affinity chromatography medium comprises a protein or ligand that binds to AAV capsid protein. In some embodiments of the method of the present disclosure, the affinity chromatography medium comprises an AAV-specific antibody or a fragment thereof. In some embodiments, the AAV-specific antibody is a camelid AAV specific antibody.

[0155] In some embodiments of the method of the present disclosure, wherein the chromatography resin is an affinity chromatography medium the at least one wash solution comprises an organic solvent or a detergent.

[0156] In some embodiments of the method of the present disclosure, the organic solvent is selected from the group consisting of acetone, acetonitrile, ethanol, ethylene glycol, ethyl acetate, methanol, dimethyl formamide, isopropanol, polypropylene / hexylene glycol and glycerol. In some embodiments of the method of the present disclosure, the wash solution comprises the organic solvent at a concentration of about 5% to about 20% w / v (e.g., about 5% to about 7.5%, about 7.5% to about 10%, about 10% to about 12.5%, about 12.5% to about 15%, about 15% to about 17.5%, about 17.5% to about 20%, about 7.5% to about 17.5%, about 10% to about 15%, about 12.5% to about 17.5%, and all percentage including and in between about 5% and about 20%).

[0157] In some embodiments of the method of the present disclosure, the wash buffer comprises anon-ionic detergent. In some embodiments of the method of the present disclosure, the at least one wash solution comprises a non-ionic detergent selected from Nonidet P-40, 3- [(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS), triton x-100, and tri (n-butyl) phosphate (TNBP). In some embodiments of the method of the present disclosure, the wash buffer comprises anon-ionic detergent at a concentration of about 0.1% to about 15% w / v (e.g., about 0.1% to about 0.5%, about 0.5% to about 1.0%, about 1.0% to about 2.5%, about 2.5% to about 5%, about 5% to about 7.5%. about 7.5% to about 10%, about 10% to about 12.5%, about 12.5% to about 15%, about 0.5% to about 12.5%, about 1% to about 10%,about 2.5% to about 7.5%, and all percentage including and in between about 0.1% to about 15%).

[0158] In some embodiments of the method of the present disclosure, the wash solution has a pH of between about 6.0 to about 9.0 (e.g., about 6.0 to about 6.5, about 6.5 to about 7.0, about 7.0 to about 7.5, about 7.5 to about 8.0, about 8.0 to about 8.5, about 8.5 to about 9.0, about 6.5 to about 8.5, about 7.0 to about 8.0, about 7.5 to about 8.5, and all integers including and in between about 6.0 and about 9.0). In one embodiment, the pH of the one or more wash solutions is in a range of 8.0 - 9.0. In some embodiments of the method of the present disclosure, the pH of the one or more wash solutions is about 8.0. In another embodiment, the pH of the one or more wash solutions is about 9.0.

[0159] In some embodiments of the method of the present disclosure, the wash solution comprises an ionic strength modifier or a non-buffering salt. As used herein, the terms "ionic strength modifier” and “non-buffering salt” are used interchangeably and refer to a salt that is present in the wash solution that is of a type, and at a concentration, such that it does not substantially contribute to retaining the pH of the wash solution(s) under the applied conditions (such as high pH) upon addition of acid or base. Typically, the ionic strength modifier is an ionic salt. Ionic strength modifier includes halogen salts, including those that comprise Cl or Br (more preferably Cl, in particular halogen salts comprising alkali metals or alkaline earth metals, including Na, K, Ca, and Mg (more preferably Na or K). The terms “ionic strength modifier” and “non-buffering salt” do not include buffering salts, such as sodium acetate, sodium phosphate and Tris, that do substantially contribute to retaining the pH of a wash solution(s) under the applied conditions. In some embodiments, the ionic strength modifier is a halogen salt (e.g., comprising Cl or Br). In some embodiments, the ionic strength modifier is a halogen salt that comprises sodium (Na), potassium (K), calcium (Ca) or magnesium (Mg), more preferably, sodium (Na) or potassium (K). In some embodiments, the ionic strength modifier is selected from the group consisting of NaCl, KC1, CaCh and MgCh. In some embodiments, the ionic strength modifier is sodium chloride (NaCl) a concentration in a range of about 0.1 M to about 2.0 M (about 0.1 M to about 0.25 M, about 0.25 M to about 0.5 M, about 0.5 M to about 0.75 M, about 0.75 M to about 1.0 M, about 1.0 M to about 1.25 M, about 1.25 M to about 1.5 M, about 1.5 M to about 1.75 M, about 1.75 M to about 2.0 M, about 0.25 M to about 1.75 M, about 0.5 M to about 1.5 M, 0.75 M to about 1.25 M, and all integers including and in between about 0. 1 M and about 1.5 M).

[0160] In some embodiments of the method of the present disclosure, the wash buffer comprises any one of Tris (tris (hydroxymethyl) methylamine), bis-Tris, bis-Tris propane,histidine, triethanolamine, diethanolamine, formate, acetate, MES (2-(N morpholino) ethanesulfonic acid), phosphate. HEPES (4-2 hy droxy ethyl- 1 -piperazineethanesulfonic acid), citrate, MOPS (3-(N- morpholino) propanesulfonic acid ), TAPS (3-{[ tris (hydroxymethyl) methylamino} propanesulfonic acid), Bicine (N, N-bis (2-hydroxyethyl) glycine), Tricine (N- tris(hydroxymethyl) methylglycine), TES (2-{[ tris (hydroxymethyl) methyl] amino} ethanesulfonic acid) . PIPES (pipera zine-N, NP-bis (2-ethanesulfonic acid), cacodylate (dimethylarsinic acid) or SSC (saline sodium citrate). In some embodiments of the method of the present disclosure, the wash buffer comprises glycine-HCl or citric acid. In some embodiments of the method of the present disclosure, the wash buffer comprises glycine-HCl.

[0161] In some embodiments of the method of the present disclosure, the elution solution further comprises an ionic strength modifier or non-buffering salt selected from the group consisting of sodium chloride, sodium iodide, sodium thiocyanate, calcium chloride, magnesium chloride, or a combination thereof. In some embodiments of the method of the present disclosure, the elution solution has a pH of 2.5 to 3.0 (e.g., about 2.5 to about 2.6, about 2.6 to about 2.7, about 2.7 to about 2.8, about 2.8 to about 2.9. about 2.9 to about 3.0, about 2.6 to about 2.9. about 2.7 to about 2.8. about 2.7 to about 2.9, and all integers including and in between about 2.5 and about 3.0).

[0162] As used herein , the term “affinity chromatography medium”, “affinity7chromatography resin”, “affinity chromatography matrix”, “AC matrix”, “AC resin” or “AC medium” are used interchangeably and intended to refer to a solid phase medium, typically a gel or resin, that allows for separation of biochemical mixtures based on a highly specific binding interaction between ligand that binds to AAV capsid protein and the AC medium, such as between an AAV-specific antibody or a fragment thereof and the AC medium. Thus, the solid phase medium comprises a ligand capable of reversibly affixing that binds to AAV capsid protein, depending upon the buffer conditions. In some embodiments, the AAV specific antibody or a fragment thereof is covalently linked to the solid phase media. Non-limiting examples of immobilized or solid phase media that can comprise the AC medium include a gel matrix, such as agarose beads (such as commercially available Sepharose matrices), and a glass matrix, such as porous glass bead. Binding of the rAAV to the hgand affixed to the AC medium typically is achieved by column chromatography. That is, the AC medium is formed into a column, a biochemical mixture containing a rAAV is flowed through the column, followed by washing of the column by flowing through the column one or more wash solutions, followed by elution of the rAAV from the column by flowing through the column an elution bufferfollowed by collecting one or more fractions of the elution buffer after flowing through the column.

[0163] Once eluted, the pH of the elution buffer collected after flowing through the column is preferably neutralized in a manner suitable for obtaining a first rAAV enriched composition with a neutral or basic pH, which includes a pH of 8.0 or above, which includes 8.5. The reason is that rAAV particles tend to lose their integrity and / or infectivity if maintained in a composition having an acid pH. According to one exemplary embodiment, the first rAAV enriched composition is neutralized in a Tris buffer having a pH of about 8.5. According to another exemplary embodiment, the first rAAV enriched composition is neutralized in a Tris buffer having a pH of about 8.0.

[0164] Alternatively, binding of the rAAV of interest to the AC medium can be achieved by batch treatment, in which the biochemical mixtures, cell lysates or cell culture supernatants containing the rAAV is incubated with the AC medium in a vessel to allow for binding of the rAAV to the AC medium, the solid phase medium is removed from the vessel (e g., by centrifugation), the solid phase medium is washed to remove impurities and again recovered (e.g., by centrifugation) and the rAAV is eluted from the solid phase medium. In yet another embodiment, a combination of batch treatment and column chromatography can be used. For example, the initial binding of the rAAV to the AC medium can be achieved by batch treatment and then the solid phase medium can be packed into a column, following by washing of the column and elution of the rAAV from the column.

[0165] In some embodiments of the method of the present disclosure, the affinity chromatography medium is selected from any one of a Protein A matrix, a Protein G matrix, a Protein A / G matrix, or a Protein L matrix. In some embodiments, the Protein A matrix is any matrix, medium or resin that is well know n in the art and suitable for use in the invention. Nonlimiting examples of commercially available Protein A resins or matrices include MabSelect™, MabSelect™ Xtra. MabSelect™ Sure, rProtein A SepharoseFF, rmpProtein A Sepharose FF, Protein A Sepharose CL-4B and nProtein A Sepharose 4 FF ( all commercially available from GE Healthcare ), ProSep™ A, ProSep-VA™ High Capacity, ProSep-VA Ultra™ and ProSep- Va Ultra™ Plus, POROS A™ and Mabcapture™ A. IPA-300, IPA-400 and IPA-500, Affigel™ protein A and Affiprep™ protein A, MABsorbent™ A1P and MABsorbent™ A2P, Protein A Ceramic Hyper DF, Ultralink™ Immobilized protein A, Agarose protein A, Protein A Cellthru300™, and Protein A Ultraflow-. In some embodiments of the method of the present disclosure, the affinity chromatography medium is selected from any one of a POROSCaptureSelect™ AAV8 (PCS-AAV8), POROS CaptureSelect™ AAV9 (PCS-AAV9), POROS CaptureSelect™ AAVX (PCS-AAVX) (Thermo Scientific), and AVB™ Sepharose High Performance (Cytiva).

[0166] In some embodiments, the method of the present disclosure further comprises contacting the affinity chromatographic resin with an equilibration buffer before applying the cell lysate and / or a cell culture supernatant to a chromatography resin or contacting the chromatography resin with the rAAV preparation. In some embodiments, the equilibration buffer comprises any one of Tris (tris (hydroxymethyl) methylamine), bis-Tris, bis-Tris propane, histidine, triethanolamine, diethanolamine, formate, acetate, MES (2-(N morpholino) ethanesulfonic acid), phosphate, HEPES (4-2 hydroxyethyl-1 -piperazineethanesulfonic acid), citrate, MOPS (3-(N- morpholino) propanesulfonic acid ). TAPS (3-{[ tris (hydroxymethyl) methylamino} propanesulfonic acid), Bicine (N, N-bis (2-hydroxyethyl) glycine), Tricine (N- tris(hydroxymethyl) methylglycine), TES (2-{[ tris (hydroxymethyl) methyl] amino} ethanesulfonic acid) , PIPES (pipera zine-N, NP-bis (2-ethanesulfonic acid), cacodylate (dimethylarsinic acid) or SSC (saline sodium citrate). In some embodiments of the method of the present disclosure, the equilibration buffer comprises Tris. In some embodiments of the method of the present disclosure, the equilibration buffer comprises Tris at a concentration of about 10 mM to 50 mM (e.g., about 10 mM to about 15 mM, about 15 mM to about 20 rnM, about 20 mM to about 25 mM, about 25 mM to about 30 mM, about 30 mM to about 35 rnM, about 35 mM to about 40 mM, about 40 mM to about 45 mM, about 45 mM to about 50 mM, about 15 mM to about 45 mM, about 20 mM to about 40 mM, about 25 mM to about 35 mM and all integers including and in between about 10 mM to 50 mM). Tris. In some embodiments of the method of the present disclosure, the equilibration buffer comprises Tris at a concentration of about 20 mM.

[0167] In some embodiments of the method of the present disclosure, the equilibration buffer comprises an ionic strength modifier selected from the group consisting of NaCl, KC1, CaCh and MgCh. In some embodiments, the ionic strength modifier is NaCl. In some embodiments, the ionic strength modifier is at a concentration in a range of about 0.1 M to about 1.0 M (about 0. 1 M to about 0.25 M, about 0.25 M to about 0.5 M, about 0.5 M to about 0.75 M, about 0.75 M to about 1.0 M, about 0.25 M to about 0.75 M, about 0.5 M to about 1.0 M, and all integers including and in between about 0. 1 M and about 1.0 M).

[0168] In some embodiments of the method of the present disclosure, the equilibration buffer comprises poloxamer 188 at a concentration of about 0.0001% (w / v) to about 0.01% (w / v) (e.g., about 0.0001% (w / v) to about 0.00025% (w / v), about 0.00025% (w / v) to about0.0005% (w / v), about 0.0005% (w / v) to about 0.001% (w / v), about 0.001% (w / v) to about0.0025% (w / v), about 0.0025% (w / v) to about 0.005% (w / v). about 0.005% (w / v) to about0.0075% (w / v), about 0.0075% (w / v) to about 0.01% (w / v), about 0.00025% (w / v) to about0.0075% (w / v), about 0.0005% (w / v) to about 0.005% (w / v), about 0.001% (w / v) to about0.01% (w / v), about 0.0025% (w / v) to about 0.0075% (w / v) and all percentages including and in between about 0.001% (w / v) to about 0.01% (w / v). In some embodiments of the method of the present disclosure, the equilibration buffer comprises poloxamer 188 at a concentration of about 0.001% (w / v).

[0169] In some embodiments of the method of the present disclosure, the equilibration buffer has a pH of between about 7.0 to about 10.0 (e.g., about 7.0 to about 7.5, about 7.5 to about 8.0, about 8.0 to about 8.5, about 8.5 to about 9.0. about 9.0 to about 9.5, about 9.5 to about 10.0, about 7.5 to about 9.5, about 8.0 to about 9.0, and all integers including and in between about 7.0 to about 10.0). In some embodiments of the method of the present disclosure, the equilibration buffer has a pH of about 7.5.

[0170] In some embodiments of the method of the present disclosure, the equilibration buffer comprises an additive that is same as the additive in the wash buffer for passing through or applying to the resin. In some embodiments, the equilibration buffer comprises an additive at a concentration that is same as compared to the concentration of the same additive in the wash buffer for passing through or applying to the resin. In some embodiments, the equilibration buffer comprises an additive at a concentration that is between at least 2x to at least l Ox (e g., at least 2x to at least 3x, at least 3x to at least 4x, at least 4x to at least 5x, at least 5x to at least 6x, at least 6x to at least 7x, at least 7x to at least 8x, at least 8x to at least 9x, at least 9x to at least lOx, at least 3x to at least 9x, at least 4x to at least 8x, at least 5x to at least 7x, and all integers including and in between about 2x to about lOx) as compared to the concentration of the same additive in the w ash buffer for passing through or applying to the resin.

[0171] In some embodiments, the method of the present disclosure comprises passing though or applying to the affinity chromatography resin about 2 to about 10 column volumes (CV) of the equilibration buffer (e.g., about 2 to about 3. about 3 to about 4. about 4 to about 5, about 5 to about 6, about 6 to about 7, about 7 to about 8, about 8 to about 9, about 9 to about 10, about 3 to about 9, about 4 to about 8, about 5 to about 7, and all integers including and in between about 2 to about 10).Method ofHCP reduction using AEX chromatographic purification

[0172] In some embodiments of the method of the present disclosure, the AEX chromatography medium is a weak ionic exchanger. In some embodiments of the method of the present disclosure, the AEX chromatography medium is a strong ionic exchanger. In some embodiments of the method of the present disclosure, the AEX chromatography medium is in the form of a packed bed. In some embodiments of the method of the present disclosure, the AEX chromatography medium is a chromatographic monolithic column.

[0173] In some embodiments of the method of the present disclosure, the AEX chromatography medium is selected from a group consisting of a combined eluent fraction CIMmultus® QA column, CIMac® QA column, NuviaQ® column, POROS™ HQ column, Eshmuno® Q column, POROS XQ™ column, FractoGel® TMAE column, and a CaptoQ® column.

[0174] In some embodiments of the method of the present disclosure, the at least one wash solution comprises a quaternary ammonium salt. As used herein, the term “quaternary ammonium salt” refers to an ionic compound having a quaternary ammonium nitrogen, four groups (e.g., alkyl or aryl groups) connected to the ammonium nitrogen, and an anionic ion (e.g.. acetate, bromide, or chloride).

[0175] In some embodiments of the method of the present disclosure, the quaternary ammonium salt is a tetraalkylammonium salt, a choline chloride, or a divalent salt. In some embodiments of the method of the present disclosure, the tetraalkylammonium salt is a tetraalkylammonium chloride or a tetraalkylammonium acetate. In some embodiments of the method of the present disclosure, the tetraal k l ammonium chloride is selected from the group consisting of tetramethyl ammonium chloride (TMAC), tetraethylammonium chloride (TEAC), tetrapropylammonium chloride (TPAC), tetrabutylammonium chloride (TBAC), benzyltributylammonium chloride (BTBAC), and any combination(s) thereof. In some embodiments of the method of the present disclosure, the tetraalkylammonium acetate is selected from the group consisting of tetramethylammonium acetate, tetraethylammonium acetate (TEA-Ac), tetrapropylammonium acetate, tetrabuty lammonium acetate, and any combination(s) thereof.

[0176] In some embodiments of the method of the present disclosure, the at least one wash solution comprises the quaternary ammonium salt at a concentration of about 30 mM to about 200 mM (e.g., about 30 mM to about 50 mM, about 50 mM to about 75 mM, about 75 mM to about 100 mM, about 100 mM to about 125 mM, about 125 mM to about 150 mM, about 150 mM and about 175 mM, about 175 mM, about 200 mM. about 50 mM to about 200 mM, about 50 mM to about 175 mM, about 75 mM to 150 mM, about 75 mM to about 125 mM, about 100mM to about 150 mM, and all integers including and in between about 30 mM and about 200 mM). In some embodiments, the at least one wash solution comprises the quaternary ammonium salt at a concentration of at least 30 mM, at least 50 mM, at least 70 mM, at least 90 mM, at least 95 mM, at least 98 mM, at least 99 mM or at least 100 mM. In some embodiments, the at least one wash solution comprises the quaternary7ammonium salt at a concentration of no more than 200 mM, no more than 180 mM, no more than 160 mM, no more than 150 mM, no more than 140 mM, no more than 130 mM. or no more than 120 mM.

[0177] In some embodiments of the method of the present disclosure, the wash solution has a pH of between about 6.0 to about 9.0 (e.g., about 6.0 to about 6.5, about 6.5 to about 7.0, about 7.0 to about 7.5. about 7.5 to about 8.0, about 8.0 to about 8.5, about 8.5 to about 9.0, about 6.5 to about 8.5, about 7.0 to about 8.0, about 7.5 to about 8.5, and all integers including and in between 6.0 and 9.0).

[0178] In some embodiments of the method of the present disclosure, the elution solution comprises a salt in the form of any one of an isocratic salt solution or a gradient salt solution. In some embodiments of the method of the present disclosure, the concentration of salt within the elution solution increases continuously over time throughout the elution step. In some embodiments of the method of the present disclosure, the concentration of salt within the elution solution increases linearly over time throughout the elution step. In some embodiments of the method of the present disclosure, the concentration of salt within the elution solution increases stepwise throughout the elution step.

[0179] In some embodiments, the elution solution comprises sodium chloride, sodium sulfate, magnesium chloride, magnesium sulfate, or any combination thereof.

[0180] In some embodiments, the elution solution comprises a divalent salt, e.g., magnesium chloride. For example, in some embodiments, the elution solution comprises 1 mM to about 10 mM (e.g., about 1 mM to about 2 mM, about 2 mM to about 3 mM, about 3 mM to about 4 mM, about 4 mM to about 5 mM, about 5 mM to about 6 mM, about 6 mM to about 7 mM, about 7 mM to about 8 mM, about 9 mM to about 10 mM, about 2 mM to about 9 mM, about 3 mM to about 8 mM, about 4 mM to about 7 mM, about 5 mM to about 7 mM, and all integers including and in between about 1 mM and about 10 mM) of a divalent salt. e.g.. magnesium chloride.

[0181] In some embodiments, the elution solution comprises about 25 mM to about 375 mM (e.g., about 25 mM to about 50 mM, about 50 mM to about 75 mM, about 75 mM to about 100 mM. about 100 mM to about 125 mM, about 125 mM to about 150 mM, about 150 mM to about 175 mM, about 175 mM to about 200 mM, about 200 mM to about 225 mM, about 225mM to about 250 mM, about 250 mM to about 275 mM, about 275 mM to about 300 mM, about 300 mM to about 325 mM, about 325 mM to about 350 mM about 350 mM to about 375 mM, about 50 mM to 350 mM, about 75 mM to about 325 mM, about 100 mM to about 300 mM, about 125 mM to about 250 mM, about 150 mM to about 225 mM, about 175 mM to about 200 mM, and all integers including and in between about 25 mM and about 375 mM) of sodium chloride.

[0182] In some embodiments of the method of the present disclosure, the elution solution comprises an anionic species, tetrafluoroborate (BF4), bromide (Br), or acetate (Ac). In some embodiments of the method of the present disclosure, the elution solution comprises an anionic species associated with tetraethylammonium (TEA), e.g., TEA-BF4, TEA-Br, or TEA-Ac.

[0183] In some embodiments of the method of the present disclosure, the elution solution comprises a stabilizer or a surfactant, e.g., a non-ionic surfactant. Non-limiting examples of suitable non-ionic surfactants include, e.g., Pluronic® F-68. In some embodiments of the method of the present disclosure, the stabilizer or surfactant is present in a solution at a concentration of about 0.0001%, about 0.0005%, or about 0.001%. In some embodiments of the method of the present disclosure, the stabilizer or surfactant is present in a solution at a concentration of no greater than about 0.001%.

[0184] In some embodiments, the elution solution comprises at least one salt in common as the at least one wash solution, but at a different concentration. In some embodiments, the elution solution comprises a different salt than that present in the at least one wash solution.

[0185] In some embodiments of the method of the present disclosure, the elution solution is a bis-Tris Propane-based buffer, a potassium phosphate-based buffer. In some embodiments of the method of the present disclosure, the elution solution has a pH of about 6.0 to about 10.0 (e.g., about 6.0 to about 6.5, about 6.5 to about 7.0. about 7.0 to about 7.5. about 7.5 to about 8.0, about 8.0 to about 8.5, about 8.5 to about 9.0, about 9.0 to about 9.5, about 9.5 to about 10.0, about 6.5 to about 9.5, about 7.0 to about 9.0, about 7.5 to about 8.5, and all integers including and in between about 6.0 to about 10.0).Method of HCP reduction using HIC chromatographic purification

[0186] In some embodiments of the method of the present disclosure, the HIC medium is selected from the group consisting of methyl HIC, Source ETH, Toyopearl1MEther-650, Toyopearl™ PPG-600, Source Iso, Source Phe, Phenyl Sepharose, Toyopearl™ Phenyl-600, Toyopearl™ Phenyl-650, Butyl Sepharose, Toyopearl™ Butyl-600, Toyopearl™ Butyl-650, Toyopearl™ SuperButyl-550, t-Butyl HIC, Toyopearl™ Hexyl-650, and Octyl Sepharose.

[0187] In some embodiments of the method of the present disclosure, the wash buffer comprises a salt selected from the group consisting of a sulfate salt, a citrate salt, ammonium sulfate, sodium sulfate, sodium phosphate, sodium chloride, ammonium chloride, sodium bromide, potassium phosphate and a combination thereof. In some embodiments of the method of the present disclosure, the wash solution comprises sodium phosphate or potassium phosphate. In some embodiments of the method of the present disclosure, the wash buffer comprises a cation selected from the group consisting of Ba2+, Ca2+, Mg2+, Li+, Cs+. Na+, K+. Rb+, NH4+and a combination thereof. In some embodiments of the method of the present disclosure, the wash buffer comprises an anion selected from the group consisting of PO43, SO42, CH3CO3 , Cl”, Br , NO3", CIO4 , I , SCN and a combination thereof. In some embodiments of the method of the present disclosure, the wash buffer comprise a salt having a concentration of between about 50 mM and about 2000 mM (e.g., about 50 mM to about 200 mM, about 200 mM to about 400 mM, about 400 mM to about 600 mM, about 600 mM to about 800 mM, about 800 mM to about 1000 mM, about 1000 mM to about 1200 mM, about 1200 mM to about 1400 mM, about 1400 mM to about 1600 mM, about 1600 mM to about 2000 mM, about 900 mM to about 1600 mM, about 200 mM to about 1600 mM, about 400 mM to about 1400 mM, about 600 mM to about 1200 mM, about 800 mM to about 1000 mM, and all integers including and in between about 50 mM and about 2000 mM).

[0188] In some embodiments of the method of the present disclosure, the wash solution has a pH of about 5.0 to about 7.0 (about 5.0 to about 5.5, about 5.5 to about 6.0, about 6.0 to about 6.5, about 6.5 to about 7.0, about 5.0 to about 6.0, about 5.5 to about 6.5, about 6.0 to about 7.0, and all integers including and in between about 5.0 and about 7.0).

[0189] In some embodiments of the method of the present disclosure, the elution solution comprises the same salt as the wash solution at a lower concentration as compared to the wash solution. In some embodiments of the method of the present disclosure, the elution solution comprises sodium phosphate or potassium phosphate at a lower concentration as compared to the wash solution.

[0190] In some embodiments of the method of the present disclosure, the elution solution has a pH of about 5.0 to about 7.0 (about 5.0 to about 5.5, about 5.5 to about 6.0, about 6.0 to about 6.5, about 6.5 to about 7.0, about 5.0 to about 6.0, about 5.5 to about 6.5, about 6.0 to about 7.0, and all integers including and in between about 5.0 and about 7.0).

[0191] In some embodiments of the method of the present disclosure, the cell culture supernatant, or the cell lysate and / or a cell culture supernatant comprising the rAAV or the rAAV preparation is concentrated via tangential flow filtration (‘TFF”) before applying to orcontacting with the chromatography resin. In some embodiments of the method of the present disclosure, the one or more fractions of the eluent flowthrough comprising said rAAV is concentrated via TFF. In some embodiments of the method of the present disclosure, the cell culture supernatant, or the cell lysate and / or a cell culture supernatant comprising the rAAV or the rAAV preparation is concentrated via tangential flow filtration (“TFF”) before applying to or contacting with the chromatography resin, and the one or more fractions of the eluent flowthrough comprising said rAAV is concentrated via TFF.

[0192] In some embodiments, the method of the present disclosure comprises concentrating the cell lysate and / or a cell culture supernatant comprising the rAAV or the rAAV preparation is concentrated via TFF before applying to or contacting with the affinity chromatography resin.

[0193] In some embodiments, the method of the present disclosure comprises concentrating the cell lysate and / or a cell culture supernatant comprising the rAAV or the rAAV preparation is concentrated via TFF before applying to or contacting with the AEX chromatography resin.

[0194] In some embodiments, the method of the present disclosure comprises concentrating the cell lysate and / or a cell culture supernatant comprising the rAAV or the rAAV preparation is concentrated via TFF before applying to or contacting with the HIC chromatography resin.

[0195] In some embodiments of the method of the present disclosure, the one or more fractions of the eluent flowthrough comprising said rAAV is concentrated via TFF. In some embodiments of the method of the present disclosure, the one or more fractions of the eluent flowthrough comprising said rAAV generated from an affinity chromatography resin is concentrated via TFF. In some embodiments of the method of the present disclosure, the one or more fractions of the eluent flow through comprising said rAAV generated from an AEX chromatography resin is concentrated via TFF. In some embodiments of the method of the present disclosure, the one or more fractions of the eluent flowthrough comprising said rAAV generated from an HIC resin is concentrated via TFF.Compositions for reducing HCP during rAAV chromatographic purification

[0196] In one aspect, the present disclosure also provides a composition comprising an effective amount of an additive selected from L-arginine or a derivative thereof and an octanoate salt, or a combination thereof, for use in reducing host cell protein (HCP) contaminants during chromatographic purification of rAAV from a rAAV preparation.

[0197] In some embodiments of the composition of the present disclosure, the additive is L-arginine or a derivative thereof at a concentration of about 50 mM to about 200 mM (e.g., about 50 mM to about 75 mM, about 75 mM to about 100 mM, about 100 mM to about 125 mM, about 125 mM to about 150 mM, about 150 mM to about 175 mM, about 175 mM to about 200 mM, about 75 mM to about 175 mM, about 100 mM to about 150 mM, about 125 mM to about 175 mM, about 150 mM to 200 mM, and all integers including and in between about 50 mM and about 200 mM).

[0198] In some embodiments of the composition of the present disclosure, the additive is an octanoate salt, e.g., sodium octanoate, at a concentration of about 10 mM to about 50 mM (e.g., about 10 mM to about 20 mM. about 20 mM to about 30 mM, about 30 mM to about 40 mM, about 40 mM to about 50 mM. about 20 mM to about 40 mM, about 30 mM to about 50 mM, about 10 mM to about 30 mM, and all integers including and in between about 10 mM and about 50 mM).

[0199] In some embodiments of the composition of the present disclosure, the composition comprises L-arginine or a derivative thereof at a concentration of about 50 mM to about 200 mM (e.g., about 50 mM to about 75 mM. about 75 mM to about 100 mM, about 100 mM to about 125 mM, about 125 mM to about 150 mM, about 150 mM to about 175 mM, about 175 mM to about 200 mM, about 75 mM to about 175 mM, about 100 mM to about 150 mM, about 125 mM to about 175 mM, about 150 mM to about 200 mM, and all integers including and in between about 50 mM and about 200 mM) and an octanoate salt at a concentration of about 10 mM to about 50 mM (e.g., about 10 mM to about 20 mM, about 20 mM to about 30 mM, about 30 mM to about 40 mM, about 40 mM to about 50 mM, about 20 mM to about 40 mM, about 30 mM to about 50 mM, about 10 mM to about 30 mM, and all integers including and in between about 10 mM and about 50 mM).

[0200] In some embodiments, the composition of the present disclosure is for use as a wash solution in a chromatographic medium post-application of a rAAV preparation to the medium.

[0201] In some embodiments of the composition of the present disclosure, the L-arginine derivative is selected from the group consisting of L-arginine hydrochloride, L-arginine dihydrochloride. L-arginine methyl ester dihydrochloride, L-arginine ethyl ester dihydrochloride, acetyl L-arginine, N-alpha-butyroyl-L-arginine, agmatine, L-arginic acid and N-alpha-pyvaloyl L-arginine.

[0202] In some embodiments of the composition of the present disclosure, the octanoate salt is selected from the group consisting of sodium octanoate, magnesium octanoate, potassium octanoate and calcium octanoate.

[0203] In some embodiments of the composition of the present disclosure, the chromatographic medium is any one of an affinity chromatography medium, an anion exchange (AEX) chromatography medium, a hydrophobic interaction chromatography (HIC) medium, a mix-mode chromatography medium, a tangential flow (TFF) chromatography medium, or a combination thereof.Composition for HCP reduction using affinity chromatographic purification

[0204] In some embodiments of the composition of the present disclosure, the chromatographic medium is an affinity chromatography medium. In some embodiments of the composition of the present disclosure, the affinity chromatography medium comprises a protein or ligand that binds to AAV capsid protein. In some embodiments of the composition of the present disclosure, the affinity chromatography medium comprises an AAV-specific antibody or a fragment thereof.

[0205] In some embodiments of the composition of the present disclosure, wherein the chromatography resin is an affinity chromatography medium the composition comprises an organic solvent or a detergent.

[0206] In some embodiments of the composition of the present disclosure, the organic solvent is selected from the group consisting of acetone, acetonitrile, ethanol, ethylene glycol, ethyl acetate, methanol, dimethyl formamide, isopropanol, polypropylene / hexylene glycol and glycerol. In some embodiments of the method of the present disclosure, the wash solution comprises the organic solvent at a concentration of about 5% to about 20% w / v (e.g., about 5% to about 7.5%, about 7.5% to about 10%, about 10% to about 12.5%, about 12.5% to about 15%, about 15% to about 17.5%, about 17.5% to about 20%, about 7.5% to about 17.5%, about 10% to about 15%, about 12.5% to about 17.5%, and all percentage including and in between about 5% and about 20%).

[0207] In some embodiments, the composition of the present disclosure comprises a nonionic detergent. In some embodiments, the composition of the present disclosure comprises a non-ionic detergent selected from Tris-HCl, Bis-Tris, MES-Na, polysorbate 80, ethylene glycol, sorbitol, mannitol, xylitol, DMSO, sucrose or trehalose, Triton X-100, or tri (n-butyl) phosphate (TNBP). In some embodiments, the composition of the present disclosure comprises a non-ionic detergent at a concentration of about 0.1% to about 15% w / v (e.g., about 0.1% to about 0.5%, about 0.5% to about 1.0%, about 1.0% to about 2.5%, about 2.5% to about 5%, about 5% to about 7.5%, about 7.5% to about 10%, about 10% to about 12.5%, about 12.5% to about 15%, about 0.5% to about 12.5%. about 1% to about 10%, about 2.5% to about 7.5%, and all percentage including and in between about 0.1% to about 15%).

[0208] In some embodiments, the composition of the present disclosure has a pH of between about 6.0 to about 9.0 (e.g., about 6.0 to about 6.5, about 6.5 to about 7.0, about 7.0 to about 7.5, about 7.5 to about 8.0, about 8.0 to about 8.5, about 8.5 to about 9.0, about 6.5 to about 8.5, about 7.0 to about 8.0, about 6.5 to about 8.5, about 7.5 to about 8.5, and all integers including and in between about 6.0 and about 9.0). In some embodiments, the composition of the present disclosure has a pH of between about 8.0 - 9.0. In some embodiments, the composition of the present disclosure has a pH of about 8.0. In some embodiments, the composition of the present disclosure has a pH of about 9.0.

[0209] In some embodiments, the composition of the present disclosure comprises an ionic strength modifier or a non-buffering salt. In some embodiments, the ionic strength modifier is a halogen salt (e.g., comprising Cl or Br). In some embodiments of the composition of the present disclosure, the ionic strength modifier is a halogen salt that comprises sodium (Na), potassium (K), calcium (Ca) or magnesium (Mg), more preferably, sodium (Na) or potassium (K). In some embodiments of the composition of the present disclosure, the ionic strength modifier is selected from the group consisting of sodium chloride, potassium chloride, calcium chloride and magnesium chloride. In some embodiments of the composition of the present disclosure, the ionic strength modifier is sodium chloride at a concentration in a range of about 0.1 M to about 2.0 M (about 0.1 M to about 0.25 M, about 0.25 M to about 0.5 M, about 0.5 M to about 0.75 M, about 0.75 M to about 1.0 M, about 1.0 M to about 1.25 M. about 1.25 M to about 1.5 M. about 1.5 M to about 1.75 M, about 1.75 M to about 2.0 M, about 0.25 to about 1 .75 M, about 0.5 M to about 1 .5 M, 0.75 M to about 1 .25 M, about 0.5 M to about 1 .5 M, and all integers including and in between about 0.1 M and about 2.0 M).

[0210] In some embodiments, the composition of the present disclosure comprises any one of Tris (tris (hydroxymethyl) methylamine), bis-Tris. bis-Tris propane, histidine, triethanolamine, diethanolamine, formate, acetate, MES (2-(N morpholino) ethanesulfonic acid), phosphate, HEPES (4-2 hy droxy ethyl- 1 -piperazineethanesulfonic acid), citrate, MOPS (3-(N- morpholino) propanesulfonic acid ), TAPS (3-{[ tris (hydroxymethyl) methylamino} propanesulfonic acid), Bicine (N, N-bis (2-hy droxy ethyl) glycine), Tricine (N- tris(hydroxymethyl) methylglycine), TES (2-{[ tris (hydroxymethyl) methyl] amino} ethanesulfonic acid) , PIPES (pipera zine-N, NP-bis (2-ethanesulfonic acid), cacodylate (dimethylarsinic acid) or SSC (saline sodium citrate). In some embodiments, the composition of the present disclosure comprises glycine-HCl or citric acid. In some embodiments, the composition of the present disclosure comprises glycine-HCl.

[0211] In some embodiments of the composition of the present disclosure, the affinity chromatography medium is selected from any one of a Protein A matrix, a Protein G matrix, a Protein A / G matrix, or a Protein L matrix. In some embodiments of the composition of the present disclosure, the Protein A matrix is any matrix, medium or resin that is well known in the art and suitable for use in the invention. Non-limiting examples of commercially available Protein A resins or matrices include Mab Select, MabSelect™ Xtra. MabSelect™ Sure, rProtein A SepharoseFF, rmpProtein A Sepharose FF. Protein A Sepharose CL-4B and nProtein A Sepharose 4 FF ( all commercially available from GE Healthcare ), ProSep™ A, ProSep-VA™ High Capacity. ProSep-VA Ultra™ and ProSep-Va Ultra™ Plus, POROS A™ and Mabcapture™ A, IPA-300, IPA-400 and IPA-500, Affigel™ protein A and Affiprep™ protein A, MABsorbent™ A1P and MABsorbent™ A2P, Protein A Ceramic Hyper DF, Ultralink™ Immobilized protein A, Agarose protein A, Protein A Cellthru300™, and Protein A Ultraflow. In some embodiments of the composition of the present disclosure, the affinity chromatography medium is selected from any one of a POROS Captures elect™ AAV8 (PCS- AAV8), POROS CaptureSelect™ AAV9 (PCS-AAV9), POROS CaptureSelect™ AAVX (PCS-AAVX) from Thermo Scientific, and AVB Sepharose High Performance from Cytiva. Composition for HCP reduction using AEX chromatographic purification

[0212] In some embodiments of the composition of the present disclosure, the AEX chromatography medium is a weak ionic exchanger. In some embodiments of the composition of the present disclosure, the AEX chromatography medium is a strong ionic exchanger. In some embodiments of the composition of the present disclosure, the AEX chromatography medium is in the form of a packed bed. In some embodiments of the composition of the present disclosure, the AEX chromatography medium is a chromatographic monolithic column.

[0213] In some embodiments of the composition of the present disclosure, the AEX chromatography medium is selected from a group consisting of a combined eluent fraction CIMmultus® QA column, CIMac QA® column, NuviaQ® column, POROS HQ™ column, Eshmuno® Q column, POROS XQ™ column, FractoGel® TMAE column, and a CaptoQ® column.

[0214] In some embodiments, the composition of the present disclosure comprises a quaternary ammonium salt. As used herein, the term ‘’quaternary ammonium salt” refers to an ionic compound having a quaternary' ammonium nitrogen, four groups (e.g., alkyd or aryl groups) connected to the ammonium nitrogen, and an anionic ion (e.g., acetate, bromide, or chloride).

[0215] In some embodiments of the composition of the present disclosure, the quaternary ammonium salt is a tetraalkylammonium salt, a choline chloride, or a divalent salt. In some embodiments of the composition of the present disclosure, the tetraalkylammonium salt is a tetraalkylammonium chloride or a tetraalkylammonium acetate. In some embodiments of the composition of the present disclosure, the tetraalkylammonium chloride is selected from the group consisting of tetramethylammonium chloride (TMAC), tetraethylammonium chloride (TEAC), tetrapropylammonium chloride (TPAC). tetrabutylammonium chloride (TBAC). benzyltributylammonium chloride (BTBAC), and any combination(s) thereof. In some embodiments, the composition of the present disclosure comprises the quaternary ammonium salt at a concentration of about 30 mM to about 200 mM (e g., about 30 mM to about 50 mM, about 50 mM to about 75 mM, about 75 mM to about 100 mM, about 100 mM to about 125 mM, about 125 mM to about 150 mM, about 150 mM and about 175 mM, about 175 mM and about 200 mM, about 50 mM to about 200 mM, about 50 mM to about 175 mM, about 75 mM to about 150 mM, about 75 mM to about 125 mM, about 100 mM to about 150 mM, and all integers including and in between about 30 mM and about 200 mM). In some embodiments, the composition of the present disclosure comprises the quaternary ammonium salt at a concentration of at least 30 mM, at least 50 mM, at least 70 mM, at least 90 mM, at least 95 mM, at least 98 mM, at least 99 mM or at least 100 mM. In some embodiments, the composition of the present disclosure comprises the quaternary ammonium salt at a concentration of no more than 200 mM, no more than 180 mM, no more than 160 mM. no more than 150 mM, no more than 140 mM, no more than 130 mM, or no more than 120 mM.

[0216] In some embodiments, the composition of the present disclosure has a pH of between about 6.0 to about 9.0 (e.g., about 6.0 to about 6.5, about 6.5 to about 7.0, about 7.0 to about 7.5, about 7.5 to about 8.0, about 8.0 to about 8.5, about 8.5 to about 9.0, about 6.5 to about 8.5, about 7.0 to about 8.0, about 7.5 to about 8.5, and all integers including and in between 6.0 and 9.0).Composition for HCP reduction using HIC chromatographic purification

[0217] In some embodiments of the composition of the present disclosure, the HIC medium is selected from the group consisting of methyl HIC, Source ETH. Toyopearl Ether-650. Toyopearl PPG-600, Source Iso, Source Phe, Phenyl Sepharose, Toyopearl Phenyl-600, Toyopearl Phenyl-650, Butyl Sepharose, Toyopearl Butyl-600, Toyopearl Butyl-650, Toy opearl SuperButyl-550, t-Butyl HIC, Toyopearl Hexyl-650, and Octyl Sepharose.

[0218] In some embodiments, the composition of the present disclosure comprises a salt selected from the group consisting of a sulfate salt, a citrate salt, ammonium sulfate, sodiumsulfate, sodium phosphate, sodium chloride, ammonium chloride, sodium bromide, potassium phosphate and a combination thereof. In some embodiments, the composition of the present disclosure comprises sodium phosphate or potassium phosphate. In some embodiments, the composition of the present disclosure comprises a cation selected from the group consisting of Ba2+, Ca2+, Mg2+, Li+, Cs+, Na+, K+, Rb+, NHr+and a combination thereof. In some embodiments, the composition of the present disclosure comprises an anion selected from the group consisting of PO43, SO42, CH3CO3 , Cl”, Br . NO3 . CIO4 , 1", SCN and a combination thereof. In some embodiments, the composition of the present disclosure comprises a salt having a concentration of between about 50 mM and about 2000 mM (e.g., about 50 mM to about 200 mM, about 200 mM to about 400 mM, about 400 mM to about 600 mM, about 600 mM to about 800 mM. about 800 mM to about 1000 mM. about 1000 mM to about 1200 mM, about 1200 mM to about 1400 mM, about 1400 mM to about 1600 mM, about 1600 mM to about 2000 mM, about 900 mM to about 1600 mM, about 200 mM to about 1600 mM, about 400 mM to about 1400 mM, about 600 mM to about 1200 mM, about 800 mM to about 1000 mM, and all integers including and in between about 50 mM and about 2000 mM).

[0219] In some embodiments, the composition of the present disclosure has a pH of about 5.0 to about 7.0 (about 5.0 to about 5.5, about 5.5 to about 6.0, about 6.0 to about 6.5, about 6.5 to about 7.0, about 5.0 to about 6.0, about 5.5 to about 6.5, about 6.0 to about 7.0, and all integers including and in between about 5.0 and about 7.0).AAV capsid serotypes

[0220] In some embodiments of the method and composition of the present disclosure, the AAV capsid is from an AAV of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, rhlO, hu37 or a variant thereof. In some embodiments, the AAV capsid is from an AAV of serotype 8. 9, or a variant thereof. In some embodiments, the AAV capsid is from an AAV of serotype hu37.Definitions

[0221] Where the use of the term “about” is before a quantitative value, the present invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” refers to a± 10% variation from the nominal value unless otherwise indicated or inferred.

[0222] Unless otherwise noted, where the term “between” is used to refer to a numerical range, the range includes the specified endpoints. For example, the range “between about 50 mM to about 200 mM” includes 50 mM, 200 mM, and values greater than 50 mM but less than 200 mM.

[0223] As used herein, the terms “contacting”, “applying”, and “passing” includes bringing together at least two substances in solution or solid phase, for example contacting a stationary phase of a chromatography material with a sample, such as a sample comprising viral particles, and allowing sufficient time.

[0224] As used herein, the term “adeno-associated virus” refers to a small, replicationdefective, non-enveloped virus that infects humans and some other primate species. AAV is not known to cause disease and elicits a very mild immune response. Gene therapy vectors that utilize AAV can infect both dividing and quiescent cells and can persist in an extrachromosomal state without integrating into the genome of the host cell. These features make AAV an attractive viral vector for gene therapy. There are currently 13 recognized seroty pes of AAV (AAV1 - 13).

[0225] As used herein, the term “adeno-associated virus”, “AAV particle” “AAV capsid” refers to a viral capsid particle that comprises a complete vector genome, that is. a vector genome that comprises a heterologous nucleic acid of interest flanked on both sides by AAV ITRs.

[0226] As used herein, the term “capsid particle” refers to a particle that comprises at least one viral capsid protein which (i) encapsidates a nucleic acid, e.g.. a vector genome or a portion thereof, and / or (ii) forms a structure surrounding a core.

[0227] As used herein, the term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, provided that they present a property of binding to one or more epitopes on the surface of AAV capsid, including but not limited to serotypes AAV 1-13.

[0228] As used herein, the term “recombinant,” may be used to describe, e.g., a nucleic acid molecule that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acid molecules, such as by genetic engineering techniques.

[0229] A “recombinant adeno-associated virus preparation” or “rAAV preparation,” refers to a product that results from a method of manufacturing recombinant AAV in a host cell (e.g., in a mammalian cell or an insect cell). In some embodiments, a recombinant AAV preparation has been subjected to one or more downstream operations after initial upstream operations,e.g., nuclease treatment, filtration to remove host cell impurities, and / or affinity purification using ligands that bind AAV capsids, as well known to those of skill in the art.

[0230] As used herein, the term “vector” is a nucleic acid molecule allowing insertion of foreign nucleic acid without disrupting the ability of the vector to replicate and / or integrate in a host cell. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains the necessary regulatory sequences to allow transcription and translation of inserted gene or genes. In some embodiments herein, the vector is an AAV vector.

[0231] The term “buffer” or "buffer substance" indicates a substance that when in solution can stabilize changes in the pH value of the solution, for example, due to the addition or release of acidic or basic substances.

[0232] As used herein, the terms “ionic strength modifier” and “non-buffering salt” are used interchangeably and refer to a salt that is present in the composition that is of a type, and at a concentration, such that it does not substantially contribute to retaining the pH of the wash solution(s) under the applied conditions (such as high pH) upon addition of acid or base. Typically, the ionic strength modifier is an ionic salt. Ionic strength modifier includes halogen salts, including those that comprise Cl or Br (more preferably Cl, in particular halogen salts comprising alkali metals or alkaline earth metals, including Na, K, Ca. and Mg (more preferably Na or K). The terms “ionic strength modifier” and “non-buffering salt” do not include buffering salts, such as sodium acetate, sodium phosphate and Tris, that do substantially contribute to retaining the pH of a wash solution(s) under the applied conditions.

[0233] As used herein, the terms “salt”, “salt composition”, and “salt concentration” when used in reference to a composition, solution, or buffer disclosed herein, refers to the identities and amounts of all salts in that composition. Thus, if a composition’s “salt concentration” is said to remain constant throughout a particular duration, it is meant that the identities and amounts of all salts in that composition remain constant throughout the particular duration.

[0234] As used herein, the terms “medium,” “chromatography medium,” “resin”, “chromatography resin”, “matrix”, “chromatography matrix” and the like, refer to a physical structure, such as column packed with resins or a monolith or a membrane, to which a rAAV preparation or a cell is applied to achieve separation of certain fractions of the preparation. For example, a rAAV preparation may be applied to a column, which column is then washed with one or more solutions to separate (and collect separated fractions) empty and full AAV particles from one another. In some embodiments, a separation medium is an anion-exchange medium.In some embodiments, a separation medium is a mixed-modal medium that can serve as an anion-exchange medium. In some embodiments, a separation medium is a column (e.g., a monolithic column or particles in a packed column). In some embodiments, a separation medium is a membrane.

[0235] As used herein, the term “chromatography” refers to the process of separating a mixture, for example a mixture containing viral capsid proteins. It involves passing a mixture through a stationary phase, which separates molecules of interest from other molecules in the mixture and allows one or more molecules of interest to be isolated.

[0236] As used herein, the terms "affinity chromatography" or "affinity purification" designate any method that uses specific binding interactions between molecules. A particular ligand is chemically immobilized or "coupled" to a solid support so that when a complex mixture is passed over the column, those molecules having specific binding affinity' to the ligand become bound. After other sample components are washed away, the bound molecule is stripped from the support, resulting in its purification from the original sample.

[0237] The term "immunoaffinity chromatography" as used herein designates any affinity chromatography method that uses immobilized antibodies, or fragments thereof, in the chromatography. The term "fragment thereof’ may encompass any fragment of an antibody that can be obtained by deleting part of the original antibody, including in a non-limitative manner any antibody of which the Fc region or parts of the variable region (including CDRs) have been deleted. The term "peptide affinity chromatography" as used herein designates any method that uses immobilized peptide in affinity chromatography. ?.

[0238] The term "antibodies or fragments thereof’ may include monoclonal and polyclonal antibodies, naturally and non-naturally occurring antibodies, whole antibodies and fragments thereof, including fragment antigen-binding components such as Fv, Fab and F(ab')2 regions, complementarity determining regions (CDRs), single-domain antibodies, nanobodies, and mixtures thereof. When immobilized onto the chromatography support, the term "antibodies or fragments thereof’ encompasses any of the whole antibodies and fragments thereof, as long as it retains its ability to bind to at least one epitope at the surface of the rAAV particles which are purified.

[0239] In particular, such antibodies or fragments thereof may include isotypes of the IgA, IgD, IgE, IgG and IgM subclasses. The antibodies or fragments thereof can be monoclonal. Antibodies which are considered by the invention may be naturally occurring or non-naturally occurring. They may be of human or non-human origin. The antibodies can be single-chainantibodies, such as the ones obtained by immunization of camelids including dromedaries, camels, llamas, and alpacas, or sharks. Antibodies and fragments thereof can be obtained and immobilized onto supports by using a variety of techniques that range from covalent attachment to adsorption-based methods, as described for instance in Moser & Hage (Immunoaffinity chromatography: an introduction to applications and recent developments; Bioanalysis; 2(4): 769-790; 2010).

[0240] A number of suitable immunoaffmity chromatography supports for use with the present invention are known and include without limitation, Affi-Gel® (Biorad); Affinica® Agarose / Polymeric Supports (Schleicher and Schuell); AvidGel (BioProbe); Bio-Gel (BioRad); Fractogel® (EM Separations); HEMA-AFC® (Alltech); Reacti-Gel® (Pierce); Sephacryl® (Pharmacia); Sepharose® (Pharmacia); Superose® (Pharmacia); Trisacryl (IBF); TSK Gel® Toyopearl® (TosoHaas); Ultragel® (IBF); AvidGel® CPG (BioProbe); HiPAC® (ChromatoChem); Protein-Pak® Affinity Packing (Waters); Ultraffinity-EP® (Bodman) and Emphaze® (3M Corp. / Pierce). Other chromatography supports include affinity monolith chromatography supports, and POROS™ affinity7chromatography supports. Preferably, antibodies and fragments thereof or peptides may be immobilized on an agarose matrix.

[0241] A number of suitable anion exchangers for use with the present invention are known and include without limitation, MACRO PREP Q® (BioRad, Hercules, Calif); UNOSPHERE Q® (BioRad. Hercules, Calif); POROS 50HQ™ (Thermo Fisher Scientific); POROS 50D™ (Thermo Fisher Scientific); POROS 50PIIM(Thermo Fisher Scientific); SOURCE 30Q® (Cytiva); DEAE SEPHAROSE® (Cytiva); Q SEPHAROSE® (Cytiva), Capto Q® and Capto Adhere® (Cytiva).

[0242] Examples of suitable monolithic chromatographic supports are known in the Art and include in a non-limitative manner the monolithic column CIMmultus® QA, CIM® QA, CIM DEAE and CIMmultus® DEAE (Sartorius).

[0243] A ‘"chromatographic monolithic column’; “chromatographic monolithic medium”, “chromatographic monolithic resin” or “chromatography monolith” is a one-piece porous solid made of fused micrometer-sized globules of silica or an organic polymer that can be synthesized directly inside a chromatography tube. This makes this support very resistant and ready -to-use. with no package parameters variability. Monoliths are homogenous columns with a continuous porous bed matrix, consisting of interconnected perfusion channels. These channels are relatively7large (1-5 gm) by comparison to the typical pore sizes of packed-bedparticle-based chromatography (5-100 nm). One benefit of this is that it increases the potential binding capacity of macromolecules such as virus because of high surface of accessibility. These supports generate low counter pressure even at high flow rates, and low shear rate. By comparison to packed-bed columns, the improved mass transport of monolithic supports results in efficient separation of the macromolecules.

[0244] From a practical point of view, the use of a chromatographic monolithic column allows one to work with a viral product (even when it is a cellular lysate) at a high flow rate compared to packed-bed columns, with a high binding capacity and a good resolution, independent of the flow rate. The high resolution of these supports contributes to the reduction of the purification steps and the scalability of the whole process.

[0245] The terms “hydrophilic interaction chromatography” or “HILIC” are intended to include a process employing a hydrophilic stationary phase and a hydrophobic organic mobile phase in which hydrophilic compounds are retained longer than hydrophobic compounds. In certain embodiments, the process utilizes a water-miscible solvent mobile phase. The term “hydrophilic,” as used herein, describes having an affinity for, attracting, adsorbing or absorbing water. The term “hydrophobic,” as used herein, describes lacking an affinity for, repelling, or failing to adsorb or absorb water. An example of a method of chromatographic separation is hydrophilic interaction liquid chromatography (HILIC).Isocratic Separation

[0246] As mentioned, in some embodiments, (i) the concentration of the quaternary salt in the elution solution remains constant throughout the step during which the elution solution is passed through or applied to the anion exchange medium and / or (ii) the elution solution’s salt composition remains constant throughout the step during which the elution solution is passed through or applied to the at least one anion exchange medium. In some embodiments, concentrations of additional components of the at least one elution solution also remain constant throughout the step.Gradient Separation

[0247] As mentioned, in some embodiments, the elution step comprises using an elution solution whose composition varies during that step or those steps. For example, in a gradient separation step, the concentration of a salt in the elution solution may gradually and continually increase (e.g., linearly, or stepwise) over time throughout the step.

[0248] As used herein, the term Log Reduction Value (LRV) is a ratio of the log to the base 10 of the challenge concentration divided by the filtrate concentration. Log reductionvalue = logio(A / B), where A is the concentration of viral particles before chromatographic purification and B is the concentration of viral particles in the eluent collected after chromatographic purification.

[0249] In some embodiments, the concentration of salt within the elution solution increases continuously over time throughout the elution step. In some embodiments, the concentration of salt within the elution solution increases linearly over time throughout the elution step. In some embodiments of the methods of the present disclosure, the concentration of salt within the elution solution increases stepwise throughout the elution step.EXAMPLES

[0250] The presence of host cell proteins (HCPs) is considered one of the most important quality attributes in biologies therapeutic drugs. There are two ways HCPs can co-purify with protein product in affinity chromatography. The first one focused on the interaction between HCPs and protein product while the second one is focused on the interaction between HCPs and affinity' ligand. The studies disclosed herein were aimed to pinpoint a process condition that could best disrupt the interaction between HCPs with either rAAV or affinity ligand in rAAV affinity chromatography and to identify additives that could be utilized in an affinity chromatography step to further reduce HCPs while maintaining rAAV genome recovery.

[0251] Materials and methods

[0252] Production and Preparation of rAAV

[0253] Recombinant AAV (rAAV) comprising an AAV8 capsid concentrate clarified harvest were obtained as load material for affinity chromatography. The rAAVs were produced using a HeLa producing cell line. The helper function for rAAV production was provided through Adenovirus (Ad5) infection of the cell culture. After Ad5 infection and prior to harvest, a nuclease treatment was performed. The nuclease treated cells were harvested and clarified through a series of depth filters, and subsequently concentrated and diafiltered via tangential flow filtration to product affinity' load material.

[0254] Instrument and Columns

[0255] The affinity runs were executed using AKTA Avant 25® (Cytiva). ImL pre-packed column with POROS Captures elect™ AAV8 resin was purchased from Thermo Fisher Scientific. A 16 mL packed bed column using POROS CaptureSelect™ AAV8 resin was prepared.

[0256] Affinity Chromatography

[0257] The POROS CaptureSelect™ AAV8 resin and pre-packed column were used to capture the AAV particle from feed stream. The column was equilibrated with equilibrationbuffer (20 mM Tris, 200 mM NaCl, 0.001% (w / v) Poloxamer 188, pH 7.5) for 5 column volumes (CV) and then was loaded with 20-fold concentrated clarified harvest for 10 L / L- column. The loaded column was washed with 10 CVs of equilibration buffer to flush away the concentration clarified harvest prior to a second 10 CVs of additive-containing buffer (equilibration buffer spiked with high concentration additive stock solution) applied. After a third 5 CVs of equilibration buffer to flush away the additive-containing buffer, the AAV was eluted by low pH elution buffer (20 mM sodium citrate, 200 mM NaCl, 0.001% Pluronic* F- 68, pH 2.5). The affinity eluates were tested for genome titer and host cell protein level to assess both process performance and product quality'. Post elution, the affinity' column was cleaned by 0.1 M Phosphoric Acid solution for 5 CVs and sanitized by 100 mM Tris Base, 2.0 M NaCl buffer for 5 CVs prior to its storage in 50 m M Tris, 150 mM NaCl, 20% Ethanol. pH 7.5 buffer.

[0258] Quantitative Polymerase Chain Reaction Assay (qPCR)

[0259] The quantity of encapsulated vector genome was determined using a qPCR method applicable to rAAV samples that contain the SV40 Poly-A sequence. To quantify only encapsulated vector genome, rAAV samples were treated with salt-activated nuclease (SAN) to remove non-encapsulated polynucleotides. The SAN were then inactivated and the rAAV samples were treated with Proteinase K to digest the capsid protein. This was followed by qPCR using the primer / probe set that is specific to the SV40 Poly-A sequence of the rAAV genomes in the rAAV therapeutic products. The qPCR was performed on an Applied Biosystems Flex 7 instrument and quantification is made using the QuantStudio 7 software. For a given experiment, the qPCR measurements were performed on the same multi-well plate.

[0260] Host Cell Protein (HCP) Assay for HeLa Cell

[0261] The HeLa HCP was quantified using a commercial colorimetric enzyme linked immunosorbent assay (ELISA) from Cygnus. Samples to be tested for HeLa HCP were added to a 96 well microtiter strip plate that were pre-coated with a blend of goat and rabbit anti-HeLa HCP antibodies. The horseradish peroxidase (HRP) labeled goat Anti-HeLa polyclonal detection antibody were added to reaction simultaneously, forming a sandwich complex of solid phase antibody-HeLa HCP-enzyme labeled antibody. The microtiter strip plate was then washed to remove any unbound material. The tetramethylbenzidine (TMB) substrate was added to hydrolyze HRP producing a colorimetric signal. The reaction was stopped by the addition of sulfuric acid and read on a microtiter plate reader. The final signal was directly proportional to the concentration of HeLa host cell proteins present in each well. Finalconcentrations were obtained from interpolation off a HeLa HCP standard curve via Four Parameter Logistic Regression.Example 1 : Determining the effect of additives in solution on rAAV stability

[0262] Objective: A total of 5 additives, including salt (sodium octanoate), chaotropic agent (urea), detergent (Pluronic® F68), hydrophobic agent (isopropyl alcohol) and amino acid (L-arginine) were evaluated in this study for their effectiveness on HCP reduction in rAAV affinity chromatography. A purified rAAV8 (named UGT-rAAV-1) was added to each additive solution and incubated for 10 minutes at room temperature before the rAAV gene of interest (GOI) recovery was measured.

[0263] Results: The results described herein show that except for urea, all the other four additives showed greater than 100% GOI genome recovery (>100% recovery was likely due to qPCR assay variability), suggesting these four additives and their working concentrations were benign to rAAV stability. For urea, however, only 67% rAAV genome was recovered after 10 min incubation, indicating that 3 M urea destabilizes rAAV capsid structure and causes GOI loss.Table 1. UGT-rAAV-1 Stability in Additive SolutionUGT-rAAV-1FinalAdditive Name Genome CopyConcentrationSpiking RecoverySodium Octanoate 20 mM 107%Urea 3 M 67%Iso-propyl alcohol 20 % 113%Pluronic® F68 1 % 129%L- Arginine 100 mM 113%

[0264] Conclusion: Based on the results of the study described herein, both L-arginine and sodium octanoate were selected for further evaluation as potential additives for reducing HCP levels in purified rAAV preparations.Example 2: Additive Screening for reducing HCP levels in Affinity Chromatography

[0265] Objective: The study described herein determined the effect of five different additives: sodium octanoate, urea. Pluronic® F68, isopropyl alcohol and L-arginine and their working concentrations were tested for their contributions on HCP reduction. To observe the maximum effectiveness of each additive, in a single affinity chromatography run, the additive was spiked into both affinity load material (concentrated clarified harvest) and affinity wash buffer (20 mM Tris-HCl, 200 mM NaCl, 0.001% Pluronic® F-68, pH 8.0), as the dissociation of HCPs from rAAV could take place in both locations in affinity chromatography. To save on affinity load material, 1 mL prepacked PCS-AAV8 column was used to perform the initial screening runs, as detailed by the running conditions described in the Materials and Methods section above.

[0266] Results: The results of the study disclosed herein showed that except for urea, all the other four additives resulted in acceptable affinity’ elution peaks areas (for both A254 nm and A280 nm trace), which are 90% to 100% to control condition (no additive). For urea, however, only -10% of elution peak area (for both A254 nm and A280 nm trace) was recovered, conoborating the observation made in Table 1 that most rAAVs were destabilized by 3 M urea and dissembled prior to affinity elution phase. (See FIG. 1). In addition, 1% Pluronic® F68 condition resulted in detectable HCP level (7 ng / mL) (See, red filled circle in FIG. 1) in its affinity eluate, comparing to the below limit-of-quantitation (BLOQ) HCP levels for other additive conditions (See, red filled triangle in FIG. 1). sho ing slightly inferior capability of 1% Pluronic® F68 in HCP reduction. Furthermore, although 20% isopropyl alcohol condition achieved BLOQ level of HCP in affinity eluate, this additive due to its flammable nature may elicit safety' concern in GMP manufacturing facility, prohibiting its use in rAAV product purification.

[0267] Conclusion: Based on the results of the study described herein, sodium octanoate and L-arginine were selected for further evaluation, as both are manufacturing-friendly components and have low effective concentration (20 mM and 100 mM, respectively), which may result in less raw' material footprint in manufacturing facility during scale-up.Example 3: Determining the effectiveness of sodium octanoate and L-arginine on removal of HCP impurities during rAAV purification and rAAV integrity

[0268] Objective: In the study described herein, the observed effectiveness of sodium octanoate and L-arginine was further confirmed by rAAV affinity chromatography using qualified scale-down model with a larger column volume (PCS-AAV8 resin packed column with 1 cm inner diameter, 20.3 cm bed height, and 15.9 mL column volume). The larger affinity column and subsequence larger volume of affinity eluate were utilized to allow for affinityeluate to be further concentrated (~ 100-fold) by 10 kDa molecular weight cut off (MWCO) centrifugal filter in an aim to increase HCP detection.

[0269] Results: The studies described herein showed that the HCP recovery was normally >90% after concentrating by 10 kDa MWCO centrifugal filter (data not shown). It was concluded that the concentration step had minimum impact on the HCP data interpretation as the control and additive-spiked runs’ affinity eluates are subjected to the same concentrating procedure. In the results described herein, the GOI genome recovery (determined by qPCR assay) as a function of HCP LRV (determined by ELISA assay), showed that both sodium octanoate and L-arginine conditions resulted in enhanced HCP reduction (>4.8 and >5.3 LRV, respectively) compared to that of negative control run (2.4 LRV), while all the GOI genome recoveries were within comparable range (from 87% to 93%). (See FIG. 2). Specifically, the affinity run with 0.1 M arginine (See, FIG. 2, square) has >5.3 HCP LRV and 87% genome recovery, and the affinity run with 20 mM sodium octanoate (See, FIG. 2, triangle) has >4.8 HCP LRV and 89% genome recovery7. In comparison, negative control (See, FIG. 2, circle) run has 2.4 HCP LRV and 93% genome recovery.

[0270] Conclusion: The results described herein indicated that both sodium octanoate and L-arginine could be used as potent additives for reducing HCP levels in rAAV chromatographic purification.Example 4: Determining the impact of additive spiking step in affinity chromatography-based purification of rAAV

[0271] Objective: As additives were spiked in both load material and wash buffer of affinity7chromatography screening runs, it is unclear which additive spiking location or step contributes to the HCP reduction. The study described herein was aimed to dissect spiking of the additives to which spiking location or step contributes to the HCP reduction. Affinity chromatography runs were performed with additive spiked only in affinity load material or only in wash buffer.

[0272] Results: The results of the study described herein showed that affinity chromatography runs with L-arginine (See, filled shapes in lower right region of FIG. 3) have relative higher HCP LRV but lower GOI genome recovery than that of sodium octanoate (See. open shapes in the upper left region of FIG. 3). Considering the best trade-off between GOI genome recovery may lie close to the diagonal line of this graph (See, dotted line of FIG. 3), the two triangle conditions, representing additive spiked in only wash buffers, stand out as optimal process conditions for affinity chromatography (See, open triangle for sodium octanoate and filled triangle for L-arginine added in wash buffer, respectively, in FIG. 3).

[0273] Conclusion: The results described herein showed that spiking of the additives to the wash buffer of rAAV chromatography was sufficient to remove HCP from purified rAAV preparations. This provided the opportunity to simplify affinity chromatography to have both additives (sodium octanoate and L-arginine) prepared in affinity wash buffer(s), rather than performing on-floor spiking operation if affinity load material was to be spiked with additives.

[0274] Based on the results of the studies described herein, sodium octanoate and L- arginine were identified as optimum additives for spiking affinity wash buffers for removal of HCP impurities in affinity chromatography. It was foreseeable that, this additive-assisted HCP reduction strategy in rAAV affinity chromatography step could be applicable for various affinity chromatography resins and different cell lines based rAAV production system.

Claims

CLAIMSWhat is claimed is:

1. A method of reducing host cell protein (HCP) contaminants in a recombinant adeno- associated virus (rAAV) preparation, the method comprising the steps of: a) providing a cell lysate and / or a cell culture supernatant comprising a rAAV; b) applying the cell lysate and / or the cell culture supernatant to a chromatography resin; c) passing at least one wash solution comprising an effective amount of an additive selected from L-arginine or a derivative thereof, an octanoate salt, and combinations thereof, through the chromatography resin; d) passing an elution solution through the washed chromatography resin to elute the rAAV to generate an eluent flow through comprising the rAAV; and e) collecting one or more fractions of the eluent flowthrough comprising said rAAV, wherein said rAAV comprises an AAV capsid and a vector genome packaged therein.

2. An elution method for separating host cell protein (HCP) from a recombinant adeno- associated virus (rAAV) preparation, the method comprising the steps of: a) contacting a chromatography resin with the rAAV preparation; b) applying at least one wash solution to the chromatography resin and allowing the wash solution to flow through, wherein the wash solution comprises an additive selected from L-arginine or a derivative thereof, an octanoate salt, and combinations thereof; c) applying an elution solution to the washed chromatography resin to elute the rAAV to generate an eluent flowthrough comprising the rAAV; and d) collecting one or more fractions of the eluent flowthrough comprising said rAAV, wherein said rAAV comprises an AAV capsid and a vector genome packaged therein.

3. The method of any one of claims 1-2, wherein the one or more fractions of the eluent flowthrough are pooled to form a combined eluent fraction.

4. The method of claim 3, further comprising determining the amount of HCP in the combined eluent fraction.

5. The method of any one of claims 3-4, wherein the combined eluent fraction comprises an HCP amount that is 4 log reduction value (LRV) or more relative to the HCP amount of the rAAV preparation.

6. The method of any one of claims 3-5, wherein the combined eluent fraction comprises HCP at a concentration of 0 ng / ml to 5 ng / ml.

7. The method of any one of claims 3-5, wherein the combined eluent fraction comprises HCP at a concentration of 0 ng / ml to 4 ng / ml.

8. The method of claim 7, wherein the combined eluent fraction comprises HCP at a concentration of 1 ng / ml to 2 ng / ml.

9. The method of any one of claims 3-8, further comprising determining the recovery of rAAV in the combined eluent fraction.

10. The method of claim 9, wherein the combined eluent fraction comprises at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the vector genome present in the rAAV preparation.

11. The method of any one of claims 1-10, wherein the at least one wash solution comprises L-arginine or a derivative thereof.

12. The method of claim 11, wherein the at least one wash solution comprises L-arginine or a derivative thereof at a concentration of about 50 mM to about 200 mM.

13. The method of any one of claims 1-10, wherein the at least one wash solution comprises an octanoate salt.

14. The method of claim 13, wherein the at least one wash solution comprises an octanoate salt at a concentration of about 10 mM to about 50 mM.

15. The method of claim 1-10, wherein the at least one wash solution comprises L-arginine or a derivative thereof and an octanoate salt.

16. The method of claim 15, wherein the at least one wash solution comprises L-arginine or a derivative thereof at a concentration of about 50 mM to about 200 mM and an octanoate salt at a concentration of about 10 mM to about 50 mM.

17. The method of any one of claims 1-10, wherein passing at least one wash solution comprises passing a first wash solution through the chromatography resin, followed by passing a second wash solution through the chromatography resin.

18. The method of claim 17, wherein the first and the second wash solutions comprise the same additive(s).

19. The method of claim 18, wherein the concentration of the additive(s) in the first and the second wash solutions is the same.

20. The method of claim 18, wherein the concentration of the additive(s) in the first and the second wash solutions is different.

21. The method of claim 20, wherein the concentration of the additive(s) in the first wash solution is less than the concentration of the additive(s) in the second wash solution.

22. The method of claim 20, wherein the concentration of the additive(s) in the first wash solution is more than the concentration of the additive(s) in the second wash solution.

23. The method of any one of claims 18-22, wherein the first and second wash solutions comprise L-arginine or a derivative thereof.

24. The method of any one of claims 18-22, wherein the first and second wash solutions comprise an octanoate salt.

25. The method of any one of claims 18-22, wherein the first and second wash solutions comprise L-arginine or a derivative thereof and an octanoate salt.

26. The method of claim 17, wherein the first and the second wash solutions comprise different additives.

27. The method of claim 26, wherein the first wash solution comprises L-arginine or a derivative thereof, and the second wash solution comprises an octanoate salt.

28. The method of claim 26, wherein the first wash solution comprises octanoate salt, and the second wash solution comprises L-arginine or a derivative thereof.

29. The method of claim 26, wherein the first wash solution comprises L-arginine or a derivative thereof, and the second wash solution comprises a combination of L-arginine or a derivative thereof and an octanoate salt.

30. The method of claim 26, wherein the first wash solution comprises a combination of L- arginine or a derivative thereof and an octanoate salt, and the second wash solution comprises L-arginine or a derivative thereof.31 . The method of claim 26, wherein the first wash solution comprises octanoate salt, and the second wash solution comprises a combination of L-arginine or a derivative thereof and an octanoate salt.

32. The method of claim 26, wherein the first wash solution comprises a combination of L- arginine or a derivative thereof and an octanoate salt, and the second wash solution comprises an octanoate salt.

33. The method of any one of claims 1-10, wherein the method comprises passing at least three wash solutions sequentially through the chromatography resin.

34. The method of claim 33, wherein each of the at least three wash solutions comprise the same additive(s).

35. The method of claim 33 or 34, wherein each of the at least three wash solutions comprise L-arginine or a derivative thereof.

36. The method of claim 35, wherein each of the at least three wash solutions comprise L- arginine or a derivative thereof at a concentration of about 50 mM to about 200 mM.

37. The method of claim 33 or 34, wherein each of the at least three wash solutions comprise an octanoate salt.

38. The method of claim 37, wherein each of the at least three wash solutions comprise an octanoate salt at a concentration of about 10 mM to about 50 mM.

39. The method of claim 33 or 34, wherein each of the at least three wash solutions comprise L-arginine or a derivative thereof and an octanoate salt.

40. The method of claim 39. wherein each of the at least three wash solutions comprise L- arginine or a derivative thereof at a concentration of about 50 mM to about 200 mM and an octanoate salt at a concentration of about 10 mM to about 50 mM.

41. The method of any one of claims 33-35, 37 and 39, wherein the concentrations of the same additive in each of the at least three wash solutions are different.

42. The method of claim 41, wherein the concentration of the additive increases in each sequentially used wash solution.

43. The method of claim 42, wherein the concentration of the additive increases linearly in each sequentially used wash solution.

44. The method of claim 42, wherein the concentration of the additive increases stepwise in each sequentially used wash solution.

45. The method of claim 41, wherein the concentration of the additive decreases in each sequentially used wash solution.

46. The method of claim 45, wherein the concentration of the additive decreases linearly in each sequentially used wash solution.

47. The method of claim 45, wherein the concentration of the additive decreases stepwise sequentially used wash solution.

48. The method of claim 33. wherein each of the at least three wash solutions comprise an additive that is different from an additive in any of the previously or subsequently used wash solutions or both.

49. The method of claim 48, wherein the first wash solution comprises L-arginine or a derivative thereof, the second wash solution comprises an octanoate salt, and the third wash solution comprises L-arginine or a derivative thereof and an octanoate salt.

50. The method of claim 48. wherein the first wash solution comprises an octanoate salt, the second wash solution comprises L-arginine or a derivative thereof, and the third wash solution comprises L-arginine or a derivative thereof and an octanoate salt.

51. The method of claim 48, wherein the first wash solution comprises L-arginine or a derivative thereof and octanoate salt, the second wash solution comprises L-arginine or a derivative thereof, and the third wash solution comprises an octanoate salt.

52. The method of claim 48, wherein the first wash solution comprises L-arginine or a derivative thereof and an octanoate salt, the second wash solution comprises an octanoate salt, and the third wash solution comprises L-arginine or a derivative thereof.

53. The method of claim 48, wherein the first wash solution comprises an octanoate salt, the second wash solution comprises L-arginine or a derivative thereof and an octanoate salt, and the third wash solution comprises L-arginine or a derivative thereof.

54. The method of claim 48, wherein the first wash solution comprises L-arginine or a derivative thereof, the second wash solution comprises L-arginine or a derivative thereof and an octanoate salt, and the third wash solution comprises an octanoate salt.

55. The method of claim 48, wherein the first and the second w ash solutions comprise L- arginine or a derivative thereof, and the third wash solution comprises an octanoate salt.

56. The method of claim 48, wherein the first and the second wash solutions comprise L- arginine or a derivative thereof, and the third wash solution comprises L-arginine or a derivative thereof and an octanoate salt.

57. The method of claim 48, wherein the first and the second wash solutions comprise an octanoate salt, and the third wash solution comprises L-arginine or a derivative thereof.

58. The method of claim 48. wherein the first and the second wash solutions comprise an octanoate salt, and the third wash solution comprises L-arginine or a derivative thereof and an octanoate salt.

59. The method of claim 48. wherein the first and the second wash solutions comprise L- arginine or a derivative thereof and an octanoate salt and the third wash solution comprises L-arginine or a derivative thereof.

60. The method of claim 48, wherein the first wash solution comprises L-arginine or a derivative thereof, and the second and the third wash solutions comprise an octanoate salt.

61. The method of claim 48, wherein the first wash solution comprises L-arginine or a derivative thereof, and the second and the third wash solutions comprise L-arginine or a derivative thereof and an octanoate salt.

62. The method of claim 48, wherein the first w ash solution comprises an octanoate salt, and the second and the third wash solutions comprise L-arginine or a derivative thereof.

63. The method of claim 48, wherein the first wash solution comprises an octanoate salt, and the second and the third w ash solutions comprise L-arginine or a derivative thereof and an octanoate salt.

64. The method of claim 48, wherein the first wash solution comprises L-arginine or a derivative thereof and an octanoate salt, and the second and the third wash solutions comprise L-arginine or a derivative thereof.

65. The method of claim 48, wherein the first wash solution comprises L-arginine or a derivative thereof and an octanoate salt, and the second and the third wash solutions compnse an octanoate salt.

66. The method of any one of claims 1-12, 15-23, and 25-65, wherein the L-arginine derivative is selected from the group consisting of L-arginine hydrochloride. L-arginine dihydrochloride, L-arginine methyl ester dihydrochloride. L-arginine ethyl ester dihydrochloride, acetyl L-arginine, N-alpha-butyroyl-L-arginine, agmatine, L-arginic acid, and N-alpha-pyvaloyl L-arginine.

67. The method of any one of claims 1-10, 13-22, and 24-65, wherein the octanoate salt is selected from the group consisting of sodium octanoate, magnesium octanoate, potassium octanoate, and calcium octanoate.

68. The method of any one of claims 1-67, wherein the chromatography resin comprises an affinity chromatography medium, an anion exchange (AEX) chromatography medium, a hydrophobic interaction chromatography (HIC) medium, a mix-mode chromatography medium, a tangential flow (TFF) chromatography medium, or a combination thereof.

69. The method of claim 68, wherein the affinity chromatography medium comprises a protein or ligand that binds to the AAV capsid.

70. The method of claim 69, wherein the affinity' chromatography medium comprises an AAV-specific antibody or a fragment thereof.

71. The method of any one of claims 68-70, wherein the at least one wash solution comprises an organic solvent or a detergent.

72. The method of claim 71, wherein the at least one wash solution comprises an organic solvent selected from any one of polysorbate 80, ethylene glycol, t-butanol. sorbitol, mannitol, xylitol, DMSO, sucrose trehalose and polyethyleneimine (PEI).

73. The method of claim 71, wherein the at least one wash solution comprises a detergent selected from the group consisting of Nonidet P-40, 3-[(3- cholamidopropyl)dimethylammonio]-l -propanesulfonate (CHAPS), triton x-100, and tri (n-butyl) phosphate (TNBP).

74. The method of any one of claims 71-73, wherein the at least one wash solution comprises Tris-HCl, Bis-Tris or MES-Na.

75. The method of claim 74. wherein the at least one wash solution has a pH of between about 6.0 to about 9.0.

76. The method of any one of claims 68-75, wherein the elution solution comprises glycine- HC1 or citric acid.

77. The method of claim 76, wherein the elution solution further comprises an ionic strength modifier selected from the group consisting of sodium chloride, sodium iodide, sodium thiocyanate, calcium chloride, magnesium chloride, and a combination thereof.

78. The method of any one of claims 75-76, wherein the elution solution has a pH of about 2.5 to about 3.0.

79. The method of claim 68, wherein the AEX chromatography medium is a weak ionic exchanger.

80. The method of claim 68, wherein the AEX chromatography medium is a strong ionic exchanger.

81. The method of any one of claims 68 and 79-80 wherein the AEX chromatography medium is in the form of a packed bed.

82. The method of any one of claims 68 and 79-80, wherein the AEX chromatography medium is a chromatographic monolithic column.

83. The method of claim 82, wherein the AEX chromatography medium comprises a combined eluent fraction CIMmultus® QA column, CIMac® QA column, NuviaQ® column, POROS HQTMcolumn, Eshmuno® Q column, POROS XQ™ column, FractoGel® TMAE column, or a CaptoQ® column.

84. The method of any one of claims 79-83, wherein the at least one wash solution comprises a quaternary ammonium salt.

85. The method of claim 84, wherein the quaternary ammonium salt comprises a tetraalkylammonium salt, a choline chloride, or a divalent salt.

86. The method of claim 85, wherein the tetraalkylammonium salt is a tetraalkylammonium chloride or a tetraalkylammonium acetate.

87. The method of claim 86, wherein the tetraalkyl ammonium chloride is selected from the group consisting of tetramethylammonium chloride (TMAC), tetraethylammonium chloride (TEAC). tetrapropylammonium chloride (TPAC). tetrabutylammonium chloride (TBAC), benzyltributylammonium chloride (BTBAC), and any combination(s) thereof.

88. The method of claim 86. wherein the tetraalkylammonium acetate is selected from the group consisting of tetramethyl ammonium acetate, tetraethylammonium acetate (TEA- Ac), tetrapropylammonium acetate, tetrabutylammonium acetate, and any combination(s) thereof.

89. The method of any one of claims 84-88, wherein the wash solution has a pH of between about 6.0 to about 9.0.

90. The method of any one of claims 84-89, wherein the elution solution comprises a salt in the form of any one of an isocratic salt solution or a gradient salt solution.

91. The method of claim 90, wherein the concentration of the salt within the elution solution increases continuously over time throughout the elution step.

92. The method of claim 91 , wherein the concentration of the salt within the elution solution increases linearly over time throughout the elution step.

93. The method of claim 91, wherein the concentration of the salt within the elution solution increases stepwise throughout the elution step.

94. The method of claim 90, wherein the concentration of the salt within the elution solution decreases continuously over time throughout the elution step.

95. The method of claim 94, wherein the concentration of the salt within the elution solution decreases linearly over time throughout the elution step.

96. The method of claim 94, wherein the concentration of the salt within the elution solution decreases stepwise throughout the elution step.

97. The method of any one of claims 90-96, wherein the salt is any one of sodium chloride, sodium sulfate, magnesium chloride, magnesium sulfate, or any combination thereof.

98. The method of any one of claims 90-97, wherein the elution solution further comprises a bis-Tris Propane-based buffer or a potassium phosphate-based buffer.

99. The method of any one of claims 90-98, wherein the elution solution has a pH of about 6.0 to about 10.0.

100. The method of claim 68, wherein the HIC medium is selected from the group consisting of methyl HIC. Source ETH, Toyopearl Ether-650, Toyopearl PPG-600, Source Iso, Source Phe, Phenyl Sepharose, Toyopearl Phenyl-600, Toyopearl Phenyl- 650, Butyl Sepharose, Toyopearl Butyl-600, Toyopearl Butyl-650, Toyopearl SuperButyl-550, t-Butyl HIC, Toy opearl Hexyl-650, and Octyl Sepharose.

101. The method of claim 100. wherein the wash solution comprises a wash buffer comprising a salt selected from the group consisting of a sulfate salt, a citrate salt,ammonium sulfate, sodium sulfate, sodium phosphate, sodium chloride, ammonium chloride, sodium bromide, potassium phosphate, and a combination thereof.

102. The method of claim 101, wherein the wash solution has a pH of about 5.0 to about 7.0.

103. The method of any one of claims 100-102, wherein the elution solution comprises the same salt as in the wash buffer, at a lower concentration as compared to a corresponding wash buffer.

104. The method of claim 103, wherein the elution solution has a pH of about 5.0 to about 7.0.

105. The method of any one of claims 1-104, wherein the AAV capsid is from an AAV of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, rhlO, hu37, or a variant thereof.

106. A composition comprising an effective amount of an additive selected from L-arginine or a derivative thereof, an octanoate salt, and combinations thereof, for use in reducing host cell protein (HCP) contaminants during chromatographic purification of rAAV from a rAAV preparation.

107. The composition for use of claim 106, wherein the additive is L-arginine or a derivative thereof at a concentration of about 50 mM to about 200 rnM.

108. The composition for use of claim 106, wherein the additive is an octanoate salt at a concentration of about 10 mM to about 50 mM.

109. The composition for use of claim 106, wherein the additive comprises L-arginine or a derivative thereof at a concentration of about 50 mM to about 200 mM and an octanoate salt at a concentration of about 10 mM to about 50 mM.1 10. The composition for use of any one of claims 106-107 and 109, wherein the L-arginine derivative is selected from the group consisting of L-arginine hydrochloride, L-arginine dihydrochloride, L-arginine methyl ester dihydrochloride, L-arginine ethyl esterdihydrochloride, acetyl L-arginine. N-alpha-butyroyl-L-arginme. agmatine, L-arginic acid, and N-alpha-pyvaloyl L-arginine.

111. The composition for use of any one of claims 106 and 108-109, wherein the octanoate salt is selected from the group consisting of sodium octanoate, magnesium octanoate, potassium octanoate, and calcium octanoate.1 12. The composition for use of any one of claims 106-111, as a wash solution in a chromatographic medium post-application of a rAAV preparation to the medium.

113. The composition for use of claim 112, wherein the chromatographic medium is any one of an affinity chromatography medium, an anion exchange (AEX) chromatography medium, a hydrophobic interaction chromatography (HIC) medium, a mix-mode chromatography medium, a tangential flow (TFF) chromatography medium, or a combination thereof.1 14. The composition for use of claim 113, wherein the affinity chromatography medium comprises a protein or ligand that binds to AAV capsid protein.

115. The composition for use of claim 114, wherein the affinity chromatography medium comprises an A AV-specific antibody or a fragment thereof.

116. The composition for use of any one of claims 114-115, further comprising an organic solvent or a detergent.

117. The composition for use of claim 116, wherein the at least one wash solution comprises an organic solvent selected from any one of polysorbate 80, ethylene glycol, t-butanol, sorbitol, mannitol, xylitol. DMSO, sucrose trehalose and polyethyleneimine (PEI).

118. The composition for use of claim 116, wherein the at least one wash solution comprises a detergent selected from the group consisting of Nonidet P-40, 3-[(3- cholamidopropyl)dimethylammonio]-l -propanesulfonate (CHAPS), triton x-100, and tri (n-butyl) phosphate (TNBP).1 19. The composition for use of any one of claim 116-118, wherein the at least one wash solution comprises Tris-HCl, Bis-Tris or MES-Na.

120. The composition for use of any one of claims 114-119, wherein the composition has a pH of between about 6.

0. to about 9.0.

121. The composition for use of claim 113, wherein the AEX chromatography medium is a weak ionic exchanger.

122. The composition for use of claim 113, wherein the AEX chromatography medium is a strong ionic exchanger.

123. The composition for use of any one of claims 113 and 121-122, wherein the AEX chromatography medium is in the form of a packed bed.

124. The composition for use of any one of claims 113 and 121-123, wherein the AEX chromatography medium is a chromatographic monolithic column.

125. The composition for use of claim 124, wherein the AEX chromatography medium comprises a CIMmultus® QA column, CIMac® QA column, NuviaQ® column, POROS HQ CIMac® QAY™ column, Eshmuno® Q column, POROS® XQ column, FractoGel® TMAE column, or a CaptoQ® column.

126. The composition for use of any one of claims 113-125, further comprising a quaternary ammonium salt.

127. The composition for use of claim 126. wherein the quaternary ammonium salt comprises a tetraalkydammonium salt, a choline chloride, or a divalent salt.

128. The composition for use of claim 127, wherein the tetraalkylammonium salt is a tetraalkylammonium chloride or a tetraalkylammonium acetate.

129. The composition for use of claim 128, wherein the tetraalkylammonium chloride is selected from the group consisting of tetramethylammonium chloride (TMAC), tetraethylammonium chloride (TEAC), tetrapropylammonium chloride (TPAC), tetrabutylammonium chloride (TBAC), benzyltributylammo um chloride (BTBAC), and any combination(s) thereof.

130. The composition for use of claim 128. wherein the tetraalkylammonium acetate is selected from the group consisting of tetramethylammonium acetate, tetraethylammonium acetate (TEA-Ac), tetrapropylammonium acetate, tetrabuty lammonium acetate, and any combination(s) thereof.

131. The composition for use of any one of claims 121-130, wherein the composition has a pH of between about 6.

0. to about 9.0.

132. The composition for use of claim 113. wherein the HIC medium is selected from the group consisting of methyl HIC. Source ETH. Toyopearl Ether-650. Toyopearl PPG- 600, Source Iso, Source Phe, Phenyl Sepharose, Toyopearl Phenyl-600, Toyopearl Phenyl-650, Butyl Sepharose, Toyopearl Butyl-600, Toyopearl Butyl-650, Toyopearl SuperButyl-550, t-Butyl HIC, Toyopearl Hexyl-650, and Octyl Sepharose.

133. The composition for use of claim 132, wherein the wash solution comprises sodium phosphate or potassium phosphate.

134. The composition for use of any one of claims 132-133, wherein the composition has a pH of about 5.0 to about 7.0.

135. The composition for use of any one of claims 106-134, wherein the AAV capsid is from an AAV of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9. 10, 11, 12, 13, rhlO, hu37 or a variant thereof.

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