WO2024038365A1 - Methods for purification of aav vectors by anion exchange chromatography - Google Patents

Abstract

The present disclosure provides methods for purifying a recombinant AAV (rAAV) vector from a solution by anion-exchange chromatography (AEX) to produce an eluate enriched for full capsids and depleted of empty capsids.

Description

METHODS FOR PURIFICATION OF AAV VECTORS BY ANION EXCHANGE CHROMATOGRAPHY

REFERENCE TO SEQUENCE LISTING

[0001] This application is being filed electronically via EFS-Web and includes an electronically submitted sequence listing in .xml format. The .xml file contains a sequence listing entitled “PC072807 Sequence Listing.xml” created on July 7, 2023 and having a size of 21 .5 KB. The sequence listing contained in this .xml file is part of the specification and is herein incorporated by reference in its entirety.

BACKGROUND

[0002] The present invention relates to the purification of AAV, and in particular recombinant AAV (rAAV) vectors by anion exchange chromatography.

[0003] Gene therapy, using a recombinant AAV (rAAV) vector to deliver a therapeutic transgene, has the potential to treat a wide range of serious diseases for which no cure, and in many cases, limited treatment exists (Wang et al. (2019) Nature Reviews 18:358-378). Manufacturing of gene therapy vectors is complex and requires specialized methods to purify the therapeutic rAAV vector from host cell impurities, and from viral capsids that do not contain a complete vector genome encoding the therapeutic transgene. In addition to development of a purification method that produces a clinical grade rAAV vector composition of high purity and with a good safety and efficacy profile, the purification method must also be scalable to high volume rAAV production to meet patient needs.

[0004] Ultracentrifugation using a cesium chloride gradient sedimentation is a robust method for removal of host cell protein and DNA, as well as separation of viral capsids that are empty (i.e., that do not contain a vector genome), partially packaged (also referred to as “intermediate capsids” and which contain a partial vector genome and/or non-transgene- related DNA) or fully packaged vectors (also referred to as “full capsids” and which contain a complete vector genome) (Burnham et al. (2015) Hum. Gene Ther. Meth. 26:228-245). However, cesium chloride gradient purification is laborious, time consuming and not amenable to large scale manufacturing. Ultracentrifugation using an iodixanol gradient is less labor intensive but generally results in vector yields of lower purity (Hermens et al. Hum. Gene Ther. (1999) 10:1885-1891 ). Chromatographic methods including affinity and/or ion exchange chromatography have proven useful for large-scale production of clinical grade rAAV, including separation of empty viral capsids from full rAAV vectors.

[0005] Empty capsids are produced by the host cells that produce and package the recombinant vector genome in the viral capsid. An excess of empty capsids are produced relative to full vectors in most mammalian expression systems, and various systems generate 1-30% full vectors (Penaud-Budloo et al. Molecular Therapy, Methods & Clinical Dev (2018) 8:166-180). The production of empty capsids may be due to an imbalance in the ratio of plasmids encoding the transgene to that of the rep/cap genes. The presence of empty capsids in a drug product may cause an undesirable immune response and/or compete with the recombinant vectors for binding sites on target cells.

[0006] Certain anion-exchange chromatographic methods, employing acetate buffers and resins such as POROS™ 50 HQ and Q-Sepharose XL, have been used to separate empty capsids from rAAV2 vector pools by relying on the slightly less anionic character of the empty capsids as compared to full vectors (US 7,261 ,544; Qu et al. (2007) J. Virol. Meth. 140(1 ):183-192). A similar approach used a combination of affinity and ion exchange chromatography (IEX) and a 10 mM to 300 mM Tris acetate gradient at pH 8 with POROS™ 50 HQ resin to enrich for full AAV vectors of various serotypes (Nass et al. (2018) Molec. Thera. Meth. & Clin. Dev. 9:33-46). Other studies have identified buffers and conditions useful for chromatographic separation of empty capsids from full AAV vectors. For instance, Urabe determined that AAV1 material could be diluted with a Tris-HCI buffer comprising MgCh and glycerol for load on an anion exchange chromatography (AEX) column and that solutions comprising antichaotropic ions were effective elution buffers for separation of the empty AAV1 capsids from full vectors (Urabe et al. (2006) Molec. Ther. 13(4):823-828).

Others have described dilution (e.g., 50-fold) of affinity chromatography eluates and the use of shallow gradient elution (e.g., 20 mM to 180 mM NaCI) from a monolithic support in AEX methods for the separation of empty capsids from full AAV vectors (US 2019-0002841 ; US 2019-0002842; US 2019-0002843; US 2018-0002844). However, these methods also employ a high pH (9.8 to 10.2) which can lead to deamidation and/or aggregation of rAAV vector and may lead to a decrease in the therapeutic potency.

[0007] Processes using a combination of methods including, for example and in no particular order, tangential flow filtration (TFF) of a host cell supernatant, precipitation of the capsid material (including rAAV vectors and empty capsids) using ammonium sulfate, AEX chromatography and size-exclusion chromatography have been developed to separate the rAAV from empty capsids (Tomono et al. (2018) Molec. Ther. Meth. Clin. Dev. 11 :180-190). [0008] There remains a need for methods for preparation of clinical grade rAAV vector (e.g., rAAV3B, other rAAVs) with optimal purity, potency and consistency. These methods include the separation of rAAV comprising a vector genome with a therapeutic transgene from empty AAV capsids at a scale necessary to meet the clinical need for treatment of disease (e.g., Wilson Disease). SUMMARY

[0009] The present disclosure provides an improved AEX method of purification of rAAV vectors including, but not limited to the separation of full rAAV vectors (e.g., rAAV3B vectors) from empty capsids. Such purified full rAAV vectors are suitable for production of a drug product for administration to a human subject, such as a subject with Wilson Disease. The disclosure also provides a novel method of preparation of a chromatography eluate comprising rAAV vectors (e.g., from affinity chromatography) for further purification by AEX. In some embodiments, the chromatography elute that is to be further purified by AEX comprises rAAV3B vectors. In some embodiments, the chromatography elute that is further purified by AEX is subject to at least one processing step including neutralization, addition of a divalent salt (e.g., MgCls), dilution and adjustment of the conductivity and pH prior to loading on the column comprising an AEX media. The disclosure also provides for the use of weak binding load conditions (also referred to as a weak binding load) wherein the solution used to dilute the solution comprising the rAAV vectors (e.g., an affinity eluate), and the subsequently diluted solution, have a high conductivity (e.g., from 3 mS/cm to 9 mS/cm, e.g., 6.4 mS/cm) and a high pH (e.g., from 7.0 to 9.2, e.g., 8.8) The weak binding load conditions disclosed herein advantageously decrease or limit empty capsid binding while maintaining a high level of full capsid binding (e.g., greater than 80%, greater than 90%, greater than 95% and higher).

[0010] This disclosure also provides for the use of a wash (also referred to as an empty capsid wash or “ECW”) that has a high pH (e.g., 9.3 or greater) and a high conductivity (e.g., 13 to 19 mS/cm). The empty capsid wash disclosed herein advantageously elutes bound empty capsids with minimal elution of bound full capsids so that the AEX eluate comprises fewer empty capsids relative to full capsids. The disclosure also provides for the use of a two wash steps, one before and one after the empty capsid wash, using a MgCI2 buffer which increases full capsid binding and reduces vg loss prior to the elution step.

[0011] In some aspects, the present disclosure provides a method of purifying an rAAV vector by AEX, the method comprising a step of: i) loading a solution comprising the rAAV vector to be purified onto a stationary phase in a column; ii) contacting the stationary phase with an empty capsid wash (ECW) solution; iii) performing gradient elution of material from the stationary phase in the column wherein a percentage of a first gradient elution buffer is varied in a manner inversely proportional to variation in a percentage of a second gradient elution buffer; and iv) collecting at least one fraction of eluate from the column during the gradient elution.

[0012] In some aspects, the present disclosure provides a method of preparing a solution comprising a rAAV vector for purification by AEX, the method comprising a step of: diluting the solution 2 to 10-fold (e.g., 5-fold) with a dilution solution comprising histidine, Tris, P188, sodium citrate, magnesium chloride and sodium chloride to form a diluted solution, wherein the ratio of sodium citrate, magnesium chloride and sodium chloride is at a molar ratio of 1 to 1.25 to 2; wherein the pH of the diluted solution is adjusted to 8.6 to 9.0; and wherein the conductivity of the diluted solution is adjusted to 6.0 mS/cm to 6.8 mS/cm.

BRIEF DESCRIPTION OF DRAWINGS

[0013] FIG. 1 depicts exemplary Screen 1 weak binding load contour plots of percent VG bound to POROS™ 50 HQ (top panel) and % VG elution yield (bottom panel).

[0014] FIG. 2 depicts exemplary Screen 1 weak binding load contour plots of SEC A260/A280 ratio of the flow-through (top panel) and elution (bottom panel) fractions.

[0015] FIG. 3 depicts exemplary SEC A260/A280 ratios of elution fractions for salts (A) NaAcetate, (B) NaAcetate plus 2 mM MgCL₂ and (C) NaPropionate.

[0016] FIG. 4 depicts exemplary SEC A260/A280 ratios of elution fractions for salts (A) NaAcetate (baseline), (D) NaCI, (E) NH₄Acetate, (F) Na₂SO₄ and (G) TMAA.

[0017] FIG. 5 depicts exemplary SEC A260/A280 ratios for AEX elution pool of the baseline AEX process and the improved AEX process, using either NaAcetate or NaSulfate as the elution salt.

[0018] FIG. 6 depicts exemplary percent VG loss in the flow-through versus the percent VG in the load by SEC A260/A280 of the baseline AEX process and the improved AEX process, using either NaAcetate or NaSulfate as the elution salt.

[0019] FIG. 7 depicts exemplary percent VG loss in the flow-through versus VP resin challenge of the baseline AEC process and the improved AEX process, using either NaAcetate or NaSulfate as the elution salt.

[0020] FIG. 8 depicts exemplary overlayed VG and VP breakthrough curves at varying VG challenge (VG/mL resin) and VP challenge (VP/mL resin).

[0021] FIG. 9 depicts exemplary correlation between percent VG yield and A260/A280 ratio to VP challenge.

[0022] FIG. 10 depicts exemplary UV data for collection of a single AEX eluate fraction.

DESCRIPTION

1. Definitions

[0023] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Numeric ranges are inclusive of the numbers defining the ranges. The terms “comprising,” “comprise,” “comprises,” “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements. It is understood that wherever embodiments are described herein with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of” and/or “consisting essentially of” are also provided. The following terms have the meanings given: [0024] As used herein, the term “about,” or “approximately” refers to a measurable value such as an amount of the biological activity, length of a polynucleotide or polypeptide sequence, percent of vector genomes (vg) or viral particles (vp), percent of full, empty or intermediate capsids, dose, time, temperature, and the like, and is meant to encompass variations of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% 1%, 0.5% or even 0.1%, in either direction (greater than or less than) of the specified amount unless otherwise stated, otherwise evident from the context, or except where such number would exceed 100% of a possible value.

[0025] As used herein, the term “associated with” refers to with one another, if the presence, level and/or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc.) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and/or form correlates with incidence of and/or susceptibility to the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example, by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and a combination thereof.

[0026] As used herein, the term “coding sequence” or “nucleic acid encoding” refers to a nucleic acid sequence which encodes a protein or polypeptide and denotes a sequence which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of (operably linked to) appropriate regulatory sequences. The boundaries of a coding sequence are generally determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences.

[0027] As used herein, the term “eluate” refers to fluid exiting from a chromatography stationary phase (e.g., a monolith, membrane, resin, media) (e.g., “eluting from the stationary phase”) comprised of mobile phase and material that passed through the stationary phase or was displaced from the stationary phase. In some embodiments, a stationary phase includes, for example, a monolith, a membrane, a resin or a media. The mobile phase may be a solution that has been loaded onto a column and has flowed through the column (i.e., “flow-through fraction”); an equilibration solution (e.g. an equilibration buffer); an isocratic elution solution; a gradient elution solution; a solution for regenerating a stationary phase; a solution for sanitizing a stationary phase; a solution for washing; and combinations thereof.

[0028] As used herein, the term “flanked,” refers to a sequence that is flanked by other elements and indicates the presence of one or more flanking elements upstream and/or downstream, i.e., 5' and/or 3', relative to the sequence. The term “flanked” is not intended to indicate that the sequences are necessarily contiguous. For example, there may be intervening sequences between a nucleic acid encoding a transgene and a flanking element. A sequence (e.g., a transgene) that is “flanked” by two other elements (e.g., ITRs), indicates that one element is located 5' to the sequence and the other is located 3' to the sequence; however, there may be intervening sequences there between.

[0029] As used herein, the term “flocculation” refers to the process by which fine particulates are caused to clump together into a floc. The fine particles may include proteins, nucleic acids, cellular fragments resulting from lysis of host cells. In some embodiments, a floc that forms in a liquid phase may float to the top of the liquid (creaming), settle to the bottom (sedimentation) of the liquid or be filtered from the liquid phase.

[0030] As used herein, the term “fragment” refers to a material or entity that has a structure that includes a discrete portion of the whole but lacks one or more moieties found in the whole. In some embodiments, a fragment consists of a discrete portion. In some embodiments, a fragment consists of or comprises a characteristic structural element or moiety found in the whole. In some embodiments, a polymer fragment comprises, or consists of, at least 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomeric units (e.g., amino acid residues, nucleotides) found in the whole polymer.

[0031] rAAV vectors are referred to as “full,” a “full capsid,” “full vector” or a “fully packaged vector” when the capsid contains a complete, or essentially complete, vector genome, including a transgene. During production of rAAV vectors by host cells, vectors may be produced that have less packaged nucleic acid than the full capsids and contain, for example a partial or truncated vector genome. These vectors are referred to as “intermediates,” an “intermediate capsid,” a “partial” or a “partially packaged vector.” An intermediate capsid may also be a capsid with an intermediate sedimentation rate, that is a sedimentation rate between that of full capsids and empty capsids, when analyzed by analytical ultracentrifugation. Host cells may also produce viral capsids that do not contain any detectable nucleic acid material. These capsids are referred to as “empty(s),” or “empty capsids.” Full capsids may be distinguished from empty capsids based on A260/A280 ratios determined by SEC-HPLC, whereby the A260/A280 ratios have been previously calibrated against capsids (i.e., full, intermediate and empty) isolated by analytical ultracentrifugation. Other methods known in the art for the characterization of capsids include CryoTEM, capillary isoelectric focusing and charge detection mass spectrometry. Calculated isoelectric points of ~6.2 and ~5.8 for empty and full AAV9 capsids, respectively have been reported (Venkatakrishnan et al., J. Virology (2013) 87.9:4974-4984).”

[0032] As used herein, the term “null capsid” refers to a capsid produced intentionally to lack a vector genome. Such null a capsid may be produced by transfection of a host cell with a rep/cap and a helper plasmid, but not a plasmid that comprises the transgene cassette sequence, also known as a vector plasmid.

[0033] As used herein, the term “functional” refers to a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized. A biological molecule may have two functions (i.e., bifunctional) or many functions (i.e., multifunctional).

[0034] As used herein, the term “gene” refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular polypeptide or protein after being transcribed and translated. “Gene transfer” or “gene delivery” refers to methods or systems for reliably inserting foreign DNA into host cells. Such methods can result in transient expression of non-integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g. episomes), and/or integration of transferred genetic material into the genomic DNA of host cells.

[0035] As used herein, the term “gradient elution” refers to application of a mixture of at least two different solutions with different pH, conductivity and/or modifier concentration to a chromatography stationary phase (including e.g., monolith, media, resin, membrane) that are gradually changed over the course of the elution. A gradient elution may be linear or nonlinear. In contrast, during an isocratic elution, the chromatography mobile phase composition is constant, and during a “step elution,” the chromatography mobile phase composition changes in a stepwise manner. Over the course of the gradient elution, a percentage of a first solution is continuously varied in a manner inversely proportional to a percentage of a second solution. For example, at the start of a gradient elution, the percentage of gradient elution buffer A (e.g., a first gradient elution buffer) in the mixture is 100% and the percentage of gradient elution buffer B (e.g., a second gradient elution buffer) in the mixture is 0% such that a continuously varying gradient in the pH, conductivity and/or modifier concentration (increasing or decreasing, depending on the embodiment) is created as the solutions are mixed and flow through the stationary phase. In some embodiments, a concentration of a salt, such as sodium acetate, will change at a constant rate over the volume of a linear gradient. For example, for a 1 mL column with a 20 mL linear gradient (i.e., 20 CV), operating at a constant flow rate of 1 mL/minute, the salt concentration will change at a rate of 5% per minute. In some embodiments, rAAV capsids (e.g., full, intermediate, empty) are bound to a stationary phase during loading of a solution comprising the rAAV capsid to be purified onto an AEX stationary phase. During a gradient elution, as a percentage of buffer B increases, such that the concentration of a salt increases (e.g., Sodium acetate) full rAAV vectors are preferentially released (eluted) from the stationary phase, and empty capsids are preferentially retained on the stationary phase. Empty capsids are released in greater amounts as the percentage of buffer B further increases. Elution of full rAAV vector from the stationary phase can be monitored during gradient elution by measuring A260 and A280 of the eluate, such that an increase in the ratio of A260/A280 is indicative of an increase in the percentage of full rAAV vector in the eluate, and conversely, a decrease in the A260/A280 ratio is indicative of a decrease in the percentage of full rAAV vector and an increase in the percentage of empty capsids. In some embodiments, an absorbance of at least one fraction of eluate is measured using a method such as analytical size exclusion chromatography (SEC) in a high performance liquid chromatography (HPLC) system, on-line UV trace, off-line UV methods, etc., and wherein the absorbance is measured at one or more wavelengths (e.g., 260 nm and/or 280 nm).

[0036] As used herein, the term “heterologous” refers to a nucleic acid inserted into a vector (e.g., rAAV vector) for purposes of vector mediated transfer/delivery of the nucleic acid into a cell. Heterologous nucleic acids are typically distinct from the vector (e.g., AAV) nucleic acid, that is, the heterologous nucleic acid is non-native with respect to the viral (e.g., AAV) nucleic acid. Once transferred or delivered into a cell, a heterologous nucleic acid, contained within a vector, can be expressed (e.g., transcribed and translated if appropriate). Alternatively, a transferred or delivered heterologous nucleic acid in a cell, contained within the vector, need not be expressed. Although the term “heterologous” is not always used herein in reference to a nucleic acid, reference to a nucleic acid even in the absence of the modifier “heterologous” is intended to include a heterologous nucleic acid. For example, a heterologous nucleic acid would be a nucleic acid encoding a ATP7B polypeptide, or a fragment thereof, for example an ATP7B transgene with deletion of the heavy metal associated domains 1-4 as described in WO 2016/097219 and WO 2016/097218, and incorporated herein by reference, for use in the treatment of Wilson disease.

[0037] A further exemplary heterologous nucleic acid comprises a wild-type coding sequence, or a fragment thereof (e.g., truncated, internal deletion), of one of the following genes, and may or may not be codon-optimized:

[0038] As used herein, the terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, and includes the progeny of such a cell. A host cell includes a “transfectant,” “transformant,” “transformed cell,” and “transduced cell,” which includes the primary transfected, transformed or transduced cell, and progeny derived therefrom, without regard to the number of passages. In some embodiments, a host cell is a packaging cell for production of a rAAV vector.

[0039] As used herein, the term “host cell DNA” or “HCDNA” refers to residual DNA, derived from a host cell culture which produced a rAAV vector, and present in a chromatography fraction (e.g., an affinity eluate, an AEX eluate, a wash) or a chromatography load (e.g., an affinity load, an AEX load). Host cell DNA may be measured by methods know in the art such as qPCR to detect a sequence unique to the host cells. General DNA concentrations may be estimated using fluorescence dyes (e.g. PicoGreen® or SYBR® Green), absorbance measurement (e.g. at 260 nm, or 254 nm) or electrophoretic techniques (e.g. agarose gel electrophoresis, or capillary electrophoresis). An amount of HCDNA present in an eluate may be expressed relative to the amount of vg present in the eluate, for example, ng HCDNA/1 x 10¹⁴ vg or pg HCDNA /1 x 10⁹ vg. An amount of HCDNA present in an eluate may be expressed relative to the amount of vg present in a volume of eluate, for example, pg HCDNA/mL eluate.

[0040] As used herein, the term “host cell protein” or “HCP” refers to residual protein, derived from a host cell culture which produced a rAAV vector, present in a chromatography fraction (e.g., an affinity eluate, an AEX eluate, a wash) or a chromatography load (e.g., an affinity load, an AEX load). Host cell protein may be measured by methods known in the art, such as ELISA. Host cell protein can be semi-quantitatively measured by various electrophoretic staining methods (e.g., silver stain SDS-PAGE, SYPRO® Ruby stain SDS- PAGE, and/or Western blot). An amount of HCP present in an eluate may be expressed relative to the amount of vg present, for example, ng HCP/1 x 10¹⁴ vg or pg HCP/1 x 10⁹ vg. [0041] As used herein, the term “identity” or “identical to” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. “Identity” measures the percent of identical matches between two or more sequences with gap alignments addressed by a particular mathematical model of computer programs (i.e. “algorithms”).

[0042] In some embodiments, polymeric molecules are considered to be “substantially identical” to one another if their sequences are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of a reference sequence. Nucleotides at corresponding positions are then compared. When a position in a first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in a second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.

[0043] To determine percent identity, or homology, sequences can be aligned using the methods and computer programs, including BLAST, available over the world wide web at ncbi.nlm.nih.gov/BLAST/. Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wis., USA. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc. Of particular interest are alignment programs that permit gaps in the sequence. Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. 70: 173- 187 (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See J. Mol. Biol. 48: 443-453 (1970).

[0044] Also of interest is the BestFit program using the local homology algorithm of Smith and Waterman (1981 , Advances in Applied Mathematics 2: 482-489) to determine sequence identity. The gap generation penalty will generally range from 1 to 5, usually 2 to 4 and in some embodiments will be 3. The gap extension penalty will generally range from about 0.01 to 0.20 and in some instances will be 0.10. The program has default parameters determined by the sequences inputted to be compared. Preferably, the sequence identity is determined using the default parameters determined by the program. This program is available also from Genetics Computing Group (GCG) package, from Madison, Wl, USA. [0045] Another program of interest is the FastDB algorithm. FastDB is described in Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1988, Alan R. Liss, Inc. Percent sequence identity is calculated by FastDB based upon the following parameters: Mismatch Penalty: 1 .00; Gap Penalty: 1 .00; Gap Size Penalty: 0.33; and Joining Penalty: 30.0.

[0046] As used herein, the term “impurity” refers to any molecule other than the full rAAV vector being purified that is also present in a solution comprising the rAAV vector being purified. Impurities include empty capsids, intermediate capsids, biological macromolecules such as DNA, RNA, non-AAV proteins (e.g., host cell proteins), AAV aggregates, damaged AAV capsids, molecules that are part of an absorbent used for chromatography that may leach into a sample during prior purification steps, endotoxins, cell debris and chemicals from cell culture, including media components, plasmid DNA from transfection, an adventitious agent, bacteria and viruses.

[0047] As used herein, the terms “inverted terminal repeat”, “ITR,” “terminal repeat,” and “TR” refer to palindromic terminal repeat sequences at or near the ends of the AAV virus genome, comprising mostly complementary, symmetrically arranged sequences. These ITRs can fold over to form T-shaped hairpin structures that function as primers during initiation of DNA replication. They are also needed for viral genome integration into host genome, for the rescue from the host genome; and for the encapsidation of viral nucleic acid into mature virions. The ITRs are required in cis for vector genome replication and its packaging into viral particles. “5’ ITR” refers to the ITR at the 5’ end of the AAV genome and/or 5’ to a recombinant transgene. “3’ ITR” refers to the ITR at the 3’ end of the AAV genome and/or 3’ to a recombinant transgene. Wild type ITRs are approximately 145 bp in length. A modified, or recombinant ITR, may comprise a fragment or portion of a wild type AAV ITR sequence. One of ordinary skill in the art will appreciate that during successive rounds of DNA replication ITR sequences may swap such that the 5’ ITR becomes the 3’ ITR, and vice versa. In some embodiments, at least one ITR is present at the 5’ and/or 3’ end of a recombinant vector genome such that the vector genome can be packaged into a capsid to produce a rAAV vector (also referred to herein as “rAAV vector particle” or “rAAV viral particle”) comprising the vector genome.

[0048] As used herein, the term “isolated” refers to a substance or composition that is 1) designed, produced, prepared, and or manufactured by the hand of man and/or 2) separated from at least one of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting). Generally, isolated compositions are substantially free of one or more materials with which they normally associate with in nature, for example, one or more protein, nucleic acid, lipid, carbohydrate and/or cell membrane. The term “isolated” does not exclude man-made combinations, for example, a recombinant nucleic acid, a recombinant vector genome (e.g., rAAV vector genome), a rAAV vector particle (e.g., such as, but not limited to, a rAAV vector particle comprising an AAV3B capsid) that packages, e.g., encapsidates, a vector genome and a pharmaceutical formulation. The term “isolated” also does not exclude alternative physical forms of the composition, such as hybrids/chimeras, multimers/oligomers, modifications (e.g., phosphorylation, glycosylation, lipidation), variants or derivatized forms, or forms expressed in host cells that are man-made.

[0049] Isolated substances or compositions may be separated from about 10%, about 20%, about 30%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated. In some embodiments, isolated agents are about 80%, about 85%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is “pure” if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may still be considered “isolated” or even “pure,” after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g., buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of the substance is calculated without including such carriers or excipients.

[0050] As used herein, the term “load chase” refers to a solution applied to a column after the load or load solution (as defined, infra) has been applied. A load chase serves to complete application of the load or load solution and to remove unbound material from the column.

[0051] As used herein, the terms “load” or “load solution” refer to any material (e.g., a solution) containing a product of interest (e.g., a full rAAV vector) that is loaded onto a chromatography stationary phase. In some embodiments, a “load solution” is exposed to a chromatography stationary phase. In some embodiments, a load solution is an affinity eluate. In some embodiments, a load solution is a diluted, and optionally filtered affinity eluate. In some embodiments, the pH, conductivity or both of a load are adjusted to optimize binding of full capsids to a stationary phase. In some embodiments, the pH, conductivity or both of a load are adjusted to reduce binding of empty capsids to a stationary phase.

[0052] As used herein, the terms “stationary phase” or “chromatography stationary phase” are used to refer to any substance that can be used for separation of a product from another substance (e.g., an impurity). In some embodiments, a chromatography stationary phase is a resin, a media, a membrane, a membrane adsorber, or a monolith. In some embodiments, a chromatography stationary phase is a media that binds to AAV capsids under certain conditions. In some embodiments, a chromatography stationary phase is an ion exchange media (e.g., an anion exchange media, a cation exchange media). In some embodiments, a chromatography stationary phase is POROS™ 50 HQ.

[0053] As used herein, the term “modifier,” or “mobile phase modifier” is a component of the mobile phase that modifies the mobile phase in order to alter the chromatography. Such altering of the chromatography results in, for example, the removal, or washing off of, impurities from the stationary phase, or elution of a product or material of interest from the stationary phase (e.g., a rAAV vector). Examples of “modifiers” include a salt, a detergent, an amino acid (e.g., arginine, histidine, citrulline, glycine), an organic solvent (e.g., ethanol, ethylene glycol), a chaotropic agent (e.g., urea), or a displacer (also referred to as a selective elution agent).

[0054] As used herein, the terms “nucleic acid sequence,” “nucleotide sequence,” and “polynucleotide” refer interchangeably to any molecule composed of or comprising monomeric nucleotides connected by phosphodiester linkages. A nucleic acid may be an oligonucleotide or a polynucleotide. Nucleic acid sequences are presented herein in the direction from the 5’ to the 3’ direction. A nucleic acid sequence (i.e., a polynucleotide) of the present disclosure can be a deoxyribonucleic acid (DNA) molecule or ribonucleic acid (RNA) molecule and refers to all forms of a nucleic acid such as, double stranded molecules, single stranded molecules, small or short hairpin RNA (shRNA), micro RNA, small or short interfering RNA (siRNA), trans-splicing RNA, antisense RNA, messenger RNA, transfer RNA, ribosomal RNA. Where a polynucleotide is a DNA molecule, that molecule can be a gene, a cDNA, an antisense molecule or a fragment of any of the foregoing molecules.

[0055] As used here, the term “nucleic acid construct,” refers to a non-naturally occurring nucleic acid molecule resulting from the use of recombinant DNA technology (e.g., a recombinant nucleic acid). A nucleic acid construct is a nucleic acid molecule, either single or double stranded, which has been modified to contain segments of nucleic acid sequences, which are combined and arranged in a manner not found in nature. A nucleic acid construct may be a “vector” (e.g., a plasmid, a rAAV vector genome, an expression vector, etc.), that is, a nucleic acid molecule designed to deliver exogenously created DNA into a host cell.

[0056] As used herein, the term “operably linked” refers to a linkage of nucleic acid sequence (or polypeptide) elements in a functional relationship. A nucleic acid is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or other transcription regulatory sequence (e.g., an enhancer) is operably linked to a coding sequence if it affects the transcription of the coding sequence. In some embodiments, operably linked means that nucleic acid sequences being linked are contiguous. In some embodiments, operably linked does not mean that nucleic acid sequences are contiguously linked, rather intervening sequences are between those nucleic acid sequences that are linked.

[0057] As used herein, the term “percent vector genome (VG) dilution yield” or “% VG dilution yield” refers to the amount of VG present in a diluted affinity pool (also referred to herein as a diluted affinity eluate) as a percentage of the amount of VG present in the affinity pool (also referred to herein as an affinity eluate) prior to dilution. For instance, % VG dilution yield = ((amount of VG in diluted affinity pool)/(amount of VG in affinity pool)) * 100.

[0058] As used herein, the term “percent VG column yield” or “% VG column yield” refers to the amount of vector genomes (VG) present in a pooled eluate collected from an AEX column (i.e., an AEX pool) as a percentage of the amount of VG present in an affinity eluate that has been diluted only, or diluted and filtered.

[0059] In some embodiments, an affinity eluate comprising a rAAV vector to be purified has been diluted only and is referred to as a “diluted affinity pool.” Optionally, the rAAV vector to be purified is harvested from a 250 L or 2000 L vessel (e.g., a single use bioreactor (SUB)). For instance, % VG column yield = ((amount of VG in AEX pool)/(amount of VG in diluted affinity pool)) * 100.

[0060] In some embodiments, an affinity eluate comprising a rAAV vector to be purified has been diluted and filtered and is referred to as an “AEX load.” Optionally, the rAAV vector to be purified is harvested from a small scale (e.g., less than 250 L) vessel (e.g., bioreactor). For instance, % VG column yield = ((amount of VG in AEX pool)/(amount of VG in diluted and filtered affinity pool)) * 100.

[0061] As used herein, the term “percent VG step yield” or “% VG step yield” refers to the amount of VG in a pooled eluate collected from an AEX column (i.e., an AEX pool) as a percentage of the amount of VG present in the affinity pool (also referred to herein as an affinity eluate) prior to dilution or filtration. For instance, % VG step yield = ((amount of VG in AEX pool)/(amount of VG in affinity pool)) * 100.

[0062] As used herein, the term “pharmaceutically acceptable” and “physiologically acceptable” refers to a biologically acceptable formulation, gaseous, liquid or solid, or mixture thereof, which is suitable for one or more routes of administration, in vivo delivery or contact.

[0063] As used herein, the term “polynucleotide” or “nucleic acid” refers to a polymeric form of nucleotides, either ribonucleotides or deoxynucleotides, or a modified form of either type of nucleotide, and may be single or double stranded forms. A “polynucleotide” or a “nucleic acid” sequence encompasses its complement unless otherwise specified. As used herein, the term “isolated polynucleotide,” “isolated nucleic acid” or “isolated recombinant nucleic acid” means a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, which by virtue of its origin or source of derivation, has one to three of the following: (1) is not associated with all or a portion of a polynucleotide with which the “isolated polynucleotide” is found in nature, (2) is operably linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence. [0064] As used herein, the terms “polypeptide,” “protein,” “peptide” or “encoded by a nucleic acid sequence” (i.e., encode by a polynucleotide sequence, encoded by a nucleotide sequence) refer to full-length native sequences, as with naturally occurring proteins, as well as functional subsequences, modified forms or sequence variants so long as the subsequence, modified form or variant retains some degree of functionality of the native full- length protein. In methods and uses of the disclosure, such polypeptides, proteins and peptides encoded by the nucleic acid sequences can be but are not required to be identical to the endogenous protein that is defective, or whose expression is insufficient, or deficient in a subject treated with gene therapy.

[0065] As used herein, the term “recombinant,” refers to a vector, polynucleotide (e.g., a recombinant nucleic acid), polypeptide or cell that is the product of various combinations of cloning, restriction or ligation steps (e.g., relating to a polynucleotide or polypeptide comprised therein), and/or other procedure that results in a construct that is distinct from a product found in nature. A recombinant virus or vector (e.g., rAAV vector) comprises a vector genome comprising a recombinant nucleic acid (e.g., a nucleic acid comprising a transgene and one or more regulatory elements). The terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct.

[0066] As used herein, the term “step elution” refers to application of a solution with a defined pH, conductivity, and/or modifier concentration to a chromatography stationary phase (including e.g., monolith, media, resin, membrane). A series of step elutions (e.g., with increasing conductivity or salt concentration) can be conducted to optimize separations. Each step elution solution has a defined composition that does not change during its application. Over the course of the step elution, as the series of solutions (e.g., a load chase, a pH stabilization solution, a wash buffer, an elution buffer) are applied to the stationary phase, the pH, conductivity and/or modifier concentration is increased, or decreased, relative to a preceding solution in the series. For example, at the start of a step elution series, the concentration of a modifier (e.g., a salt, e.g., sodium acetate) in the first solution is low, e.g., 0 to 10 mM, e.g., about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM) . In each subsequent solution in the series, the concentration of the salt is increased, such that over the course of 2 to 20 solutions, the concentration of the salt is increased to, for example, 50 mM to 300 mM (e.g., about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 120 mM, about 140 mM, about 160 mM, about 180 mM, about 200 mM). The salt concentration in the series of 2 to 20 (or more) solutions is not necessarily varied in equal or proportional increments.

[0067] In some embodiments, a step elution comprises 2 to 20 solutions, 2 to 10 solutions, 10 to 20 solutions, for example 2, 3, 4, 5,6 7, 8 19, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more solutions. In some embodiments, rAAV capsids (e.g., full, intermediate, empty) are bound to a stationary phase during loading of a solution comprising the rAAV capsid onto an AEX stationary phase. During a step elution, as a pH, conductivity and/or modifier concentration is varied, full rAAV vectors are preferentially released (eluted) from the stationary phase, and empty capsids are preferentially retained on the stationary phase. Empty capsids are released in greater amounts as the concentration of modifier (e.g., salt) increases. Elution of full rAAV vector from the stationary phase can be monitored during step elution by measuring A260 and A280 of the eluate, such that an increase in the ratio of A260/A280 is indicative of an increase in the percentage of full rAAV vector in the eluate, and conversely, a decrease in the A260/A280 ratio is indicative of a decrease in the percentage of full rAAV vector and an increase in the percentage of empty capsids. In some embodiments, an absorbance of at least one fraction of eluate is measured using a method such as analytical size exclusion chromatography (SEC) in a high performance liquid chromatography (HPLC) system, on-line UV trace, off-line UV methods, etc., and wherein the absorbance is measured at one or more wavelengths (e.g., 260 nm and/or 280 nm). [0068] As used herein, the term “subject” refers to an organism, for example, a mammal (e.g., a human, a non-human mammal, a non-human primate, a primate, a laboratory animal, a mouse, a rat, a hamster, a gerbil, a cat, a dog). In some embodiments, a human subject is an adult, adolescent, or pediatric subject. In some embodiments, a subject is suffering from a disease, disorder or condition, e.g., a disease, disorder or condition that can be treated as provided herein. In some embodiments, a subject is suffering from a disease, disorder or condition associated with deficient or dysfunctional copper-transporting ATPase 2 (ATP7B), e.g., Wilson disease. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a susceptible subject is predisposed to and/or shows an increased risk (as compared to the average risk observed in a reference subject or population) of developing a disease, disorder or condition. In some embodiments, a subject displays one or more symptoms of a disease, disorder or condition. In some embodiments, a subject does not display a particular symptom (e.g., clinical manifestation of disease) or characteristic of a disease, disorder, or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is a human patient. In some embodiments, a subject is an individual to whom diagnosis and/or therapy is and/or has been administered (e.g., gene therapy for Wilson disease). In some embodiments, a subject is a human patient with Wilson disease.

[0069] Disease, disorders and conditions that can be treated using a rAAV vector purified according to the methods set forth herein include, for example a metabolic disease or disorder (e.g., Fabry disease, Gaucher disease, Pompe disease, phenylketonuria, a glycogen storage disease); a urea cycle disease or disorder (e.g., ornithine transcarbamylase deficiency); a lysosomal storage disease or disorder (e.g., metachromatic leukodystrophy, mucopolysaccharidosis); a liver disease or disorder (e.g., progressive familial intrahepatic cholestasis type 1-3); a blood disease or disorder (Hemophilia A, Hemophilia B, a thalassemia); a cancer (e.g., a carcinoma, a sarcoma, a blood cancer); a genetic disease or disorder (e.g., cystic fibrosis); or an infectious disease (e.g., HIV).

[0070] Diseases, disorders and conditions that can be treated using a rAAV vector purified according to the methods set forth herein include, for example: 21 -hydroxylase- deficient congenital adrenal hyperplasia, achondrogenesis Type 1 B, achondroplasia, achromatopsia, acid sphingomyelinase deficiency (Niemann-Pick disease type A or B), acute intermittent porphyria, adenosine deaminase 2 deficiency, adenosine deaminase deficiency (e.g., severe combined immunodeficiency, X-linked), adrenoleukodystrophy (e.g., X-linked), age-related macular degeneration (e.g., neovascular, wet), Alagille syndrome, alkaptonuria, alpha-1 antitrypsin deficiency, alpha-thalassemia, Alport syndrome, Alzheimer disease, Apert syndrome, arginase deficiency, argininosuccinate lyase (ASL) deficiency, argininosuccinate synthase (ASS1 ) deficiency (citrullinemia type 1), aromatic L-amino acid decarboxylase deficiency, autosomal recessive congenital ichthyosis, Becker muscular dystrophy, beta-thalassemia, carbamoylphosphatase synthetase I deficiency, ceroid lipofuscinosis, Charcot-Marie-Tooth neuropathy, choroideremia, chronic granulomatous disease, citrin deficiency, Crigler-Najjar syndrome type 1 and 2, critical limb ischemia, cystic fibrosis, cystinosis, Danon disease, diabetic macular retinopathy, dominant inherited short stature, Dravet syndrome, Duchenne muscular dystrophy, dysferlinopathy (e.g., Miyoshi myopathy, limb-girdle muscular dystrophy 2B), dystrophic epidermolysis bullosa, Fabry disease, familial hypercholesterolemia, familial lipoprotein lipase deficiency, Fanconia anemia (e.g., Fanconia anemia A), Friedreich’s ataxia, frontotemporal dementia, Gaucher disease, glycogen storage disease type 1A and 1 B (Von Gierke’s disease), glycogen storage disease type II (Pompe disease), glycogen storage disease type III, glycogen storage disease type IV, glycogen storage disease type V, glycogen storage disease type VI, glycogen storage disease type XV, GM1 gangliosidosis, gyrate atrophy, hemophilia A, hemophilia B, hereditary angiodema, types l-lll, Huntington’s disease, inclusion body myositis, junctional epidermolysis bullosa, Kabuki Syndrome, Leber congenital amaurosis, leukocyte adhesion defect type 1 , limb girdle muscular dystrophy, limb girdle muscular dystrophy type 2C (gamma-sarcoglycanopathy), limb girdle muscular dystrophy type 2D, metachromatic leukodystrophy, mucopolysaccharidosis Type I, mucopolysaccharidosis type II (Hunter syndrome), mucopolysaccharidosis type 11 IA, mucopolysaccharidosis type II IB, mucopolysaccharidosis type NIC, mucopolysaccharidosis type HID, mucopolysaccharidosis type IVA (Morquio A syndrome), mucopolysaccharidosis type IVB (Morquio B syndrome), mucopolysaccharidosis type VI (Maroteaux-Lamy), myotonic dystrophy type 1 , myotonic dystrophy type 2, N-acetylglutamate synthase (NAGS) deficiency, Netherton syndrome, neuronal ceroid lipofuscinosis, ornithine translocase deficiency, ornithine transcarbamylase deficiency disease, Parkinson’s disease, phenylketonuria, Pompe, progressive familial intrahepatic cholestasis type 1 -3, progressive myofibrillar myopathy, pyruvate kinase deficiency, retinitis pigmentosa, RPE65-related Leber congenital amaurosis, Sandhoff disease, sickle cell disease, spinal muscular atrophy, Tay Sachs disease, Wilson disease, Wiskott-Aldrich syndrome, Wiskott-Aldrich syndrome 2, X-linked adrenoleukodystrophy, X- linked chronic granulomatous disease, X-linked myotubular myopathy, X-linked retinitis pigmentosa, X-linked retinoschisis and X-linked severe combined immunodeficiency. [0071] As used herein, the term “substantially” refers to the qualitative condition of exhibition of total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the art will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0072] As used herein, the term “therapeutic polypeptide” is a peptide, polypeptide or protein (e.g., enzyme, structural protein, transmembrane protein, transport protein) that may alleviate or reduce symptoms that result from an absence or defect in a protein in a target cell (e.g., an isolated cell) or organism (e.g., a subject). A therapeutic polypeptide or protein encoded by a transgene is one that confers a benefit to a subject, e.g., to correct a genetic defect, to correct a deficiency in a gene related to expression or function. Similarly, a “therapeutic transgene” is the transgene that encodes the therapeutic polypeptide. In some embodiments, a therapeutic polypeptide, expressed in a host cell, is an enzyme expressed from a transgene (i.e., an exogenous nucleic acid that has been introduced into the host cell). In some embodiments, a therapeutic polypeptide is a copper-transporting ATPase 2 protein, or fragment thereof, expressed from a therapeutic transgene transduced into a liver cell .

[0073] As used herein, the term “therapeutically effective amount” refers to an amount that produces the desired therapeutic effect for which it is administered. In some embodiments, the term refers to an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and/or severity of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition. Those of ordinary skill in the art will appreciate that the term “therapeutically effective amount” does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment.

[0074] A nucleic acid of interest can be introduced into a host cell by a wide variety of techniques that are well-known in the art, including transfection and transduction.

[0075] As used herein, the term “transduction” refers to transfer of a nucleic acid (e.g., a vector genome) by a viral vector (e.g., rAAV vector) to a cell (e.g., a target cell, including, but not limited to, a cell within a mammal). In some embodiments, a gene therapy for the treatment disease includes transducing a vector genome comprising a modified nucleic acid encoding a therapeutic protein into a target cell. In some embodiments, a gene therapy for Wilson disease includes transducing a vector genome comprising a modified nucleic acid encoding copper-transporting ATPase 2 into a hepatocyte. A cell into which a transgene has been introduced by a virus or a viral vector is referred to as a “transduced cell.” In some embodiments, a transduced cell is an isolated cell and transduction occurs ex vivo. In some embodiments, a transduced cell is a cell within an organism (e.g., a subject) and transduction occurs in vivo. A transduced cell may be a target cell of an organism which has been transduced by a recombinant AAV vector such that the target cell of the organism expresses a polynucleotide (e.g., a transgene encoding a therapeutic protein, e.g., a modified nucleic acid encoding copper-transporting ATPase 2).

[0076] A cell that may be transduced includes a cell of any tissue or organ type, or any origin (e.g., mesoderm, ectoderm or endoderm). Non-limiting examples of cells include liver (e.g., hepatocytes, sinusoidal endothelial cells), pancreas (e.g., beta islet cells, exocrine), lung, central or peripheral nervous system, such as brain (e.g., neural or ependymal cells, oligodendrocytes) or spine, kidney, eye (e.g., retinal), spleen, skin, thymus, testes, lung, diaphragm, heart (cardiac), muscle or psoas, or gut (e.g., endocrine), adipose tissue (white, brown or beige), muscle (e.g., fibroblasts, myocytes), synoviocytes, chondrocytes, osteoclasts, epithelial cells, endothelial cells, salivary gland cells, inner ear nervous cells or hematopoietic (e.g., blood or lymph) cells. Additional examples include stem cells, such as pluripotent or multipotent progenitor cells that develop or differentiate into liver (e.g., hepatocytes, sinusoidal endothelial cells), pancreas (e.g., beta islet cells, exocrine cells), lung, central or peripheral nervous system, such as brain (e.g., neural or ependymal cells, oligodendrocytes) or spine, kidney, eye (e.g., retinal), spleen, skin, thymus, testes, lung, diaphragm, heart (cardiac), muscle or psoas, or gut (e.g., endocrine), adipose tissue (white, brown or beige), muscle (e.g., fibroblast, myocytes), synoviocytes, chondrocytes, osteoclasts, epithelial cells, endothelial cells, salivary gland cells, inner ear nervous cells or hematopoietic (e.g., blood or lymph) cells.

[0077] In some embodiments, cells present within particular areas of a tissue or organ (e.g., liver) may be transduced by vector (e.g., a rAAV comprising a therapeutic transgene, a reporter transgene) that is administered to the tissue or organ. In some embodiments, cells present within particular areas of a tissue or organ (e.g., liver) may be transduced by a rAAV vector (e.g., a rAAV comprising an ATP7B transgene) that is administered to the tissue or organ.

[0078] As used herein, the term “transfection” refers to transfer of a recombinant nucleic acid (e.g., an expression plasmid) into a cell (e.g., a host cell) without use of a viral vector. A cell into which a recombinant nucleic acid has been introduced is referred to as a “transfected cell.” A transfected cell may be a host cell (e.g., a CHO cell, Pro10 cell, HEK293 cell) comprising an expression plasmid/vector for producing a recombinant AAV vector. In some embodiments, a transfected cell (e.g., a packing cell) may comprise a plasmid comprising a transgene (e.g., a transgene encoding a therapeutic protein), a plasmid comprising an AAV rep gene and an AAV cap gene and a plasmid comprising a helper gene. Many transfection techniques are known in the art, which include, but are not limited to, electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and liposomes in combination with a nuclear localization signal.

[0079] As used herein, the term “transgene” is used to mean any heterologous polynucleotide for delivery to and/or expression in a host cell, target cell or organism (e.g., a subject). Such “transgene” may be delivered to a host cell, target cell or organism using a vector (e.g., rAAV vector). A transgene may be operably linked to a control sequence, such as a promoter. It will be appreciated by those of skill in the art that expression control sequences can be selected based on an ability to promote expression of the transgene in a host cell, target cell or organism. Generally, a transgene may be operably linked to an endogenous promoter associated with the transgene in nature, but more typically, the transgene is operably linked to a promoter with which the transgene is not associated in nature. An example of a transgene is a nucleic acid encoding a therapeutic polypeptide, for example a copper-transporting ATPase 2 polypeptide or fragment thereof, and an exemplary promoter is one not operable linked to a nucleotide encoding copper- transporting ATPase 2in nature. Such a non-endogenous promoter can include an alpha- 1 -antitrypsin promoter or a liver specific promoter, among many others known in the art.

[0080] As used herein, the term “vector” refers to a plasmid, virus (e.g., a rAAV), cosmid, or other vehicle that can be manipulated by insertion or incorporation of a nucleic acid (e.g., a recombinant nucleic acid). A vector can be used for various purposes including, e.g., genetic manipulation (e.g., cloning vector), to introduce/transfer a nucleic acid into a cell, to transcribe or translate an inserted nucleic acid in a cell. In some embodiments a vector nucleic acid sequence contains at least an origin of replication for propagation in a cell. In some embodiments, a vector nucleic acid includes a heterologous nucleic acid sequence, an expression control element(s) (e.g., promoter, enhancer), a selectable marker (e.g., antibiotic resistance), a poly-adenosine (polyA) sequence and/or an ITR. In some embodiments, when delivered to a host cell, the nucleic acid sequence is propagated. In some embodiments, when delivered to a host cell, either in vitro or in vivo, the cell expresses the polypeptide encoded by the heterologous nucleic acid sequence. In some embodiments, when delivered to a host cell, the nucleic acid sequence, or a portion of the nucleic acid sequence is packaged into a capsid. A host cell may be an isolated cell or a cell within a host organism. In addition to a nucleic acid sequence (e.g., transgene) which encodes a polypeptide or protein, additional sequences (e.g., regulatory sequences) may be present within the same vector (i.e., in cis to the gene) and flank the gene. In some embodiments, regulatory sequences may be present on a separate (e.g., a second) vector which acts in trans to regulate the expression of the gene. Plasmid vectors may be referred to herein as “expression vectors.”

[0081] As used herein, the term “vector genome” refers to a nucleic acid that is packaged/ encapsidated in an AAV capsid to form a rAAV vector. Typically, a vector genome includes a heterologous polynucleotide sequence (e.g., a transgene, regulatory elements, etc.) and at least one ITR. In cases where a recombinant plasmid is used to construct or manufacture a recombinant vector (e.g., rAAV vector), the vector genome does not include the entire plasmid but rather only the sequence intended for delivery by the viral vector. This non-vector genome portion of the recombinant plasmid is referred to as the “plasmid backbone,” which is important for cloning, selection and amplification of the plasmid, a process that is needed for propagation of recombinant viral vector production, but which is not itself packaged or encapsidated into a rAAV vector. Typically, the heterologous sequence to be packaged into the capsid is flanked by the ITRs such that when cleaved from the plasmid backbone, it is packaged into the capsid.

[0082] As used herein, the term “viral vector” generally refers to a viral particle that functions as a nucleic acid delivery vehicle and which comprises a vector genome (e.g., comprising a transgene which has replaced the wild type rep and cap) packaged within the viral particle (i.e., capsid) and includes, for example, lenti- and parvo- viruses, including AAV serotypes and variants (e.g., rAAV vectors). As noted elsewhere herein, a recombinant viral vector does not comprise a virus genome with a rep and/or a cap gene; rather, these sequences have been removed to provide capacity for the vector genome to carry a transgene of interest.

[0083] The present disclosure provides methods for purification of rAAV vectors (e.g., full rAAV vectors) from host cell harvests. In particular, the disclosure provides methods for the separation of empty capsids from full rAAV vectors (e.g., rAAV vectors comprising a vector genome) using a weak binding load. Furthermore, the disclosure provides methods for the separation of empty capsids from full rAAV vectors (e.g., rAAV vectors comprising a vector genome) using an empty capsid wash. Each of these aspects of the disclosure is discussed further in the ensuing sections.

2. General Techniques

[0084] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et aL, 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.L Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).

3. AAV and rAAV Vectors

A. AAV

[0085] “Adeno-associated virus” and/or “AAV” refer to parvoviruses with a linear singlestranded DNA genome and variants thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise. Parvoviruses, including AAV, are useful as gene therapy vectors as they can penetrate a cell and introduce a nucleic acid (e.g., transgene) into the nucleus. In some embodiments, the introduced nucleic acid (e.g., rAAV vector genome) forms circular concatemers that persist as episomes in the nucleus of transduced cells. In some embodiments, a transgene is inserted in specific sites in the host cell genome, for example at a site on human chromosome 19. Site-specific integration, as opposed to random integration, is believed to likely result in a predictable long-term expression profile. The insertion site of AAV into the human genome is referred to as AAVS1 . Once introduced into a cell, polypeptides encoded by the nucleic acid can be expressed by the cell. Because AAV is not associated with any pathogenic disease in humans, a nucleic acid delivered by AAV can be used to express a therapeutic polypeptide for the treatment of a disease, disorder and/or condition in a human subject.

[0086] The canonical AAV wild-type genome comprises 4681 bases (Berns et al. (1987) Advances in Virus Research 32:243-307) and includes terminal repeat sequences (e.g., inverted terminal repeats ( ITRs)) at each end which function in cis as origins of DNA replication and as packaging signals for the virus. The genome includes two large open reading frames, known as AAV replication (“AAV rep” or “rep”) and capsid (“AAV cap” or “cap”) genes, respectively. AAV rep and cap may also be referred to herein as AAV “packaging genes.” These genes code for the viral proteins involved in replication and packaging of the viral genome.

[0087] Wild type AAV comprises a small (20-25 nm) icosahedral virus capsid composed of three proteins, VP1 , VP2 and VP3, with 60 capsid proteins comprising the capsid. The three capsid genes VP1 , VP2 and VP3 overlap each other within a single open reading frame and alternative splicing leads to production of VP1 , VP2 and VP3 (Grieger et al.

(2005) J. Virol. 79(15):9933-9944.). A single P40 promoter allows all three capsid proteins to be expressed at a ratio of about 1 :1 :10 for VP1 , VP2, VP3, respectively, which complements AAV capsid production. More specifically, VP1 is the full-length protein, with VP2 and VP3 being increasingly shortened due to increasing truncation of the N-terminus. A well-known example is the capsid of AAV9 as described in US Patent No. 7,906,111 , wherein VP1 comprises amino acid residues 1 to 736 of a sequence identified as number 123, VP2 comprises amino acid residues 138 to 736 of a sequence identified as number 123, and VP3 comprises amino acid residues 203 to 736 of a sequence identified as number 123. The AAV2 capsid protein sequences are available in Genbank: VP1 (735 aa; Genbank Accession No. AAC03780), VP2 (598 aa; Genbank Accession No. AAC03778) and VP3 (533 aa; Genbank Accession No. AAC03779). As used herein, the term “AAV Cap” or “cap” refers to AAV capsid proteins VP1 , VP2 and/or VP3, and variants and analogs thereof.

[0088] A second open reading frame of the capsid gene encodes an assembly factor, called assembly-activating protein (AAP), which is essential for the capsid assembly process (Sonntag et al. (2011) J. Virol. 85(23):12686-12697).

[0089] At least four viral proteins are synthesized from the AAV rep gene - Rep 78, Rep 68, Rep 52 and Rep 40 - named according to their apparent molecular weights. As used herein, “AAV rep” or “rep” means AAV replication proteins Rep 78, Rep 68, Rep 52 and/or Rep 40, as well as variants and analogs thereof. As used herein, rep and cap refer to both wild type and recombinant (e.g., modified chimeric, and the like) rep and cap genes as well as the polypeptides they encode. In some embodiments, a nucleic acid encoding a rep will comprise nucleotides from more than one AAV serotype. For instance, a nucleic acid encoding a rep protein may comprise nucleotides from an AAV2 serotype and nucleotides from an AAV3 serotype (Rabinowitz et al. (2002) J. Virology 76(2):791-801).

[0090] Multiple serotypes of AAV exist in nature with at least fifteen wild type serotypes having been identified from humans thus far (i.e., AAV1 -AAV15). Naturally occurring and variant serotypes are distinguished by having a protein capsid that is serologically distinct from other AAV serotypes. Naturally occurring and non-naturally occurring AAV serotypes include: AAV type 1 (AAV1 ), AAV type 2 (AAV2), AAV type 3 (AAV3), including AAV type 3A (AAV3A) and AAV type 3B (AAV3B), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), AAV type 10 (AAV10), AAV type 12 (AAV12), AAVrhIO, AAVrh74 (see WO 2016/210170), AAV1 .1 , AAV2.5, AAV6.1 , AAV6.3.1 , AAV9.45, RHM4-1 (SEQ ID N0:5 of WO 2015/013313), RHM15-1 , RHM15-2, RHM15-3/RHM15-5, RHM15-4, RHM15-6, AAV hu.26, AAV2i8, AAV29G, AAV2,8G9, AAV-LK03, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV, AAV type 2i8 (AAV2i8), NP4, NP22, NP66, AAVDJ, AAVDJ/8, AAVDJ/9, among many others (see, e.g., Fields et al., “Virology”, volume 2, chapter 69 (4^th ed., Lippincott-Raven Publishers); US Patent No. 7,906,111 ; Gao et al. (2004) J. Virol. 78:6381 ; Morris et al. (2004) Virol. 33:375; WO 2013/063379; WO 2014/194132; WO 2015/121501 ; WO 2015/013313, all of which are hereby incorporated by reference). AAV variants isolated from human CD34+ cell include AAVHSC1 , AAVHSC2, AAVHSC3, AAVHSC4, AAVHSC5, AAVHSC6, AAVHSC7, AAVHSC8, AAVHSC9, AAVHSC10, AAVHSC11 , AAVHSC12, AAVHSC13, AAVHSC14 and AAVHSC15 (Smith et al. (2014) Molecular Therapy 22(9):1625-1634, which is hereby incorporated by reference).

[0091] Serotype distinctiveness is determined on the basis of the lack of cross-reactivity between antibodies to one AAV as compared to another AAV. Such cross-reactivity differences are usually due to differences in capsid protein sequences and antigenic determinants (e.g., due to VP1 , VP2, and/or VP3 sequence differences of AAV serotypes). However, some naturally occurring AAV or man-made AAV mutants (e.g., recombinant AAV) may not exhibit serological difference with any of the currently known serotypes. These viruses may then be considered a subgroup of the corresponding type, or more simply a variant AAV. Thus, as used herein, the term “serotype” refers to both serologically distinct viruses, e.g., AAV, as well as viruses, e.g., AAV, that are not serologically distinct but that may be within a subgroup or a variant of a given serotype.

[0092] A comprehensive list and alignment of amino acid sequences of capsids of known AAV serotypes is provided by Marsic et al. (2014) Molecular Therapy 22(11 ):1900- 1909, especially at supplementary Figure 1 ; the entire publication is hereby incorporated by reference.

[0093] Genomic sequences of various serotypes of AAV, as well as sequences of the native inverted terminal repeats (ITRs), rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as GenBank. See, e.g., GenBank Accession Numbers NC_002077 (AAV1), AF063497 (AAV1), NC 001401 (AAV2), AF043303 (AAV2), NC_001729 (AAV3), AF028705.1 (AAV3B), NC 001829 (AAV4), U89790 (AAV4), NC_006152 (AAV5), AF028704 (AAV6), AF513851 (AAV7), AF513852 (AAV8), NC_006261 (AAV8), AY530579 (AAV9), AY631965 (AAV10), AY631966 (AAV11 ), and DQ813647 (AAV12); the disclosures of which are incorporated by reference herein. See also, e.g., Srivistava et al. (1983) J. Virology 45:555; Chiorini etal. (1998) J. Virology 71 :6823; Chiorini et al. (1999) J. Virology 73: 1309; Bantel-Schaal etal. (1999) J. Virology 73:939; Xiao et al. (1999) J. Virology 73:3994; Muramatsu et al. (1996) Virology 221 :208; Shade et al. (1986) J. Virol. 58:921 ; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99: 11854; Moris et al. (2004) Virology 33:375-383; International Patent Publications WO 00/28061 , WO 99/61601 , WO 98/11244; WO 2013/063379; WO 2014/194132; WO 2015/121501 , and US Patent No. 6,156,303 and US Patent No.

7,906,111 , all of which are hereby incorporated by reference.

[0094] In one embodiment of the method of purifying a rAAV vector disclosed herein, a rAAV vector comprises an AAV3B VP1 polypeptide (also referred to herein as a “capsid protein”) comprising the amino acid sequence of SEQ ID NO:10. AAV3B VP2 and VP3 encompass about amino acids 138 to 736 and about amino acids 203 to 736 of SEQ ID NO:10 (GenBank accession no. AAB95452.1), respectively. In one embodiment of the method of purifying a rAAV vector disclosed herein, a rAAV vector comprises an AAV3B VP1 polypeptide (also referred to herein as a “capsid protein”) encoded by the nucleic acid sequence of SEQ ID NOU 1 (nucleotides 2208-4418 of GenBank accession no. AF028705.1 ).

B. Recombinant AA V (rAA V)

[0095] A “recombinant adeno-associated virus,” or “rAAV” (also referred to herein as a “rAAV vector,” “rAAV viral particle,” and/or “rAAV vector particle”) refers to an AAV capsid comprising a vector genome, unless specifically noted otherwise. The vector genome comprises a polynucleotide sequence that is not, at least in part, derived from a naturally- occurring AAV (e.g., a heterologous polynucleotide not present in wild type AAV), and wherein the rep and/or cap genes of the wild type AAV genome have been removed from the vector genome. ITRs from an AAV have been added or remain in the vector genome. Therefore, the term rAAV vector encompasses a rAAV viral particle that comprises a capsid but does not comprise a complete AAV genome; instead the recombinant viral particle can comprise a heterologous, i.e., not originally present in the capsid, nucleic acid, the vector genome. Thus, a “rAAV vector genome” (or “vector genome”) refers to a heterologous polynucleotide sequence (including at least one ITR) that may, but need not, be contained within an AAV capsid. A rAAV vector genome may be double-stranded (dsAAV), singlestranded (ssAAV) or self-complementary (scAAV). Typically, a vector genome comprises a heterologous nucleic acid often encoding a therapeutic transgene, for example an ATP7B gene, or fragment thereof, as provided in SEQ ID NO:2. In some embodiments, a vector genome comprises a heterologous nucleic acid encoding a copper-transporting ATPase 2 protein, or fragment thereof, as provided in SEQ ID NO:1 .

[0096] A rAAV vector, and those terms provided above, are to be distinguished from an “AAV viral particle” or “AAV virus” that is not recombinant, contains a virus genome encoding rep and cap genes, and which AAV virus is capable of replicating when present in a cell also comprising a helper virus, such as an adenovirus and/or herpes simplex virus, and/or required helper genes therefrom. Thus, production of a rAAV vector necessarily includes production of a recombinant vector genome using recombinant DNA technologies, and wherein the recombinant vector genome is contained within an AAV capsid to form the rAAV vector.

[0097] The present disclosure provides for methods of purifying a rAAV vector by AEX. In some embodiments, the rAAV vector comprises an AAV3B capsid and optionally, a transgene encoding a polypeptide that is a target for therapeutic treatment (e.g., a nucleic acid encoding a copper-transporting ATPase 2, or a fragment thereof, for the treatment of Wilson disease, e.g., SEQ ID NO:2). Delivery or administration of a rAAV vector to a subject (e.g. a patient) provides encoded proteins and peptides to the subject. Thus, a rAAV vector can be used to transfer/deliver a heterologous polynucleotide for expression for the treatment of diseases, disorders and/or conditions. In some embodiments, a rAAV vector transfers a copy of an ATP7B, or fragment thereof (e.g., an ATP7B with deletion of the MBS1 -4 coding regions) to hepatocytes which is expressed as a shortened coppertransporting ATPase 2 for the treatment of Wilson disease.

[0098] A rAAV vector genome generally retains 130 to 145 base ITRs in cis to the heterologous nucleic acid sequence that replaces the viral rep and cap genes. Such ITRs are necessary to produce a recombinant AAV vector as they mediate AAV genome replication and packaging. However, modified AAV ITRs and non-AAV terminal repeats including partially or completely synthetic sequences can also serve this purpose. ITRs form hairpin structures and function to, for example, serve as primers for host-cell-mediated synthesis of the complementary DNA strand after infection. ITRs also play a role in viral packaging, integration, etc. ITRs are the only AAV viral elements which are required in cis for AAV genome replication and packaging into rAAV vectors. A rAAV vector genome optionally comprises two ITRs which are generally at the 5’ and 3’ ends of the vector genome comprising a heterologous sequence (e.g., a transgene encoding a gene of interest). A 5’ and a 3’ ITR may both comprise the same sequence, or each may comprise a different sequence (e.g., SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8). In some embodiments, a rAAV vector genome of the disclosure comprises an ITR comprising or consisting of the nucleic acid sequence of any one of SEQ ID NO:5-8. In some embodiments, a rAAV vector genome of the disclosure comprises an ITR comprising a nucleic acid sequence that is, or is at least, or is at most 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to any one of SEQ ID NO:5-8. An AAV ITR may be from any AAV, including but not limited to, serotypes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 or any other AAV. In some embodiments, an AAV ITR may be a AAV2 ITR or modification thereof.

[0099] A rAAV vector genome of the disclosure may comprise an ITR from an AAV serotype (e.g., wild-type AAV2, a fragment or variant thereof) that differs from the serotype of the capsid (e.g., AAV3B or other). Such a rAAV vector genome comprising at least one ITR from one serotype, but comprising a capsid from a different serotype, may be referred to as a hybrid viral vector (see U.S. Patent No. 7,172,893). An rAAV ITR may include the entire wild type ITR sequence, or be a variant, fragment, or modification thereof, but will retain functionality.

[0100] In addition to a transgene and at least one ITR, a vector genome may also include various regulatory or control elements. Typically, regulatory elements are nucleic acid sequence(s) that influence expression of an operably linked polynucleotide (e.g., a transgene). The precise nature of regulatory elements useful for gene expression will vary from organism to organism and from cell type to cell type including, for example, a promoter, enhancer, intron etc., with the intent to facilitate proper heterologous polynucleotide transcription and translation. Regulatory control can be affected at the level of transcription, translation, splicing, message stability, etc.

[0101] In some embodiments, a method of purifying a rAAV vector of the disclosure comprises an rAAV vector comprising a recombinant nucleic acid comprising at least one ITR, a transgene, a promoter and a polyadenylation signal (polyA) sequence. In some embodiments, a transgene encodes a copper-transporting ATPase 2, or a fragment thereof. In some embodiments, a transgene encodes a copper-transporting ATPase 2, or a fragment thereof comprising or consisting of the amino acid sequence of SEQ ID NOU . In some embodiments, a transgene encodes a copper-transporting ATPase 2, or a fragment thereof comprising an amino acid that is, or is at least, or is at most 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NOU .

[0102] In some embodiments, an ATP7B transgene is disclosed in WQ2016/097219 (nucleotides 473-3580 of SEQ ID NO:6, incorporated herein by reference). In some embodiments, a transgene comprises or consists of the nucleic acid of SEQ ID NO:2 which encodes a copper-transporting ATPase 2, or a fragment thereof. In some embodiments, a transgene is an ATP7B gene, or fragment thereof (e.g., SEQ ID NO:2). In some embodiments, a transgene comprises a nucleic acid sequence that is, or is at least, or is at most 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO:2.

[0103] In some embodiments, a promoter is a minimal AAT promoter. In some embodiments, a minimal AAT promoter is disclosed in WQ2016/097219 (nucleotides 156- 460 of SEQ ID NO:1 ; incorporated herein by reference). In some embodiments, a promoter comprises or consists of the nucleic acid sequence of SEQ ID NO:3. In some embodiments, a promoter comprises a nucleic acid sequence that is, or is at least, or is at most 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO:3.

[0104] In some embodiments, a polyA signal sequence is from a rabbit p-globin gene. In some embodiments, a polyA signal sequence is disclosed in WQ2016/097219 (nucleotides 4877-4932 of SEQ ID NO:1 ; incorporated herein by reference). In some embodiments, a polyA signal sequence comprises or consists of the nucleic acid sequence of SEQ ID NO:4. In some embodiments, a polyA signal sequence comprises a nucleic acid sequence that is, or is at least, or is at most 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO:4.

[0105] In some embodiments, an exemplary vector genome comprises a nucleic acid encoding a copper-transporting ATPase 2, a minimal AAT promoter, a polyA sequence and two ITR sequences. In some embodiments, a vector genome comprises: a nucleic acid encoding a copper-transporting ATPase 2 with a deletion of metal binding sites (MBS) 1 -4 and/or encoding a copper-transporting ATPase 2 comprising or consisting of the amino acid sequence of SEQ ID NO:1 , a minimal AAT promoter comprising the nucleic acid sequence of SEQ ID NO:3, a polyA comprising the nucleic acid sequence of SEQ ID NO:4 and two AAV2 ITR sequences. In some embodiments, the ITR sequences comprise the nucleic acid sequence of any one of SEQ ID NO:5-8.

[0106] A viral capsid of a rAAV vector may be, but not limited to, any of the wild type AAV and variant AAV, described above. In some embodiments, a viral capsid polypeptide is of an AAV serotype selected from the group consisting of AAV1 , AAV2, AAV3 (including AAV3A and AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhIO, AAVrh74, AAV12, AAV2i8, NP4, NP22, NP66, AAVDJ, AAVDJ/8, AAVDJ/9, AAVLK03, AAV1.1 , AAV2.5, AAV6.1 , AAV6.3.1 , AAV9.45, RHM4-1 , RHM15-1 , RHM15-2, RHM15-3/RHM15-5, RHM15-4, RHM15-6, AAV hu.26, AAV1 .1 , AAV2.5, AAV6.1 , AAV6.3.1 , AAV9,45, AAV2i8, AAV29G, AAV2,8G9, AVV-LK03, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N, AAVHSC1 , AAVHSC2, AAVHSC3, AAVHSC4, AAVHSC5, AAVHSC6, AAVHSC7, AAVHSC8, AAVHSC9, AAVHSC10, AAVHSC11 , AAVHSC12, AAVHSC13, AAVHSC14 and AAVHSC15.

[0107] In some embodiments, a viral capsid of a rAAV vector is an AAV3B capsid. In some embodiments, a viral capsid of a rAAV vector comprises a polypeptide encoded by at least a portion of the sequence of GenBank accession no. AF028705.1 (e.g., nucleotides 2208-4418). In some embodiments, a viral capsid of a rAAV vector comprises a polypeptide encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO:11 . In some embodiments, a viral capsid of a rAAV vector comprises a polypeptide encoded by a nucleic acid sequence that is, or is at least, or is at most 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO:10.

[0108] In some embodiments, a viral capsid of a rAAV vector comprising a polypeptide comprising or consisting of the amino acid sequence of GenBank accession no.

AAB95452.1 . In some embodiments, a viral capsid of a rAAV vector comprises a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:10. In some embodiments, a viral capsid of a rAAV vector is a polypeptide that is, or is at least, or is at most 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NQ:10.

[0109] In some embodiments, a rAAV vector comprises i) a vector genome comprising: a nucleic acid encoding a copper-transporting ATPase 2 with a deletion of metal binding sites (MBS) 1-4 and/or encoding a copper-transporting ATPase 2 comprising the amino acid sequence of SEQ ID NO:1 , a minimal AAT promoter comprising the nucleic acid sequence of SEQ ID NO:3, a polyA sequence comprising the nucleic acid sequence of SEQ ID NO:4 and two AAV2 ITR sequences, optionally comprising the nucleic acid sequence of any one of SEQ ID NO:5-8 and ii) a capsid comprising an AAV3B capsid (e.g., the amino acid sequence of SEQ ID NQ:10).

[0110] In some embodiments, the present disclosure provides for the use of ancestral AAV vectors for use in rAAV vectors for in vivo gene therapy. Specifically, in silico-derived sequences may be synthesized de novo and characterized for biological activities. Prediction and synthesis of ancestral sequences, in addition to assembly into a rAAV vector, may be accomplished using methods described in WO 2015/054653, the contents of which are incorporated by reference herein. Notably, rAAV vectors assembled from ancestral viral sequences may exhibit reduced susceptibility to pre-existing immunity in human populations as compared to contemporary viruses or portions thereof.

[0111] In some embodiments, a rAAV vector comprising a capsid protein encoded by a nucleotide sequence derived from more than one AAV serotype (e.g., wild type AAV serotypes, variant AAV serotypes) is referred to as a “chimeric vector” or “chimeric capsid” (See US Patent No. 6,491 ,907, the entire disclosure of which is incorporated herein by reference). In some embodiments, a chimeric capsid protein is encoded by a nucleic acid sequence derived from 2, 3, 4, 5, 6, 7, 8, 9, 10 or more AAV serotypes. In some embodiments, a recombinant AAV vector includes a capsid sequence derived from e.g., AAV1 , AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAVrh74, AAVrhl 0, AAV2i8, or variant thereof, resulting in a chimeric capsid protein comprising a combination of amino acids from any of the foregoing AAV serotypes (see, Rabinowitz et al. (2002) J. Virology 76(2):791-801). Alternatively, a chimeric capsid can comprise a mixture of a VP 1 from one serotype, a VP2 from a different serotype, a VP3 from yet a different serotype, and a combination thereof. For example, a chimeric virus capsid may include an AAV1 cap protein or subunit and at least one AAV2 cap protein or subunit. A chimeric capsid can, for example include an AAV capsid with one or more B19 cap subunits, e.g., an AAV cap protein or subunit can be replaced by a B19 cap protein or subunit. For example, in one embodiment, a VP3 subunit of an AAV capsid can be replaced by a VP2 subunit of B19.

[0112] Chimeric capsids my comprise capsids with substitutions of the VP1 variable regions (e.g., VR I - VR IX) and p-sheet regions (e.g., A though I). The amino acid sequence between p-sheet G and p-sheet H (also referred to herein as the “GH loop”), encompasses variable region IV through variable region VIII and contains the highest level of diversity among AAV serotypes as well as among all Parvoviruses. The GH loop is at the 3-fold axis of symmetry, constitutes about 30% of the capsid and interacts with primary glycan attachment receptor. In some embodiments, a chimeric AAV capsid polypeptide comprises an amino acid sequence of a parental AAV VP1 polypeptide, comprising a substitution of amino acids from a region between p-sheet G and p-sheet H with amino acids from a region between p-sheet G and p-sheet H of an alternative AAV VP1 polypeptide. In some embodiments, the substitution includes amino acids from the p-sheet G and/or p-sheet H of either the parental AAV VP1 polypeptide or the alternative AAV VP1 polypeptide. Such chimeric capsids may be referred to as an “AAV capsid with a GH loop substitution” or a “GH loop substitution capsid.”

[0113] In some embodiments, a chimeric AAV capsid polypeptide comprises an amino acid sequence of a parental AAV VP1 polypeptide, comprising a substitution of amino acids from a region between p-sheet G and p-sheet I with amino acids from a region between p- sheet G and p-sheet I of an alternative AAV VP1 polypeptide. In some embodiments, the substitution includes amino acids from the p-sheet G and/or p-sheet I. Such chimeric capsids may be referred to an “AAV capsid with a Gl loop substitution” or a “Gl loop substitution capsid.”

[0114] In some embodiments, chimeric vectors have been engineered to exhibit altered tropism or tropism for a particular tissue or cell type. The term “tropism” refers to preferential entry of the virus into certain cell or tissue types and/or preferential interaction with the cell surface that facilitates entry into certain cell or tissue types. AAV tropism is generally determined by the specific interaction between distinct viral capsid proteins and their cognate cellular receptors (Lykken et al. (2018) J. Neurodev. Disord. 10:16). Preferably, once a virus or viral vector has entered a cell, sequences (e.g., heterologous sequences such as a transgene) carried by the vector genome (e.g., a rAAV vector genome) are expressed.

[0115] A “tropism profile” refers to a pattern of transduction of one or more target cells, tissues and/or organs. For example, an AAV capsid may have a tropism profile characterized by efficient transduction of muscle cells with only low transduction of, for example, liver cells.

C. Exemplary Recombinant AA V

[0116] In exemplary embodiments, the present disclosure provides a method of purifying a rAAV vector for treatment of Wilson Disease (WD) and to restore normal biliary secretion of copper, fecal excretion of copper or both, and to normalize loading of copper into ceruolplasmin. The rAAV vector comprises a AAV3B capsid and a vector genome with AAV2 ITRs flanking an AAT promoter, a ATP7B transgene, with deletion of MBS1 -4, and a polyA signal sequence (see, e.g., WO2016/097219, and WO2016/097218, each of which are incorporated herein by reference).

[0117] In some embodiments, the rAAV vector comprises i) a vector genome comprising: a nucleic acid encoding a copper-transporting ATPase 2 with a deletion of metal binding sites (MBS) 1-4, encoding a copper-transporting ATPase 2 comprising the amino acid sequence of SEQ ID NO:1 , or both, a minimal AAT promoter comprising the nucleic acid sequence of SEQ ID NO:3, a polyA comprising the nucleic acid sequence of SEQ ID NO:4 and two AAV2 ITR sequences, optionally comprising the nucleic acid sequence of any one of SEQ ID NO:5-8 and ii) a capsid comprising an AAV3B capsid. In some embodiments, the AAV3B capsid polypeptide comprises the amino acid sequence set forth in SEQ ID NQ:10 and at GenBank accession no. AAB95452.1 and/or encoded by the nucleotide sequence set forth in SEQ ID NO:11 and at nucleotides 2208-4418 of GenBank accession no. AF028705.1.

4. Assembly of Viral Vectors

[0118] A viral vector (e.g., rAAV vector) carrying a transgene (e.g., encoding a coppertransporting ATPase 2 with a deletion of MBS 1 -4) is assembled from a polynucleotide encoding a transgene, suitable regulatory elements and elements necessary for production of viral proteins which mediate cell transduction. Examples of a viral vector include but are not limited to adenoviral, retroviral, lentiviral, herpesvirus and adeno-associated virus (AAV) vectors, and in particular rAAV vector (as discussed, supra).

[0119] In an exemplary non-limiting embodiment, a vector genome includes a portion of a parvovirus genome, such as an AAV genome with rep and cap deleted and/or replaced by a modified nucleic acid (e.g., transgene) and its associated expression control sequences. A modified nucleic acid encoding copper-transporting ATPase 2, or a fragment thereof, is typically inserted adjacent to one or two (i.e., is flanked by) AAV ITRs or ITR elements adequate for viral replication (Xiao et al. (1997) J. Virol. 71 (2): 941 -948), in place of the nucleic acid encoding viral rep and cap proteins. Other regulatory sequences suitable for use in facilitating tissue-specific expression of an ATP7B transgene in the target cell (e.g., liver cell) may also be included.

A. Packaging cell

[0120] One skilled in the art would appreciate that a rAAV vector comprising a transgene, and lacking virus proteins needed for viral replication (e.g., cap and rep), cannot replicate since such proteins are necessary for virus replication and packaging. Cap and rep genes may be supplied to a cell (e.g., a host cell, e.g., a packaging cell) as part of a plasmid that is separate from a plasmid supplying the vector genome with the transgene.

[0121] “Packaging cell” or “producer cell” means a cell or cell line which may be transfected with a vector, plasmid or DNA construct, and provides in trans all the missing functions which are required for the complete replication and packaging of a viral vector. The required genes for rAAV vector assembly include the vector genome (e.g., an ATP7B transgene, regulatory elements, and ITRs), AAV rep gene, AAV cap gene, and certain helper genes from other viruses such as, e.g., adenovirus. One of ordinary skill would understand that the requisite genes for AAV production can be introduced into a packaging cell in various ways including, for example, transfection of one or more plasmids. However, in some embodiments, some genes (e.g., rep, cap, helper) may already be present in a packaging cell, either integrated into the genome or carried on an episome. In some embodiments, a packaging cell expresses, in a constitutive or inducible manner, one or more missing viral functions.

[0122] Any suitable packaging cell known in the art may be employed in the production of a packaged viral vector. Mammalian cells or insect cells are preferred. Examples of cells useful for the production of a packaging cell in the practice of the disclosure include, for example, human cell lines, such as PER.C6, WI38, MRC5, A549, HEK293 (which express functional adenoviral E1 under the control of a constitutive promoter), B-50 or any other HeLa cell, HepG2, Saos-2, HuH7, and HT1080 cell lines. Suitable non-human mammalian cell lines include, for example, VERO, COS-1 , COS-7, MDCK, BHK21-F, HKCC or CHO cells.

[0123] In some embodiments, a packaging cell is capable of growing in suspension culture. In some embodiments, a packaging cell is capable of growing in serum-free media. For example, HEK293 cells are grow in suspension in serum free medium. In another embodiment, a packaging cell is a HEK293 cell as described in U.S. Patent No. 9,441 ,206 and deposited as American Type Culture Collection (ATCC) No. PTA 13274. Numerous rAAV packaging cell lines are known in the art, including, but not limited to, those disclosed in WO 2002/46359.

[0124] A packaging cell generally includes one or more viral vector functions along with helper functions and packaging functions sufficient to result in replication and packaging of the viral vector. These various functions may be supplied together, or separately, to the packaging cell using a genetic construct such as a plasmid or an amplicon, and they may exist extrachromosomally within the cell line, or integrated into the host cell’s chromosomes. In some embodiments, a packaging cell is transfected with at least i) a plasmid comprising a vector genome comprising a transgene and AAV ITRs and further comprising at least one of the following regulatory elements: an enhancer, a promoter, an exon, an intron and a poly A and ii) a plasmid comprising a rep gene (e.g., AAV2 rep) and a cap gene (e.g., AAV3B or other cap).

[0125] In some embodiments, a host cell is supplied with one or more of the packaging or helper functions incorporated within, e.g., a host cell line with one or more vector functions incorporated extrachromosomally or integrated into the cell’s chromosomal DNA.

B Helper function

[0126] AAV is a dependovirus in that it cannot replicate in a cell without co-infection of the cell by a helper virus. Helper functions include helper virus elements needed for establishing active infection of a packaging cell, which is required to initiate packaging of the viral vector. Helper viruses include, typically, adenovirus or herpes simplex virus. Adenovirus helper functions typically include adenovirus components adenovirus early region 1 A (E1a), E1 b, E2a, E4, and viral associated (VA) RNA. Helper functions (e.g., E1 a, E1b, E2a, E4, and VA RNA) can be provided to a packaging cell by transfecting the cell with one or more nucleic acids encoding various helper elements. Alternatively, a host cell (e.g., a packaging cell) can comprise a nucleic acid encoding the helper protein. For instance, HEK293 cells were generated by transforming human cells with adenovirus 5 DNA and now express a number of adenoviral genes, including, but not limited to E1 and E3 (see, e.g., Graham et al. (1977) J. Gen. Virol. 36:59-72). Thus, those helper functions can be provided by the HEK 293 packaging cell without the need of supplying them to the cell by, e.g., a plasmid encoding them. In some embodiments, a packaging cell is transfected with at least i) a plasmid comprising a vector genome comprising a transgene and AAV ITRs and further comprising at least one of the following regulatory elements: an enhancer, a promoter, an exon, an intron and a poly A and ii) a plasmid comprising a rep gene (e.g., AAV2 rep) and a cap gene (e.g., AAVB3B or other cap) and iii) a plasmid comprising a helper function.

[0127] Any method of introducing a nucleotide sequence carrying a helper function into a cellular host for replication and packaging may be employed, including but not limited to, electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, a carrier molecule (e.g., polyethylenimine (PEI)) and liposomes in combination with a nuclear localization signal. In some embodiments, helper functions are provided by transfection using a virus vector, or by infection using a helper virus, standard methods for producing viral infection may be used.

[0128] The vector genome may be any suitable recombinant nucleic acid, such as a DNA or RNA construct and may be single stranded, double stranded, or duplexed (i.e., self- complementary as described in WO 2001/92551).

5. Production of Packaged Viral Vector

[0129] Viral vectors can be made by several methods known to skilled artisans (see, e.g., WO 2013/063379). An exemplary non-limiting method is described in Grieger, et al. (2015) Molecular Therapy 24(2):287-297, the contents of which are incorporated by reference herein for all purposes. Briefly, efficient transfection of HEK293 cells is used as a starting point, wherein an adherent HEK293 cell line from a qualified clinical master cell bank is used to grow in animal component-free suspension conditions in flasks and bioreactors that allow for rapid and scalable rAAV production. Using a triple transfection method (e.g., WO 96/40240), a HEK293 cell line suspension can generate greater than 1 x10⁵ vector genome containing particles (VG)/cell, or greater than 1 x10¹⁴ VG/L of cell culture, when harvested 48 hours post-transfection. More specifically, triple transfection refers a method whereby a packaging cell is transfected with three plasmids: one plasmid encodes the AAV rep and cap (e.g., AAV9 cap) genes, another plasmid encodes various helper functions (e.g., adenovirus or HSV proteins such as E1 a, E1 b, E2a, E4, and VA RNA, and another plasmid encodes a transgene (e.g., ATP7B, or a fragment thereof) and various elements to control expression of the transgene.

[0130] To achieve the desired vector yields, a number of variables are optimized such as selection of a compatible serum-free suspension media that supports both growth and transfection, selection of a transfection reagent, transfection conditions and cell density. [0131] A rAAV vector may be purified by methods standard in the art such as by column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors are known in the art and include methods described in Clark et al. (1999) Human Gene Therapy 10(6):1031 -1039; Schenpp and Clark (2002) Methods Mol. Med. 69:427-443; U.S. Patent No. 6,566,118 and WO 98/09657.

[0132] After rAAV vectors of the present disclosure have been purified according to methods disclosed herein, they can be titered (e.g., the amount of rAAV vector in a sample can be quantified) to prepare compositions for administration to subjects, such as human subjects with Wilson Disease. rAAV vector titering can be accomplished using methods know in the art.

[0133] In some embodiments, the number of viral particles, including particles containing a vector genome and “empty” capsids that do not contain a vector genome, can be determined by electron microscopy, e.g., transmission electron microscopy (TEM). Such a TEM-based method can provide the number of vector particles (or virus particles in the case of wild type AAV) in a sample. In some embodiments, the amount of particles, containing a vector genome (full capsids), and “empty” capsids that do not contain a vector genome, can be determined by charge detection mass spectrometry, analytical ultracentrifugation (AUC), and/or measurement of absorbance at 260 nm and 280 nm to determine A260/A280 ratio.

[0134] In some embodiments, rAAV vector genomes can be titered using quantitative PCR (qPCR) using primers against any sequence in the vector genome, for example ITR sequences, and/or sequences in the transgene (or regulatory elements). By performing qPCR in parallel on dilutions of a standard of known concentration, such as a plasmid containing the sequence of the vector genome, a standard curve can be generated permitting the concentration of the rAAV vector to be calculated as the number of vector genomes (VG) per unit volume such as microliters or milliliters. By comparing the number of vector particles as measured by, e.g., SEC or ELISA, to the number of vector genomes in a sample, the percentage of empty capsids can be estimated. Because the vector genome contains the therapeutic transgene, vg/kg or vg/ml of a vector sample may be more indicative of the therapeutic amount of the vector that a subject will receive than the number of vector particles, some of which may be empty and not contain a vector genome. Once the concentration of rAAV vector genomes in the stock solution is determined, it can be diluted into or dialyzed against suitable buffers for use in preparing a composition (e.g., a drug substance) for administration to subjects (e.g., subjects with Wilson Disease).

6. rAAV Vector Purification by Anion-exchange Chromatography (AEX)

[0135] A novel, universal purification strategy, based on ion exchange chromatography methods, may be used to generate high purity rAAV vector preparations of various AAV serotypes or from chimeric capsids (e.g., AAV1 , AAV2, AAV3, including AAV3A and AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV12, AAVrhIO, AAVrh74, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV, and recombinantly produced variants (e.g., capsid variants with insertions, deletions and substitutions, etc.), such as variants referred to as AAV2i8, NP4, NP22, NP66, AAVDJ, AAVDJ/8, AAVDJ/9, AAVLK03, RHM4-1 , AAVHSC1 , AAVHSC2, AAVHSC3, AAVHSC4, AAVHSC5, AAVHSC6, AAVHSC7, AAVHSC8, AAVHSC9, AAVHSC10, AAVHSC11 , AAVHSC12, AAVHSC13, AAVHSC14 and AAVHSC15). In some embodiments, this process can be completed in less than a week, result in high full to empty capsid ratios (up to 70% full capsids), provide step yields up to 70% and purity suitable for clinical use. In some embodiments, such a method is universal with respect to AAV serotype and/or chimerism of the capsid. Scalable manufacturing technology, as described herein, may be used to manufacture GMP clinical and commercial grade rAAV vectors to treat disease (e.g., Wilson Disease etc.).

[0136] Production of recombinant AAV vector (rAAV) for gene therapy requires purification of the rAAV vector from a host cell (e.g., host cell debris including but not limited to host cell DNA, RNA, proteins, lipids, membrane and organelles) that produced the vector, as well as removal of capsids that do not contain a complete vector genome (e.g., intermediate and/or empty capsids) and thus, do not comprise a therapeutic transgene. [0137] Such purification methods generally comprise multiple steps including, for example, lysis of the host cell, precipitation of cellular protein and DNA, separation of the rAAV vector from host cell protein and nucleic acids, and separation of the rAAV vector from empty and intermediate capsids by column purification, low speed centrifugation, ultracentrifugation, normal flow filtration, ultrafiltration/diafiltration or any combination of these methods. Column purification may include, for example, at least one of ion exchange chromatography (e.g., anion, cation), affinity chromatography, size exclusion chromatography, multimodal chromatography, and hydrophobic interaction chromatography. Centrifugation methods may include, for example, ultracentrifugation or low speed centrifugation (e.g., for removal of solids and clarification). Filtration methods may include, for example, at least one of diafiltration, depth filtration, nominal filtration, and absolute filtration.

[0138] AEX employs a positively charged stationary phase (e.g., a resin) to separate substances (e.g., AAV capsids, DNA, protein, high molar mass species, amino acids) based on charge differences of said substances, and is useful for separating rAAV capsids from impurities based on charge differences at moderately acidic to alkaline pH (e.g., greater than pH 6). AEX can also separate empty capsids from rAAV vectors containing a complete vector genome (i.e., full capsid) by relying on the charge differences of empty capsids as compared to full capsids.

[0139] Without wishing to be bound by theory, the tightness of binding between an AAV capsid and an AEX chromatography stationary phase is related to the strength of the negative charge of the capsid, including the charge contribution from any nucleic acid within the capsid, solution pH and solution conductivity (Qu, G. et aL, J. VirologicaL Methods (2007) 140:183-192). In some embodiments, an AEX chromatography stationary phase is a resin comprising polystyrenedivinylbenzene particles modified with covalently bound quaternized polyethyleneimine, and optionally OH groups (e.g., POROS™ 50 HQ resin). Polystyrenedivinylbenzene particles may comprise pores of 500-10,000 Angstroms (A). [0140] In some embodiments, an AEX chromatography stationary phase is a resin comprising agarose particles with a cationic ligand (e.g. Capto Q ImpRes, Q Sepharose High Performance). In some embodiments, an AEX chromatography stationary phase is a resin selected from the group consisting of Capto Q, Capto Q XP, Q Sepharose XL, STREAMLINE Q XL, Capto HiRes Q, RESOURCE Q, SOURCE 15 Q, SOURCE 30 Q, Q Sepharose HP, Q Sepharose FF, Q Sepharose™ BB, POROS™ 20 HQ, POROS™ XQ, TOYOPEARL QAE-550C, TOYOPEARL Q-600C AR, TOYOPEARL GigaCap Q-650S, TOYOPEARL GigaCap Q-650M, TOYOPEARL SuperQ-650S, TOYOPEARL SuperQ-650M, TOYOPEARL SuperQ-650C, TSKgel SuperQ-5PW (20), TSKgel SuperQ-5PW (30), Q Ceramic HyperD F, ESHMUNO® Q, Fractogel® EMD TMAE (S), Fractogel® EMD TMAE (M), Fractogel® EMD TMAE Hicap (M), Fractogel® EMD TMAE (S), Fractogel® EMD TMAE (M), Fractogel® EMD TMAE Hicap (M), Nuvia Q, Nuvia HP-Q, UNOsphere Q, Macro-Prep High Q, Macro-Prep 25 Q, BioRad AG® 1-X2, WorkBeads™ 40Q, WorkBeads™ 100Q, Cellufine MAX Q-r, Cellufine MAX Q-h, Praesto™ Q65, Praesto™ Q90, Praesto™ Jetted Q35, BAKERBOND™ POLYQUAT, BAKERBOND™ POLYPEI, YMC - BioPro Q30, YMC - BioPro Q75, YMC - BioPro SmartSep Q10, YMC - BioPro SmartSep Q30, DEAE Sepharose FF, ANX Sepharose 4 FF (high sub), POROS™ 50 PI, POROS™ 50 D, TOYOPEARL NH₂- 750F, TOYOPEARL GigaCap DEAE-650M, TOYOPEARL DEAE-650S, TOYOPEARL DEAE-650M, TOYOPEARL DEAE-650C, TSKgel DEAE-5PW (20), TSKgel DEAE-5PW (30), Ceramic HyperD DEAE, Hypercel Star AX, Fractogel® EMD DEAE (M), Fractogel® EMD DMAE (M) Resin, Macro-Prep DEAE, WorkBeads™ 40 DEAE, Cellufine MAX DEAE, DEAE PuraBead HF and WorkBeads™ 40 TREN.

[0141] In some embodiments, an AEX chromatography stationary phase is a monolith comprising porous poly-methacrylate with a cationic ligand (e.g. CIMmultus™ QA). In some embodiments, an AEX chromatography stationary phase is a membrane adsorber comprising polyethersulfone with a cationic ligand (e.g. Mustang Q, Mustang E, Sartobind® Q, Sartobind STIC® PA).

[0142] In some embodiments, a rAAV vector can be purified by AEX from a solution exiting from an affinity chromatography stationary phase (e.g., “eluting from the stationary phase”) comprised of a mobile phase and material such as rAAV vector or capsid that passed through the stationary phase or was displaced from the stationary phase. This solution may be referred to as an affinity eluate or an “affinity pool.”

[0143] In some embodiments, a rAAV vector can be purified by AEX from a “supernatant from a cell lysate” (also known as a “clarified lysate”), which, as used herein, refers to a solution collected following sedimentation of lysed host cells from a host cell culture.

[0144] In some embodiments, a rAAV vector can be purified by AEX from a “postharvest solution”, which, as used herein, refers to solution resulting from a cell lysis that has undergone flocculation, depth filtration and/or nominal filtration.

[0145] In some embodiments, a rAAV vector can be purified from a solution having undergone at least one other purification or processing step (e.g., cell lysis, flocculation, filtration, dilution, pH adjustment, chromatography).

[0146] In some embodiments, a rAAV vector can be purified by AEX from an affinity eluate, optionally having undergone at least one other purification or processing step (e.g., cell lysis, flocculation, filtration, dilution, pH adjustment, conductivity adjustment, chromatography). In some embodiments, a rAAV vector can be purified by AEX from a cell lysate, optionally having undergone at least one other purification or processing step (e.g., cell lysis, flocculation, filtration, dilution, pH adjustment, conductivity adjustment, chromatography). In some embodiments, a rAAV vector can be purified by AEX from a postharvest solution, optionally having undergone at least one other purification or processing step (e.g., cell lysis, flocculation, filtration, dilution, pH adjustment, conductivity adjustment, chromatography).

[0147] As a solution comprising a substance to be purified (e.g., a rAAV vector) and impurities, flows through an AEX stationary phase, a substance that binds (e.g., negatively charged proteins such as an AAV capsid or rAAV vector) to a positively charged AEX stationary phase are retained within the stationary phase. Unbound substances pass through the column and are collected in a flow-through, and/or during a subsequent wash step.

[0148] In some embodiments, a solution comprising a rAAV vector to be purified (e.g., an affinity elute) is subjected to at least one additional processing step including neutralization, addition of a divalent salt (e.g., MgCh), dilution and adjustment of the conductivity and pH prior to loading on a column comprising an AEX media. The present disclosure provides for the use of weak binding load conditions (also referred to as a weak binding load) wherein the solution used to dilute the solution comprising the rAAV vectors (e.g., an affinity eluate), and the subsequently diluted solution, have a high conductivity (e.g., from 3 mS/cm to 9 mS/cm) and a high pH (e.g., from 7.0 to 9.2) relative to the solution comprising a rAAV vector prior to dilution. The weak binding load conditions disclosed herein advantageously decrease or limit empty capsid binding while maintaining a high level of full capsid binding (e.g., greater than 80%, greater than 90%, greater than 95%, greater than 98% and higher) allowing a greater percentage of empty capsids to be collected in the flow through rather than the eluate

[0149] The partitioning of bound and unbound substances between the stationary and mobile phases can be modulated by both pH and conductivity of solutions applied to the column after a solution comprising a rAAV vector to be purified is loaded onto the stationary phase.

[0150] This disclosure provides for the use of a wash (also referred to as an empty capsid wash or “ECW”) that has a high pH (e.g., 9.3 or greater) and a high conductivity (e.g., 13 to 19 mS/cm). The empty capsid wash step follows the load step (though not necessarily directly after the load) and advantageously elutes bound empty capsids with minimal elution of bound full capsids so that the AEX eluate comprises fewer empty capsids relative to full capsids. The disclosure also provides for the use of a two wash steps, one before and one after the empty capsid wash, using a MgCh buffer which increases full capsid binding and reduces vg loss prior to the elution step. In some embodiments, these wash steps are referred to together as a “sandwich” wash as they are performed before and after the empty capsid wash.

[0151] Bound substances may be eluted from the stationary phase by adjusting a salt concentration and/or pH within the column. For example, and without wishing to be bound by any particular theory of operation, a salt concentration of an elution buffer is gradually increased such that anions in the salt (e.g., acetate (C2H3O2 ), CI SO4²) compete with and displace (i.e., elute) a substance bound to the resin. In another embodiment, the pH of the solution within the column can be gradually decreased to decrease the negative charge of a bound substance and cause it to be released (i.e., eluted) from the stationary phase. Upon release from the stationary phase, a substance may be collected as a column eluate.

[0152] Without wishing to be bound by theory, separation of substances, such as a mixture of AAV capsids , or more specifically a mixture of a rAAV vector (i.e., a full capsid), an AAV capsid (e.g., an empty capsid, an intermediate capsid) and host cell proteins, will depend on the total charge difference of the substances. The charge composition of ionizable side groups will determine the total charge of a protein at a particular pH. At the isoelectric point (pl), the total charge on a protein is 0 and it will not bind to a matrix. If the pH is above the pl, a protein will have a negative charge and bind to an anion exchange column stationary phase.

[0153] An AEX protocol for separation of full rAAV vectors from empty capsids includes multiple steps, for example, at least one of pre-use flushing of a column media to displace storage solution, pre-use sanitizing of a column stationary phase, post-use sanitizing of a column stationary phase, equilibrating a column stationary phase, loading a solution (e.g., a diluted affinity eluate) comprising a rAAV vector onto a column stationary phase, washing a column stationary phase (e.g., using an empty capsid wash), eluting a substance to be purified from a stationary phase (e.g., by gradient elution, by step elution), applying a gradient hold to a column stationary phase, regenerating a column stationary phase, and applying a storage solution to a column stationary phase. One of skill in the art will understand that an AEX protocol for purification of rAAV vectors may comprise all, or only some of these steps. One of skill in the art will also understand that the order of these steps may vary, and that certain steps may be performed more than once, and not necessarily in sequence.

A. AEX Column Preparation

[0154] AEX methods of the disclosure may be performed at various scales utilizing columns ranging in volume from 1 .0 mL to 20 L. In some embodiments, an AEX method includes use of a column with a column volume (CV) of about 1 .0 mL, about 5.1 mL, about 49 mL, about 52 mL, about 6.67 mL, about 1 .256 L, about 1 .3 L, about 6.0 L, about 6.1 L, about 6.2 L, about 6.3 L, about 6.4 L, about 6.5 L, about 6.6 L, about 6.7 L, about 6.8 L, about 6.9 L, or about 7.0 L. In some embodiments, an AEX method of the disclosure includes use of a column with a CV of 1 .0 mL to 20 L, e.g., 1 .0 ml to 10 mL, 30 mL to 70 mL, 44 mL to 54 mL, 10 mL to 100 mL, 100 mL to 1000 mL, 1 L to 1.5 L, 1.5 L to 2.0 L, 1.8 L to 2.2 L, 2.0 L to 5 L, 5 L to 7.5 L, 7.5 L to 10 L, 10 L to 15 L or 15 L to 20 L. In some embodiments, an AEX method of the disclosure includes use of a column with a CV of 1 .0 mL to 10 L, 10 mL to 10 L, 100 mL to 20 L, 100 mL to 10 L, 1 L to 20 L, 1 L to 10 L, 1 L to 5 L, 1 L to 2 L or 1 I to 1 .5 L. In some embodiments, an AEX method of the disclosure includes use of a column with a CV of 6.0 L to 6.6 L (e.g., 6.4 L). In some embodiments, an AEX method of the disclosure includes use of a column with a CV of 1 .8 L to 2.2 L (e.g., 2.0 L) [0155] A volume of solution applied to a column to, for example, to equilibrate a stationary phase therein, is generally expressed in terms of a “column volume” (CV), with one CV equivalent to the volume of the column.

[0156] In some embodiments, an AEX chromatography stationary phase (also referred to herein as “resin” or “media”) of the disclosure is a polystyrenedivinylbenzene particle with covalently bound quaternized polyethyleneimine (e.g., POROS™ 50 HQ resin).

[0157] Generally, prior to application (i.e., loading) of a solution to be purified (e.g., an affinity chromatography eluate, also referred to herein as an “affinity eluate” or an “affinity pool”) to a column comprising a chromatography stationary phase, at least one solution is applied to the stationary phase to, for example, flush, sanitize, regenerate and/or equilibrate the stationary phase. In some embodiments, an “affinity eluate” or an “affinity pool” has been diluted, and optionally filtered prior to loading of the solution onto the AEX column. In some embodiments, a diluted affinity eluate is filtered in-line with the AEX column.

[0158] As disclosed herein, a method of preparing an AEX stationary phase for use in a method of purifying a rAAV (e.g., rAAV3B or others) vector from a solution (e.g., an affinity eluate) by AEX comprises pre-use flushing of the AEX stationary phase in a column. In some embodiments, pre-use flushing of the AEX stationary phase is intended to displace a storage solution (e.g., a solution comprising ethanol) from the stationary phase. In some embodiments, pre-use flushing of a column precedes loading a solution comprising a rAAV vector to be purified onto the column. In some embodiments, pre-use flushing comprises application of water (e.g., water for injection) to AEX stationary phase in a column. In some embodiments, pre-use flushing comprises an upward flow of water (“up flow”). During upward flow of pre-use flushing, the flow direction is opposite that of chromatographic separation steps (e.g., loading, washing or eluting), such that the solution (e.g., water) flows from the bottom of the column to the top of the column, whereas during a chromatographic separation step (e.g., loading) the solution flows from the top of the column to the bottom of the column (“down flow”). In some embodiments, pre-use flushing comprises application of 1 to 10 column volumes (CV) (e.g., about 5 CV) of water to AEX stationary phase in a column, at a linear velocity of 10 cm/hr to 1000 cm/hr. In some embodiments, pre-use flushing comprises application of >4.5 CV (e.g., about 5 CV) of water for injection to AEX stationary phase in a column, at a linear velocity of 350 cm/hr to 430 cm/hr (e.g., about 390 cm/hr), a residence time (i.e., a contact time) of 1 .5 min/CV to 2.5 min/CV (e.g., about 2 min/CV) or both.

[0159] A method of preparing an AEX stationary phase for use in a method of purifying a rAAV (e.g., rAAV3B or others) vector from a solution (e.g., an affinity eluate) by AEX comprises sanitizing the AEX stationary phase in a column. Sanitizing an AEX stationary phase serves to reduce the bioburden (including, but not limited to bacteria) and/or inactivate microbes and viruses within the column, and more generally to remove contaminants such as proteins, particulates, etc. In some embodiments, sanitizing precedes loading a solution comprising a rAAV vector to be purified onto a column. In some embodiments, sanitizing comprises application of a solution comprising NaOH, ethanol, acetic acid, phosphoric acid, guanidine HCI, urea, PAB (phosphoric acid, acetic acid, benzyl alcohol), peracetic acid etc. to an AEX stationary phase in a column. In some embodiments, sanitizing comprises application of a solution comprising 0.1 M to 1.0 M, about 0.1 M to about 0.8 M, about 0.1 M to about 0.6 M, about 0.2 M to about 0.8 M, about 0.2 M to about 0.6 M or about 0. 4 M to about 0.6 M (e.g., about 0.5 M) NaOH to AEX stationary phase in a column. In some embodiments, sanitizing comprises application of a solution comprising about 0.5 M NaOH to AEX stationary phase in a column using an upward flow (i.e., that is the flow direction is opposite that of chromatographic separation steps, e.g., loading, washing or eluting). In some embodiments, sanitizing comprises application of 5 CV to 10 CV (e.g., about 8 CV) of a solution comprising about 0.5 M NaOH to AEX stationary phase in a column. In some embodiments, sanitizing comprises application of 5 CV to 10 CV (e.g. about 8 CV ) of a solution comprising about 0.5 M NaOH to AEX stationary phase in a column at a linear velocity of 350 cm/hr to 430 cm/hr (e.g., about 390 cm/hr), a residence time (i.e., the amount of time per column volume that the solution is in contact with the stationary phase within the column, and also referred to herein as the contact time) of 1 .5 min/CV to 2.5 min/CV (e.g., about 2 min/CV) or both.

[0160] A method of preparing an AEX stationary phase for use in a method of purifying a rAAV (e.g., rAAV3B or others) vector from a solution (e.g., an affinity eluate) by AEX comprises regenerating (also referred to herein as “a rinse”) an AEX stationary phase in a column. One of skill in the art will understand that regenerating an ion exchange stationary phase serves to replace ions taken up in the exchange process with the original ions that occupied the exchange sites. In some embodiments, regeneration can also refer to bringing back a stationary phase to its original state by, for example, the removal of impurities using a strong solvent. In some embodiments, regenerating precedes loading a solution comprising a rAAV vector to be purified onto a stationary phase. In some embodiments, regenerating may be performed on a stationary phase more than once.

[0161] In some embodiments, regenerating comprises application of a solution comprising a salt and/or a buffering agent, with a pH ranging from 8 to 10, to an AEX stationary phase in a column. In some embodiments, a salt is selected from the group consisting of sodium chloride (NaCI), sodium acetate (NaAcetate,CH₃COONa), ammonium acetate (NH₄Acetate), magnesium chloride (MgCh) or sodium sulfate (NasSO^. In some embodiments, a concentration of a salt in a solution (e.g., NaCI) ranges from 1 M to 5 M (e.g., about 1 M to about 4.5 M, about 1 to about 4M, about 1 M to about 3.5 M, about 1 M to about 3 M, about 1 M to about 2.5 M or about 1 .5 M to about 2.5 M. In some embodiments, a concentration of a salt in a solution (e.g., NaCI) is about 1 M, about 2 M, about 3 M, about 4 M or about 5 M. In some embodiments, regenerating comprises application of a solution comprising 1 M to 3 M (e.g., 2 M) NaCI to the stationary phase in the column.

[0162] In some embodiments, a buffering agent is selected from the group consisting of Tris (e.g., a mixture of Tris Base and Tris-HCI), BIS-Tris propane, and/or bicine. In some embodiments, the concentration of the buffering agent (e.g., Tris) in a solution ranges from 10 mM to 500 mM (e.g., about 10 mM to about 150 mM, about 10 mM to about 200 mM, about 10 mM to about 250 mM, about 10 mM to about 300 mM, about 10 mM to about 350 mM, about 10 mM to about 400 mM, about 10 mM to about 450 mM, or about 50 mM to about 150 mM. In some embodiments, the concentration of the buffering agent (e.g., Tris) in a solution is about 10 mM, about 20 mM about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 300 mM, about 400 mM or about 500 mM. In some embodiments, regenerating comprises application of a solution comprising 50 mM to 150 mM (e.g., 100 mM) Tris to a stationary phase in a column.

[0163] In some embodiments, regenerating a stationary phase in a column comprises application of a solution with a pH of about 7 to 11 (e.g., about 7.5 to 10.5, about 8 to 10, or about 7, 7.5, 8, 8,5, 9, 9.5, 10, 10.5 or 11 ) to a stationary phase in a column. In some embodiments, regenerating a stationary phase in a column comprises application of a solution with a pH of about 9 to a stationary phase in a column.

[0164] In some embodiments, regenerating comprises application of a solution comprising about 1 M to 3 M (e.g., about 2 M) NaCI, 50 mM to 150 mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., about 9) to AEX stationary phase in a column. In some embodiments, regenerating comprises application of 4.5 to 5.5 CV (e.g., about 5 CV) of a solution comprising about 2 M NaCI, 100 mM Tris, pH 9 to AEX stationary phase in a column. In some embodiments, regenerating comprises application of 1 to 10 CV of a solution comprising about 2 M NaCI, 100 mM Tris, pH 9 to AEX stationary phase in a column at a linear velocity of 100 to 1000 cm/hr. In some embodiments, regenerating comprises application of 4.5 to 5.5 (e.g., about 5) CV of a solution comprising about 2 M NaCI, 100 mM Tris, pH 9 to AEX stationary phase in a column at a linear velocity of 350 to 430 cm/hr (e.g., about 390 cm/hr,), a residence time (i.e., a contact time) of 1 .5 to 2.5 min/CV (e.g., about 2 min/CV) or both.

[0165] In some embodiments, the present disclosure provides a method of preparing an AEX stationary phase for use in a method of purifying a rAAV (e.g., rAAV3B or others) vector by AEX, the method comprising at least one step of: i) pre-use flushing comprising application of >4.5 CV (e.g., about 5 CV) of water for injection to AEX stationary phase in a column; ii) sanitizing comprising application of about 5 CV to 10 CV (e.g., about 8 CV) of a solution comprising 0.1 M to 1 .0 M (e.g., about 0.5 M NaOH) to the AEX stationary phase in the column, optionally by upward flow; and iii) regenerating comprising application of 4.5 to 5.5 CV (e.g., about 5 CV) of a solution comprising 1 M to 3 M (e.g., about 2 M) NaCI, 50 mM to 150 mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., about 9) to the AEX stationary phase in the column; wherein at least one of steps i) - iii) is performed at a linear velocity of 350 cm/hr to 430 cm/hr (e.g., about 390 cm/hr), a residence time of 1 .5 min/CV to 2.5 min/CV (e.g., about 2 min/CV) or both; optionally wherein at least one step is performed prior to loading a solution comprising the rAAV vector to be purified onto the column; and optionally wherein the AEX stationary phase is POROS™ 50 HQ. One of ordinary skill will understand that the above steps may be performed in any order and may be performed more than once.

B. Equilibration

[0166] A method of preparing an AEX stationary phase for use in a method of purifying a rAAV (e.g., rAAV3B or others) vector from a solution (e.g., an affinity eluate) by AEX comprises equilibration of the AEX stationary phase in a column. In some embodiments, equilibration of an AEX stationary phase in a column serves to adjust the pH, conductivity, modifier (e.g., salt, detergent, amino acid etc.) concentration, or other condition, of the mobile and stationary phase such that some substances loaded onto the column will bind to the stationary phase, and others will flow through with the mobile phase. For example, conditions within the column may be adjusted by the application of a series of equilibration buffers to the column such that full rAAV vectors bind to the stationary phase, and at least a portion of the empty capsids do not bind. In some embodiments, AEX stationary phase in a column is equilibrated prior to application of a solution comprising a substance to be purified (e.g., a rAAV vector) to the column. In some embodiments, AEX stationary phase in a column is equilibrated by application of an equilibration buffer (e.g., a first equilibration buffer, a second equilibration buffer, a third equilibration buffer, a fourth equilibration buffer, etc.). An equilibration buffer may also be referred to herein as a “wash buffer,” a “post- sanitization rinse,” a “rinse,” or a “regeneration buffer.” Reference to an equilibration buffer as a first, second, third, fourth, etc. equilibration buffer does not necessarily imply the order in which the buffers are applied to a column.

[0167] In some embodiments, an equilibration buffer (e.g., a first equilibration buffer, a second equilibration buffer, a third equilibration buffer, a fourth equilibration buffer, etc.) comprises at least one component selected from the group consisting of at least one of a buffering agent, a salt, an amino acid and a detergent. In some embodiments, a buffering agent is Tris (e.g., a mixture of Tris Base and Tris-HCI), BIS-Tris propane, diethanolamine, diethylamine, tricine, triethanolamine and/or bicine. One of ordinary skill in the art would understand that a Tris buffer with a desired pH can be prepared using Tris Base, Tris-HCI or both. In some embodiments, a salt is sodium chloride (NaCI), sodium acetate (NaAcetate (CH₃COONa)), ammonium acetate (NH₄Acetate), magnesium chloride (MgCh), sodium citrate (NaCitrate) or sodium sulfate (NasSO^. In some embodiments, an amino acid is histidine, arginine, glycine or citrulline. In some embodiments, a detergent is poloxamer 188 (P188), Triton X-100, Polysorbate 80, Brij-35 or nonyl phenoxypolyethoxylethanol (NP-40). [0168] In some embodiments, an equilibration buffer comprises 10 mM to 350 mM of a buffering agent selected from the group consisting of Tris (e.g., a mixture of Tris Base and Tris-HCI), BIS-Tris propane, diethanolamine, diethylamine, tricine, triethanolamine and bicine.

[0169] In some embodiments, an equilibration buffer comprises 10 mM to 50 mM, 10 mM to 100 mM Tris, 10 mM to 150 mM Tris, 10 mM to 200 mM Tris, 10 mM to 250 mM Tris, 10 mM to 300 mM Tris or 10 mM to 350 mM Tris. In some embodiments, an equilibration buffer comprises 50 mM to 100 mM Tris, 50 mM to 150 mM Tris, 50 mM to 200 mM Tris, 50 mM to 250 mM Tris, 100 mM to 200 mM Tris, 100 mM to 250 mM Tris, 100 mM to 300 mM Tris or 100 mM to 150 mM Tris.

[0170] In some embodiments, an equilibration buffer comprises about 10 mM Tris, about 20 mM Tris, about 30 mM Tris, about 40 mM Tris, about 50 mM Tris, about 60 mM Tris, about 70 mM Tris, about 80 mM Tris, about 90 mM Tris, about 100 mM Tris, about 110 mM Tris, about 120 mM Tris, about 130 mM Tris, about 140 mM Tris, about 150 mM Tris, about 160 mM Tris, about 170 mM Tris, about 180 mM Tris, about 190 mM Tris, about 200 mM Tris, about 220 mM Tris, about 240 mM Tris or about 250 mM Tris. In some embodiments, an equilibration buffer comprises about 100 mM Tris or about 190 mM Tris.

[0171] In some embodiments, an equilibration buffer comprises 1 mM to 1 M salt. In some embodiments, an equilibration buffer comprises about 10 mM to about 550 mM, about 10 mM to about 600 mM, about 10 mM to about 650 mM, about 10M to about 700 mM, 10 mM to about 750 mM, about 10 mM to about 800 mM, about 10 mM to about 850 mM, about 10 mM to about 900 mM, about 10 mM to about 950 mM, about 50 mM to about 550 mM, about 50 mM to about 600 mM, about 50 mM to about 650 mM, about 50 mM to about 700 mM, about 50 mM to about 750 mM, about 100 mM to about 600 mM, about 200 mM to about 600 mM, about 300 mM to about 600 mM or about 400 mM to about 600 mM salt. In some embodiments, an equilibration buffer comprises about 500 mM salt. In some embodiments, an equilibration buffer comprises a salt selected from the group consisting of sodium chloride (NaCI), sodium acetate (NaAcetate,CH₃COONa), ammonium acetate (NH₄Acetate), magnesium chloride (MgCh), sodium citrate or sodium sulfate (NasSC ). In some embodiments, an equilibrium buffer comprises about 500 mM sodium acetate.

[0172] In some embodiments, an equilibration buffer comprises 1 mM to 50 mM salt (e.g., sodium chloride (NaCI), sodium acetate (NaAcetate,CH₃COONa), ammonium acetate (NH₄Acetate), magnesium chloride (MgCh), sodium citrate or sodium sulfate (NasSC ). In some embodiments, an equilibration buffer comprises about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM or 50 mM salt. In some embodiments, an equilibration buffer comprises a salt selected from the group consisting of sodium chloride (NaCI), sodium acetate (NaAcetate,CH₃COONa), sodium citrate, ammonium acetate (NH₄Acetate), magnesium chloride (MgCh) or sodium sulfate (NasSC ). In some embodiments, an equilibration buffer comprises about 18 mM NaCI. In some embodiments, an equilibration buffer comprises about 9 mM sodium citrate. In some embodiments, an equilibration buffer comprises about 11 mM MgCh. In some embodiments, an equilibration buffer comprises about 2 mM MgCh.

[0173] In some embodiments, an equilibration buffer comprises an amino acid, e.g., histidine, arginine, glycine or citrulline. In some embodiments, an equilibration buffer comprises 100 mM to 300 mM of an amino acid (e.g., histidine arginine, glycine or citrulline). In some embodiments, an equilibration buffer comprises about 10 mM to about 300 mM, about 10 mM to about 350 mM, about 10 mM to about 400 mM, about 10 mM to about 450 mM about 10 mM to about 500 mM, about 10 mM to about 550 mM, about 10 mM to about 600 mM, about 50 mM to about 300 mM, about 50 mM to about 350 mM, about 50 mM to about 400 mM, about 50 mM to about 450 mM, about 50 mM to about 500 mM, about 50 mM to about 550 mM, about 50 mM to about 600 mM, about 100 mM to about 300 mM, about 100 mM to about 400 mM, about 100 mM to about 500 mM, about 100 mM to about 600 mM salt, or about 150 mM to about 250 mM of an amino acid (e.g., histidine). In some embodiments, an equilibration buffer comprises about 190 mM histidine.

[0174] In some embodiments, an equilibration buffer comprises a detergent, e.g., P188, Triton X-100, Polysorbate 80, Brij-35 or NP-40. In some embodiments, an equilibration buffer comprises 0.005% to 1.0% of a detergent (e.g., P188). In some embodiments, an equilibration buffer comprises about 0.005% to about 1 .0%, about 0.005% to about 0.05%, about 0.005% to about 0.1%, about 0.005% to about 0.5%, about 0.007% to about 0.07%, 0.008% to about 0.05% or about 0.008% to about 0.03% of P188. In some embodiments, an equilibration buffer comprises about 0.01 % to about 0.75%, about 0.01 % to about 1 .0%. about 0.01 % to about 1 .5%, about 0.05% to about 1 .5%, about 0.05% to about 1 .0%, about 0.05% to about 0.75%, about 0.1% to about 1 .5%, about 0.1 % to about 1 .0%, about 0.1% to about 0.75%, or about 0.25% to about 0.75% P188.

[0175] In some embodiments, an equilibration buffer comprises about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03% about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, 0.95% or about 1 .0% of a detergent (e.g., P188). In some embodiments, an equilibration buffer comprises about 0.01% P188. In some embodiments, an equilibration buffer comprises about 0.5% P188.

[0176] In some embodiments, an equilibration buffer has a pH of 8 to 10. In some embodiments, an equilibration buffer has a pH of 8.7 to 9.3. In some embodiments, an equilibration buffer has a pH of about 8.0, about 8.1 , about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1 , about 9.2, about 9.3, about 9.4, about 9.5 or about 10.0. In some embodiments, an equilibration buffer has a pH of about 8.8. In some embodiments, an equilibration buffer has a pH of about 8.9. In some embodiments, an equilibration buffer has a pH of about 9.0.

[0177] In some embodiments, an equilibration buffer comprises 50 mM to 150 mM Tris, 400 mM to 600 mM sodium acetate, 0.005% to 0.015% P188 and has a pH of 8.5 to 9.5. In some embodiments, an equilibration buffer comprises about 100 mM Tris, about 500 mM sodium acetate, about 0.01% P188 and has a pH of about 8.9.

[0178] In some embodiments, an equilibration buffer comprises 100 mM to 300 mM histidine, 100 mM to 300 mM Tris, 10 mM to 30 mM NaCI, 1 mM to 20 mM sodium citrate, 1 mM to 20 mM MgCh, 0.1% to 1.0% P188, pH 8.5 to 9.5. In some embodiments, an equilibration buffer comprises about 190 mM histidine, about 190 mM Tris, about 18 mM NaCI, about 9 mM sodium citrate, about 11 mM MgCh, about 0.5% P188 and has a pH of about 8.8.

[0179] In some embodiments, an equilibration buffer comprises 50 mM to 150 mM Tris, 0.5 mM to 5 mM MgCh, 0.005% to 0.015% P188, and has a pH of 8.5 to 9.5. In some embodiments, an equilibrium buffer comprises about 100 mM Tris, about 2 mM MgCh, about 0.01% P188 and has a pH of 8.9. [0180] In some embodiments, an equilibration buffer comprises 50 mM to 150 mM Tris and has a pH of 8.5 to 9.5. In some embodiments, an equilibration buffer comprises 100 mM Tris and has a pH of 9.

[0181] In some embodiments, an equilibration buffer (e.g., a first equilibration buffer) comprises 100 mM Tris, 500 mM sodium acetate, 0.01% P188, pH 8.9. In some embodiments, an equilibration buffer (e.g., a second equilibration buffer) comprises 190 mM histidine, 190 mM Tris, 18 mM NaCI, 9 mM sodium citrate, 11 mM MgCh, 0.5% P188, pH 8.8. In some embodiments, an equilibration buffer (e.g., a third equilibration buffer) comprises 100 mM Tris, 2 mM MgCh, 0.01% P188, pH 8.9. In some embodiments, an equilibration buffer (e.g., a fourth equilibration buffer) comprises 100 mM Tris, pH 9.

[0182] In some embodiments, an equilibration buffer described above may be a first, second, third and fourth equilibration buffer. In some embodiments, a first, second, third or fourth equilibration buffer is applied to a column stationary phase in sequential order. In some embodiments, a solution (e.g., an affinity eluate) is applied to the column after application of two equilibration buffers. For example, a first and second equilibration buffer may be applied to a column, followed by application of an affinity eluate, which is followed by application of a third and a fourth equilibration buffer.

[0183] In some embodiments, an amount of equilibration buffer applied to a column is 1 CV to 5 CV, 4 CV to 6 CV 4 CV to 10 CV or 1 CV to 10 CV. In some embodiment, an amount of equilibration buffer applied to a column is > 4.5 CV. In some embodiments, an amount of an equilibration buffer applied to a column is 4.5 CV to 5.5 CV. In some embodiments, an amount of equilibration buffer applied to a column is about 2 CV, about 5 CV or about 10 CV. In some embodiments, an amount of equilibration buffer applied to a column is about 5 CV.

[0184] A solution, including but not limited to an equilibration buffer, applied to a column is set to flow through the stationary phase at a particular rate (e.g., cm/hr, mL/min) so that the solution within the column is in contact with the stationary phase, for a particular period of time (referred to herein as “residence time” or “contact time”). In some embodiments, a residence time of a solution in a column 1 .5 to 2.5 min/CV (e.g., 2.0 min/CV).

[0185] One of ordinary skill will understand that linear velocity (also referred to herein as “linear flow velocity” or “velocity”) of a solution through a column is related, at least in part, to a volume and or dimension of the column and the stationary phase therein. In some embodiments, a linear velocity of a solution, including but not limited to an equilibration buffer, through a stationary phase in a column is 100 cm/hr to 1800 cm/hr, e.g., 100 cm/hr to 200 cm/hr, 200 cm/hr to 400 cm/hr, 350 cm/hr to 430 cm/hr, 400 cm/hr to 600 cm/hr, 600 cm/hr to 800 cm/hr, 800 cm/hr to 1000 cm/hr, 1000 cm/hr to 1500 cm/hr, or 1500 cm/hr to 1800 cm/hr. In some embodiments, a linear velocity of a solution through a stationary phase in a column is about 100 cm/hr, about 240 cm/hr, about 298 cm/hr, about 300 cm/hr, about 390 cm/hr, about 600 cm/hr, about 611 cm/hr or about 1790 cm/hr. In some embodiments, a linear velocity of a solution, including but not limited to an equilibration buffer, through a stationary phase in a column is 350 cm/hr to 430 cm/hr (e.g., about 390 cm/hr).

[0186] A method of preparing an AEX stationary phase for use in a method of purifying a rAAV (e.g., rAAV3B or others) vector from a solution (e.g., an affinity eluate) by AEX comprises equilibrating the AEX stationary phase in a column. In some embodiments, equilibrating precedes loading a solution comprising a rAAV vector to be purified onto a column. In some embodiments, equilibrating follows loading a solution comprising a rAAV vector to be purified onto a column.

[0187] In some embodiments, equilibrating comprises application of 4.5 CV to 5.5 CV (e.g., about 5 CV) of an equilibration buffer comprising about 100 mM Tris, about 500 mM sodium acetate, about 0.01% P188 and has a pH of about 8.9 to a column comprising AEX stationary phase at a linear velocity of 350 cm/hr to 430 cm/hr (e.g., about 390 cm/hr), a residence time of 1 .5 min/CV to 2.5 min/CV (e.g., about 2 min/CV) or both.

[0188] In some embodiments, equilibration comprises application of > 4.5 CV (e.g., about 5 CV) of an equilibration buffer comprising about 190 mM histidine, about 190 mM Tris, about 18 mM NaCI, about 9 mM sodium citrate, about 11 mM MgCls, about 0.5% P188 and has a pH of about 8.8 to a column comprising an AEX stationary phase at a linear velocity of 350 cm/hr to 430 cm/hr (e.g., about 390 cm/hr), a residence time of 1 .5 min/CV to 2.5 min/CV (e.g., about 2 min/CV) or both.

[0189] In some embodiments, equilibrating comprises application of 4.5 CV to 5.5 CV (e.g., about 5 CV) of an equilibration buffer comprising about 100 mM Tris, about 2 mM MgCh, about 0.01% P188 and has a pH of 8.9 to a column comprising an AEX stationary phase at a linear velocity of 350 cm/hr to 430 cm/hr (e.g., about 390 cm/hr), a residence time of 1 .5 min/CV to 2.5 min/CV (e.g., about 2 min/CV) or both.

[0190] In some embodiments, equilibrating comprises application of 4.5 CV to 5.5 CV (e.g., about 5 CV) of an equilibration buffer comprising about 100 mM Tris and has a pH of 9.0 to a column comprising an AEX stationary phase at a linear velocity of 350 cm/hr to 430 cm/hr (e.g., about 390 cm/hr), a residence time of 1 .5 min/CV to 2.5 min/CV (e.g., about 2 min/CV) or both.

[0191] In some embodiments, the present disclosure provides a method preparing an AEX stationary phase for use in a method of purifying a rAAV (e.g., rAAV3B or others) vector by AEX, the method comprising at least one step of: i) pre-use flushing comprising application of >4.5 CV (e.g., about 5 CV) of water for injection to the AEX stationary phase in a column; ii) sanitizing comprising application of about 5 CV to 10 CV (e.g., about 8 CV) of a solution comprising 0.1 M to 1 .0 M (e.g., about 0.5 M) NaOH to the AEX stationary phase in the column; iii) regenerating comprising application of 4.5 CV to 5.5 CV (e.g., about 5 CV) of a solution comprising 1 M to 3 M (e.g., about 2 M) NaCI, 50 mM to 150 mM (e.g., about 100 mM Tris), pH 8.5 to 9.5 (e.g., about 9) to the AEX stationary phase in the column; iv) equilibration comprising application of 4.5 CV to 5.5 CV (e.g., about 5 CV) of a solution comprising 50 mM to 150 mM (e.g., about 100 mM) Tris, 400 mM to 600 mM (e.g., about 500 mM) sodium acetate, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., about 8.9) to the AEX stationary phase in the column; v) equilibration comprising application of >4.5 CV (e.g., about 5 CV) of an equilibration buffer comprising 100 mM to 300 mM (e.g., about 190 mM) histidine, 100 mM to 300 mM (e.g., about 190 mM) Tris, 10 mM to 30 mM (e.g., 18 mM) NaCI, 1 mM to 20 mM (e.g., about 9 mM) sodium citrate, 1 mM to 20 mM (e.g., about 11 mM) MgCh, 0.1% to 1.0% (e.g., about 0.5%) P188, pH 8.5 to 9.5 (e.g., 8.8) to the AEX stationary phase in the column; vi) equilibration comprising application of 4.5 CV to 5.5 CV (e.g., about 5 CV) of an equilibration buffer comprising 100 mM to 300 mM (e.g., about 200 mM) Tris, 0.5 mM to 5 mM (e.g., about 2 mM) MgCI₂, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., 8.9) to the AEX stationary phase in the column; and vii) equilibration comprising application of 4.5 CV to 5.5 CV (e.g., about 5 CV) of an equilibration buffer comprising 50 mM to 150 mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., 9.0) to the AEX stationary phase in the column; optionally wherein at least one of steps i) - vii) is performed at a linear velocity of 350 cm/hr to 430 cm/hr (e.g., about 390 cm/hr), a residence time of 1 .5 min/CV to 2.5 min/CV (e.g., about 2 min/CV) or both; optionally wherein the AEX stationary phase is POROS™ 50 HQ; optionally wherein the rAAV vector is a rAAV3B vector, and optionally wherein a step (e.g., a load step, an empty capsid wash step) may occur between any step of equilibration. One of ordinary skill will understand that the order of the above steps may be varied.

C. Dilution and Filtration

[0192] A method of purifying a rAAV (e.g., rAAV3B or others) vector from a solution (e.g., an affinity eluate) by AEX comprises preparation of the solution by diluting, and optionally filtering, the solution. A solution comprising a rAAV vector to be purified may be an affinity eluate, a supernatant from a cell lysate and/or a post-harvest solution having undergone at least one purification or processing step. A solution comprising a rAAV vector to be purified may be diluted, and optionally filtered prior to loading onto an AEX column in order to make the solution compatible with processing through the AEX column. In some embodiments, diluting, and optionally filtering, a solution comprising a rAAV vector to be purified results in a change in pH, conductivity or both of the solution. The disclosure also provides for the use of weak binding load conditions (also referred to as a weak binding load) wherein the solution used to dilute the solution comprising the rAAV vectors (e.g., an affinity eluate), and the subsequently diluted solution, have a high conductivity (e.g., from 3 mS/cm to 9 mS/cm, e.g., 6.4 mS/cm) and a high pH (e.g., from 7 to 9.2, e.g., 8.8) The weak binding load conditions disclosed herein advantageously decrease or limit empty capsid binding while maintaining a high level of full capsid binding (e.g., greater than 80%, greater than 90%, greater than 95% and higher).

[0193] In some embodiments, a solution comprising a rAAV vector to be purified is an eluate resulting from affinity chromatography purification of a rAAV vector produced in a 1 L to 2000 L (or greater) single use bioreactor (SUB).

[0194] In some embodiments, an affinity eluate is generated from affinity purification of a rAAV vector produced in a vessel (e.g., a single use bioreactor (SUB)) with a volume of 1 mL to 2000 L, or greater than 2000 L. In some embodiments, an affinity eluate is generated from affinity chromatography purification of a rAAV vector produced in a vessel (e.g., a SUB) with a volume of about 1 mL, about 10 mL, about 50 mL, about 100 mL, about 250 mL, about 500 mL, about 750 mL, about 1 L, about 50 L, about 100 L, about 250 L, about 500 L, about 1000 L, about 2000 L or greater. In some embodiments, an affinity eluate is generated from affinity purification of a rAAV vector produced in a vessel (e.g., a SUB) with a volume of 1 mL to 100 mL, 100 mL to 500 mL, 500 mL to 750 mL, 750 mL, to 1 L, 1 L to 10 L, 10 L to 50 L, 50 L to 100 L, 100 L to 250 L, 250 L to 500 L, 500 L to 750 L, 750 L to 1000 L, 1000 L to 1500 L, 1500 L to 2000 L, 2000 L to 3500 L, 3500 L to 4000 L or 4500 L to 5000 L. In some embodiments, an affinity eluate is generated from affinity purification of a rAAV vector produced in a vessel (e.g., a SUB) with a volume of 1 mL to 5000 L, 100 mL to 1000 L, 100 mL to 2000 L, 100 mL to 3000 L, 100 mL to 4000 L, 100 mL to 5000 L, 1 L to 1000 L, 1 L to 2000 L, 1 L to 3000 L, 1 L to 4000 L, 1 L to 5000 L, 500 mL to 1000 L, 500 mL to 2000 L, 500 mL to 3000 L, 500 mL to 4000 L or 500 mL to 5000 L.

[0195] Prior to dilution of an affinity eluate for processing by AEX, in some embodiments, the eluate is neutralized to raise the pH to a range of 7.4 to 7.8 (e.g., 7.6). In some embodiments, an affinity eluate is neutralized to raise the pH of the eluate to a range of 7.4 to 7.8 (e.g., 7.6) by titration with 5% (v/v) of 1 M Tris base. Prior to dilution of an affinity eluate for processing by AEX, in some embodiments, the eluate is neutralized to lower the pH to a range of 7.4 to 7.8 (e.g., 7.6). In some embodiments, an affinity eluate is neutralized to lower the pH of the eluate to a range of 7.4 to 7.8 (e.g., 7.6) by titration with 1% (v/v) of 2 M glycine, pH 2.7.

[0196] Prior to dilution of an affinity eluate for processing by AEX, in some embodiments, the eluate is spiked with 1 M MgCh. Prior to dilution of an affinity eluate for processing by AEX, in some embodiments, the eluate is spiked with 1 M MgCh to achieve a final concentration of 23 mM to 27 mM (e.g., about 25 mM) MgCh. In some embodiments, MgCh stabilizes rAAV vectors in a solution. In some embodiments, an affinity eluate that has been neutralized (e.g., to a pH of 7.4 to 7.8) and spiked with MgCh (e.g., to a concentration of 23 mM to 27 mM MgCh) is stable at 2°C to 8°C for 5 days or fewer.

[0197] A method of preparing a solution comprising a rAAV vector for purification by AEX comprises i) diluting an affinity eluate, and optionally ii) filtering the affinity eluate from step i) to produce the diluted affinity eluate (also referred to herein as a “diluted affinity pool,” “load,” or “AEX load”). In some embodiments, pH of an affinity eluate after dilution, and optional filtration is increased as compared to pH of the affinity eluate before the dilution. In some embodiments, pH of an affinity eluate after dilution is 8.6 to 9.0 (e.g., 8.8). In some embodiments, a pH of an affinity eluate is adjusted to 8.6 to 9.0 (e.g., 8.8) by dilution.

[0198] In some embodiments, conductivity of an affinity eluate after dilution, and optional filtration is increased as compared to conductivity of the affinity eluate before the dilution. In some embodiments, conductivity of an affinity eluate after dilution is 6.0 mS/cm to 6.8 mS/cm (e.g., 6.4 mS/cm). In some embodiments, conductivity of an affinity eluate is adjusted to 6.0 mS/cm to 6.8 mS/cm (e.g., 6.4 mS/cm) by dilution. In some embodiments, the diluted, and optionally filtered affinity eluate is loaded on an AEX stationary phase.

[0199] In some embodiments, diluting a solution comprising a rAAV vector to be purified (e.g., an affinity eluate) comprises diluting the solution about 2 to 25-fold or about 2 to 10- fold, or about 4 to 7-fold (e.g., about 2-fold, about 3-fold, about 4-fold, about 5-fold, about, 6- fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11 -fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 20-fold, about 25-fold) to produce a diluted affinity eluate. In some embodiments, diluting a solution comprising a rAAV vector to be purified (e.g., an affinity eluate) comprises diluting the solution about 5-fold.

[0200] In some embodiments, diluting a solution comprising a rAAV vector to be purified (e.g., an affinity eluate) comprises diluting the solution until a target pH, target conductivity or both of the solution is reached. In some embodiments, diluting a solution comprising a rAAV vector to be purified (e.g., an affinity eluate) comprises diluting the solution until a target pH of 8.6 to 9.0, a target conductivity of 6.0 to 6.8 mS/cm, or both of the solution is reached. In some embodiments, diluting a solution comprising a rAAV vector to be purified (e.g., an affinity eluate) comprises diluting the solution until a target pH of 8.8, a target conductivity of 6.4 mS/cm, or both of the solution is reached.

[0201] In some embodiments, diluting a solution comprising a rAAV vector to be purified (e.g., an affinity eluate) comprises starting with an initial dilution of 4.5-fold and increasing the dilution until a target pH, a target conductivity, or both is reached. In some embodiments, diluting a solution comprising a rAAV vector to be purified (e.g., an affinity eluate) comprises starting with an initial dilution of 4.5-fold and increasing the dilution until a target pH of 8.6 to 9.0, a target conductivity of 6.0 to 6.8 mS/cm, or both is reached. In some embodiments, diluting a solution comprising a rAAV vector to be purified (e.g., an affinity eluate) comprises starting with an initial dilution of 4.5-fold and increasing the dilution until a target pH of 8.8, a target conductivity of 6.4 mS/cm, or both is reached.

[0202] In some embodiments, diluting a solution comprising a rAAV vector to be purified (e.g., an affinity eluate) is performed “in-line” with the column, and wherein a dilution solution (diluent) is delivered through a first tubing to a Y-connector, and the solution comprising a rAAV vector to be purified is delivered through a second tubing to the Y-connector, and optionally wherein a static mixer is contained within a third tubing located after the Y- connector.

[0203] In some embodiments, diluting a solution comprising a rAAV vector to be purified (e.g., an affinity eluate) is performed “in-line” and directed into a holding vessel (e.g., a break tank). For example, a dilution solution (diluent) is delivered through a first tubing to a Y- connector, and a solution comprising a rAAV vector to be purified is delivered through a second tubing to the Y-connector, wherein the end of the Y-connector is connected to a holding vessel which is optionally, connected to a chromatography column (e.g., an AEX column).

[0204] In some embodiments, diluting comprises delivery of a dilution solution through a first tubing to a Y-connector at a flow rate of 1 to 5 mL/min (e.g., about 3.5 mL/min) and delivery of a solution comprising a rAAV vector to be purified (e.g., an affinity eluate) through a second tubing at a flow rate of 0.1 to 2 mL/min (e.g., about 0.25 mL/min).

[0205] In some embodiments, diluting comprises delivery of a dilution solution through a first tubing to a Y-connector at a flow rate of about 3.5 mL/min and delivery of an affinity eluate through a second tubing at a flow rate of about 0.25 mL/min, such that the affinity eluate is diluted about 15-fold.

[0206] In some embodiments, diluting comprises dilution of a solution comprising a rAAV vector to be purified (e.g., an affinity eluate) with a dilution solution comprising a buffering agent (Tris, BIS-Tris propane, diethanolamine, diethylamine, tricine, triethanolamine and/or bicine). In some embodiments, a solution comprising a rAAV vector to be purified (e.g., an affinity eluate) is diluted with a dilution solution comprising 10 mM to 500 mM buffering agent (e.g., Tris). In some embodiments, a dilution solution comprises about 10 mM to about 250 mM, about 10 mM to about 300 mM, about 10 mM to about 350 mM, about 10 mM to about 400 mM, about 10 mM to about 450 mM, about 50 mM to about 250 mM, about 50 mM to about 300 mM, about 50 mM to about 350 mM, about 50 mM to about 400 mM, about 50 mM to about 450 mM, about 100 mM to about 300 mM, about 100 mM to about 350 mM, about 100 mM to about 400 mM, about 100 mM to about 450 mM, or about 150 mM to about 250 mM Tris. In some embodiments, a dilution solution comprises about 190 mM Tris.

[0207] In some embodiments, dilution solution comprises an amino acid, e.g., histidine, arginine, glycine or citrulline. In some embodiments, an dilution solution comprises 10 mM to 500 mM of an amino acid (e.g., histidine arginine, glycine or citrulline). In some embodiments, a dilution solution comprises about 10 mM to about 250 mM, about 10 mM to about 300 mM, about 10 mM to about 350 mM, about 10 mM to about 400 mM, about 10 mM to about 450 mM, about 50 mM to about 250 mM, about 50 mM to about 300 mM, about 50 mM to about 350 mM, about 50 mM to about 400 mM, about 50 mM to about 450 mM, about 100 mM to about 300 mM, about 100 mM to about 350 mM, about 100 mM to about 400 mM, about 100 mM to about 450 mM, or about 150 mM to about 250 mM histidine. In some embodiments, a dilution solution comprises about 190 mM histidine.

[0208] In some embodiments, a dilution solution comprises a detergent, e.g., P188, Triton X-100, Polysorbate 80, Brij-35 or NP-40. In some embodiments, dilution solution comprises 0.005% to 1.5% detergent (e.g., P188). In some embodiments, a dilution solution comprises 0.1% to 1.0% detergent (e.g., P188). In some embodiments, a dilution solution comprises about 0.01% to about 0.75%, about 0.01 % to about 1 .0%. about 0.01% to about 1 .5%, about 0.05% to about 0.75%, about 0.05% to about 1 .0%, about 0.05% to about 1 .5%, about 0.1% to about 0.75%, about 0.1% to about 1 .0%, about 0.1% to about 1 .5%, or about 0.25% to about 0.75% detergent (e.g., P188). In some embodiments, a dilution solution comprises about 0.5% P188.

[0209] In some embodiments, an dilution solution comprises 1 mM to 50 mM salt. In some embodiments, a dilution solution comprises 1 mM to 10 mM, 1 mM to 15 mM, 1 mM to 20 mM, 1 mM to 30 mM or 1 mM to 40 mM salt. In some embodiments, an equilibration buffer comprises about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 1 1 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM or 50 mM salt. In some embodiments, a dilution solution comprises a salt selected from the group consisting of sodium chloride (NaCI), sodium acetate (NaAcetate,CH₃COONa), sodium citrate, ammonium acetate (NH₄Acetate), magnesium chloride (MgCh) or sodium sulfate (NasSC ). In some embodiments, a dilution solution comprises about 18 mM NaCI. In some embodiments, a dilution solution comprises about 9 mM sodium citrate. In some embodiments, a dilution solution comprises about 1 1 mM MgCh. In some embodiments, a dilution solution comprises about 2 mM MgCh.

[0210] In some embodiments, a dilution solution has a pH of 7 to 9.2. In some embodiments, a dilution solution has a pH of 8.5 to 9.5. In some embodiments, a dilution solution has a pH of 8.6 to 9.0. In some embodiments, a dilution solution has a pH of about 8.0, about 8.1 , about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1 , about 9.2, about 9.3, about 9.4, about 9.5 or about 10.0. In some embodiments, a dilution solution has a pH of about 8.8. [0211] In some embodiments, a dilution solution has a conductivity of 3 mS/cm to 9 mS/cm. In some embodiments, a dilution solution has a conductivity of 6.0 mS/cm to 6.8 mS/cm. In some embodiments, a dilution solution has a conductivity of about 6.0 mS/cm, about 6.1 mS/cm, about 6.2 mS/cm, about 6.3 mS/cm, about 6.4 mS/cm, about 6.5 mS/cm, about 6.6 mS/cm, about 6.7 mS/cm or about 6.8 mS/cm. In some embodiments, a dilution solution has a conductivity of about 6.4 mS/cm.

[0212] In some embodiments, diluting comprises dilution of a solution comprising a rAAV vector to be purified (e.g., an affinity eluate) with a buffer comprising 100 mM to 300 mM (e.g., about 190 mM) histidine, 100 mM to 300 mM (e.g., about 190 mM) Tris, 10 mM to 30 mM (e.g., about 18 mM) NaCI, 1 mM to 20 mM (e.g., about 9 mM) sodium citrate, 1 mM to 20 mM (e.g., about 11 mM MgCh), 0.1% to 1 .0% (e.g., 0.5%) P188 and having a pH of 8.6 to 9.0 (e.g., 8.8) and a conductivity of 6.0 to 6.8 mS/cm (e.g., about 6.4 mS/cm). In some embodiments, diluting comprises dilution of a solution comprising a rAAV vector to be purified (e.g., an affinity eluate) 4 to 7-fold by weight (e.g., about 5-fold) with a buffer comprising about 190 mM histidine, 190 mM Tris, 18 mM NaCI, 9 mM sodium citrate, 11 mM MgCh, 0.5% P188, and having a pH of 8.6 to 9.0 (e.g., about 8.8) and a conductivity of 6.0 to 6.8 mS/cm (e.g., about 6.4 mS/cm). In some embodiments, diluting comprises dilution of an affinity eluate comprising a rAAV vector to be purified about 5-fold by weight with a buffer comprising about 190 mM histidine, 190 mM Tris, 18 mM NaCI, 9 mM sodium citrate, 11 mM MgCh, 0.5% P188, and having a pH of 8.7 to 9.0 (e.g., about pH 8.8) and a conductivity of 6.0 to 6.8 mS/cm (e.g., about 6.4 mS/cm), and thereby forming a diluted affinity eluate.

[0213] In some embodiments, filtering comprises filtration of a solution comprising a rAAV vector to be purified (e.g., an affinity eluate, a diluted affinity eluate) prior to loading the solution onto an AEX column. In some embodiments, prior to filtering, a filter is pre-wet with water for injection and/or a dilution solution. In some embodiments, filtering comprises filtration of a solution comprising a rAAV vector to be purified (e.g., an affinity eluate, a diluted affinity eluate) through a filter which collects aggregates, such as nucleic acid or protein aggregates, or other high molecular mass species, but allows AAV capsids to flow through. In some embodiments, a filter is an 0.1 pm to 0.45 pm filter (e.g., a 0.2 pm polyethersulfone (PES) filter or a 0.45 pm PES filter). In some embodiments, filtering comprises filtration of a diluted affinity eluate comprising a rAAV vector to be purified through an 0.2 pm filter prior to loading onto an AEX column. A filter used to filter a solution comprising a rAAV vector to be purified (e.g., an affinity eluate, a diluted affinity eluate) may be separate from the column, or may be in-line with the column or chromatography apparatus (also referred to as a chromatography skid).

[0214] In some embodiments, filtering comprises filtration of a diluted affinity eluate comprising a rAAV vector to be purified through an in-line 0.2 pm filter before loading the eluate onto an AEX column.

[0215] In some embodiments, pH of a solution comprising a rAAV vector to be purified (e.g., an affinity eluate) is 7.4 to 7.8 prior to diluting, and optionally filtering, and pH of the solution comprising a rAAV vector to be purified (e.g., an affinity eluate) after diluting, and optionally filtering, is 8.7 to 8.9 (e.g., about pH 8.8).

[0216] In some embodiments, conductivity of a solution comprising a rAAV vector to be purified (e.g., an affinity eluate) is 5.0 mS/cm to 7.0 mS/cm prior to diluting, and optionally filtering, and conductivity of the solution comprising a rAAV vector to be purified (e.g., an affinity eluate) after diluting, and optionally filtering, 6.0 mS/cm to 6.8 mS/cm (e.g., about 6.4 mS/cm).

[0217] As used herein, the term “percent VG dilution yield” or “% VG dilution yield” refers to the amount of VG present in a diluted affinity pool (also referred to herein as a diluted affinity eluate) as a percentage of the amount of VG present in the affinity pool (also referred to herein as an affinity eluate) prior to dilution. For instance, % VG dilution yield = ((amount of VG in diluted affinity pool)/(amount of VG in affinity pool)) * 100.

[0218] In some embodiments, a percentage of VG recovered in a diluted, and optionally filtered solution (% VG dilution yield) comprising a rAAV vector to be purified (e.g., an affinity eluate) is 60% to 100% of the VG present in a solution (e.g., an affinity eluate) prior to diluting, and optionally filtering. In some embodiments, a % VG yield of a diluted, and optionally filtered solution comprising a rAAV vector to be purified (e.g., an affinity eluate) is 60% to 70%, 70% to 80%, 80% to 90%, 90% to 100% of the VG present in a solution (e.g., an affinity eluate) prior to diluting, and optionally filtering. In some embodiments, a % VG yield of a diluted, and optionally filtered solution comprising a rAAV vector to be purified (e.g., an affinity eluate) is about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99% or about 100% of the VG present in a solution prior to diluting, and optionally filtering.

[0219] A method of purifying a rAAV (e.g., rAAV3B or others) vector from a solution (e.g., an affinity eluate) by AEX comprises diluting the solution by 4 to 7-fold (e.g., about 5- fold) with a buffer comprising 100 mM to 300 mM (e.g., about 190 mM) histidine, 100 mM to 300 mM (e.g., about 190 mM) Tris, 10 mM to 30 mM (e.g., about 18 mM) NaCI, 1 mM to 20 mM (e.g., about 9 mM) sodium citrate, 1 mM to 20 mM (e.g., about 11 mM) MgCI₂, 0.1% to 1 .0% (e.g., 0.5%) P188 and having a pH of 8.7 to 9.0 (e.g., 8.8) and a conductivity of 6.0 to 6.8 mS/cm (e.g., about 6.4 mS/cm); and optionally comprises filtration of the diluted solution through a 0.1 pm to 0.45 pm (e.g., about 0.2 pm) filter, and wherein the diluted, and optionally filtered solution has a pH of about 8.7 to 9.0 (e.g., about pH 8.8) and a conductivity of 6.0 mS/cm to 6.8 mS/cm.

[0220] A method of preparing a weak binding load comprising a rAAV vector for purification by AEX chromatography, as disclosed herein, comprises i) diluting the affinity eluate 4 to 7-fold (e.g., about 5-fold) with a buffer comprising 190 mM histidine, 190 mM Tris, 18 mM NaCI, 9 mM sodium citrate, 11 mM MgCL, 0.5% P188 and having a pH of 8.8 and a conductivity of 6.4 mS/cm; and ii) optionally filtering the affinity eluate from step i) through a 0.2 pm filter to produce the diluted affinity eluate; wherein the pH of the diluted affinity eluate is increased as compared to the pH of the affinity eluate; wherein the conductivity of the diluted affinity eluate is increased as compared to the conductivity of the affinity eluate; optionally wherein the rAAV vector is an AAV3B vector; and optionally wherein the affinity eluate is produced by affinity purification of a rAAV vector produced in a vessel (e.g., a SUB) with a volume of 250 L to 2000 L.

D. Load

[0221] A method of purifying a rAAV (e.g., rAAV3B or others) vector by AEX disclosed herein comprises loading a solution comprising a substance to be purified (e.g., a rAAV vector) onto an AEX stationary phase in a column. Loading may be performed by gravity feeding the load onto the column or pumping the load onto the chromatography column. In some embodiments, a solution comprising a rAAV vector to be purified by AEX is selected from the group consisting of an affinity eluate, a supernatant from a cell lysate and a postharvest solution, each having undergone at least one other purification or processing step (e.g., cell lysis, flocculation, filtration, dilution, pH adjustment, conductivity adjustment, chromatography). A solution comprising a rAAV vector to be purified may be further processed by any one of dilution, filtration, pH adjustment, conductivity adjustment prior to loading the solution onto an AEX column in order to make the solution compatible with processing through the AEX column. Such further processing used to make a weak binding load, wherein the solution comprising the rAAV vector to be purified has a high conductivity (e.g., from 3 mS/cm to 9 mS/cm, e.g., 6.4 mS/cm) and a high pH (e.g., from 7.0 to 9.2, e.g., 8.8) The weak binding load conditions disclosed herein advantageously decrease or limit empty capsid binding while maintaining a high level of full capsid binding (e.g., greater than 80%, greater than 90%, greater than 95% and higher).

[0222] In some embodiments, a solution comprising a rAAV vector to be purified is an eluate resulting from affinity chromatography purification of a rAAV vector produced in a 100 L to 500 L (e.g., about 250 L), 1000 L to 3000 L (e.g., about 2000 L) or larger vessel (e.g., a single use bioreactor (SUB)), and wherein the eluate has been diluted and filtered.

[0223] In some embodiments, loading comprises application of a diluted, and optionally filtered solution (e.g., an affinity eluate) comprising about 1.0E+12 vector genomes (VG)/mL resin to 1 .0E+15 VG/mL, e.g., 5.0E+12 VG/mL to 5.0E+14 VG/mL, 1 .0E+13 VG/mL to 5.0E+14 VG/mL, 1.0E+13 VG/mL to 1 .OE+15 VG/mL, 5.0E+13 VG/mL to 5.0E+15 VG/mL, 5.0E+14 VG/mL to 5. OE+15 VG/mL, or more of column volume (also referred to as a “column challenge VG/mL resin”) onto an AEX column, as measured by qPCR analysis of a sequence within the vector genome. In some embodiments, loading comprises application of a diluted solution (e.g., an affinity eluate) comprising 0.4E+14 VG/mL to 1.3E+14 VG/mL (e.g., 5.0E+13 VG/mL) of column volume onto an about 2 L AEX column as measured by qPCR analysis of a transgene sequence within the vector genome (e.g., wherein the transgene is an ATP7B transgene). In some embodiments, loading comprises application of a diluted solution (e.g., an affinity eluate) comprising 1 .OE+14 VG/mL to 3.0E+14 VG/mL (e.g., 2.5E+14 VG/mL) of column volume onto an about 2 L AEX column as measured by qPCR analysis of a transgene sequence within the vector genome (e.g., wherein the transgene is an ATP7B transgene). In some embodiments, loading comprises application of a diluted and, optionally filtered solution (e.g., an affinity eluate) comprising 1.05E+14 VG/mL of column volume onto an about 2 L AEX column as measured by qPCR analysis of ITR sequences within the vector genome.

[0224] In some embodiments, loading comprises application of a diluted and, optionally filtered solution (e.g., an affinity eluate) comprising 8.0E+12 total VG to 2.0E+18 total VG, e.g., 8.0E+12 total VG to 8.0E+13 total VG, 8.0E+13 to 8.0E+14 total VG, 8.0E+14 total VG to 8.0E+15 total VG, 8.0E+15 total VG to 8.0E+16 total VG, 8.0E+16 total VG to 8.0E+17 total VG, 8.0E+17 total VG to 2.0E+18 total VG, or more onto an AEX column. In one embodiment, loading comprises application of a diluted and, optionally filtered solution (e.g., an affinity eluate) comprising 2.1 E+17 total VG onto an AEX column, and optionally wherein the VG are measured by quantitative polymerase chain reaction (qPCR) analysis of the transgene.

[0225] When a solution comprising a rAAV vector to be purified (e.g., an affinity eluate) is loaded onto a column, the solution flows through the column stationary phase at a particular rate (e.g., cm/hr, mL/min) and is in contact with the stationary phase for a particular period of time (i.e., residence time).

[0226] In some embodiments, a residence time of a solution comprising a rAAV vector loaded onto a column is 0.1 min/CV to 10 min/CV, e.g., 0.1 min/CV to 1.0 min/CV, 1 .0 min/CV to 5 min/CV, 1 min/CV to 8 min/CV, 1 min/CV to 10 min/CV, 2 min/CV to 6 min/CV, 2 min/CV to 8 min/CV, 2 min/CV to 10 min/CV, 3 min/CV to 8 min/CV, 3 min/CV to 10 min/CV, 4 min/CV to 8 min/CV, 4 min/CV to 10 min/CV or more. In some embodiments, a residence time of a solution comprising a rAAV vector loaded onto a column is about 6.0 min/CV. In some embodiments, a residence time of a solution comprising a rAAV vector loaded onto a column is 4.0 min/CV to 8.0 min/CV. In some embodiments, a residence time of a diluted and/or filtered affinity eluate comprising a rAAV vector loaded on a 2.0 L AEX column is 4.0 min/CV to 8.0 min/CV (e.g., about 6 min/CV).

[0227] In some embodiments, a linear velocity of a solution comprising a rAAV vector loaded onto a column is 10 cm/hr to 500 cm/hr, e.g., 10 cm/hr to 200 cm/hr, 10 cm/hr to 300 cm/hr, 10 cm/hr to 400 cm/hr, 50 cm/hr to 200 cm/hr, 50 cm/hr to 300 cm/hr, 50 cm/hr to 400 cm/hr, 100 cm/hr to 200 cm/hr. In some embodiments, a linear velocity of a solution comprising a rAAV vector loaded onto a column is 110 cm/hr to 150 cm/hr (e.g. about 130 cm/hr). In some embodiments, a linear velocity of a solution comprising a rAAV vector loaded onto a column is about 130 cm/hr. In some embodiments, a linear velocity of a diluted, and optionally filtered affinity eluate comprising a rAAV vector loaded on a 2.0 L AEX column is 110 cm/hr to 150 cm/hr (e.g., about 130 cm/hr).

[0228] In some embodiments, a method of purifying a rAAV vector (e.g., rAAV3B or others) from an affinity eluate comprises i) diluting the affinity eluate with a buffer comprising a detergent (e.g., P188), an amino acid (e.g., histidine), a buffer (e.g., Tris) and a salt (e.g., NaCI, MgCI₂, sodium citrate); ii) optionally filtering the diluted affinity eluate; and iii) loading the diluted, and optionally filtered affinity eluate onto a column comprising an AEX stationary phase wherein the AEX stationary phase has been flushed, sanitized, rinsed and/or equilibrated prior to loading, and optionally wherein the AEX stationary phase is POROS™ 50 HQ.

[0229] In some embodiments, a method of purifying a rAAV vector (e.g., rAAV3B or others) from an affinity eluate comprises i) diluting the affinity eluate 4 to 7-fold (e.g., about 5-fold) with a buffer comprising about 100 mM to 300 mM (e.g., about 190 mM) histidine, 100 mM to 300 mM (e.g., about 190 mM) Tris, 10 mM to 30 mM (e.g., about 18 mM) NaCI, 1 mM to 20 mM (e.g., about 9 mM) sodium citrate, 1 mM to 20 mM (e.g., about 11 mM) MgCI₂, 1.0% to 1 .5% (e.g., about 0.5%) P188, pH 8.7 to 9.0; ii) optionally filtering the diluted affinity eluate through an in-line 0.1 to 0.45 pm (e.g., about 0.2 pm) filter; and iii) loading the diluted and filtered affinity eluate onto a column comprising an AEX stationary phase; optionally wherein at least one step is performed at a linear velocity of 110 cm/hr to 150 cm/hr (e.g., about 130 cm/hr), a residence time of 4 min/CV to 8 min/CV (e.g., about 6 min/CV) and, optionally wherein the AEX stationary phase is POROS™ 50 HQ. In some embodiments, a column is a 2.0L column.

E. Empty Capsid Wash

[0230] This disclosure provides for the use of a wash (also referred to as an empty capsid wash or “ECW”) that has a high pH (e.g., 9.3 or greater) and a high conductivity (e.g., 9.3 mS/cm to 11 .6 mS/cm). The empty capsid wash step follows the load step (though not necessarily directly after the load) and advantageously elutes bound empty capsids with minimal elution of bound full capsids so that the AEX eluate comprises fewer empty capsids relative to full capsids. The disclosure also provides for the use of two wash (or equilibration) steps, one before and one after the empty capsid wash, using a MgCI₂ buffer which increases full capsid binding and reduces vg loss prior to the elution step. In some embodiments, these wash steps are referred to together as a “sandwich” wash as they are performed before and after the empty capsid wash.

[0231] In some embodiments, an empty capsid wash solution comprises a buffering agent, a salt, or both. In some embodiments, a buffering agent is selected from the group consisting of Tris (e.g., a mixture of Tris Base and Tris-HCI), BIS-Tris propane (BTP), diethanolamine, diethylamine, tricine, triethanolamine, bicine and a combination thereof. One of ordinary skill in the art would understand that a Tris buffer with a desired pH can be prepared using Tris Base, Tris-HCI or both. In some embodiments, a salt is sodium chloride (NaCI), sodium acetate (NaAcetate (CH₃COONa)), ammonium acetate (NH₄Acetate), magnesium chloride (MgCls), sodium citrate (NaCitrate) or sodium sulfate (NasSO^.

[0232] In some embodiments, an empty capsid wash solution comprises 1 mM to 50 mM of a buffering agent selected from the group consisting of Tris (e.g., a mixture of Tris Base and Tris-HCI), BIS-Tris propane, diethanolamine, diethylamine, tricine, triethanolamine and bicine.

[0233] In some embodiments, an empty capsid wash solution comprises 1 mM to 20 mM, 1 mM to 30 mM, 1 mM to 40 mM, 1 mM to 50 mM, 5 mM to 20 mM, 5 mM to 30 mM, 5 mM to 40 mM, 5 mM to 50 mM, 10 mM to 20 mM, 10 mM to 30 mM, 10 mM to 40 mM, 10 mM to 50 mM, 15 mM to 30 mM, 15 mM to 40 mM, 15 mM to 50 mM, 20 mM to 30 mM, 20 mM to 40 mM, 20 mM to 50 mM or ranges therein of BlS-tris propane. In some embodiments, an empty capsid wash solution comprises 20 mM to 30 mM BlS-tris propane. In some embodiments, an empty capsid wash solution comprises 25 mM BlS-tris propane. [0234] In some embodiments, an empty capsid wash solution comprises 1 mM to 250 mM of a salt selected from the group consisting of sodium chloride (NaCI), sodium acetate (NaAcetate, CH₃COONa), ammonium acetate (NH₄Acetate), magnesium chloride (MgCh), sodium citrate, sodium sulfate (Na₃SO₄) and a combination thereof.

[0235] In some embodiments, an empty capsid wash solution comprises 1 mM to 250 mM salt. In some embodiments, an empty capsid wash solution comprises 1 mM to 50 mM, 1 mM to 60 mM, 1 mM to 70 mM, 1 mM to 80 mM, 1 mM to 90 mM, 1 mM to 100 mM, 1 mM to 120 mM, 1 mM to 140 mM, 1 mM to 160 mM, 1 mM to 180 mM, 1 mM to 200 mM, 1 mM to 220 mM, 1 mM to 240 mM, 1 mM to 250 mM, 50 mM to 75 mM, 50 mM to 100 mM, 50 mM to 150 mM, 50 mM to 200 mM, 50 mM to 250 mM, 75 mM to 100 mM, 75 mM to 150 mM, 75 mM to 200 mM, 75 mM to 250 mM, 100 mM to 200 mM, 100 mM to 250 mM, or ranges therein of NaCI. In some embodiments, an empty capsid wash solution comprises 80 mM to 100 mM NaCI. In some embodiments, an empty capsid wash solution comprises 90 mM NaCI.

[0236] In some embodiments, an empty capsid wash solution has a pH of 8.6 to 9.6. In some embodiments, an empty capsid wash solution has a pH of 9.3 to 9.5. In some embodiments, an empty capsid wash solution has a pH of 8.6, 8.7, 8.8, 8.9, 9.0, 9.1 , 9.2, 9.3, 9.4, 9.5, or 9.6. In some embodiments, an empty capsid wash solution has a pH of 9.4 [0237] In some embodiments, an empty capsid wash solution has a conductivity of less than 1 1 mS/cm. In some embodiments, an empty capsid wash solution has a conductivity of 9.3 mS/cm to 11 .6 mS/cm. In some embodiments, an empty capsid wash solution has a conductivity of 13.5 mS/cmto 18.9 mS/cm.

[0238] In some embodiments, an amount of an empty capsid wash solution applied to a column is 1 CV to 5 CV, 4 CV to 6 CV 4 CV to 10 CV or 1 CV to 10 CV. In some embodiment, an amount of an empty capsid wash solution applied to a column is 4.5 CV to 5.5 CV. In some embodiments, an amount of an empty capsid wash solution applied to a column is about 2 CV, about 5 CV or about 10 CV. In some embodiments, an amount of an empty capsid wash solution applied to a column is about 5 CV.

[0239] In some embodiments, a residence time of an empty capsid wash solution in a column 1 .5 to 2.5 min/CV (e.g., 2.0 min/CV).

[0240] In some embodiments, a linear velocity of an empty capsid wash solution through a stationary phase in a column is 100 cm/hr to 500 cm/hr, e.g., 100 cm/hr to 200 cm/hr, 100 cm/hr to 300 cm/hr, 100 to 400 cm/hr, 200 cm/hr to 400 cm/hr, 200 cm/hr to 500 cm/hr, 350 cm/hr to 430 cm/hr, 400 cm/hr to 500 cm/hr. In some embodiments, a linear velocity of an empty capsid wash solution through a stationary phase in a column is 350 cm/hr to 430 cm/hr (e.g., about 390 cm/hr). In some embodiments, a linear velocity of an empty capsid wash solution through a stationary phase in a column is about 390 cm/hr. [0241] In some embodiments, a stationary phase is contacted with 4.5 CV to 5.5 CV (e.g., about 5 CV) of an empty capsid wash solution comprising about 25 mM bis-tris propane, about 90 mM NaCI and having a pH of 9.3 to 9.5 (e.g., 9.4) and a conductivity of 9.3 mS/cm to 11 .6 mS/cm at a linear velocity of 350 cm/hr to 430 cm/hr (e.g., about 390 cm/hr), a residence time of 1 .5 min/CV to 2.5 min/CV (e.g., about 2 min/CV) or both.

[0242] In some embodiments, before contacting a stationary phase with an empty capsid wash solution, the stationary phase is contacted with 4.5 CV to 5.5 CV (e.g., about 5 CV) of an equilibration buffer comprising about 100 mM Tris, about 2 mM MgCI₂, about 0.01% P188 and having a pH of about 8.9 at a linear velocity of 350 cm/hr to 430 cm/hr (e.g., about 390 cm/hr), a residence time of 1 .5 min/CV to 2.5 min/CV (e.g., about 2 min/CV) or both.

[0243] In some embodiments, after contacting a stationary phase with an empty capsid wash solution, the stationary phase is contacted with 4.5 CV to 5.5 CV (e.g., about 5 CV) of an equilibration buffer comprising about 100 mM Tris, about 2 mM MgCI₂, about 0.01 % P188 and having a pH of about 8.9 at a linear velocity of 350 cm/hr to 430 cm/hr (e.g., about 390 cm/hr), a residence time of 1 .5 min/CV to 2.5 min/CV (e.g., about 2 min/CV) or both. [0244] In some embodiments, before and after contacting a stationary phase with an empty capsid wash solution, the stationary phase is contacted with 4.5 CV to 5.5 CV (e.g., about 5 CV) of an equilibration buffer comprising about 100 mM Tris, about 2 mM MgCI₂, about 0.01 % P188 and having a pH of about 8.9 at a linear velocity of 350 cm/hr to 430 cm/hr (e.g., about 390 cm/hr), a residence time of 1 .5 min/CV to 2.5 min/CV (e.g., about 2 min/CV) or both (also referred to as a “sandwich wash”).

[0245] In some embodiments, the present disclosure provides a method of purifying a rAAV (e.g., rAAV3B or others) vector by AEX, the method comprising a step of: i) pre-use flushing comprising application of >4.5 CV (e.g., about 5 CV) of water for injection to an AEX stationary phase in a column; ii) sanitizing comprising application of 5 CV to 10 CV (e.g., about 8 CV) of a solution comprising 0.1 M to 1 .0 M (e.g., about 0.5 M) NaOH to the AEX stationary phase in the column, optionally by upward flow; iii) regenerating comprising application of 4.5 CV to 5.5 CV (e.g., about 5 CV) of a solution comprising 1 M to 3 M (e.g., about 2 M) NaCI), 50 mM to 150 mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., about 9) to the AEX stationary phase in the column; iv) equilibration comprising application of 4.5 CV to 5.5 CV (e.g., about 5 CV) of a solution comprising 50 mM to 150 mM (e.g., about 100 mM) Tris, 600 mM to 700 mM (e.g., about 500 mM) sodium acetate, 0.005% to 0.015% (e.g., about 0.01 %) P188, pH 8.5 to 9.5 (e.g., about 8.9) to the AEX stationary phase in the column; v) equilibration comprising application of > 4.5 CV (e.g., about 5 CV) of an equilibration buffer comprising 100 mM to 300 mM (e.g., about 190 mM) Tris, 100 mM to 300 mM (e.g., about 190 mM) histidine, 1 mm to 20 mM (e.g., 9 mM) sodium citrate, 1 mM to 20 mM (e.g., 11 mM) MgCh, 10 mM to 30 mM (e.g., 18 mM) NaCI, 0.1 % to 1 .0% (e.g., about 0.5%) P188, pH 8.5 to 9.5 (e.g., about 8.8) to the AEX stationary phase in the column; vi) loading the affinity eluate to an AEX stationary phase in the column, optionally wherein the eluate has been a) diluted about 4 to 7-fold (e.g., about 5-fold) with a buffer comprising 100 mM to 300 mM (e.g., about 190 mM) histidine, 100 mM to 300 mM (e.g., about 190 mM) Tris, 1 mm to 20 mM (e.g., 9 mM) sodium citrate, 1 mM to 20 mM (e.g., 11 mM) MgCh, 10 mM to 30 mM (e.g., 18 mM) NaCI, 0.1 %to 1.0% (e.g., about 0.5%) P188, pH 8.5 to 9.5 (e.g., about 8.8), and optionally b) filtered through an in-line 0.1 pm to 0.45 pm (e.g., about 0.2 pm) filter prior to application to the stationary phase; vii) equilibration comprising application of 4.5 CV to 5.5 CV (e.g., about 5 CV) of an equilibration buffer comprising 50 mM to 150 mM (e.g., 100 mM) Tris, 0.5 mM to 5 mM (e.g., about 2 mM) MgCh, 0.005% to 0.015% (e.g., about 0.01 %) P188, pH 8.5 to 9.5 (e.g., 8.9) to the AEX stationary phase in the column; viii) washing comprising application of 4.5 CV to 5.5 CV (e.g., about 5 CV) of an empty capsid wash solution comprising 50 mM to 150 mM (e.g., about 100 mM) bis-tris propane, 80 mM to 100 mM (e.g., about 90 mM) NaCI pH 9.0 to 9.6 (e.g., about 9.4) to the AEX stationary phase in the column; ix) equilibration comprising application of 4.5 CV to 5.5 CV (e.g., about 5 CV) of an equilibration buffer comprising 50 mM to 150 mM (e.g., 100 mM) Tris, 0.5 mM to 5 mM (e.g., about 2 mM) MgCls, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., 8.9) to the AEX stationary phase in the column; optionally wherein at least one of steps i) - ix) is performed at a linear velocity of 350 cm/hr to 430 cm/hr (e.g., about 390 cm/hr), a residence time of 1 .5 min/CV to 2.5 min/CV (e.g., about 2 min/CV) or both; optionally wherein the rAAV vector is a rAAV3B vector; and optionally wherein the AEX stationary phase is POROS™ 50 HQ.

[0246] In some embodiments, at least step vi) is performed at a linear velocity of 110 cm/hr to 150 cm/hr (e.g., about 130 cm/hr), a residence time of 4.0 min/CV to 8.0 min/CV (e.g., about 6 min/CV) or both. One of ordinary skill will understand that the order of the above steps may be varied.

F. Gradient Elution

[0247] A method of purifying a rAAV vector (e.g., rAAV3B or others) from a solution (e.g., an affinity eluate) comprises recovery of full, intermediate and/or empty capsids by gradient elution. Gradient elution may comprise use of at least 2 different solutions (e.g., gradient elution buffers) with different pH, conductivity, and/or modifier concentration. Over the course of a gradient elution, a percentage of a first solution is varied in a manner inversely proportional to variation of a percentage of a second solution such that a gradient in the pH, conductivity, and/or modifier concentration is created as the solutions are mixed and flow through the column stationary phase. For example, at the start of a gradient elution, a percentage of a first solution (e.g., a first gradient elution buffer, buffer A) is 100% and a percentage of a second solution (e.g., a second gradient elution, buffer B) is 0% and at the end of the gradient elution the percentage of the first solution is 0% and the percentage of the second solution is 100%. In another embodiment, at the start of a gradient elution, a percentage of a first solution (e.g., a first gradient elution buffer, buffer A) is 100% and a percentage of a second solution (e.g., a second gradient elution, buffer B) is 0% and at the end of the gradient elution the percentage of the first solution is 25% and the percentage of the second solution is 75%. One of ordinary skill would understand that the percentage of each solution at the start of the gradient and at the end of the gradient can be anywhere between 0% and 100%. For instance, in some embodiments, at the start of the elution, at the end of the elution, or at any point over the course of the elution, a percentage of a first gradient elution buffer relative to a second gradient elution buffer is about 100%/0%, about 99%/1%, about 98%/2%, about 97%3%, about 96%/4%, about 95%/5%, about 90%10%, about 80%20%, about 75%/25%, about 70%/30%, about 60%/40%, about 50%/50%, about 40%/60%, about 30%/70%, about 25%/75%, about 20%/80%, about 10%/90%, about 5%/95%, about 4%/96%, about 3%/97%, about 2%/98%, about 1 %/99% or about 0%/100%. [0248] In some embodiments, at the start of the elution, at the end of the elution, or at any point over the course of the elution, a percentage of a first gradient elution buffer relative to a percentage of a second gradient elution buffer is about 100% to 90%/0% to 10%, 90% to 80%/10% to 20%, 80% to 70%/20% to 30%, 70% to 60%/30% to 40%, 60% to 50%/40% to 50%, 50% to 40%/50% to 60%, 40% to 30%/60% to 70%, 30% to 20%/70% to 80%, 20% to 10%/ 80% to 90%, 10% to 0%/90% to 100%.

[0249] In some embodiments, over the course of application of 10 to 60 CV of a solution to the column, a percentage of buffer A (e.g., a first gradient elution buffer) is decreased, and a percentage of buffer B (e.g., a second gradient elution buffer) is increased such that at the end of the gradient elution, the percentage of gradient elution buffer A is 0%, and the percentage of gradient elution buffer B is 100%. In some embodiments, over the course of application of about 37.5 CV of a solution to the column, the percentage of buffer A (e.g., a first gradient elution buffer) is decreased, and the percentage of buffer B (e.g., a second gradient elution buffer) is increased such that the rate of increase of Buffer B is about 2.67% of buffer B per CV and such that the final percentage of buffer B in the solution is 100%. In some embodiments, over the course of application of about 37.5 CV of a solution to the column, the percentage of buffer A (e.g., a first gradient elution buffer) is decreased, and the percentage of buffer B (e.g., a second gradient elution buffer) is increased such that the rate of increase of Buffer B is about 2% of buffer B per CV, and such that the final percentage of buffer B in the solution is 75%.

[0250] In some embodiments, over the course of application of 10 to 60 CV of a solution to the column, the percentage of buffer A (e.g., a first elution buffer) is increased, and the percentage of buffer B (e.g., a second elution buffer) is decreased such that at the end of the gradient elution, the percentage of gradient elution buffer A is 100%, and the percentage of gradient elution buffer B is 0%. One of skill in the art with recognize that a gradient elution may be run to different percentages of buffer (e.g., from 0% to 75% buffer B, corresponding to 100% to 25% buffer A; from 0% to 50% buffer B, corresponding to 100% to 50% buffer A). [0251] In some embodiments, a method of purifying a rAAV vector by AEX of the disclosure comprises performing gradient elution of a material from a stationary phase in a column wherein a concentration of a component of a first gradient elution buffer or a second gradient elution buffer increases or decreases continuously during the gradient elution. In some embodiments, a material eluted from the stationary phase comprises a rAAV vector to be purified. A rate of increase or decrease of a concentration of a component of a first gradient elution buffer or a second gradient elution buffer may be equivalent to a change in concentration of the component per total CV. In some embodiments, a rate of increase of a concentration of sodium acetate during a gradient elution is equivalent to a change in concentration of the sodium acetate per total CV applied to a stationary phase during the elution. In some embodiments, a change in concentration of a component is relative to a concentration of the component at the start of a elution as compared to a concentration of the component at the end of the elution. For example, a concentration of a component (e.g., a salt such as sodium acetate) at the start of a gradient elution is 0 mM to 100 mM, and the concentration of the component at the end of the elution is 100 mM to 1 M. In some embodiments, a concentration of a salt (e.g., sodium acetate) at the start of a gradient elution is 0 mM and the concentration of the salt at the end of the gradient elution is 400 mM to 600 mM (e.g., about 500 mM). In some embodiments, a change in a concentration of a component is 2 mM to 1 M from the start of a gradient to the end of a gradient elution, over the course of 2 CV to 100 CV of elution buffer. In some embodiments, a change in concentration of a salt is from about 0 mM to about 500 mM from the start a gradient to the end of a gradient elution over the course of 10 CV to 60 CV, 10 CV to 50 CV, 10 CV to 40 CV, 10 CV to 30 CV or 15 CV to 25 CV (e.g., 20 CV) of elution buffer, such that when the elution gradient comprises 20 CV of solution, the rate of change of sodium acetate concentration is about 500 mM per 20 CV, or 25 mM/CV. In some embodiments, a change in concentration of a salt is from about 0 mM to about 375 mM from the start a gradient to the end of a gradient elution over the course of 10 CV to 60 CV, 10 CV to 50 CV, 10 CV to 40 CV, 10 CV to 30 CV or 15 CV to 25 CV (e.g., 37.5 CV) of elution buffer, such that when the elution gradient comprises 37.5 CV of solution, the rate of change of concentration of sodium acetate is about 375 mM per 37.5 CV, or 10 mM/CV.

[0252] In some embodiments, during a gradient elution, a concentration of sodium acetate of a first gradient elution buffer, a second gradient elution buffer or a mixture of both increases continuously during the gradient elution; wherein a rate of increase of the sodium acetate is equivalent to a change in concentration of the sodium acetate per total CV applied to the stationary phase; and wherein the rate of change in concentration of the sodium acetate over the gradient elution is about 5 mM/CV to 15 mM/CV, 10 mM/CV to 40 mM/CV, 10 mM/CV to 30 mM/CV, 10 mM to 40 mM/CV, 10 mM/CV to 50 mM/CV or 20 mM/CV to 30 mM/CV (e.g., about 10 mM/CV, about 25 mM/CV).

[0253] In some embodiments, a change in concentration of a component over a gradient elution is about 1 mM/CV to 1 M/CV, e.g., 1 mM/CV to 10 mM/CV, 1 mM/CV to 25 mM/CV, 5 mM/CV to 15 mM/CV, 10 mM/CV to 50 mM/CV, 50 mM/CV to 100 mM/CV, 100 mM/CV to 500 mM/CV, 500 mM/CV to 1 M/CV, 1 mM/CV to 750 mM/CV, 1 mM/CV to 500 mM/CV, 1 mM/CV to 100 mM/CV, 10 mM/CV to 750 mM/CV or 50 mM/CV to 500 mM/CV.

[0254] In some embodiments, over the course of a gradient elution, a concentration of a salt in the gradient solution may vary. In some embodiments, over the course of a gradient elution, a concentration of a salt (e.g., sodium chloride, sodium acetate, ammonium acetate, magnesium chloride, sodium sulfate and a combination thereof) in the gradient solution may increase or decrease. For example, at the start of a gradient elution, a concentration of a salt in the gradient solution may be 0 mM to 100 mM, and increase to 50 mM to 1 M, e.g., 50 mM to 100 mM , 100 mM to 150 mM, 150 mM to 200 mM, 200 mM to 250 mM, 250 mM to 300 mM, 300 mM to 400 mM, 400 mM to 500 mM, 500 mM to 600 mM, 600 mM to 700 mM, 700 mM to 800 mM, 800 mM to 900 mM,900 mM to 1 M, 50 mM to 750 mM, 50 mM to 500 mM, 50 mM to 400 mM, 50 mM to 200 mM, 100 mM to 1 M, 100 mM to 750 mM, 100 mM to 500 mM, 100 mM to 400 mM or 100 mM to 200 mM over the course of the gradient elution. [0255] In a further example, at the start of a gradient elution, a concentration of salt in the gradient solution may be 50 mM to 1 M, e.g., 50 mM to 100 mM , 100 mM to 150 mM, 150 mM to 200 mM, 200 mM to 250 mM, 250 mM to 300 mM, 300 mM to 400 mM, 400 mM to 500 mM, 500 mM to 600 mM, 600 mM to 700 mM, 700 mM to 800 mM, 800 mM to 900 mM, 900 mM to 1 M, 50 mM to 750 mM, 50 mM to 500 mM, 50 mM to 400 mM, 50 mM to 200 mM, 100 mM to 1 M, 100 mM to 750 mM, 100 mM to 500 mM, 100 mM to 400 mM or 100 mM to 200 mM and decrease to 0 mM to 100 mM over the course of the gradient elution. In some embodiments, at the start of a gradient elution a concentration of sodium acetate in the gradient solution is about 0 mM and, at the end of the gradient elution the concentration of sodium acetate is about 500 mM. In some embodiments, at the start of a gradient elution a concentration of sodium acetate in the gradient elution solution is about 0 mM and, at the end of the gradient elution the concentration of sodium acetate in the gradient elution solution is about 375 mM. In some embodiments, at the start of a gradient elution a concentration of sodium sulfate in the gradient solution is about 0 mM and, at the end of the gradient elution the concentration of sodium sulfate is about 500 mM. In some embodiments, at the start of a gradient elution a concentration of sodium sulfate in the gradient elution solution is about 0 mM and, at the end of the gradient elution the concentration of sodium sulfate in the gradient elution solution is about 375 mM.

[0256] In some embodiments, over the course of a gradient elution, a pH of the gradient solution may vary. In some embodiments, over the course of a gradient elution, a pH of the gradient solution may increase or may decrease. In some embodiments, at the start of a gradient elution, a pH of the gradient solution may be between 7.0 and 1 1 .0 (e.g., 7.0 to 7.5,

7.5 to 8.0, 8.0 to 8.5, 8.5 to 9.0, 9.0 to 9.5, 10.0 to 10.5, 10.5 to 1 1 , 7.5 to 10.5, 8.0 to 10.0,

8.5 to 9.5 or 8.0 to 9.0). In some embodiments, at the end of a gradient elution, a pH of the gradient solution may be between 7.0 and 11 .0 (e.g., 7.0 to 7.5, 7.5 to 8.0, 8.0 to 8.5, 8.5 to 9.0, 9.0 to 9.5, 10.0 to 10.5, 10.5 to 1 1 , 7.5 to 10.5, 8.0 to 10.0, 8.5 to 9.5 or 8.0 to 9.0).

[0257] In some embodiments, over the course of a gradient elution, a conductivity of the gradient solution may vary. In some embodiments, over the course of a gradient elution, a conductivity of the gradient solution may increase or may decrease. In some embodiments, at the start of a gradient elution, a conductivity of the gradient solution may be between 1 .0 mS/cm and 2.5 mS/cm, e.g., 1 .2 mS/cm and 2.0 mS/cm. In some embodiments, at the end of a gradient elution, a conductivity of the gradient solution may be between 20 mS/cm and 35 mS/cm, e.g., 27 mS/cm and 33 mS/cm. In some embodiments, at the start of a gradient elution a conductivity of the gradient solution is about 1 .6 mS/cm and at the end of the gradient elution the conductivity of the gradient solution is about 30 mS/cm.

[0258] In some embodiments, over the course of a gradient elution, a concentration of a buffer in the gradient solution may vary. In some embodiments, over the course of a gradient elution, a concentration of a buffer (e.g., Tris (e.g., a mixture of Tris Base and Tris-HCI), BIS- Tris propane, diethanolamine, diethylamine, tricine, triethanolamine and/or bicine of) in the gradient solution may increase or decrease. For example, at the start of a gradient elution a concentration of a buffer in the gradient solution may range from 10 mM to 500 mM, e.g., from 10 mM to 50 mM, from 10 mM to 200 mM, from 10 mM to 300 mM, from 10 mM to 400 mM, from 50 mM to 100 mM, from 50 mM to 150 mM, from 100 mM to 200 mM, from 100 mM to 400 mM, from 200 mM to 300 mM, from 300 mM to 400 mM, from 400 mM to 500 mM, or more. At the end of a gradient elution a concentration of a buffer in the gradient solution may range froml 0 mM to 500 mM, e.g., from 10 mM to 50 mM, from 10 mM to 200 mM, from 10 mM to 300 mM, from 10 mM to 400 mM, from 50 mM to 100 mM, from 50 mM to 150 mM, from 100 mM to 200 mM, from 100 mM to 400 mM, from 200 mM to 300 mM, from 300 mM to 400 mM, from 400 mM to 500 mM, or more.

[0259] In some embodiment, over the course of a gradient elution, a concentration of a detergent in the gradient solution may vary. In some embodiments, over the course of a gradient elution, a concentration of a detergent (e.g., poloxamer 188 (P188), Triton X-100, polysorbate 80 (PS80), Brij-35, nonyl phenoxypolyethoxylethanol (NP-40) and a combination thereof) in the gradient solution, may increase or decrease. For example, at the start of a gradient elution a concentration of a detergent (e.g., P188) in the gradient solution may range from 0.005% to 1 .0%, e.g., from 0.005% to 0.01%, from 0.005% to 0.5%, from 0.01% to 1 .0%, from 0.01% to 0.5%, from 0.01% to 0.02%, from 0.02% to 0.03%, from 0.03% to 0.04%, from 0.04% to 0.05%, from 0.05% to 0.06%, from 0.05% to 1 .0%, from 0.05% to 0.5%, from 0.07% to 0.08%, from 0.08% to 0.09%, from 0.09% to 0.1%, from 0.1% to 0.5%, from 0.1% to 1 .0%, from 0.5% to 1 .0%.

[0260] In some embodiments, at the end of a gradient elution, a concentration of a detergent (e.g., P188) in the gradient solution may range from 0.005% to 1 .0%, e.g., from 0.005% to 0.01%, from 0.005% to 0.5%, from 0.01% to 1 .0%, from 0.01% to 0.5%, from 0.01% to 0.02%, from 0.02% to 0.03%, from 0.03% to 0.04%, from 0.04% to 0.05%, from 0.05% to 0.06%, from 0.05% to 1 .0%, from 0.05% to 0.5%, from 0.07% to 0.08%, from 0.08% to 0.09%, from 0.09% to 0.1%, from 0.1% to 0.5%, from 0.1% to 1 .0%, from 0.5% to 1 .0%.

[0261] Over the course of a gradient elution, while one or more aspects of the gradient solution (e.g., a salt concentration) may be varied, other aspects of the gradient, such as conductivity, pH, buffer concentration, detergent concentration etc., may remain constant. For example, pH of a gradient solution may range from 7.0 to 11 .0, e.g., from 7.5 to 10.5, from 8.0 to 10.0, from 8.5 to 9.5 or from 8.0 to 9.0, from 7.0 to 7.5, from 7.5 to 8.0, from 8.0 to 8.5, from 8.5 to 9.0, from 9.0 to 9.5, from 9.5 to 10, from 10.0 to 10.5 or from 10.5 to 11 .0, but be constant throughout the gradient elution (e.g., a pH of about 8.8, about 8.9, about 9). In some embodiments, a pH of a gradient elution solution is about 8.9.

[0262] In some embodiments, a concentration of a buffer, such as Tris, BIS-Tris propane, bicine and a combination thereof, in a gradient elution may range from 10 mM to 500 mM, e.g., from 10 mM to 30 mM, from 10 mM to 50 mM, from 50 mM to 100 mM, from 100 mM to 200 mM, from 200 mM to 300 mM, from 300 mM to 400 mM, from 400 mM to 500 mM, from 10 mM to 400 mM, from 10 mM to 300 mM, about 10 mM to 200 mM, about 50 mM to about 150 mM or more, but be constant throughout the gradient elution (e.g., about 20 mM, about 100 mM). In some embodiments, a concentration of a buffer, such as Tris, in a gradient elution is 50 mM to 150 mM. In some embodiments, a concentration of a buffer, such as Tris, in a gradient elution solution is about 100 mM.

[0263] In some embodiments, a concentration of a detergent, such as poloxamer 188 (P188), Triton X-100, polysorbate 80 (PS80), Brij-35, nonyl phenoxypolyethoxylethanol (NP- 40) and a combination thereof, in a gradient elution may range from 0.005% to 0.01%, from 0.005% to 0.5%, from 0.01% to 1 .0%, from 0.01% to 0.5%, from 0.01% to 0.02%, from 0.02% to 0.03%, from 0.03% to 0.04%, from 0.04% to 0.05%, from 0.05% to 0.06%, from 0.05% to 1 .0%, from 0.05% to 0.5%, from 0.07% to 0.08%, from 0.08% to 0.09%, from 0.09% to 0.1%, from 0.1% to 0.5%, from 0.1% to 1 .0%, from 0.5% to 1 .0%but be constant throughout the gradient elution. In some embodiments, a concentration of P188 during a gradient elution is 0.05% to 0.1%. In some embodiments, a concentration of P188 during a gradient elution is about 0.01%.

[0264] During a gradient elution, as conditions within the column change, for example, pH, conductivity, salt concentration and/or modifier concentration, substances loaded onto the column elute from the column at varying points during the gradient.

[0265] In some embodiments, AAV capsids (e.g., full, intermediate, empty) are bound to a stationary phase during loading a solution comprising the capsids to be purified. During a gradient elution, as a percentage of a gradient elution buffer increases, such that the concentration of a salt increases (e.g., sodium acetate, sodium sulfate), full rAAV vectors are preferentially released (eluted) from the stationary phase, and empty capsids are preferentially retained on the stationary phase. Empty capsids are released in greater amounts as the percentage of the gradient elution buffer further increases (along with the salt concentration). Empty capsids may also be recovered in an AEX column flow-through that is, the unbound fraction. In some embodiments, empty capsids may be recovered in a flow-through of an empty capsid wash solution (e.g., 25 mM bis-tris propane, 90 mM NaCI, pH 9.4). In some embodiments, full capsids, intermediate capsids or both are recovered in a first elution peak and in a portion of a second elution peak (e.g., the first 2/3s of a second elution peak) from an AEX column. In some embodiments, full capsids, intermediate capsids or both are recovered in in the center of the elution peak and empty capsids are recovered in the shoulders of the elution peak. Elution of full rAAV vector from the stationary phase can be monitored during a gradient elution by measuring an A260 and A280 of the eluate, such that an increase in the A260/A280 ratio is indicative of an increase in the presence of full rAAV vector in the eluate.

[0266] In some embodiments, performing a gradient elution comprises application of about 37.5 CV of a solution to a column, wherein the solution is buffer A, buffer B or a mixture of buffer A and buffer B and wherein at the start of the gradient elution, the solution is 100% buffer A and at the end of the step the solution is 100% B, such that a gradient between buffer A and buffer B is created over the course of the elution phase, optionally, wherein the rate of increase of buffer B is about 2.67% of buffer B per CV, and optionally, when buffer B comprises sodium acetate or sodium sulfate, the concentration of sodium acetate or sodium acetate increases at a rate of 13.3 mM per CV.

[0267] In some embodiments, performing a gradient elution comprises application of about 37.5 CV of a solution to a column, wherein the solution is buffer A, buffer B or a mixture of buffer A and buffer B and wherein at the start of the gradient elution, the solution is 100% buffer A and at the end of the step the solution is 75% buffer B and 25% buffer A, such that a gradient between buffer A and buffer B is created over the course of the elution phase, optionally, wherein the rate of increase of buffer B is about 2% of buffer B per CV, and optionally, when buffer B comprises sodium acetate or sodium sulfate, the concentration of sodium acetate or sodium sulfate increases at a rate of 10 mM per CV.

[0268] In some embodiments, buffer A (e.g., a first gradient elution buffer) comprises 50 mM to 150 mM (e.g., about 100 mM) Tris, 0.005% to 0.015 % (e.g., about 0.01%) P188, pH

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

9.5 (e.g., about 8.9).

[0269] In some embodiments, a gradient elution begins with application of 100% buffer A to the column and ends with application of 100% buffer B to the column over the course of 30 CV to 40 CV (e.g., about 37.5 CV), such that a gradient between buffer A and buffer B is created over the course of the elution phase, and wherein buffer A comprises about 100 mM Tris, 0.01% P188, pH 8.9 and buffer B comprises about 500 mM sodium acetate, 100 mM Tris, 0.01% P188, pH 8.9. In some embodiments, a gradient elution begins with application of 100% buffer A to the column and ends with application of 75% buffer B and 25% buffer A to the column over the course of 30 CV to 40 CV (e.g., about 37.5 CV), such that a gradient between buffer A and buffer B is created over the course of the elution phase, and wherein buffer A comprises about 100 mM Tris, 0.01% P188, pH 8.9 and buffer B comprises about 500 mM sodium acetate, 100 mM Tris, 0.01% P188, pH 8.9.

[0270] In some embodiments, a residence time of a gradient elution buffer (e.g., buffer, A, buffer B or a mixture of buffer A and buffer B) in an AEX column is 0.1 min/CV to 15 min/CV, e.g., 0.1 min/CV to 1 min/CV, 1 min/CV to 2 min/CV, 1.5 min/CV to 2.5 min/CV, 2 min/CV to 4 min/CV, 4 min/CV to 8 min/CV, 6 min/CV to 8 min/CV, or 8 min/CV to 12 min/CV, 10 min/CV to 12 min/CV, 10 min/CV to 15 min/CV. In some embodiments, a residence time of a gradient elution buffer in an AEX column is 0.1 min/CV, about 0.5 min/CV, about 1 .5 min/CV, about 2.0 min/CV, about 2.5 min/CV, about 3 min/CV, about 3.6 min/CV or about 4 min/CV, about 5 min/CV, about 6 min/CV, about 7 min/CV, about 8 min/CV, about 9 min/CV or about 10 min/CV. In some embodiments, a residence time of a gradient elution buffer (e.g., buffer A, buffer B or a mixture of buffer A and buffer B) in an AEX column is about 2 min/CV.

[0271] In some embodiments, a residence time of a gradient elution buffer (e.g., buffer A, buffer B or a mixture of buffer A and buffer B) in a column is 1 .5 to 2.5 min/CV (e.g., about 2 min/CV).

[0272] In some embodiments, a linear velocity of a gradient elution buffer (e.g., buffer A, buffer B or a mixture of buffer A and buffer B) through a stationary phase in a column is 50 to 1800 cm/hr, e.g., 50 cm/hr to 100 cm/hr, 100 cm/hr to 200 cm/hr, 200 cm/hr to 400 cm/hr, 400 cm/hr to 600 cm/hr, 600 cm/hr to 800 cm/hr, 800 cm/hr to 1000 cm/hr, 1000 cm/hr to 1500 cm/hr, or 1500 cm/hr to 1800 cm/hr. In some embodiments, a linear velocity of a gradient elution buffer (e.g., buffer A, buffer B or a mixture of buffer A and buffer B) through a stationary phase in a column is about 350 cm/hr to 430 cm/hr (e.g., about 390 cm/hr).

[0273] In some embodiments, a method of purifying a rAAV vector (e.g., rAAV9, rAAV3B or others) from a solution (e.g., an affinity eluate) comprises application of a gradient elution buffer to a column comprising POROS™ 50 HQ stationary phase. In some embodiments, a method of purifying a rAAV vector (e.g., AAV3B or others) from an affinity eluate comprises performing a gradient elution beginning with application of 100% buffer A (e.g., comprises 50 mM to 150 mM (e.g., about 100 mM) Tris, 0.005% to 0.015 % (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., about 8.9)) and ending with application of 75% to 100% buffer B (e.g., 50 mM to 150 mM (e.g., about 100 mM) Tris, 400 mM to 600 mM (e.g., about 500 mM) sodium acetate or sodium sulfate, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., about 8.9)) over 15 to 40 CV (e.g., about 20 CV, about 37.5 CV) to a column comprising an AEX stationary phase, wherein the rate of change of the percentage of buffer B during the gradient elution is 2% buffer B per CV to 5% buffer B per CV. In some embodiments, the column is a 2 L column.

[0274] In some embodiments, a method of purifying a rAAV vector (e.g., AAV3B or others) from an affinity eluate comprises performing a gradient elution beginning with application of 100% of a first buffer comprising about 100 mM Tris, 0.01% P188, pH 8.9 and ending with application of 75% to 100% of a second buffer comprising 500 mM sodium acetate or sodium sulfate, 100 mM Tris, 0.01% P188, pH 8.9 over 15 CV to 40 CV (e.g., about 37.5 CV) to a column comprising an AEX stationary phase at a linear velocity of 350 cm/hr to 430 cm/hr (e.g., about 390 cm/hr), a residence time of 1 .5 min/CV to 2.5 min/CV (e.g., 2 min/CV), or both, such that a gradient between the first buffer and the second buffer is created over the course of the elution, and wherein the rate of change of the percentage of buffer B during the gradient elution is 2% buffer B per CV to 5% buffer B per CV (e.g., 2% buffer B per CV, 2.67% buffer B per CV) .

[0275] In some embodiments, the present disclosure provides a method of purifying a rAAV (e.g., rAAV3B or other rAAV) vector by AEX, the method comprising at least one step of: i) pre-use flushing comprising application of >4.5 CV (e.g., about 5 CV) of water for injection to an AEX stationary phase in a column; ii) sanitizing comprising application of 5 CV to 10 CV (e.g., about 8 CV) of a solution comprising 0.1 M to 1 .0 M (e.g., about 0.5 M) NaOH to the AEX stationary phase in the column, optionally by upward flow; iii) regenerating comprising application of 4.5 CV to 5.5 CV (e.g., about 5 CV) of a solution comprising 1 M to 3 M (e.g., about 2 M) NaCI), 50 mM to 150 mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., about 9) to the AEX stationary phase in the column; iv) equilibration comprising application of 4.5 CV to 5.5 CV (e.g., about 5 CV) of a solution comprising 50 mM to 150 mM (e.g., about 100 mM) Tris, 600 mM to 700 mM (e.g., about 500 mM) sodium acetate, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., about 8.9) to the AEX stationary phase in the column; v) equilibration comprising application of > 4.5 CV (e.g., about 5 CV) of an equilibration buffer comprising 100 mM to 300 mM (e.g., about 190 mM) Tris, 100 mM to 300 mM (e.g., about 190 mM) histidine, 1 mm to 20 mM (e.g., 9 mM) sodium citrate, 1 mM to 20 mM (e.g., 1 1 mM) MgCh, 10 mM to 30 mM (e.g., 18 mM) NaCI, 0.1% to 1 .0% (e.g., about 0.5%) P188, pH 8.5 to 9.5 (e.g., about 8.8) to the AEX stationary phase in the column; vi) loading the affinity eluate to an AEX stationary phase in the column, optionally wherein the eluate has been a) diluted about 4 to 7-fold (e.g., about 5- fold) with a buffer comprising 100 mM to 300 mM (e.g., about 190 mM) histidine, 100 mM to 300 mM (e.g., about 190 mM) Tris, 1 mm to 20 mM (e.g., 9 mM) sodium citrate, 1 mM to 20 mM (e.g., 11 mM) MgCh, 10 mM to 30 mM (e.g., 18 mM) NaCI, 0.1 %to 1.0% (e.g., about 0.5%) P188, pH 8.5 to 9.5 (e.g., about 8.8), and optionally b) filtered through an in-line 0.1 pm to 0.45 pm (e.g., about 0.2 pm) filter prior to application to the stationary phase; vii) equilibration comprising application of 4.5 CV to 5.5 CV (e.g., about 5 CV) of an equilibration buffer comprising 50 mM to 150 mM (e.g., 100 mM) Tris, 0.5 mM to 5 mM (e.g., about 2 mM) MgCI₂, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., 8.9) to the AEX stationary phase in the column; viii) washing comprising application of 4.5 CV to 5.5 CV (e.g., about 5 CV) of an empty capsid wash solution comprising 50 mM to 150 mM (e.g., about 100 mM) bis-tris propane, 80 mM to 100 mM (e.g., about 90 mM) NaCI, pH 9.0 to 9.6 (e.g., about 9.4) to the AEX stationary phase in the column; ix) equilibration comprising application of 4.5 CV to 5.5 CV (e.g., about 5 CV) of an equilibration buffer comprising 50 mM to 150 mM (e.g., 100 mM) Tris, 0.5 mM to 5 mM (e.g., about 2 mM) MgCI₂, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., 8.9) to the AEX stationary phase in the column; x) performing gradient elution of material from the stationary phase in the column beginning with application of 100% of a first buffer comprising 50 mM to 150 mM (e.g., about 100 mM) Tris, 0.005% to 0.015 % (e.g., about 0.01%) P188, pH 8.5 to 9.0 (e.g., about 8.9) and ending with application of 75% to 100% of a second buffer comprising 400 mM to 600 mM (e.g., about 500 mM) sodium acetate or sodium sulfate, 50 mM to 150 mM (e.g., about 100 mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., pH 8.9) to the stationary phase over 30 CV to 40 CV (e.g., about 37.5 CV); optionally wherein at least one of steps i) - x) is performed at a linear velocity of 350 cm/hr to 430 cm/hr (e.g., about 390 cm/hr), a residence time of 1 .5 min/CV to 2.5 min/CV (e.g., about 2 min/CV) or both; optionally wherein the rAAV vector is a rAAV3B vector; and optionally wherein the AEX stationary phase is POROS™ 50 HQ. In some embodiments, at least step vi) (i.e., loading) is performed at a linear velocity of 110 cm/hr to 150 cm/hr (e.g., about 130 cm/hr), a residence time of 4.0 min/CV to 8.0 min/CV (e.g., about 6 min/CV) or both. In some embodiments, material eluted from the stationary phase during gradient elution comprises a rAAV vector to be purified. One of ordinary skill will understand that the order of the above steps may be varied.

G. Fraction Collection, Neutralization and Pooling

[0276] A method of purifying a rAAV vector (e.g., rAAV3B or other rAAV) from a solution (e.g., an affinity eluate) by AEX comprises collecting at least one fraction of eluate from an AEX column to recover and enrich for full capsids, optionally during a gradient elution. Empty capsids may be recovered in an AEX column flow-through, that is, the unbound fraction. In some embodiments, empty capsids may be recovered in a flow-through of an empty capsid wash solution (e.g., 25 mM bis-tris propane, 90 mM NaCI, pH 9.4). In some embodiments, full capsids, intermediate capsids or both are recovered in in the center of the elution peak (e.g., from 32% buffer B to 52% buffer B, e.g., from 160 mM to 260 mM sodium acetate) and empty capsids are recovered in the shoulders of the elution peak.

[0277] During an elution (e.g., a gradient elution) of an AEX method of purifying a rAAV vector, eluate from an AEX column may be collected in discrete fractions of a particular volume, and/or with a particular attribute (e.g., absorbance at a particular wavelength). For example, a volume of eluate such as 1 mL to 4 L, e.g., 1 mL to 10 mL, 1 mL to 3 L, 1 mL to 2 L, 1 mL to 1 L, 1 mL to 100 mL, 10 mL to 50 mL, 50 mL to 100 mL, 100 mL to 250 mL, 250 mL to 500 mL, 500 mL to 1 L, 1 L to 1 .5 L, 1 .5 L. to 2 L, 2 L to 3 L, 3 L to 4 L, or more (e.g., about 1 mL, 5 mL, 10 mL, 100 mL, 500 mL, 1 L, 2 L, 3 L, 4 L etc.) or specific CV equivalents such as 1/8 of a CV to 10 CV, e.g., 1/8 of a CV to 1 CV, 1 CV to 2 CV, 2 CV to 5 CV, 5 CV to 8 CV, 8 CV to lO CV or more (e.g., 1/8 of a CV, 1/4 of a CV, 1/3 of a CV, 1/2 of a CV, 1 CV, 2 CV, 3 CV, 4 CV, 5 CV, 6 CV, 7 CV, 8 CV, 9 CV or more) of eluate may be collected from an AEX column during a chromatography step (e.g., gradient elution). In some embodiments, a volume of eluate of about 1/2 CV may be collected from an AEX column during a chromatography step. In some embodiments, collecting at least one fraction of eluate from an AEX column during a chromatography step (e.g., a gradient elution) comprises collecting the eluate when an absorbance (e.g., absorbance at 260 nm and/or 280 nm) of a column-flow through reaches an absorbance threshold (e.g., > 0.5 mAU/mm path length, e.g., 5 mAU/mm path length, e.g., 10 mAU/mm path length). In some embodiments, collecting at least one fraction of eluate from an AEX column during a chromatography step (e.g., a gradient elution) comprises collecting the eluate when a gradient elution solution comprises a particular percentage of an elution buffer, for example when the gradient elution solution comprises about 30% to about 35% (e.g., about 32%) to about 50% to about 55% (e.g., about 52%) of the second elution buffer (e.g., buffer B). In some embodiments, a second elution buffer (e.g., buffer B) comprises 500 mM sodium acetate, 100 mM Tris, 0.01% P188, pH 8.9. In some embodiments, 20 fractions of 0.5 CV are collected when i) a UV signal about 5 mAU/mm path length is detected or ii) a gradient reaches 32% buffer B (e.g., 500 mM sodium acetate, 100 mM tris, 0.01% P188, pH 8.9), whichever comes first.

[0278] In some embodiments, an eluate is collected in multiple fractions (e.g., 5 fractions, 10 fractions, 20 fractions or more) of a particular volume (e.g., 1/3 CV, 1/2 CV). In some embodiments, an eluate is collected as a single fraction. In some embodiments, an eluate is collected in a single fraction when the A280 of the eluate is > 0.5 mAU, and optionally collected for about 2.3 CV.

[0279] In some embodiments, collecting at least one fraction eluate from an AEX column comprises measuring an absorbance at 260 nm (A260) and/or absorbance at 280 nm (A280) of the eluate collected from the column, optionally during a gradient elution. In some embodiments, measuring an absorbance (e.g., at A260 or A280) of an AEX eluate is performed in-line with collecting the at least one fraction eluate. In some embodiments, when an eluate collected from an AEX column during a chromatography elution (e.g., a gradient elution) has an A280 of 0.5 to 10 mAU/mm path length, at least one fraction of eluate is collected. In some embodiments, collecting eluate from an AEX column comprises collecting at least one fraction of eluate with a volume of > 1/3 of a CV. In some embodiments, collecting at least one fraction of eluate (e.g., a first fraction of eluate) from an AEX column, optionally during a gradient elution, comprises collecting at least one fraction of eluate when the A280 of the eluate is > 0.5 mAU/mm path length, and wherein a volume of the at least one fraction of eluate is >1/3 of a CV.

[0280] In some embodiments, one to 25 fractions, e.g., 1 to 5 fractions, 1 to 10 fraction, 5 to 15 fractions, 10 to 20 fractions or 15 to 25 fractions of eluate are collected from an AEX column, optionally during a gradient elution. In some embodiments, at least one, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, or more, fractions of eluate are collected from an AEX column. In some embodiments, at least 20 fractions of eluate, each with a volume of about 1/2 of a CV, are collected from an AEX column, optionally during a gradient elution.

[0281] In some embodiments, a method of purifying a rAAV vector (e.g., rAAV3B or other rAAV) from an affinity eluate by AEX comprises collecting the first of about 20 fractions of eluate from an AEX column, optionally during a gradient elution, when a percentage of a second elution buffer (e.g., buffer B) of the gradient elution solution is about 30% to about 35% (e.g., about 32%) and continuing the collecting until the percentage of a second elution buffer (e.g., buffer B) is about 50% to 55% (e.g., about 52%) of the gradient elution solution, and wherein each fraction has a volume of about 1/2 of a CV.

[0282] In some embodiments, a method purifying a rAAV vector (e.g., rAAV3B or other rAAV) from an solution (e.g., an affinity eluate) by AEX comprises adjusting a pH of at least one fraction of eluate collected from an AEX column, optionally during a gradient elution. In some embodiments, adjusting a pH of at least one fraction of eluate is referred to as a neutralization step. In some embodiments, a pH of at least one fraction of eluate collected from an AEX column is pH 8.5 to 9.1 prior to pH adjustment. In some embodiments, a pH of at least one fraction of eluate is adjusted to a pH of 7.5 to 7.7 (e.g., about pH 7.6).

[0283] In some embodiments, a pH of at least one fraction of eluate collected from an AEX column is adjusted to a pH of about 7.5 to 7.7 by collecting the at least one faction into a vessel comprising about 0.01 CV to 0.1 CV (e.g., about 0.066 CV) of a solution comprising 50 mM to 500 mM, e.g., about 50 mM to 100 mM, 50 mM to 400 mM, 50 mM to 300 mM, 50 mM to 200 mM, 100 mM to 200 mM, 100 mM to 300 mM, 200 mM to 300 mM, 300 mM to 400 mM, or 400 mM to 500 mM sodium citrate, pH 3.0 to 4.0 (e.g., about 3.5). In some embodiments, adjusting a pH of at least one fraction of eluate collected from an AEX column, optionally during a gradient elution, comprises adjustment of the pH to 7.5 to 7.7 (e.g., about pH 7.6) by collecting the at least one fraction into a vessel comprising about 0.01 CV to 0.1 CV (e.g., about 0.066 CV) of a solution comprising about 250 mM sodium citrate, pH 3.5.

[0284] In some embodiments, a method of purifying a rAAV vector (e.g., rAAV3B or other rAAV) from a solution (e.g., an affinity eluate) by AEX comprises measuring an absorbance of at least one fraction of eluate collected from an AEX column, optionally during a gradient elution. In some embodiments, an absorbance of at least one fraction of eluate is measured using analytical size exclusion chromatography (SEC) in a high performance liquid chromatography (HPLC) system, and measuring the absorbance at one or more wavelengths (e.g., 260 nm and/or 280 nm).

[0285] In some embodiments, measuring an absorbance of at least one fraction of eluate collected from an AEX column comprises measuring the absorbance at 260 nm (A260) and 280 nm (A280), and optionally determining an A260/A280 ratio (when measured by SEC, the measurement may be referred to as SEC A260/A280 or A260/A280 (SEC)). An A260/A280 ratio of at least one fraction of eluate collected from an AEX column is at least 0.5 to 2.0, e.g., at least 0.5 to 0.75, 0.75 to 1 .25, 1 .0 to 1 .5, 1 .25 to 2.0, 0.5 to 1 .5, 1 .5 to 2.0 or more. An A260/A280 ratio of at least one fraction of eluate collected from an AEX column is at least 0.5, at least 0.55, at least 0.6, at least 0.65, at least 0.7, at least 0.75, at least 0.8, at least 0.85, at least 0.9, at least 0.95, at least 1 .0, at least 1.10, at least 1.11 , at least 1.12, at least 1.13, at least 1.14, at least 1.15, at least 1.16, at least 1.17, at least 1.18, at least 1 .19, at least 1 .20, at least 1.21 , at least 1 .22, at least 1 .23, at least 1 .24, at least 1 .25, at least 1 .26, at least 1 .27, at least 1 .28, at least 1 .29, at least 1 .30, at least 1 .31 , at least 1 .32, at least 1 .33, at least 1 .34, at least 1 .35, at least 1 .36, at least 1 .37, at least, 1 .38, at least 1 .39, at least 1 .40 or greater). In some embodiments, an A260/A280 ratio of at least one fraction of eluate collected from an AEX column, optionally during a gradient elution, is at least 1 .25.

[0286] In some embodiments, a method of purifying a rAAV vector (e.g., rAAV9, rAAV3B or others) from a solution (e.g., an affinity eluate) by AEX comprises measuring a % of high molecular mass species (HMMS) of at least one fraction of eluate collected from an AEX column, optionally during a gradient elution. In some embodiments, a % of HMMS is measured by SEC. In some embodiments, a % HMMS of at least one fraction of eluate collected during AEX purification of rAAV vectors produced in a 250 L to 2000 L SUB ranges from 0% to 10% (e.g., about 5%).

[0287] In some embodiments, a method of purifying a rAAV vector (e.g., rAAV9, rAAV3B or others) from a solution (e.g., an affinity eluate) by AEX comprises determining a % purity of at least one fraction of eluate collected from an AEX column, optionally during a gradient elution. In some embodiments, a % purity is determined by RP-HPLC. In some embodiments, a % purity of at least one fraction of eluate collected during AEX purification of rAAV vectors produced in a 250 L to 2000 L SUB ranges from 90% to 100 %.

[0288] In some embodiments, a method of purifying a rAAV vector (e.g., rAAV3B or other rAAV) from a solution (e.g., an affinity eluate) by AEX comprises measuring an amount of host cell DNA (HC-DNA) of at least one fraction of eluate collected from an AEX column, optionally during a gradient elution. In some embodiments, an amount of HC-DNA is measured by qPCR.

[0289] In some embodiments, a method of purifying a rAAV vector (e.g., rAAV3B or other rAAV) from a solution (e.g., an affinity eluate) by AEX comprises measuring an amount of host cell protein (HCP) of at least one fraction of eluate collected from an AEX column, optionally during a gradient elution. In some embodiments, an amount of HCP is measured by ELISA.

[0290] In some embodiments, a method of purifying a rAAV vector (e.g., rAAV3B or other rAAV) from a solution (e.g., an affinity eluate) comprises combining at least two fractions of eluate collected from an AEX column (e.g., during a gradient elution) to form a pooled eluate (also referred to herein as an “AEX pool”). In some embodiments, at least two fractions of eluate from an AEX column, each having an A260/A280 ratio (e.g., measured by SEC) of at least 0.5 to 2.0, e.g., at least 0.5 to 1 .0, 0.75 to 1 .25, 1 .0 to 1 .5, 1 .25 to 2.0, 0.5 to 1 .5, 1 .5 to 2.0 or more are pooled. In some embodiments, at least two fractions of eluate from an AEX column, each having an A260/A280 ratio (e.g., measured by SEC) of at least at least 0.9, at least 0.95, at least 1.0, at least 1.10, at least 1.11 , at least 1.12, at least 1.13, at least 1.14, at least 1.15, at least 1.16, at least 1.17, at least 1.18, at least 1.19, at least 1.20, at least 1 .21 , at least 1 .22, at least 1 .23, at least 1 .24, at least 1 .25, at least 1 .26, at least 1 .27, at least 1 .28, at least 1 .29, at least 1 .30 or greater, are combined to form a pooled eluate. In some embodiments, combining at least two fractions of eluate collected from an AEX column, optionally during a gradient elution, comprises combining at least two fractions of eluate, each having an A260/A280 ratio of > 0.99 to form a pooled eluate. In some embodiments, all consecutive fractions that would account for >2% of the total vg of the theoretical pool are pooled. The percent vg of the fraction contribution to the theoretical pool is equal to (fraction titer x fraction volume)/(summed theoretical pool titer x summer theoretical pool volume) x 100.

[0291] In some embodiments, combining at least two fractions of eluate to form a pooled eluate comprises pooling 2 to 7, 2 to 10, 2 to 15, 2 to 20 or 2 to 50 fractions of eluate collected from an AEX column, optionally during a gradient elution. In some embodiments, an A260/A280 ratio of a pooled eluate is at least 0.90 to 1 .25, 1 .0 to 1 .5, 1 .25 to 1 .75, 0.5 to 1 .5, 1 .5 to 2.0 or more. In some embodiments, an A260/A280 ratio of a pooled eluate is at least 0.9, at least 0.95, at least 1.0, at least 1.10, at least 1.11 , at least 1.12, at least 1.13, at least 1.14, at least 1.15, at least 1.16, at least 1.17, at least 1.18, at least 1.19, at least 1.20, at least 1 .21 , at least 1 .22, at least 1 .23, at least 1 .24, at least 1 .25, at least 1 .26, at least 1 .27, at least 1 .28, at least 1 .29, at least 1 .30 or greater). In some embodiments, an A260/A280 ratio of a pooled eluate is >1 .16 and is enriched for full capsids as compared to the affinity eluate or diluted affinity eluate prior to purification by AEX. In some embodiments, an A260/A280 ratio of a pooled eluate is >1 .25, and is enriched for full capsids as compared to the affinity eluate or diluted affinity eluate prior to purification by AEX.

[0292] In some embodiments, a pooled eluate comprises only a single fraction, for example, when only a single fraction meets a predetermined criterion, such as a A280 value or A260/A280 ratio. In some embodiments, a pooled eluate comprises only a single fraction, for example, when a single fraction is collected over the course of performing a gradient elution, starting at a particular point (e.g., when a particular A280 value is measured) and ending at a particular point (e.g., a particular A280 value is measured, a specific volume of eluate is collected).

[0293] In some embodiments, a pooled eluate has a pH of about 7.5 to 7.7. In some embodiments a pooled eluate has a pH of about 7.6.

[0294] In some embodiments, a method of purifying a rAAV vector (e.g., rAAV3B or other rAAV) from an affinity eluate comprises at least one step of i) collecting the first of 20 fractions of eluate from an AEX column during a gradient elution step when the first of either a) or b) occurs with a) occurring when a gradient elution solution comprises about 32% to 52% of an elution buffer (e.g., buffer B) comprising about 500 mM sodium acetate, about 100 mM Tris, about 0.01% P188, pH 8.9 and b) occurring when the A280 of the eluate is >0.5 mAU/mm path length; ii) adjusting the pH of the 20 fractions of eluate from the column to a pH of 7.5 to 7.7 (e.g., 7.6) by collecting each fraction of eluate into a vessel comprising 0.01 CV to 0.1 CV (e.g., about 0.066 CV) of a solution comprising about 200 mM to 300 mM (e.g., about 250 mM) sodium citrate, pH 3.0 to 4.0 (e.g., about 3.5); and iii) measuring an absorbance of the 20 fractions of eluate collected from the column and determining an A260/A280 ratio; iv) combining at least two fractions of eluate collected from the column to form a pooled eluate, wherein an A260/A280 of the first fraction is > 0.99 and all other fractions are all consecutive fractions that would account for >2% of the total VG titer of the theoretical pool; wherein an A260/A280 of the pooled eluate is >1 .0; wherein the volume of each fraction is equal to 0.5 CV, and wherein the at least one fraction of eluate or the pooled eluate is enriched for full capsids, and/or depleted of empty capsids, as compared to an affinity eluate or a diluted affinity eluate prior to purification by AEX.

[0295] In some embodiments, the present disclosure provides a method of purifying a rAAV (e.g., rAAV3B or other rAAV) vector by AEX, the method comprising at least one step of: i) pre-use flushing comprising application of >4.5 CV (e.g., about 5 CV) of water for injection to an AEX stationary phase in a column; ii) sanitizing comprising application of 5 CV to 10 CV (e.g., about 8 CV) of a solution comprising 0.1 M to 1 .0 M (e.g., about 0.5 M) NaOH to the AEX stationary phase in the column, optionally by upward flow; iii) regenerating comprising application of 4.5 CV to 5.5 CV (e.g., about 5 CV) of a solution comprising 1 M to 3 M (e.g., about 2 M) NaCI), 50 mM to 150 mM (e.g., about 100 mM) Tris, pH 8.5 to 9.5 (e.g., about 9) to the AEX stationary phase in the column; iv) equilibration comprising application of 4.5 CV to 5.5 CV (e.g., about 5 CV) of a solution comprising 50 mM to 150 mM (e.g., about 100 mM) Tris, 600 mM to 700 mM (e.g., about 500 mM) sodium acetate, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., about 8.9) to the AEX stationary phase in the column; v) equilibration comprising application of > 4.5 CV (e.g., about 5 CV) of an equilibration buffer comprising 100 mM to 300 mM (e.g., about 190 mM) Tris, 100 mM to 300 mM (e.g., about 190 mM) histidine, 1 mm to 20 mM (e.g., 9 mM) sodium citrate, 1 mM to 20 mM (e.g., 1 1 mM) MgCh, 10 mM to 30 mM (e.g., 18 mM) NaCI, 0.1% to 1 .0% (e.g., about 0.5%) P188, pH 8.5 to 9.5 (e.g., about 8.8) to the AEX stationary phase in the column; vi) loading the affinity eluate to an AEX stationary phase in the column, optionally wherein the eluate has been a) diluted about 4 to 7-fold (e.g., about 5-fold) with a buffer comprising 100 mM to 300 mM (e.g., about 190 mM) histidine, 100 mM to 300 mM (e.g., about 190 mM) Tris, 1 mm to 20 mM (e.g., 9 mM) sodium citrate, 1 mM to 20 mM (e.g., 11 mM) MgCh, 10 mM to 30 mM (e.g., 18 mM NaCI), 0.1 %to 1.0% (e.g., about 0.5%) P188, pH 8.5 to 9.5 (e.g., about 8.8), and optionally b) filtered through an in-line 0.1 pm to 0.45 pm (e.g., about 0.2 pm) filter prior to application to the stationary phase; vii) equilibration comprising application of 4.5 CV to 5.5 CV (e.g., about 5 CV) of an equilibration buffer comprising 50 mM to 150 mM (e.g., 100 mM) Tris, 0.5 mM to 5 mM (e.g., about 2 mM) MgCh, 0.005% to 0.015% (e.g., about 0.01 %) P188, pH 8.5 to 9.5 (e.g., 8.9) to the AEX stationary phase in the column; viii) washing comprising application of 4.5 CV to 5.5 CV (e.g., about 5 CV) of an empty capsid wash solution comprising 50 mM to 150 mM (e.g., about 100 mM) bis-tris propane, 80 mM to 100 mM (e.g., about 90 mM) NaCI pH 9.0 to 9.6 (e.g., about 9.4) to the AEX stationary phase in the column; ix) equilibration comprising application of 4.5 CV to 5.5 CV (e.g., about 5 CV) of an equilibration buffer comprising 50 mM to 150 mM (e.g., 100 mM) Tris, 0.5 mM to 5 mM (e.g., about 2 mM) MgCh, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., 8.9) to the AEX stationary phase in the column; x) performing gradient elution of material from the stationary phase in the column beginning with application of 100% of a first buffer comprising 50 mM to 150 mM (e.g., about 100 mM) Tris, 0.005% to 0.015 % (e.g., about 0.01 %) P188, pH 8.5 to 9.0 (e.g., about 8.9) and ending with application of 75% to 100% of a second buffer comprising 400 mM to 600 mM (e.g., about 500 mM) sodium acetate or sodium sulfate, 50 mM to 150 mM (e.g., about 100 mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.5 to 9.5 (e.g., pH 8.9) to the stationary phase over 30 CV to 40 CV (e.g., about 37.5 CV); xi) collecting the first of 20 fractions of eluate of 0.5 CV from the AEX column during the gradient elution step when the first of either a) or b) occurs with a) occurring when the gradient elution solution comprises about 32% to 52% of an elution buffer (e.g., buffer B) comprising 400 mM to 600 mM (e.g., about 500 mM) sodium acetate or sodium sulfate, 50 mM to 150 mM (e.g., about 100 mM) Tris, 0.005% to 0.015% (e.g., about 0.01%) P188, pH 8.9 and b) occurring when the A280 of the eluate is >0.5 mAU/mm path length; xii) adjusting the pH of the 20 fractions of eluate from the column to a pH of 7.5 to 7.7 (e.g., 7.6) by collecting each fraction of eluate into a vessel comprising 0.01 CV to 0.1 CV (e.g., about 0.066 CV) of a solution comprising about 200 mM to 300 mM (e.g., about 250 mM) sodium citrate, pH 3.0 to 4.0 (e.g., about 3.5); xiii) measuring an absorbance of the 20 fractions of eluate collected from the column and determining an A260/A280 ratio; xiv) combining at least two fractions of eluate collected from the column to form a pooled eluate, wherein an A260/A280 of the first fraction is > 0.99 and all other fractions are all consecutive fractions that would account for >2% of the total VG titer of the theoretical pool; optionally wherein at least one of steps i) - xiv) is performed at a linear velocity of 350 cm/hr to 430 cm/hr (e.g., about 390 cm/hr), a residence time of 1 .5 min/CV to 2.5 min/CV (e.g., about 2 min/CV) or both; optionally wherein the rAAV vector is a rAAV3B vector; optionally wherein an A260/A280 of the pooled eluate is >1 .0; and optionally wherein the AEX stationary phase is POROS™ 50 HQ. In some embodiments, at least step vi) (i.e., loading) is performed at a linear velocity of 110 cm/hr to 150 cm/hr (e.g., about 130 cm/hr), a residence time of 4.0 min/CV to 8.0 min/CV (e.g., about 6 min/CV) or both. In some embodiments, material eluted from the stationary phase during gradient elution comprises a rAAV vector to be purified. In some embodiments, at least one fraction of eluate or the pooled eluate is enriched for full capsids, and/or depleted of empty capsids, as compared to an affinity eluate or a diluted affinity eluate prior to purification by AEX. One of ordinary skill will understand that the order of the above steps may be varied.

H. Characterization of Pooled Eluate and Drug Substance

[0296] A method of purifying a rAAV vector (e.g., rAAV3B or other rAAV) from a solution (e.g., an affinity eluate) by AEX comprises collecting at least one fraction of eluate from an AEX column during an elution step (e.g., a gradient elution) and forming a pooled eluate enriched for full capsids as compared to a percentage of full capsids in the solution. A method of purifying a rAAV vector from a solution by AEX comprising collecting at least one fraction of eluate from the AEX column during an elution step and forming a pooled eluate further comprises filtering the pooled eluate by a method selected from the group consisting of viral filtration, ultrafiltration/diafiltration (UF/DF), filtration through a 0.2 pm filter and a combination thereof, to produce a drug substance. In some embodiments, quality attributes, including A260/A280 (e.g., as measured by SEC), percentages of full capsid, intermediate capsid and empty capsid, % purity, % HMMS, amount of HCP and/or amount of HC-DNA of a pooled eluate are not substantially different from the same quality attribute of a drug substance produced from the pooled eluate.

[0297] In some embodiments, a pooled eluate or drug substance prepared by methods disclosed herein is enriched for full capsids such that full capsids comprise 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 98%, 50% to 99%, 50% to greater than 99%, of total capsids in the pooled eluate or drug substance, and optionally wherein the capsids are measured by analytical ultracentrifugation (AUC) (Burnham B. et al. Human Gene Therapy Methods (2015) 26; 228-242). In some embodiments, a pooled eluate or drug substance prepared by methods disclosed herein is enriched for full capsids such that full capsids comprise > 70% of total capsids in the pooled eluate or drug substance. In some embodiments, a method of purifying a rAAV vector from an affinity eluate comprises increasing the percentage of full capsids from less than 50% in an affinity eluate to greater than 50% of total capsids in a pooled AEX eluate or drug substance.

[0298] A method of purifying a rAAV vector (e.g., rAAV3B or other rAAV) from a solution (e.g., an affinity eluate) by AEX comprises collecting at least one fraction of eluate from an AEX column during an elution step (e.g., a gradient elution) and forming a pooled eluate with a depleted percentage of empty capsids as compared to the percentage of empty capsids in the solution, and wherein the pooled eluate is further subjected to a method of filtration selected from the group consisting of viral filtration, ultrafiltration/diafiltration (UF/DF), filtration through a 0.2 pm filter and a combination thereof, to produce a drug substance. In some embodiments, a percentage of empty capsids in an affinity eluate comprising an rAAV vector to be purified is 40% or greater of total capsids. In some embodiments, a pooled eluate or drug substance prepared by methods disclosed herein is depleted of empty capsids such that empty capsids comprise 1 .0% to 5%, 1 .0% to 10%, 1 .0% to 15%, 1 .0% to 20%, 1 .0% to 30%, or 1 .0% to 40% of total capsids in the pooled eluate or drug substance, and optionally wherein the capsids are measured by analytical ultracentrifugation (AUC). In some embodiments, a pooled eluate or drug substance prepared by methods disclosed herein is depleted of empty capsids such that empty capsids comprise 15% to 20% of total capsids in the pooled eluate or drug substance. In some embodiments, a method of purifying a rAAV vector from an affinity eluate comprises reducing a percentage of empty capsids from 40% to 50% in an affinity eluate, to 15% to 20% of total capsids in a pooled AEX eluate or drug substance.

[0299] A method of purifying a rAAV vector (e.g., rAAV9, AAV3B or others) from a solution (e.g., an affinity eluate) by AEX comprises collecting at least one fraction of eluate from an AEX column during an elution step (e.g., a gradient elution) and forming a pooled eluate comprising intermediate capsids, and wherein the pooled eluate is further subjected to a method of filtration selected from the group consisting of viral filtration, ultrafiltration/diaf iltration (UF/DF), filtration through a 0.2 pm filter and a combination thereof, to produce a drug substance. In some embodiments, intermediate capsids comprise 1 .0% to 5%, 1 .0% to 10%, 1 .0% to 15%, 1 .0% to 20%, 1 .0% to 30%, or 1 .0% to 40% of total capsids in a pooled eluate or drug substance, and optionally wherein the capsids are measured by analytical ultracentrifugation (AUC). In some embodiments, intermediate capsids comprise 4% to 6% of total capsids in a pooled eluate or drug substance.

[0300] A method of purifying a rAAV vector (e.g., rAAV3B or other rAAV) from a solution (e.g., an affinity eluate) by AEX comprises collecting at least one fraction of eluate from an AEX column during an elution step (e.g., a gradient elution) and forming a pooled eluate or drug substance that is enriched for full capsids and depleted of empty capsids as compared to the percentage of full capsids and empty capsids in the solution comprising the rAAV vector to be purified. In addition to full capsids and empty capsids, capsids which contain a partial vector genome (also referred to as a truncated, or fragmented vector genome) and/or non-transgene-related DNA (i.e., intermediate capsids) may, in certain non-limiting exemplary embodiments, make up the balance of capsid species in a pooled eluate (e.g., a pooled AEX eluate) or drug substance.

[0301] In some embodiments, a method of purifying a rAAV vector from an affinity eluate by AEX produces a pooled eluate comprising about 80% full rAAV capsids, about 4% intermediate capsids and about 16% empty capsids of total capsids.

[0302] In some embodiments, a method of purifying a rAAV vector from an affinity eluate by AEX produces a drug substance comprising about 77% full rAAV capsids, about 7% intermediate capsids and about 16% empty capsids of total capsids.

[0303] A method of purifying a rAAV vector (e.g., rAAV3B or other rAAV) from a solution (e.g., an affinity eluate) by AEX comprises collecting at least one fraction of eluate from an AEX column during an elution step (e.g., a gradient elution) and forming a pooled eluate, and optionally a drug substance, comprising rAAV vectors that may be quantified by quantitative polymerase chain reaction (qPCR) analysis of vector genomes (VG or vg). qPCR analysis may measure copies of ITR sequence, copies of transgene sequence and/or copies of any other nucleotide sequence present in an intact vector genome.

[0304] An amount of VG present in a pooled eluate from an AEX column may be expressed as a % VG step yield which refers to the amount of VG present in a pooled eluate collected from an AEX column (i.e., an AEX pool) as a percentage of the amount of VG present in an affinity eluate prior to dilution or filtration.

[0305] A method of purifying a rAAV vector according to methods disclosed herein results in % VG step yield of >60%. A method of purifying a rAAV vector according to methods disclosed herein results in % VG step yield of 60% to 70%, 60% to 80%, 60% to 90%, 60% to 95%, 60% to 98% or 60% to 100%.

[0306] A method of purifying a rAAV vector (e.g., rAAV3B or other rAAV) from a solution (e.g., an affinity eluate) by AEX comprises collecting at least one fraction of eluate from an AEX column during an elution step (e.g., a gradient elution) and forming a pooled eluate, or drug substance, with a reduced amount of host cell protein (HCP) as compared to the amount of HCP in the solution. In some embodiments, a reduced amount of HCP in a pooled eluate, in at least one fraction of eluate, or in a drug substance, is lower than a level of quantification (LLOQ), as measured by ELISA. In some embodiments, a reduced amount of HCP in a pooled eluate, in at least one fraction of eluate, or in a drug substance, is 10 ng to 2000 ng / 1 x 10⁹ VG, 50 ng to 200 ng/ 1 x 10⁹ VG, 100 ng to 1000 ng/ 1 x 10⁹ VG or 200 to 2000 ng/ 1 x 10⁹ VG.

[0307] A method of purifying a rAAV vector (e.g., rAAV3B or other rAAV) from a solution (e.g., an affinity eluate) by AEX comprises collecting at least one fraction of eluate from an AEX column during an elution step (e.g., a gradient elution) and forming a pooled eluate, or drug substance, comprising the rAAV vector and wherein the purity of the rAAV vector is at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% as measured by, e.g., analytical reverse phase HPLC, capillary gel electrophoresis, .

[0308] A method of purifying a rAAV vector (e.g., rAAV3B or other rAAV) from a solution (e.g., an affinity eluate) by AEX comprises collecting at least one fraction of eluate from an AEX column during an elution step (e.g., a gradient elution) and forming a pooled eluate, or drug substance, with a percentage of HMMS of 0% to 10% (e.g., 4.7%). In some embodiments, a percentage of HMMS is measured by size exclusion chromatography (SEC).

[0309] A method of purifying a rAAV vector (e.g., rAAV3B or other rAAV) from a solution (e.g., an affinity eluate) by AEX comprises collecting at least one fraction of eluate from an AEX column during an elution step (e.g., a gradient elution) and forming a pooled eluate, or drug substance, with about 1 .0 pg to 25 pg residual HC-DNA / 1 x 10⁹ VG. In some embodiments, an amount of HC-DNA is measured by qPCR.

[0310] A method of purifying a rAAV vector (e.g., rAAV3B or other rAAV) from a solution (e.g., an affinity eluate) by AEX comprises collecting at least one fraction of eluate from an AEX column during an elution step (e.g., a gradient elution) and forming a pooled eluate, or drug substance, with an A260/A280 of > 1.16 (e.g., about 1.33). In some embodiments, an A260/A280 is measured by size exclusion chromatography (SEC).

[0311] A method of purifying a rAAV vector (e.g., rAAV9, rAAV3B or others) from a solution (e.g., an affinity eluate) by AEX comprises collecting at least one fraction of eluate from an AEX column during an elution step (e.g., a gradient elution) and forming a pooled eluate, wherein the pooled eluate is subjected to a method of filtration selected from the group consisting of viral filtration, ultrafiltration/diafiltration (UF/DF), filtration through a 0.2 pm filter, and a combination thereof, to produce a drug substance suitable for production of a therapeutic drug product. In some embodiments, the drug substance is suitable for administration to a human subject to treat a disease, disorder or condition (e.g., Wilson disease). In some embodiments, the rAAV vector is an AAV3B vector.

I. AEX Stationary phase Regeneration

[0312] Following elution (e.g., gradient elution) and collection of at least one fraction of eluate comprising a full rAAV capsid from an AEX column, additional steps may be performed to prepare the column stationary phase for further rAAV purification runs. Such steps may include, for example, sanitization, equilibration, regeneration, flush and/or storage. One of skill in the art will understand that one or more steps may be performed, in varying order and frequency.

[0313] A method of regenerating AEX stationary phase in a column for use in further rAAV purification runs comprises post-use sanitizing of the stationary phase. In some embodiments, post use sanitizing of the stationary phase follows an elution step (e.g., a gradient elution). In some embodiments, sanitizing comprises application of a solution comprising about 0.1 M to 1 M NaOH to AEX stationary phase in a column. In some embodiments, sanitizing comprises application of a solution comprising about 0.5 M NaOH to AEX stationary phase in a column. In some embodiments, post-use sanitizing comprises application of a solution comprising about 0.5 M NaOH to AEX stationary phase in a column and use of an upward flow. In some embodiments, post-use sanitizing comprises application of 7 to 9 CV (e.g., about 8 CV) of a solution comprising 0.5 M NaOH to AEX stationary phase in a column. In some embodiments, post-use sanitizing comprises application of 7 to 9 CV (e.g. about 8 CV) of a solution comprising 0.5 M NaOH to AEX stationary phase in a column at a linear velocity of 350 to 430 cm/hr (e.g., about 390 cm/hr), a residence time of 1 .5 to 2.5 min/CV (e.g., about 2 min/CV), or both.

[0314] A method of regenerating a column stationary phase for further rAAV purification runs comprises regenerating the stationary phase (in some embodiments, such a step may be referred to as a “equilibration”). In some embodiments, regenerating a column stationary phase follows an elution step (e.g., a gradient elution). In some embodiments, regenerating comprises application of a solution comprising a salt (e.g., NaCI, sodium acetate, ammonium acetate (NH₄Acetate), MgCh and NasSC ) and buffering agent (e.g., Tris, BIS-Tris propane, diethanolamine, diethylamine, tricine, triethanolamine and/or bicine) to a stationary phase in a column. In some embodiments, regenerating comprises application of a solution comprising about 1 M to 5 M of a salt to the stationary phase. In some embodiments, regenerating comprises application of a solution comprising about 2 M NaCI to the stationary phase. In some embodiments, regenerating comprises application of a solution comprising about 1 mM to 500 mM of a buffering agent to the stationary phase. In some embodiments, regenerating comprises application of a solution comprising about 100 mM Tris to the stationary phase.

[0315] In some embodiments, regenerating comprises application of a solution with a pH of about 7.0 and 11 .0 (e.g., 9.0) to the stationary phase.

[0316] In some embodiments, regenerating comprises application of a solution comprising about 2 M NaCI, 100 mM Tris, pH 9 to AEX stationary phase in a column. In some embodiments, regeneration comprises application of 1 to 10 CV (e.g., about 5 CV) of a solution (e.g., a regeneration solution) to AEX stationary phase in a column. In some embodiments, regeneration comprises application of 4.5 to 5.5 CV (e.g., about 5 CV) of a solution comprising 2 M NaCI, 100 mM Tris, pH 9 to AEX stationary phase in a column at a linear velocity of 350 to 430 cm/hr (e.g., about 390 cm/hr), a residence time of 1 .5 min/CV to 2.5 min/CV (e.g., 2 min/CV), or both.

[0317] A method of regenerating a column stationary phase for further rAAV purification runs comprises equilibration of the stationary phase (in some embodiments, such a step may be referred to as a “regeneration step”). In some embodiments, equilibration of stationary phase in a column follows an elution step (e.g., a gradient elution). In some embodiments, equilibration of media in a column comprises application of a solution comprising about 100 mM Tris, pH 9 to AEX stationary phase in a column. In some embodiments, equilibration of a column comprises application of 1 to 10 CV (e.g., about 5 CV) of a solution (e.g., an equilibration solution) to AEX media in a column. In some embodiments, equilibration of a column comprises application of 4.5 to 5.5 CV (e.g., about 5 CV) of a solution comprising 100 mM Tris, pH 9 to AEX stationary phase in a column at a linear velocity of 350 to 430 cm/hr (e.g., about 390 cm/hr), a residence time of 1 .5 min/CV to 2.5 min/CV (e.g., 2 min/CV), or both.

[0318] A method of regenerating a column stationary phase for further rAAV purification runs comprises post-use flushing of the stationary phase. In some embodiments, post-use flushing of a column follows an elution step (e.g., a gradient elution). In some embodiments, post-use flushing of a column comprises application of water for injection (e.g. purified water) to an AEX stationary phase in a column. In some embodiments, post-use flushing of a column comprises application of >4.5 CV (e.g., about 5 CV) of water for injection to an AEX stationary phase in a column. In some embodiments, post-use flushing of a column comprises application of 1 to 10 CV (e.g., about 5 CV) of a solution comprising water for injection to an AEX stationary phase in a column at a linear velocity of 350 to 430 cm/hr (e.g., about 390 cm/hr), a residence time of 1 .5 min/CV to 2.5 min/CV (e.g., 2 min/CV) or both.

[0319] A method of regenerating a column stationary phase for further rAAV purification runs comprises applying a storage solution to the stationary phase. In some embodiments, applying a storage solution to a column follows an elution step (e.g., a gradient elution). In some embodiments, a storage solution comprising 16% to 20% ethanol (e.g., about 17.5%) is applied to an AEX stationary phase in a column. In some embodiments, 1 to 10 CV (e.g., about 5 CV) of a storage buffer are applied to an AEX stationary phase in a column. In some embodiments, applying a storage solution to a column comprises application of 4.5 to 5.5 CV (e.g., about 5 CV) of a solution comprising 17.5% ethanol to AEX stationary phase in a column, at a linear velocity of 230 to 290 cm/hr (e.g., about 260 cm/hr), a residence time of 1 .5 to 2.5 min/CV (e.g., about 2 min/CV), or both.

[0320] A method of regenerating a column stationary phase for further rAAV purification runs, the method comprising a step of: i) post-use sanitizing comprising application of 14.4 to 17.6 CV (e.g. about 16 CV) of a solution comprising about 0.5 M NaOH to the stationary phase; ii) regenerating comprising application of 4.5 to 5.5 CV (e.g., about 5 CV) of a solution comprising about 2 M NaCI, 100 mM Tris, pH 9 to the stationary phase; iii) equilibration comprising application of 4.5 to 5.5 CV (e.g., about 5 CV) of a solution comprising about 100 mM Tris, pH 9 to the stationary phase; iv) post-use flushing comprising application of 4.5 to 5.5 CV (e.g., about 5 CV) of water for injection to the stationary phase; and/or v) applying a storage solution to the stationary phase comprising application of 2.7 to 3.3 CV (e.g., about 3 CV) of a storage solution comprising about 17.5% ethanol to the column; wherein at least one of steps i) - v) is performed at a linear velocity of 270 to 330 cm/hr (e.g., about 300 cm/hr), a flow rate of 1 .5 to 2.0 L/min (e.g., about 1 .8 L/min) through a 6.0 to 6.6 L (e.g., 6.4 L) column or about 314 mL/min through a 1 .3 L column, and/or a residence time of 3.5 to 4.5 min/CV (e.g., about 4 min/CV) and wherein the stationary phase is AEX stationary phase, optionally POROS™ 50 HQ stationary phase.

[0321] A method of regenerating AEX stationary phase for further rAAV purification runs comprises application of an ethanol washout solution to the stationary phase prior to the first step of a method of purifying a rAAV vector (i.e., prior to sanitization, prior to equilibration, etc.). In some embodiments, an ethanol washout solution comprises about 20 mM Tris, pH 9. In some embodiments, application of an ethanol washout solution to the column stationary phase comprises application of 8 to 12 CV (e.g., about 10 CV) of a solution comprising about 20 mM Tris, pH 9 to AEX stationary phase. In some embodiments, application of an ethanol washout solution to AEX stationary phase comprises application of 8 to 12 CV (e.g., about 10 CV) of a solution comprising about 20 mM Tris, pH 9 to AEX stationary phase at a velocity of 100 to 1000 cm/hr (e.g., about 600 cm/hr) and/or with a residence time of 1 to 10 min/CV (e.g., about 1.5 min/CV). 7. Equivalents

[0322] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the disclosure. The foregoing description and Examples detail certain exemplary embodiments of the disclosure. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the disclosure may be practiced in many ways and the disclosure should be construed in accordance with the appended claims and any equivalents thereof.

[0323] All references cited herein, including patents, patent applications, papers, textbooks, and the like, and the references cited therein, to the extent that they are not already, are hereby incorporated herein by reference in their entirety.

EMBODIMENTS

[0324] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following embodiments (E).

E1 . A method of purifying an rAAV vector by AEX, the method comprising a step of: i) loading a solution comprising the rAAV vector to be purified onto a stationary phase in a column; ii) contacting the stationary phase with an empty capsid wash (ECW) solution; iii) performing gradient elution of material from the stationary phase in the column wherein a percentage of a first gradient elution buffer is varied in a manner inversely proportional to variation in a percentage of a second gradient elution buffer; and iv) collecting at least one fraction of eluate from the column during the gradient elution.

E2. The method of E1 , wherein the solution comprising a rAAV vector to be purified comprises full capsids and empty capsids.

E3. The method of E1 or E2, wherein the ECW solution removes bound empty capsids from the stationary phase.

E4. The method of any one of E1 -E3, wherein the ECW solution removes 10% or more of the bound empty capsids from the stationary phase.

E5. The method of any one of E1 -E5, wherein the ECW solution removes 10% or more of the VP from the stationary phase.

E6. The method of any one of E1 -E4, wherein the ECW solution does not remove bound full capsids from the stationary phase.

E7. The method of any one of E1 -E6, wherein the ECW solution removes 5% or less of the bound full capsids from the stationary phase.

E8. The method of any one of E1 -E7, wherein the ECW solution removes less than 5% of VG from the stationary phase.

E9. The method of any one of E1 -E8, wherein the ECW solution comprises a buffer and a salt. E10. The method of E9, wherein the buffer is selected from the group consisting of Tris (e.g., a mixture of Tris Base and Tris-HCI), BIS-Tris propane (BTP), diethanolamine, diethylamine, tricine, triethanolamine and a combination thereof.

E11 . The method of any one of E1 -E10, wherein the ECW solution comprises 1 mM to 20 mM, 1 mM to 30 mM, 1 mM to 40 mM, 1 mM to 50 mM, 5 mM to 20 mM, 5 mM to 30 mM, 5 mM to 40 mM, 5 mM to 50 mM, 10 mM to 20 mM, 10 mM to 30 mM, 10 mM to 40 mM, 10 mM to 50 mM, 15 mM to 30 mM, 15 mM to 40 mM, 15 mM to 50 mM, 20 mM to 30 mM, 20 mM to 40 mM or 20 mM to 50 mM of the buffer.

E12. The method of any one of E1 -E11 , wherein the ECW solution comprises 20 mM to 30 mM bis-tris-propane.

E13. The method of any one of E1 -E12, wherein the ECW solution comprises about 25 mM bis-tris propane.

E14. The method of E9, wherein the salt is selected from the group consisting of sodium chloride (NaCI), sodium acetate (NaAcetate,CH₃COONa), ammonium acetate (NH₄Acetate), magnesium chloride (MgCh), sodium citrate, sodium sulfate (NasSC ) and a combination thereof.

E15. The method of any one of E1 -E14, wherein the ECW solution comprises 1 mM to 50 mM, 1 mM to 60 mM, 1 mM to 70 mM, 1 mM to 80 mM, 1 mM to 90 mM, 1 mM to 100 mM, 1 mM to 120 mM, 1 mM to 140 mM, 1 mM to 160 mM, 1 mM to 180 mM, 1 mM to 200 mM, 1 mM to 220 mM, 1 mM to 240 mM, 1 mM to 250 mM, 50 mM to 75 mM, 50 mM to 100 mM, 50 mM to 150 mM, 50 mM to 200 mM, 50 mM to 250 mM, 75 mM to 100 mM, 75 mM to 150 mM, 75 mM to 200 mM, 75 mM to 250 mM, 80 mM to 100 mM, 100 mM to 200 mM or 100 mM to 250 mM of salt.

E16. The method of any one of E1 -E15, wherein the ECW solution comprises 80 mM to 100 mM NaCI.

E17. The method of any one of E1 -E16, wherein the ECW solution comprises about 95 mM NaCI.

E18. The method of any one of E1 -E17, wherein the ECW solution comprises 0 mM to 10 mM, 0 mM to 7.5 mM, 2 mM to 8 mM or 4 mM to 6 mM MgCh.

E19. The method of E17, wherein the ECW solution comprises about 0 mM or about 5 mM MgCh.

E20. The method of any one of E1 -E19, wherein the ECW solution has a pH of 8.6 to 9.6.

E21 . The method of any one of E1 -E20, wherein the ECW solution has a pH of 9.3 to 9.5.

E22. The method of any one of E1 -E21 , wherein the ECW solution has a pH of about 9.4

E23. The method of any one of E1 -E22, wherein the ECW solution has a conductivity of <

11 mS/cm.

E24. The method of any one of E1 -E23, wherein the ECW solution has a conductivity of 9.3 mS/cm to 11 .6 mS/cm.

E25. The method of any one of E1 -E24, wherein the ECW solution comprises about 20 mM to about 30 mM bis-tris propane, about 80 mM to 100 mM NaCI and has a pH of about 9.3 to 9.5 and a conductivity of 9.3 mS/cm to 11 .6 mS/cm.

E26. The method of any one of E1 -E25, wherein the ECW solution comprises about 25 mM bis-tris propane, about 90 mM NaCI and has a pH of about 9.4.

E27. The method of any one of E1 -E26, wherein the stationary phase is contacted with 1 to 10 column volumes (CV) of ECW solution.

E28. The method of any one of E1-E27, wherein the stationary phase is contacted with 4 to 6 column volumes (CV) of ECW solution

E29. The method of any one of E1 -E28, wherein the stationary phase is contacted with about 5 CV of ECW solution.

E30. The method of any one of E1 -E29, wherein the stationary phase is contacted with the ECW solution for a residence time of 1 to 10 minutes/column volume (min/cv) of ECW solution.

E31 . The method of any one of E1 -E30, wherein the stationary phase is contacted with the ECW solution for a residence time of about 2 minutes/column volume (min/cv).

E32. The method of any one of E1 -E31 , wherein the stationary phase is contacted with the ECW solution at a linear velocity of about 350 to 430 cm/hr.

E33. The method of any one of E1 -E32, wherein the stationary phase is contacted with the ECW solution at a linear velocity of about 390 cm/hr.

E34. The method of any one of E1 -E33, wherein the stationary phase is contacted with an equilibration solution before the stationary phase is contacted with the ECW solution.

E35. The method of any one of E1 -E34, wherein the stationary phase is contacted with an equilibration solution after the stationary phase is contacted with the ECW solution.

E36. The method of any one of E1 -E35, wherein the stationary phase is contacted with an equilibration solution before and after the stationary phase is contacted with the ECW solution.

E37. The method of any one of E34-E36, wherein the equilibration solution comprises a buffer, a salt and a detergent.

E38. The method of E37, wherein the buffer is Tris.

E39. The method of E37 or E38, wherein the salt is MgCh.

E40. The method of any one of E37 to E39, wherein the detergent is poloxamer 188 (P188).

E41 . The method of any one of E34-E40, wherein the equilibration solution comprises 50 mM to 150 mM Tris.

E42. The method of any one of E34-E41 , wherein the equilibration solution comprises 0.5 mM to 5 mM MgCh.

E43. The method of any one of E34-E42, wherein the equilibration solution comprises 0.005% to 0.015% P188.

E44. The method of any one of E34-E43, wherein the equilibration solution has a pH of 8.5 to 9.9.

E45. The method of any one of E34-E44, wherein the equilibration solution comprises about 100 mM Tris, about 2 mM MgCh, and about 0.01% P188 and has a pH of about 8.9.

E46. The method of any one of E1 -E45, wherein the SEC A260/A280 of the ECW solution after contact with the stationary phase is less than 0.80, less than 0.75, less than 0.70, less than 0.65, less than 0.60, less than 0.55 or less than 0.50.

E47. The method of any one of E1 -E46, wherein the % VP in the ECW solution after contact with the stationary phase is greater than 5%, greater than 10%, greater than 15%, greater than 20%, greater than 25% of the total VP in the solution comprising the rAAV capsid.

E48. The method of any one of E1 -E47, wherein the % VG in the ECW solution after contact with the stationary phase is less than 10%, less than 8%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% of the total VG in the solution comprising the rAAV capsid.

E49. The method of any one of E1 -E48, wherein the VP titer of the ECW solution after contact with the stationary phase is less than 1 .5E+12 VP/mL, less than 1 .OE+12 VP/mL, less than 9.0E+11 VP /mL, less than 8.0E+11 VP /mL, less than 7.0E+11 VP /mL, less than 6. OE+11 VP /mL, less than 5. OE+11 VP /mL or less than 4. OE+11 VP /mL.

E50. The method of any of E1 -E49, wherein the VP titer of the ECW solution after contact with the stationary phase is less than 1.12E+12 VP/mL.

E51 . The method of any one of E1 -E50, wherein the at least one fraction of eluate is 10 to 20 fractions.

E52. The method of any one of E1 -E51 , wherein the at least one fraction of eluate has a volume of 0.5 CV.

E53. The method of any one of E1 -E52, wherein the at least one fraction of eluate is collected into a vial comprising 0.066 CV of 250 mM sodium citrate, pH 3.5.

E54. The method of E53, wherein the at least one fraction of eluate may be stored at 2- 8°C for up to 8 days.

E55. The method of any one of E1 -E54, wherein the at least one fraction of eluate meets predefined pooling criteria.

E56. The method of E55, wherein the predefined pooling criteria comprises pooling i) a first fraction of eluate that has an SEC A260/A280 ratio greater than or equal to 0.99 and ii) all subsequent fractions of eluate that would account for greater than or equal to 2% of a total VG titer of a theoretical pool.

E57. The method of E55 or E56, wherein a number of fractions of eluate that meet predefined pooling criteria is increased as compared to a number of factions of eluate that meet predefined pooling criteria when the stationary phase is not contacted with the ECW.

E58. The method of E57, wherein the number of fractions of eluate that meet the predefined pooling criteria are combined to form a pooled eluate.

E59. The method of E58, wherein the % of full capsids in the pooled eluate is increased as compared to the % of full capsids in a pooled eluate when the stationary phase is not contacted with the ECW solution.

E60. The method of E59, wherein the % of full capsids in the pooled eluate is greater than or equal to 50%.

E61 . The method of any one of E58-E60, wherein the pooled eluate has an A260/A280 ratio of at least 1 .0, at least 1 .1 , at least 1 .16, at least 1 .2, at least 1 .25, at least 1 .3, at least 1 .35, at least 1 .4, at least 1 .45 or at least 1 .5.

E62. The method of any one of E58-E61 , wherein the pooled eluate has an A260/A280 ratio of 1.16 or greater.

E63. The method of any one of E58-E62, wherein the pooled eluate has a % VG step yield of at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%.

E64. The method of any one of E58-E63, wherein the pooled eluate has a %VG step yield of 60% or greater.

E65. The method of any one of E58-E64, wherein the pooled eluate comprises greater than 4.0E+13 VG/L. E66. The method of any one of E1 -E65, wherein the solution comprising the rAAV vector to be purified is an affinity eluate.

E67. The method of E66, wherein the affinity eluate is generated from purification of a rAAV vector produced in a single use bioreactor with a volume of 10 mL to 2000 L.

E68. The method of E66 or E67, wherein the eluate is generated from purification of a rAAV vector produced in a single use bioreactor with a volume of 250 mL.

E69. The method of any one of E66-E68, wherein the eluate is generated from purification of a rAAV vector produced in a single use bioreactor with a volume of 2000 L.

E70. The method of E69, wherein the diluted affinity eluate is spiked with 1 M MgCh to achieve a final concentration of 23 mM to 27 mM (e.g., 25 mM) MgCh.

E71 . The method of any one of E1 -E70, wherein the solution comprising the rAAV vector to be purified is a diluted affinity eluate, wherein the affinity eluate has been diluted 4 to 7-fold with a dilution solution comprising 100 mM to 300 mM histidine, 100 mM to 300 mM Tris, 1 mM to 20 mM sodium citrate, 1 mM to 20 mM MgCh, 10 mM to 30 mM NaCI, and 0.1 % to 1% P188 and has a pH of 7 to 9.2 and a conductivity of 6.0 mM to 6.4 mS/cm.

E72. The method of any one of E1 -E70, wherein the solution comprising the rAAV vector to be purified is a diluted affinity eluate, wherein the affinity eluate has been diluted 4 to 7-fold with a dilution solution comprising 190 mM histidine, 190 mM Tris, 9 mM sodium citrate, 11 mM MgCh and 18 mM NaCI, and 0.5% P188 and has a pH of 8.8 and a conductivity of 6.4 mS/cm.

E73. The method of E71 or E72, wherein the diluted affinity eluate is further filtered.

E74. The method of E73, wherein the diluted affinity eluate is further filtered with an in-line filter.

E75. The method of any one of E1 -E74, wherein performing gradient elution of material from the stationary phase comprises application of 30 to 40 CV at least the first gradient elution buffer, the second gradient elution buffer or a mixture of both to the stationary phase.

E76. The method of E75, wherein over the course of the gradient elution, a percentage of a first gradient elution buffer is varied in a manner inversely proportional to a percentage of a second gradient elution buffer.

E77. The method of E75 or E76, wherein the first gradient elution buffer and the second gradient elution buffer comprise a component selected from the group consisting of a buffering agent, a salt, a detergent, and a combination thereof.

E78. The method of E77, wherein the buffering agent is selected from the group consisting of Tris, BIS-Tris propane, diethanolamine, diethylamine, tricine, triethanolamine and/or bicine.

E79. The method of E77 or E78, wherein the salt is selected from the group consisting of sodium chloride, sodium acetate, ammonium acetate, magnesium chloride, sodium sulfate and a combination thereof.

E80. The method of any one of E77-E79, wherein the detergent is selected from the group consisting of poloxamer 188 (P188), Triton X-100, polysorbate 80 (PS80), Brij-35, nonyl phenoxypolyethoxylethanol (NP-40) and a combination thereof.

E81 . The method of any one of E77-E80, wherein the first gradient elution buffer (e.g., buffer A) comprises about 50 mM to about 150 mM (e.g., about 100 mM) Tris, about 0.005% to about 0.015% (e.g., about 0.01%) P188 and has a pH of about pH 8.5 to 9.5 (e.g., about 8.9). E82. The method of any one of E77-E81 , wherein the second gradient elution buffer (e.g., buffer B) comprises about 400 mM to about 600 mM (e.g., about 500 mM) sodium acetate, about 50 mM to about 150 mM (e.g., about 100 mM) Tris, about 0.005% to about 0.015% (e.g., about 0.01%) P188 and has a pH of about pH 8.5 to 9.5 (e.g., about 8.9).

E83. The method of any one of E77-E82, wherein performing the gradient elution comprises application of about 37.5 CV of the first gradient elution buffer, the second gradient elution buffer or both to the stationary phase.

E84. The method of any one of E75-E83, wherein at the start of the gradient elution the percentage of the first gradient elution buffer (e.g., buffer A) is 50% to 100% and at the end of the gradient elution the percentage of the second gradient elution buffer, (e.g., buffer B) is 50% to 100%, and wherein optionally 30 to 40 CV of the first gradient elution buffer, the second gradient elution buffer or a mixture of both are applied to the stationary phase during the gradient elution.

E85. The method of any one of E75-E84, wherein at the start of the gradient elution the percentage of the first gradient elution buffer (e.g., buffer A) is 100% and at the end of the gradient elution the percentage of the second gradient elution buffer, (e.g., buffer B) is 75%, and wherein optionally 30 to 40 CV of the first gradient elution buffer, the second gradient elution buffer or a mixture of both are applied to the stationary phase during the gradient elution.

E86. The method of any one of E75-E85, wherein at the start of the gradient elution the percentage of the first gradient elution buffer (e.g., buffer A) is 100% and at the end of the gradient elution the percentage of the second gradient elution buffer, (e.g., buffer B) is 75%, wherein the percentage of the second gradient elution buffer increases at a rate of 2% CV; and wherein 37.5 CV of the first gradient elution buffer, the second gradient elution buffer or a mixture of both are applied to the stationary phase during the gradient elution.

E87. The method of any one of E75-E86, wherein a concentration of a component of the first gradient elution buffer or second gradient elution buffer increases or decreases continuously during the gradient elution; wherein a rate of increase or decrease of a concentration of a component of the first gradient elution buffer or second gradient elution buffer is equivalent to a change in concentration of a component per total CV; and wherein the rate of change in concentration of a component over a gradient elution is about 5 mM/CV to 25 mM/CV.

E88. The method of any one of E75-E87, wherein a concentration of sodium acetate increases continuously during the gradient elution; wherein a rate of increase of the concentration of sodium acetate is equivalent to a change in concentration of the sodium acetate per total CV; and wherein the rate of change in concentration of the sodium acetate during the gradient elution is about 10 mM/CV.

E89. The method of any one of E75-E88, wherein at least one fraction of eluate is collected when the gradient comprises from about 32% to 52% of the second gradient elution buffer.

E90. The method of any one of E75-E89, wherein full capsids are eluted from the stationary phase in the center of the elution peak.

E91 . The method of any one of E75-E90, wherein intermediate capsids are eluted from the stationary phase in the center of the elution peak.

E92. The method of any one of E75-E89, wherein empty capsids are recovered in a column flow-through, in the unbound and fronting shoulder of the elution peak or both. E93. The method of any one of E1 -E92, wherein the stationary phase is a resin.

E94. The method of any one of E1 -E93, where in the stationary phase is positively charged.

E95. The method of any one of E1 -E94, wherein the stationary phase is a polystyrenedivinylbenzene particle with covalently bound quaternized polyethyleneimine.

E96. The method of any one of E1 -E95, wherein the stationary phase is POROS™ 50 HQ.

E97. A method of preparing a solution comprising a rAAV vector for purification by AEX, the method comprising a step of: diluting the solution 2 to 10-fold (e.g., 5-fold) with a dilution solution comprising histidine, tris-base, tris-HCL, P188, sodium citrate, magnesium chloride and sodium chloride to form a diluted solution, wherein the ratio of sodium citrate, magnesium chloride and sodium chloride is at a molar ratio of 1 to 1 .25 to 2; wherein the pH of the diluted solution is adjusted to 8.6 to 9.0; and wherein the conductivity of the diluted solution adjust to 6.0 mS/cm to 6.8 mS/cm.

E98. The method of E98, further comprising the step of filtering the solution comprising a rAAV vector through a filter to produce a diluted and filtered solution.

E99. The method of E97 or E98, wherein the solution comprising the rAAV vector is selected from the group consisting of an affinity eluate, a supernatant from a cell lysate and a post-harvest solution.

E100. The method of any one of E97-E99, wherein the solution is neutralized prior to the step of diluting.

E101 . The method of E100, wherein the solution is neutralized by raising the pH of the solution to a range of 7.4 to 7.8.

E102. The method of E100 or E101 , wherein the solution is neutralized by raising the pH of the solution to 7.6.

E103. The method of any one of E100-E102, wherein the solution is neutralized by titration of the solution with 5% (v/v) of 1 M Tris base.

E104. The method of E100, wherein the solution is neutralized by lowering the pH of the solution to a range of 7.4 to 7.8.

E105. The method of E100 or E104, wherein the solution is neutralized by lowering the pH of the solution to 7.6.

E106. The method of E100, E104 or E105, wherein the solution is neutralized by titration of the solution with 1% (v/v) of 2 M glycine, pH 2.7.

E107. The method of any one of E97-E106, wherein the solution is spiked with MgCh prior to the step of diluting.

E108. The method of E107, wherein the solution is spiked with 1 M MgCh to achieve a final concentration of 23 mM to 27 mM MgCh prior to the step of diluting.

E109. The method of E107 or E108, wherein the solution is spiked with 1 M MgCh to achieve a final concentration of 25 mM MgCh prior to the step of diluting.

E110. The method of any one of E97-E109, wherein the solution is held at 2-8°C for 5 days or less. E111 . The method of any one of E97-E110, wherein the dilution solution comprises about 100 mM to 300 mM histidine (e.g., about 190 mM).

E112. The method of any one of E97-E111 , wherein the dilution solution comprises about 100 mM to 300 mM Tris (e.g., about 190 mM).

E113. The method of preparing a solution comprising a rAAV vector for purification by AEX of any one of E97-E112, wherein the dilution solution comprises about 1 mM to 20 mM sodium citrate (e.g., about 9 mM).

E114. The method of any one of E97-E113, wherein the dilution solution comprises about 1 mM to 20 mM MgCh (e.g., about 11 mM).

E115. The method of any one of E97-E114, wherein the dilution solution comprises 10 mM to 30 mM NaCI (e.g., about 18 mM).

E116. The method of any one of E97-E115, wherein the dilution solution comprises about 0.1% to 1 .0% P188 (e.g., about 0.5%).

E117. The method of any one of E97-E116, wherein the dilution solution comprises about 190 mM histidine, about 190 mM Tris, about 9 mM sodium citrate, about 11 mM MgCh, about 18 mM NaCI, and about 0.5% P188.

E118. The method of any one of E97-E117, wherein the dilution solution has a pH of 7 to 9.2.

E119. The method of any one of E97-E118, wherein the dilution solution has a pH of 8.6 to 9.0.

E120. The method of any one of E97-E119, wherein the dilution solution has a pH of 8.8.

E121 . The method of any one of E97-E120, wherein the dilution solution has a conductivity of 3 to 9 mS/cm.

E122. The method of any one of E97-E121 , wherein the dilution solution has a conductivity of 6.0 to 6.8 mS/cm.

E123. The method of any one of E97-E122, wherein the dilution solution has a conductivity 6.4 mS/cm.

E124. The method of any one of E97-E123, wherein the dilution solution has pH of 7.0 to 9.2 and a conductivity of 3 to 9 mS/cm.

E125. The method of any one of E97-E124, wherein the dilution solution has a pH of 8.6 to 9.0 and a conductivity of 6.0 to 6.8 mS/cm.

E126. The method of E97-E125, wherein the dilution solution has a pH of 8.8 and conductivity of 6.4 mS/cm.

E127. The method of any one of E97-E126, wherein the dilution solution comprises about 190 mM histidine, about 190 mM Tris, about 9 mM sodium citrate, about 11 mM MgCh and about 18 mM NaCI, and about 0.5% P188, has a pH of 8.6 to 9.0 and a conductivity of 6.0 to 6.8 mS/cm.

E128. The method of any one of E97-E127, wherein the dilution solution comprises about 190 mM histidine, about 190 mM Tris, about 9 mM sodium citrate, about 11 mM MgCh and about 18 mM NaCI, and about 0.5% P188, has a pH of about 8.8 and a conductivity of about 6.4 mS/cm.

E129. The method any one of E100-E128, wherein the solution comprising the rAAV vector to be purified has pH of 7.4 to 7.8 and the diluted solution has a pH of 8.6 to 9.0 (e.g., 8.8).

E130. The method of any one of E100-E130, wherein the solution comprising the rAAV vector to be purified has a conductivity of 5.0 to 7.0 mS/cm and the diluted solution has a conductivity of 6.0 to 6.8 mS/cm (e.g., 6.4 mS/cm).

E131 . The method any one of E97-E130, wherein the diluted solution has a conductivity of 6.0 to 6.8 mS/cm and a pH of 8.6 to 9.0.

E132. The method of any one of E97-E131 , wherein the solution comprising the rAAV vector is diluted 4 to 7-fold.

E133. The method of any one of E97-E132, wherein the solution comprising a rAAV vector is diluted 5-fold.

E134. The method of any one of E97-E133, wherein the solution comprising a rAAV vector is diluted in a 20 L WAVE bag or a 50 L single use mixer.

E135. The method of any one of E97-E134, wherein the solution comprising a rAAV vector is diluted until the conductivity of the diluted solution reaches a conductivity range of 6.0 to 6.8 mS/cm.

E136. The method of any one of E97-E135, wherein the solution comprising a rAAV vector is diluted until the conductivity of the diluted solution is 6.4 mS/cm.

E137. The method of any one of E97-E136, wherein the solution comprising a rAAV vector is diluted until the pH of the diluted solution reaches a pH range of 8.6 to 9.0.

E138. The method of any one of E97-E137, wherein the solution comprising a rAAV vector is diluted until the pH of the diluted solution is 8.8.

E139. The method of any one of E97-E138, further comprising a step of loading the diluted solution onto a stationary phase in a column.

£140. The method of E139, wherein the diluted solution (e.g., the load) comprises about 1 .0E+13 VG/mL resin to about 5.0E+14 vg/mL resin.

E141 . The method of E139 or £140, wherein the diluted solution comprises about 0.4E+14 VG/mL resin to about 1.3E+14 VG/mL resin.

£142. The method of any one of E139-E141 , wherein the diluted solution comprises about 5.0E+13 VG/mL resin.

£143. The method of £139 or £140, wherein the diluted solution comprises about 1 .0E+14 VG/mL to about 3.0E+14 VG/mL resin.

E144. The method of any one of E139, £140 and E143, wherein the diluted solution comprises about 2.5E+14 VG/mL resin.

E145. The method of E139, wherein the diluted solution comprises about 1 .OE+17 to 1.0E+18 (e.g., about 2.1 OE+17) total VG.

E146. The method any one of E139-E146, wherein the residence time of the loading step is about 1 to 10 min/CV.

E147. The method of any one of E139-E146, wherein the residence time of the loading step is about 6 min/CV.

E148. The method of any one of E139-E147, wherein the flow rate of the loading step is about 0.01 to 0.5 mL/min.

E149. The method of any one of E139-E148, wherein the flow rate of the loading step is about 0.03 mL/min.

E150. The method of any one of E139-E149, wherein the linear velocity of the loading step is 350 cm/hr to 430 cm/hr.

£151 . The method of any one of E139-E150, wherein the linear velocity of the loading step is 390 cm/hr. E152. The method of any one of E97-E151 , wherein greater than 95% of VG in the solution binds the stationary phase in the column.

E153. The method any one of E97-E152, wherein a column flow-through has a SEC A260/A280 ratio of less than 0.70.

E154. The method of any one of E1 -E153, wherein the rAAV vector comprises a capsid protein from an AAV serotype selected from the group consisting of AAV1 , AAV2, AAV3 (including AAV3A, AAV3B) AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhIO, AAVrh74, AAV12, AAV2i8, NP4, NP22, NP66, AAVDJ, AAVDJ/8, AAVDJ/9, AAVLK03, AAV1 .1 , AAV2.5, AAV6.1 , AAV6.3.1 , AAV9.45, RHM4-1 (SEQ ID NO:5 of WO 2015/013313), RHM15-1 , RHM15-2, RHM15-3/RHM15-5, RHM15-4, RHM15-6, AAV hu.26, AAV1 .1 , AAV2.5, AAV6.1 , AAV6.3.1 , AAV9,45, AAV2i8, AAV29G, AAV2,8G9, AVV-LK03, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N, AAVHSC1 , AAVHSC2, AAVHSC3, AAVHSC4, AAVHSC5, AAVHSC6, AAVHSC7, AAVHSC8, AAVHSC9, AAVHSC10, AAVHSC11 , AAVHSC12, AAVHSC13, AAVHSC14 and AAVHSC15.

E155. The method of any one of E1 -E154, wherein a purified rAAV vector is produced.

E156. The method of E155, wherein the purified rAAV vector is a drug substance.

E157. The method of E156, wherein the drug substance and a pharmaceutically acceptable excipient are combined to form a drug product.

E158. The method of any one of E155- E157, wherein the purified rAAV vector, the drug substance and/or the drug product is suitable for administration to a subject to treat a disease, disorder or condition.

E159. The method of E158, wherein the disease, disorder or condition is Wilson disease .

E160. The method of any one of E1 -E159, wherein the rAAV vector comprises a vector genome comprising a modified nucleic acid encoding a copper-transporting ATPase 2 with deletion of metal binding sites 1-4.

E161 . The method of E160, wherein the modified nucleic acid encodes a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:1 .

E162. The method of E160 or 161 , wherein the modified nucleic acid comprises a nucleic acid sequence 99% identical to the nucleic acid sequence of SEQ ID NO:2.

E163. The method of any one of E160-E162, wherein the vector genome comprises a minimal alpha 1 -antitrypsin promoter.

E164. The method of E163, wherein the promoter comprises or consists of the nucleic acid sequence of SEQ ID NO:3.

E165. The method of any one of E160-E164, wherein the vector genome comprises a polyadenylation (polyA) signal sequence.

E166. The method of E165, wherein the polyA signal sequence comprises or consists of the nucleic acid sequence of SEQ ID NO:4.

E167. The method of any one of E160-E166, wherein the vector genome comprises at least one ITR.

E168. The method of E167, wherein the at least one ITR comprises or consists of a nucleic acid sequence selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and a combination thereof.

E169. The method of any one of E160-E168, wherein the vector genome comprises an expression cassette, and wherein the expression cassette comprises or consists of the nucleic acid sequence of SEQ ID NO:9. E170. The method of any one of E1 -E169, wherein the rAAV vector comprises a VP1 polypeptide of AAV3B.

E171 . The method of E170, wherein the VP1 polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:10.

E172. The method of any one of E1 -E171 , wherein the rAAV vector comprises an AAV3B capsid protein and a transgene encoding the amino acid sequence of SEQ ID NO:1 .

EXAMPLES

[0325] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein

[0326] The following Examples describe development of an anion exchange chromatography (AEX) process for purification of full rAAV3B vectors.

[0327] The AEX process was developed to achieve higher full capsid enrichment (and thereby a reduction in empty capsids) of the drug substance as well as drug substance productivity (e.g., greater than 4.0E+13 vg purified /L bioreactor) and step yield (e.g., a % VG step yield greater than 60%), while maintaining all other product quality attributes (e.g., % Purity). Furthermore, the process conditions that were developed were scalable to meet rAAV production levels suitable for use of the rAAV vector in the clinic.

[0328] AEX is the most critical unit operation within the downstream process for the enrichment of full rAAV capsids in the drug substance, and thereby the therapeutic drug product. The goal was to increase the percentage of full capsids present in the drug substance to > 50%. The Examples provided below exemplify the successful development of an AEX process that produced a final drug substance with a high percentage of full capsids (e.g., > 50%) an A260/A280 of >1.16, and a high step yield (e.g., > 60%).

[0329] Development of the AEX process focused on three essential aspects to increase full capsid enrichment and yield. First, the column loading step was evaluated to determine if modulation of the buffer conditions would improve separation of empty and full capsids. It was discovered that empty capsid binding and full capsid binding to the column resin during the load phase could be individually adjusted by modulating buffer composition, pH and conductivity such that the elution pool was enriched for full rAAV capsids.

[0330] Second, an additional wash step (referred to herein as an empty capsid wash or “ECW”) was developed which removed empty capsids that bound to the column resin during the load phase and prior to the elution step. Thus, the ECW reduced the level of empty capsids that were collected in the eluate, as they were washed off the column resin prior to the elution step. The ECW was further optimized by the addition of a “sandwich wash” which comprised a wash step (also referred to as an equilibration step) immediately before and after the ECW with a solution comprising 2 mM MgCI₂. It was discovered that the MgCh in the solution stabilized AAV3B capsids, induced a confirmational change to the AAV3B capsids, or both, thereby increasing their binding affinity for POROS™ 50 HQ, and preventing capsid loss during the ECW.

[0331] Finally, elution salts were screened to evaluate potential for increased separation of empty and full capsids during the elution step. This utilized the tropic properties and other capabilities of different salts to further the separation during the elution step.

Example 1 : Empty capsid wash

[0332] An empty capsid wash (ECW) was developed to remove empty capsids that bound to the column resin prior to the elution gradient step in order to increase the percentage of full capsids in the elution pool. The pH and salt composition of the ECW were formulated to remove the weaker bound empty capsids while maintaining the binding of the more negatively charged full capsids to the resin. In prior studies, empty capsids eluted from POROS™ 50 HQ at 11 ms/cm and above in a sodium acetate gradient. Initial salt concentrations shown in Table 2 were chosen so that the empty capsid wash would have a conductivity below 11 ms/cm to preserve the %VG step yield of the eluate. Bis-Tris Propane (BTP) was employed for ECW pH buffering to enable the testing of pH conditions from 8.7 to pH 9.3 using the same buffering reagent. Dickerson et aL, (BiotechnoL J. (2021) 16, 2000015) described an AEX method using an empty capsid wash and an isocratic elution. Dickerson’s method differed significantly from the method described here, in that their method was used to purify AAV2 vectors, used a sodium chloride/magnesium chloride wash at pH 9 and a CIMmultus QA column resin.

[0333] HEK 293 cells were grown in suspension culture and transfected with 3 plasmids to produce rAAV3B vector per standard methods known in the art. HEK 293 cells were harvested, lysed, flocculated, and the resulting lysate was filtered. rAAV3B vector was purified from the clarified lysate by affinity chromatography. An affinity column was equilibrated, loaded with clarified lysate, washed, and the purified rAAV3B vector was eluted with 20 mM sodium citrate, 5 mM MgCI₂, 40 mM NaCI, 0.01% P188, pH 2.5. The rAAV3B vector comprised a vector genome with a transgene encoding the amino acid of SEQ ID NO:1 (copper transporting ATPase 2 with deletion of metal binding sites 1-4). The affinity elution pool was used in the screening studies described below.

[0334] 1.1 Loose Resin Screening

[0335] The wash conditions listed in Table 2 were screened using a loose resin format. An excess of POROS™ 50 HQ resin was buffer exchanged from 17.5% ethanol to 2 M NaCI, 100 mM Tris, pH 8.9 at a resin slurry of 50%. 50 pL of slurry were added into each well of a 96-well 0.45 pm PTFE filter plate and the loose resin procedure described in Table 1 was executed. The filter plate was centrifuged between each buffer addition and liquid was collected into a 96-well collection plate. The wash samples were assayed for viral particle (VP) titer and A₂₆o/A_28o via the size exclusion chromatography AAV (SEC_AAV) test. Table 1. Loose Resin Procedure

[0336] The results of the loose resin screen are summarized in Table 2. Within salt and pH groups, washing with a higher ionic strength solution removed more bound VP. The ECW using NaCI, at the most alkaline condition (pH 9.3) and the highest ionic strength (90 mM, Table 2, row 12) enabled high wash VP titer (1.12E+12) and low wash A₂₆o/A_28o (0.74).

Conversely, a pH 9.0 wash with the same salt (NaCI) and ionic strength (90 mM Table 2, row 6), increased the A₂₆o/A_28o up to 0.90, indicating higher relative loss of full capsids, compared to the pH 9.3 wash. These two results indicate that ECWs at higher pH force tighter full capsid binding, enabling selective empty capsid washing with more stringent solutions (e.g. higher ionic strength). Furthermore, the interplay between wash pH, ionic strength, and salt are critical determinants of wash performance. The wash composition of 25 mM Bis-Tris Propane (BTP), 90 mM NaCI pH 9.3 shown promise as a condition for selectively removing empty capsids due to the low A₂₆o/A_28o and high VP titer in this study.

[0337] Table 2. Empty Capsid Wash Loose Resin Screening Results

[0338] 1 .2 Scale up of Empty Capsid Wash

[0339] The empty capsid wash was further developed using flowing chromatography and directly compared to a baseline AEX process. The empty capsid wash was added to the baseline AEX method after Equilibration 3 and before Equilibration 4 as shown in Table 3. All experiments in this section were performed using a pre-packed 1 ml POROS™ 50 HQ column with a 5 cm bed height and a load preparation comprising a 25 mM MgCh spiked, neutralized affinity eluate pool diluted 10-20 fold with 0.5% P188, 200 mM Histidine, 200 mM Tris Base, pH 8.8, to obtain a conductivity < 2.5 mS/cm. [0340] Table 3. Empty Capsid Wash with Baseline AEX Method

[0341] The wash composition of 25 mM BTP, 90 mM NaCI pH 9.3 from the loose resin experiment described above was evaluated using affinity eluate as described above. The wash condition was loaded at a lower vector genome challenge than the control run due to limited availability of material. A summary of the results are shown in Table 4. The empty capsid wash removed 3.5% of the loaded VP but did not remove any quantifiable VG. The empty capsid wash SEC A₂₆o/A_28o was 0.57 and provided further evidence of selective empty capsid removal with minimal full capsid loss. The elution pool for the run which included the wash condition had an A₂₆o/A_28o of 1 .03, while the control run enriched to an A₂₆o/A_28o of 0.97. The control run gave a 21% VG elution step yield which was higher than the 13% elution step yield from the run which included the ECW condition. This difference in VG elution yield was likely due to the 13-fold lower column challenge on the wash run, and is inline with previous work that has shown that underloading the AEX column leads to lower yields. Although the original pH target for this condition was 9.3, the pH of the wash was measured to be 9.4 after the experiment. For this reason, a pH target of 9.4 was used for future experiments.

Table 4. Evaluation of Empty Capsid Wash using Flowing Chromatography

[0342] The NaCI concentration of the empty capsid wash was increased from 90 mM to 95 mM to increase empty capsid removal. The improvement in enrichment was not evaluated against a control, but this change was deemed low-risk due to the minimal VG loss in the 90 mM NaCI wash. An example of this wash condition is shown in Table . The empty capsid wash removed 14.9% of the loaded VPs and 0.1% of the loaded VGs and had an SEC A260/A280 of 0.60.

Table 5. Evaluation of 25 mM BTP, 95 mM NaCI pH 9.4 Wash

[0343] The pH and conductivity range of the empty capsid wash were tested further using two different compositions. A wash composition of 25 mM BTP, 105 mM NaCI, pH 9.3 was chosen to determine AEX yield when pH and conductivity conditions are not ideal for VG loss. A wash composition of 25 mM BTP, 85 mM NaCI pH 9.5 was chosen to determine enrichment when pH and conductivity conditions are not ideal for empty capsid removal. A summary of this experiment can be found in Table able 6. Both boundary conditions produced a higher yield and an increased A260/A280 in the elution pool when compared to the control run (no ECW). This study demonstrated that the performance of the 25 mM BTP, 95 mM NaCI pH 9.4 wash was robust over the designated pH and conductivity range.

Table 6. Evaluation of 25mM BTP, 95mM NaCI pH 9.4 Wash, pH and Conductivity Ranges

[0344] A wash composition of 25 mM Tris, 90 mM NaCI, pH 8.7 was also evaluated due to concerns of potential capsid deamidation at pH 9.4. This wash condition was chosen from the loose resin screening described above. Similar to the previous experiment, a wash composition of 25 mM Tris, 100 mM NaCI, pH 8.6 was chosen to determine AEX yield when pH and conductivity conditions are not ideal for VG loss and a wash composition of 25 mM BTP, 80 mM NaCI, pH 8.8 was chosen to determine enrichment when pH and conductivity conditions are not ideal for empty capsid removal. The results of this experiment are shown in Table 7. The low conductivity and high pH condition did not increase the A₂6o/A₂8oof the elution pool when compared to the control (no ECW), while the high conductivity and low pH condition had an elevated level of VGs in the wash (12%). For this reason, the wash composition of 25 mM Tris, 90 mM NaCI pH 8.7 was not considered an improvement over other conditions that were tested and the wash composition comprising 25 mM BTP, 95 mM NaCI pH 9.4 was selected.

Table 7. Evaluation of 25 mM BTP, 90 mM NaCI pH 8.7 Wash, pH and Conductivity

Ranges

Example 2: Screening, optimization and implementation of a weak binding load

[0345] 2.1 Introduction to Weak Binding Load (WBL)

[0346] AEX processes for the purification of rAAV vectors for gene therapy described in the Examples utilized POROS™ 50 HQ’s positively charged quaternary amine group to bind the negatively charged rAAV3B capsid. A baseline process involved loading a diluted affinity eluate at pH > 8.6 and < 2.5 mS/cm, resulting in binding of all full and empty capsids to the resin (no VG or VP was recovered in the unbound fraction). Gradient elution, which exploited the difference in charge between empty and full capsids, was used to separate the two capsid species. Full capsids, which are more negatively charged due to the presence of the negatively charged packaged DNA, bind more tightly to the resin than empty capsids, and elute from the column at higher conductivities. However, due to the large excess of empty capsids which bound to the POROS™ 50 HQ under load conditions of pH > 8.6 and < 2.5 mS/cm, there was incomplete separation of empty capsids from full capsids, even during shallow gradient elution.

[0347] In order to solve this problem, development of the improved AEX method described herein, focused in part on reducing empty capsid binding while maintaining full capsid binding at the load step in order to improve separation of empty capsids from full capsids during the elution step. It was discovered that by altering load conductivity and pH, empty capsids would bind weakly, or not at all, to the resin and thus the separation of empty capsids from full capsids would be improved during the AEX process. This approach is referred to herein as a weak binding load (WBL). Initially, WBL conditions were screened in a loose resin format, and top performers were selected for scale-up to column chromatography for further evaluation.

[0348] 2.2 Loose Resin Plate Screening

[0349] 2.2.1 Loose Resin Plate Screening Method Development

[0350] To enable transfer of WBL conditions from screening to scale-up, a loose resin, filter plate method was developed that closely matched flowing AEX chromatography. An Agilent CaptiVac vacuum collar and manifold was employed to drive flow through the 96-well 0.2 pm Agilent PP filter plate. Filtration was controlled using the utility vacuum pump. The manifold included a valve that could control the vacuum pressure applied to the plate. For method simplicity reasons, there was no set vacuum pressure for each step, and visual inspection of liquid flowing through the filter was completed to ensure proper filtration occurred. After resin was loaded into the 96-well filter plate, a plate shaker was used during load, equilibration, wash, and elution steps for proper mixing. Table 8 details the process in which the resin was prepared, loaded into each well, equilibrated, washed, eluted, and stripped. POROS™ 50 HQ resin was prepared in a bulk 15 mL conical tube at a 50% slurry, cleaned, and equilibrated. After the resin was resuspended at a 50% slurry in the equilibration buffer, 100 pL of slurry was added to each well for a total resin volume of 50 pL per well. AEX loads were prepared with dilution buffers of varying pH and conductivity in 1 .5 mL sample tubes, mixed, sampled, and loaded into the 96-well filter plate containing the resin. After each step as detailed in Table 8, solution from the filter plate was filtered into a 96-well collection plate. Flowthroughs (unbound load fractions), washes, elutions and strips were sampled and submitted for analysis by qPCR to determine VG titer and SEC to determine A₂₆o/A₂₈o ratio. This filter plate method was executed for experiments described in Section 2.2.3 and Section 2.2.4. Table 8. Loose Resin Filter Plate Method

[0351] 2.2.2 Dilution Buffer Preparations

[0352] Formulation of dilution buffers that achieved target load pH and conductivity was critical to the WBL approach. It was hypothesized that molar ratios of salts in the AEX load could impact vector stability and/or resin binding. Therefore, dilution buffers were designed to maintain load molar ratios of sodium citrate: magnesium chloride: sodium chloride to 1 :1 .25:2, as was used in the baseline process. To facilitate processing, a 5x dilution was used for each WBL condition, except for the baseline condition, which maintained a 15x dilution. One alternative approach for WBL screening was to use the dilution buffer from the baseline process at varying dilution factors to tune load conductivity. However, this option would not enable screening of load pH, and involved volumetric constraints that were impractical in the filter plate format. While the 5x dilution factor facilitated filter plate processing and enabled pH screening, it required formulation of a unique dilution buffer for each WBL condition. A summary of the dilution buffer preparations used in each WBL screen condition is found below in Table 9.

[0353] 2.2.3 Loose Resin Plate Screen 1 Design

[0354] Dilution buffer conductivity and pH were initially screened in a loose resin format to determine limits of empty and full capsid binding and to evaluate the utility of a WBL approach for the improved AEX process. Ranges of dilution buffer pH (7-9.2) and conductivity (3-9 mS/cm) were selected based on insights gathered from the baseline process and from the development of the ECW described in Example 1 . Notably, in the baseline AEX process load, full and empty capsids bound tightly to POROS™ 50 HQ at pH > 8.6 and < 2.5 mS/cm, and suggested selection of the lowest screened conductivity of 3 mS/cm. Empty capsids begin to elute from POROS™ 50 HQ in the baseline AEX process at a constant pH of 8.8 during sodium acetate gradient elution at 14 mS/cm, and informed the highest screened WBL conductivity of 9 mS/cm. The maximum pH of 9.2 was selected based on findings from development of the ECW (Example 1), and the hypothesis that empty/full separation is facilitated at higher pH. Therefore, pH 9.2 (more alkaline than pH 8.8 used in the baseline AEX process) was set as a screening maximum. Surprisingly, the screening WBL dilution buffer ranges of pH (7-9.2) and conductivity (3-9 mS/cm) demonstrated synergy between these parameters, revealed where full capsid binding was lost, and showed the interplay between full vs. empty capsid binding to build a %VG yield vs % Full capsid profile.

[0355] 2.2.3.1 Loose Resin Plate Screen 1 Dilution Buffer Formulations

[0356] Thirteen novel dilution buffers were formulated to bracket the desired WBL ranges of pH (7.0-9.2) and conductivity (3-9 mS/cm). Each dilution buffer was comprised of Histidine, Tris Base, Tris-HCI, P188, Sodium Citrate, Magnesium Chloride, and Sodium Chloride. As discussed above, dilution buffers were formulated to maintain constant molar ratios of sodium citrate: magnesium chloride: sodium chloride to 1 :1 .25:2, the same ratios as used in the baseline AEX process. The 13 novel buffers consisted of 3 pH levels (7.0, 8.2, and 9.2) at 4 conductivity levels (3.0, 5.0, 7.0, and 9.0 mS/cm) yielding 12 unique buffers, and a center point (pH 8.2, 6.0 mS/cm) (Table 9). Initially, 4 corner buffers and the center point were formulated, and then mixed together to make the remaining WBL buffers at desired pH and conductivity levels. Finally, to mimic the baseline AEX load preparation, 200 mM histidine, 200 mM Tris, 0.5% P188, pH 8.8 was used as the baseline dilution buffer (pH 8.8, 2.0 mS/cm).

[0357] 2.2.3.2 Loose Resin Screen 1 Results and Discussion

[0358] Table 9 reports loose resin screen conditions and results, including dilution buffer pH and conductivity, resin challenge, binding characteristics, and elution yield and SEC A₂6o/A₂8o ratio. Triplications of the centerpoint condition (pH 8.2, 6.0 mS/cm) yielded near identical results and demonstrated robustness of the WBL plate screening method. A negative control was run on POROS™ OH resin, which contains a hydroxylated functional group immobilized on the same base matrix as POROS™ 50 HQ. The negative control was fed a pH 8.8, 3.0 mS/cm load, and returned 10% VG bound, with 0% VG yield in ECW and elution fractions, and thus showed the method closed VG mass balance. The baseline AEX process run returned values of 100% VG bound to the resin, 56% VG elution step yield, and a 1 .08 elution SEC A₂₆o/ A₂₈o. These readouts were within the expected ranges, except the %VG elution yield was lower in the screening format (56%) than in an at-scale iteration of the process (e.g., 84%). Collectively, these control runs showed that the loose resin plate screening technique was robust, reproducible, and could be leveraged with confidence to screen WBL conditions.

[0359] Results from Table 9 were plotted in JMP 14 software to generate contour plots (FIG. 1 and FIG. 2) that revealed important WBL trends across the screened ranges of dilution buffer pH and conductivity. FIG. 1 (top panel) shows %VG bound to POROS™ 50 HQ and demonstrated that conductivity is a critical determinant of vector binding, while pH is less impactful. Independent of pH, conductivities < 6 mS/cm enabled high vector binding, while conductivities > 7 mS/cm significantly reduced vector binding. This phenomenon may be explained by increased competition to binding incurred at higher concentration of ions in elevated conductivities. The high % VG binders in FIG. 1 (top panel) translated to high % VG elution yields, reported in FIG. 1 (bottom panel). The data demonstrated that to obtain a satisfactory %VG elution yield (e.g., > 60%), dilution buffer conductivity should be < 6 mS/cm and dilution buffer pH can range from pH 7 to pH 9.2. [0360] FIG. 2 shows Screen 1 WBL contour plots of SEC A₂₆o/A_28o of flow through (top panel) and elution (bottom panel) fractions. FIG. 2 (top panel) shows that dilution buffer pH and conductivity can be tuned to modulate the selectivity of the binding step. Flow through fraction SEC A₂₆o/A_28o was lowest (0.60-0.72) in the range of 5 mS/cm, indicating the highest relative concentration of unbound empty capsids (highest binding selectivity). At 5 mS/cm, binding in pH 9.2 dilution buffer enabled higher selectivity than binding at pH 7.0, leading to flow through SEC A₂₆o/A_28o of 0.60 to 0.64 and 0.76 to 0.80, respectively. This phenomenon might be attributable to synergy between binding competition (higher at higher conductivity) and higher anionic nature of capsid and genome at more alkaline environments, which mediates selective binding. FIG. 2 (bottom panel) shows elution SEC A₂₆o/A_28o and that dilution buffer conductivity is a key driver to achieving AEX-based empty capsid reduction, with minimal impact from pH. Dilution buffer conductivity of 5-6 mS/cm yielded an elution pool SEC A₂₆O/A_28O of 1 .16 to 1.18, the 2^nd highest range of full capsid enrichment in the study. Maximum enrichment (SEC A₂₈o/A₂₈o of 1 .22-1 .26) was achieved via 7 mS/cm dilution buffer at pH 7, pH 8.2 and pH 9.2.

[0361] These data informed the dilution buffer specification for the high performance WBL AEX process. Leveraging dilution buffers with conductivity from 5-6 mS/cm and pH 7- 9.2 enabled sufficiently high VG step yield (> 60%), and moderately high full capsid enrichment (SEC A₂₈o/A₂₈o of 1.16-1 .20) which exceed those same metrics for the baseline process. Based on these findings, a second WBL screen was executed to elucidate the impact of a constricted dilution buffer range and resin challenge on AEX performance.

Table 9. Comprehensive Results Table - Loose Resin Plate Screen 1

[0362] 2.2.4 Loose Resin Plate Screen 2 [0363] A 2^nd WBL loose resin filter plate screen was conducted on narrowed dilution buffer ranges of pH (8.0-8.8) and conductivity (3.75-5.25 mS/cm). Additionally, resin challenges were evaluated at the pH condition of 8.4 at low, medium and high levels of 1 E+13, 5E+13 and 1 E+14 VG/mL resin, respectively. Compared to screen 1 , a simpler approach to buffer preparation was taken by creating initial dilution buffers (200 mM Histidine, 200 mM Tris, 0.5%P188) at pH of 8.0, 8.4, and 8.8, by varying Tris Base and Tris- HCI concentrations. To achieve target dilution buffer conductivity, these three solutions were spiked with 230 mM Sodium Citrate, 280 mM MgCh, 460 mM NaCI (same salt ratios as found in affinity eluate).

[0364] Table 10 shows Screen 2 data, which revealed a narrower understanding of WBL effects, compared to Screen 1 . %VG bound, %VG elution yield, and flow through SEC A₂6o/A₂8o were generally unaffected across VG resin challenges and the narrower pH and conductivity ranges. An exception to this was that more acidic dilution buffers (pH 8.0) yielded lower % VG elution yields than pH 8.4 and pH 8.8 counterparts. In-line with Screen 1 , Screen 2 data demonstrated that higher dilution buffer conductivity generated higher elution SEC A260/A280, up to a maximum of 1 .21 -1 .23 at 5.25 mS/cm. Collectively, Screen 2 confirmed the impact of dilution buffer conductivity on full vector enrichment and demonstrated robustness to narrowed dilution buffer pH and conductivity ranges and column challenge.

Table 10. Comprehensive Results Table - Loose Resin Plate Screen 2

[0365] 2.2.5 Loose Resin Plate Screen Conclusion

[0366] Loose resin WBL AEX screens proved to be a powerful tool to accelerate process development and minimize material consumption. Screen 1 and 2 revealed important AEX load behaviors. Binding selectivity was increased via careful tuning of WBL pH and conductivity. Dilution buffer conductivity was found to be a critical determinant of AEX performance, while pH was a less impactful factor. Notably, elevated conductivities increased competition of binding to the POROS™ 50 HQ resin, leading to decreased empty capsid adsorption. [0367] AEX parameters were calibrated to achieve process development goals of > 52

% full capsid (SEC A₂6o/A₂so of > -1.21) and > 60 % VG Step Yield. With these goals in mind, AEX dilution buffer specifications of 5.8 mS/cm and pH 8.8 were set. It was hypothesized that a 5.8 mS/cm dilution buffer would offer a favorable balance of high full capsid enrichment and reasonable %VG step yield. Because pH was found to be a less impactful driver of WBL performance, a dilution buffer pH of 8.8 was maintained from the baseline version of the AEX process. A resin challenge of 5.0E+13 VG/mL resin was set to afford maximum robustness. Concerted efforts were taken to formulate the novel WBL dilution buffer and scale-up the AEX process in a traditional chromatography column format (Example 3).

[0368] 2.3 Weak Binding Load AEX Dilution Buffer Formulation

WBL screening activities involved preparation of dilution buffers using stock solutions. This approach facilitated screening but would be unacceptable for large scale GMP manufacturing. Therefore, once the target dilution buffer conductivity and pH were determined, a dilution buffer from powder components had to be formulated.

To obtain a formulation for the AEX dilution buffer at 5.8 mS/cm and pH 8.8, three preparations were made via the stock solution approach. Briefly, stock solutions 230 mM Sodium Citrate, 280 mM MgCls, 460 mM NaCI (affinity salt spike buffer) and 200 mM Histidine, 200 mM Tris, 0.5%, pH 8.8 (earlier process AEX dilution buffer) were mixed together to reach the target conductivity of 5.8 mS/cm. Mixing ratios from the triplicate preparations were used to calculate an initial formulation of 190 mM Histidine, 190 mM Tris, 9 mM sodium citrate, 11 mM MgCI2, 18 mM NaCI, 0.5% P188, pH 8.8. Three additional preps in volumetric flasks were made to ensure consistency and accuracy of the buffer formulation and preparation.

A WBL dilution buffer was also formulated from powder raw materials. To test the “from powder” formulated dilution buffer, it was used in an AEX process carried out on a 0.2 mL POROS™ 50 HQ column (0.5 cm ID x 1 cm BH).

Table 11 shows results from the AEX functional assessment and demonstrates that using the “from powder” dilution buffer provided performance comparable to AEX run from stock solution AEX dilution buffer.

Table 11. AEX Functional Assessment of From Powder Dilution Buffer

a: Difference in SEC ratio from AEX load to weighted theoretical AEX pool ^p: Fractions to be included in theoretical pools were determined using baseline Pooling rules

Example 3: Scale up of WBL Method to Flowing Chromatography and Evaluation of Magnesium Chloride in Buffers Adjacent to ECW

[0369] After WBL screening experiments were completed (Example 2), the method was scaled-up to flowing chromatography. An initial AEX run was carried out on a 0.2 mL prepacked POROS™ 50 HQ column and leveraged a 5.8 mS/cm dilution buffer and a 6 min residence time during the load phase. The method used the same product contacting buffers from screening experiments (Table 8), except washes before and after the ECW did not contain 2 mM MgCh. The chromatogram revealed the ECW peak (A260 and A280 traces) was larger than prior runs, and implied that a significant amount of capsid or VG was being lost in this wash step. Conversely, in screening runs that used 2 mM MgCh in buffers before and after the ECW (Table 8), minimal VG loss was observed in said ECWs (Table 9 and Table 10).

[0370] Low concentrations (e.g. 2 mM) of Mg²⁺ has demonstrated the ability to modulate AAV empty/full selectivity in AEX-based separations (Wang et aL, Molec. Therap. Meth. Clin. DeveL (2019) 15:27-263; Khatwani et aL, Molec. Therap. Meth. Clin. DeveL (2021) 21 :548- 558), and stabilize AAV at lower concentrations compared to other salts (Wright et aL, Molec. Therap. (2005) 12(1 ):171 -178). It was discovered that 2 mM MgCh in the wash (referred to as “Wash 1” in Table 8) prior to the ECW stabilized and/or induced a confirmational change to the AAV3B capsids, increasing their binding affinity for POROS™ 50 HQ, and prevented capsid loss during said ECW. The ECW was originally developed with the ‘strong binding’ load approach in the baseline process. The ECW involves sharp increases in conductivity and pH, which strain vector binding considerably. Weakening of capsid binding in the WBL version of the AEX process offered less tolerance during the ECW as compared to the strong binding load baseline version of the AEX process.

[0371] Based on these discoveries, 2 mM MgCh was added to “sandwich” washes before and after the ECW (referred to as “Wash 1” and “Wash 2” in Table 8) to tighten capsid binding and avoid product loss (complete formula: 100 mM Tris, 2 mM MgCh, 0.01% P188, pH 8.9).

[0372] An affinity eluate comprising rAAV3B vector was spiked with MgCh, diluted 5X with a 5.8 mS/cm, pH 8.8 dilution buffer made from stock solutions, (approximate formulation was: about 190 mM Histidine, about 190 mM Tris, about 9 mM sodium citrate, about 11 mM MgCI2, about 18 mM NaCI, 0.5% P188, pH 8.8) and loaded onto a 0.2 mL pre-packed column. Table 12 shows the AEX method utilized for this run, which included the added 2 mM MgCI2 “sandwich” washes (Washes 3 and 4).

[0373] Table 12. AEX Method with 2 mM MgCh “Sandwich” Washes Adjacent to ECW

[0374] Table 13 shows results of the runs carried out with and without the 2 mM MgCh “sandwich washes”. Without the 2 mM MgCh sandwich washes, 28.7% of the loaded VG was lost during the ECW from AEX load. Addition of 2 mM MgCI₂ to “sandwich” washes reduced % VG loss in the ECW by 26.8%. Maintaining bound VG during the ECW led to an improved % VG elution yield from 45.1 % to 58.4%, in the absence and presence of 2 mM MgCI2 sandwich washes, respectively. Addition of the 2 mM MgCI₂ sandwich wash did not impact elution SEC A₂6o/A₂8o, (1 .31 , and 1 .33 for AEX runs with and without the added 2 mM MgCI2 sandwich washes, respectively). In conclusion, the 2 mM MgCI₂ “sandwich” washes reduced the % VG loss in the ECW, increased %VG elution yield and maintained elution SEC A₂₆O/A₂8O, and thus was incorporated into the improved AEX process described in following Examples.

[0375] Table 13. Analytical Comparison of AEX Runs with and without 2 mM MgCI2 Sandwich Washes

Example 4: Screening of Elution Salts

[0376] 4.1 Introduction to Elution Salt Screening

[0377] To improve the full capsid enrichment across the AEX process, seven different elution salts were chosen for screening (Designated A-G and listed in Table 15). To determine which elution salts were better performers, the maximum SEC A₂6o/A₂so ratio in the elution fractions as well as the number of elution fractions with an SEC A₂₆o/A₂₈o ratio above a threshold of 0.80 (based off an affinity pool SEC A₂₆o/A₂₈o ratio of 0.75) were compared across the conditions.

[0378] Most of the salts for screening were suggested by the literature (Urabe et aL, Molec. Therap. (2006) 13(4):823-828; Qu et aL, J Virol. Meth. (2007) 140(1 -2):183-192; Wang et aL, Molec. Therap. -Meth. Clin. DeveL (2019) 15:257-263; Dickerson et aL, BiotechnoL J. (2020) 16(1 ) :E2000015; Joshi et aL, Molec. Therap. -Meth. Clin. Devel (2021) 21 :341-356), as well as previous process development work. Sodium acetate as the baseline elution salt was chosen to compare with new potential elution salts. Salts with different properties were selected for screening, including kosmotropic nature, stabilizing nature, and compounds with different alkyl chains. Among the salts screened, sodium propionate was selected for its methyl aliphatic chain. Magnesium chloride was selected because it has demonstrated the ability to modulate AAV empty/full selectivity in AEX-based separations Wang (Molec. Therap.-Meth. Clin. DeveL (2019) 15:257-263)and stabilizes AAV at lower concentrations compared to other salts (Wright et aL, Molec. Therap. (2005) 12(1 ):171 -178). Ammonium acetate, sodium sulfate, and tetramethylammonium acetate were selected for their kosmotropic nature.

[0379] 4.2 Method

[0380] An affinity pool comprising rAAV vector was aliquoted and frozen at -80 °C post elution. Aliquots were thawed at room temperature for AEX development studies. After thawing, an aliquot was spiked with 1 M MgCI₂ to a final concentration of 25 mM MgCI₂. A 0.5 cm inner diameter (ID) x 1 cm bed height (BH) prepacked POROS™ 50 HQ 0.2 mL CV column was used for the salt screen at a target resin challenge of 1 E+13 VG/mL resin and a load dilution of 14.7X with 200 mM histidine, 200 mM tris, 0.5% P188, pH 8.8 buffer (baseline dilution process). All elution buffers were formulated to 500 mM salt concentration. However, for sodium sulfate the elution gradient length was changed to normalize for a higher buffer conductivity. For 500 mM sodium sulfate, the elution gradient length was changed from 50 to 100 column volumes (CVs) to adjust for the 61 mS/cm elution buffer conductivity compared to 27 mS/cm for the 500 mM sodium acetate baseline condition. For all other salt screening runs, the elution phase was collected over 50 CV. One CV elution fractions were collected into 96 deep well plates as shown in Table 14. The wells were precharged with 0.132 CV of 250 mM sodium citrate pH 3.5 for elution neutralization.

[0381] Table 14. Elution Salt Screen AEX Method

[0382] 4.3 Results and Discussion

[0383] For the elution salt screens, elution fractions were not pooled for analytical testing but the analytical results from the individual elution fractions were used for a theoretical pool for each of the runs based on a set pooling criteria. With an affinity elution pool ratio of 0.75, an SEC A₂₆o/A_28o ratio equal to or greater than 0.80 was used as the pooling criteria for the theoretical AEX elution pool calculations. See Table 15 for details of the theoretical pool calculations for each elution salt condition. For the baseline condition sodium acetate (NaAcetate), five fractions were included in the theoretical elution pool which resulted in a pure average SEC A₂₆o/A_28o ratio of 0.826 and average SEC A₂₆o/A_28o ratio weighted by VG yield of 0.830. For sodium acetate with magnesium chloride (NaAcetate + 2mM MgCI2), nine fractions were included in the theoretical elution pool, which resulted in a slight improvement by 2% of VG step yield, but slightly lower unweighted and weighted average SEC A₂₆o/A_28o ratios compared to the sodium acetate baseline condition. For sodium propionate (NaPropionate), only four fractions were included in the theoretical pool for a slight improvement of VG step yield in the theoretical pool, with similar unweighted and much lower weighted average SEC A₂₆o/A_28o ratios compared to the baseline condition. For sodium chloride (NaCI), only three fractions were included in the theoretical pool for a decrease in VG step yield and only slightly higher unweighted and weighted average SEC A₂₆o/A_28o ratios compared to the baseline condition. For ammonium acetate (NH₄Acetate), only four fractions were included in the theoretical pool, resulting in a slight improvement to VG step yield but lower unweighted and weighted average SEC A₂₆o/A_28o ratios compared to the baseline condition. For sodium sulfate (Na₂SO4), six fractions were included in the theoretical elution pool, leading to a similar VG step yield, and significantly improved unweighted and weighted average SEC A₂₆o/A_28o ratios by about 0.05 compared to the baseline condition. For tetramethylammonium acetate (TMAA), twelve fractions were included, but the six fractions from the third peak maximum were segregated into a second theoretical pool from the six fractions under the first two peak maxima. The high SEC A₂₆o/A_28o ratio and low VP step yield for the theoretical pool under the third peak maximum suggests that this peak is not enriched in full capsids. Tetramethylammonium acetate resulted in a drastic loss of VG step yield, with similar unweighted and weighted average SEC A₂₆o/A_28o ratios of the first theoretical pool compared to the sodium acetate baseline condition.

[0384] FIG. 3 and FIG. 4 compare the seven elution salts in terms of enrichment of the SEC A₂₆O/A_28O ratio in the elution fractions back to the SEC A₂₆o/A_28o ratio of the affinity pool. Of the seven elution salts screened, two new top performers were identified for additional development. From FIG. 3, it can be observed that sodium acetate with magnesium chloride spiked in the elution buffer performed similarly in terms of the SEC A₂₆o/A_28o ratio in the elution fractions and that sodium sulfate resulted in elution fractions with a higher SEC A₂₆O/A_28O ratio compared to the baseline elution condition. Sodium acetate spiked with magnesium chloride and sodium sulfate gradient elutions offered the most promise for the process moving forward, while all other salts were not investigated further as they did not offer significant benefits over the baseline condition. Sodium acetate spiked with magnesium chloride in both elution buffers and sodium sulfate showed comparable VG yield in the theoretical elution pool compared to the baseline condition of sodium acetate. Sodium acetate spiked with magnesium chloride resulted in a larger number of elution fractions included in the theoretical pool but with a similar SEC A₂₆o/A_28o ratio in the theoretical pool compared to the baseline. On the other hand, sodium sulfate resulted in a similar number of elution fractions in the theoretical pool, but with about a 0.05 higher SEC A₂₆o/A_28o of the theoretical pool compared to the baseline. See full details in Table 15.

Table 15. Elution Salt Screen Results

Example 5: Impact of full capsids in Load on baseline process versus improved process and re-evaluation of sodium sulfate in weak binding load format

[0385] 5.1 Purpose [0386] Prior Examples describe work to develop an AEX process with improved separation of empty rAAV capsids from full capsids with a target % full capsids of greater than 50% and an A260/A280 of greater than 1 .16 in a drug substance. In this example, the improved AEX process was further studied to: (1) determine the impact of load material SEC A₂6o/A₂8o on performance of the baseline process and the improved process; (2) directly compare the baseline process and the improved process performance using common feed materials; (3) evaluate the improved process performance with two gradient elution salts, namely sodium acetate and sodium sulfate.

[0387] 5.2 Method

[0388] Nine AEX runs were executed using a mixture of affinity pools according to Table 16. The AAVx affinity pools were thawed to room temperature, spiked with 1 M MgCl2 to a final concentration of 25 mM MgCh and mixed according to the study design. Resulting affinity pools were aliquoted for each AEX run and re-frozen at -80 °C. Prior to each AEX run, aliquots were thawed at room temperature and sampled prior to dilution. AEX runs were executed on 0.7 mL CV POROS™ 50 HQ columns (0.3 cm ID x 10.0 cm BH) at a target resin challenge of 5E+13 VG/mL resin. Table 17 and Table 18 detail the baseline and improved AEX processes, respectively. For the baseline process runs, a 15X (v/v) dilution was performed with 200 mM histidine, 200 mM tris, 0.5% P188, pH 8.8. For the improved process runs, a 5X (v/v) dilution was performed with a modified dilution buffer (pH 8.8, 5.8 mS/cm). The modified dilution buffer was previously made by mixing an salt stock solution (230 mM sodium citrate, 280 mM MgCh, 460 mM NaCI) with baseline process dilution buffer (200 mM histidine, 200 mM tris, 0.5% P188, pH 8.8) until a target conductivity of 5.8 mS/cm was achieved. The formulation of the modified dilution buffer was 191 .9 mM Histidine, 191 .9 mM Tris, 9.3 mM sodium citrate, 11.3 mM MgCI2, 18.5 mM NaCI, 0.5% P188, pH 8.8. For the AEX runs with 500 mM sodium acetate as the elution salt, the gradient elution phase was collected over 50 column volumes (CV) in 1 CV fractions into 96 deep well plates. For the AEX runs with 200 mM sodium sulfate as the elution salt, the elution phase was collected over 60 column volumes (CV) in 1 CV fractions into 96 deep well plates. The wells were pre-charged with 0.132 CV of 250 mM sodium citrate pH 3.5 for elution neutralization.

[0389] Table 16. Head-to-Head-to-Head Study Design

[0390] Table 17. Baseline AEX Method

[0391] Table 18. Improved AEX Method

[0392] 5.3 Results and Discussion

[0393] Across three different AEX loads (with SEC A₂₆o/A_28o ratio of 0.75, 0.86, and 0.95), the improved AEX process generated elution pools with SEC A₂₆o/A_28o ratios that were significantly higher than the baseline process. FIG. 5 shows enrichment of SEC A₂₆o/A_28o ratio in AEX elution pools for the three different AEX methods using three different starting materials. The improved AEX methods, using either sodium acetate or sodium sulfate as the elution salt, enriched elution pool SEC A₂₆o/A_28o ratio to 1 .20 - 1 .25, significantly higher than the baseline process (SEC A₂₆o/A_28o ratio of 0.93 - 1.10). [0394] Table 19 describes the VG yield of the AEX elution pool for each of the three

AEX methods using three different starting materials. Notably, VG yield of the improved AEX methods were like the baseline process.

[0395] Table 20 reports % full capsid enrichment, as measured by analytical ultracentrifugation (AUC), for starting materials with SEC A₂₆o/A_28o ratios of 0.75, 0.86, and 0.95, respectively. The improved AEX process, leveraging sodium acetate for elution, enriched % full (AUC) from 9% to 21% in affinity pools, to 45% to 52% in AEX elution pools. Additionally, the nominated improved AEX process offered a significant increase in % full at the AEX elution pool (45% to 52%), compared to the earlier process (19% to 36%).

[0396] Table 19. Comprehensive Results Summary

% Capsid Species (AUC)

[0397] The improved AEX process, eluted with sodium sulfate, demonstrated similar yield and similar enrichment of the SEC A₂₆o/A_28o ratio across all starting materials. In the case of the AEX starting material with an SEC A₂₆o/A_28o ratio of 0.95, the improved process, with sodium sulfate as an elution salt, enriched to a slightly higher SEC A₂₆o/A_28o ratio (1 .23) than the improved process eluted with sodium acetate (1.20) (Table 19). Collectively, sodium sulfate achieved process performance similar to sodium acetate. Sodium acetate was used in the baseline process and demonstrated robust in-process stability and has been used extensively as a UF/DF feed material. Conversely, the in-process stability and UF/DF performance of a sodium sulfate elution salt is unknown. Therefore, sodium acetate offers substantially reduced risk relative to sodium sulfate, and thus the former was nominated as the elution salt for the improved AEX process.

[0398] Interestingly, the % VG loss of the AEX flow-through was larger when lower SEC A₂₆O/A_28O ratio load material was employed (FIG. 6). Less than 1 % VG loss was observed in the flow-through when the SEC A₂₆o/A_28o ratio of the AEX load was 0.95. For the improved process, when the SEC A₂₆o/A_28o ratio of the AEX load was 0.86, the flow-through accounted for 6-10% VG yield. When the SEC A₂₆o/A_28o ratio of the AEX load was 0.75 the flow-through accounted for 22-23% VG yield (all compared to the affinity pool).

[0399] The VG resin challenge was constant, but VG loss in the flow-through correlated with the VP resin challenge. A larger VG loss was observed in the flow-through at a higher VP resin challenge (FIG. 7). One explanation for this finding is that as the ratio of loaded empty: full capsids increases, the growing excess of empty capsids in the load begins to outcompete binding of full capsids to POROS™ 50 HQ, leading to loss of VG. To better understand this finding, further work was executed to understand the impact of VP resin challenge on the Phase 3 AEX process yield at a fixed SEC A₂₆o/A_28o ratio of the AEX load and is described in Example 6.

Example 6: Viral Particle Column Challenge Study

[0400] Example 5 showed higher VG loss was observed in flow-through fractions when the VP resin challenge was higher, and when the load SEC A₂₆o/A_28owas lower. This Example was designed to gain a better understanding of the impact of VP challenge at constant load SEC A₂₆o/A_28o by evaluating the improved AEX process performance across increasing VP challenge targets, ranging from 1.25E+13 to 8E+14 VP/mL resin. The results of this Example were used as a starting point of evaluating the loading challenge range to be recommended further for scale up. [0401] 6.1 Viral Particle Challenge Study Methods

[0402] An affinity eluate with a VP titer of 3.23E+13 VP/mL was used for this Example to prevent variability caused by the use of different starting materials. Using a 0.3 cm x 10.0 cm (0.707 mL column volume) POROS™ 50 HQ AEX column, the targeted VP/mL resin challenges were 1.25E+13, 2.5E+13, 5+E13, 1 E+14, 2E+14, 4E+14, and 8E+14. In addition to collecting load, ECW, and elution fractions, the load flowthrough was fractionated into 10 mL aliquots where possible (this was impractical at low challenges). These samples were submitted for qPCR transgene titer and SEC_AAV assays for viral particle titer and A₂6o/A₂8o ratio. Elution fractions were pooled based on predefined pooling rules. Resulting elution pools were additionally submitted for qPCR transgene titer and SEC_AAV for analysis. The analysis of the flowthrough fractions was used to determine breakthrough of full capsids during the column loading step.

[0403] 6.2 Viral Particle Challenge Study Results and Discussion

[0404] The viral particle challenge study offered preliminary insight on the operating space for the AEX column loading in terms of % yield and full capsid enrichment. Table 20 reports results of the viral particle challenge study and reveals impactful binding and elution yield trends. According to AKTA absorbance data, elution peak heights correlated with VP challenge until the 8E+14 VP/mL resin challenge condition, where the elution peak absorbance and A₂₆o/A₂₈o ratio decreased relative to the 4E+14 VP/mL resin challenge condition. Percent breakthrough of VG and VP (FIG. 8) in the AEX flowthrough (F/T) was plotted against the VP challenge and demonstrated the VG and VP loading challenge at which 10% breakthrough occurred. For this material, the 10% breakthrough occurred at 2.0E+13 VG/mL resin challenge and 5.0E+13 VP/mL resin challenge. The overlayed VG and VP breakthrough curves demonstrated that challenges of 2.0E+13 to 5.0E+13 VG/mL resin provide selective full capsid binding, with %VG breakthrough less than 60% (FIG. 8). The correlation between %VG yield and ASEC A260/280 ratio with VP challenge is shown in FIG. 9. All conditions demonstrated an increase in SEC Ratio of 0.32 and higher. Regarding %VG yield, an acceptable operating space was observed between 2.5E+13 to 4.0E+14 VP/mL resin challenge, that generated a %VG yield between 63% and 1 16%. This Example successfully identified an acceptable operating space for viral particle challenge for the AEX process.

[0405] Table 20. Viral Particle Challenge Study Results

[0406] Example 7: AEX process run

[0407] HEK 293 cells were grown in a 250 L suspension culture and transfected with 3 plasmids to produce rAAV3B vector per standard methods known in the art. HEK 293 cells were harvested, lysed, flocculated, and the resulting lysate was filtered. rAAV3B vector was purified from the clarified lysate by affinity chromatography. An affinity column was equilibrated, loaded with clarified lysate, washed, and the purified rAAV3B vector was eluted. The rAAV3B vector comprised a vector genome with a transgene encoding the amino acid of SEQ ID NO:1 (copper transporting ATPase 2 with deletion of metal binding sites 1-4). [0408] The affinity Pool was spiked with 2.5% v/v with 1 M MgCI₂ (to yield a final MgCh concentration of 25 mM) and diluted 5-7 fold (X) with dilution buffer (190 mM Histidine, 190 mM Tris, 9 mM Sodium Citrate, 11 mM MgCh, 18 mM NaCI, 0.50% P188 pH 8.7-9.0, conductivity 5.1 -6.1), to obtain a conductivity of 6.4 ± 0.4 mS/cm. The load pH was 8.6 to 9.0. The load capacity target was 5E+13 VG/mL resin with a range of 0.4E+13 to 1.3E+14 VG/mL resin.

[0409] The spiked, diluted affinity pool was then loaded onto a Poros™ 50 HQ Anion Exchange column (target column height 9 to 11 cm) and eluted via a Sodium Acetate gradient according to the process of Table 21 . Eighteen 0.5 CV elution fractions were collected from 32% to 50% B in vessels pre-charged with 0.066 CV of 250 mM sodium citrate, pH 3.5. AAV3B empty capsids were recovered in the unbound load fraction, empty capsid wash and shoulders of the elution peak. Enriched full/intermediate capsids were eluted from the column in the center of the elution peak.

[0410] Table 21. AEX process parameters

[0411 ] Neutralized elution fractions were assayed via SEC A₂₆o/A₂₈o and qPCR.

[0412] Table 22. AEX Performance Summary

Example 8: AEX Process run at 2000 L scale

[0413] HEK 293 cells were grown in a 2000 L suspension culture and transfected with 3 plasmids to produce rAAV3B vector per standard methods known in the art. HEK 293 cells were harvested, lysed, flocculated, and the resulting lysate was filtered. rAAV3B vector was purified from the clarified lysate by affinity chromatography. An affinity column was equilibrated, loaded with clarified lysate, washed, and the purified rAAV3B vector was eluted. The rAAV3B vector comprised a vector genome with a transgene encoding the amino acid of SEQ ID NO:1 (copper transporting ATPase 2 with deletion of metal binding sites 1-4).

[0414] The affinity pool was neutralized to a pH of 7.74 (target: 7.6; range: 7.4 to 7.8) using 6.96% v/v (target: 5% v/v) of 1 M Tris Base. To reduce the pH, 1 .01% v/v (target: 1% v/v) of 2 M glycine was added to the affinity pool. The affinity pool was then spiked with 2.37% v/v (target: 2.5% v/v, range: 2.3% to 2.7% v/v) of 1 M MgCh to yield a final MgCh target concentration of 25 mM (range: 23 to 27 mM). The neutralized and spiked affinity eluate was stored at 2°C to 8°C for 21 .4 hours, though it may be stored for up to 5 days. [0415] Before loading on the AEX column to separate remaining impurities and empty capsids from full capsids, the neutralized and spiked affinity eluate was diluted 4.6 fold (target: 5 fold, range: 4 to 7 fold) by weight with a dilution buffer (190 mM Histidine, 190 mM Tris, 9 mM Sodium Citrate, 11 mM MgCh, 18 mM NaCI, 0.50% P188 with a pH of 8.8 (range: 8.7 to 9.0) to obtain a conductivity of 6.58 mS/cm (target: 6.4 mS/cm, range: 6.0 to 6.8 mS/cm) and a pH of 8.77 (target: 8.8, range: 8.6 to 9.0). The load capacity target was 5.0E+13 VG/mL with a range of 4.0E+13 to 1 .3E+14 VG/mL. The actual load was 1 .05E+14 VG/mL for a total vg loaded of 2.1 E+17.

[0416] The diluted affinity pool was filtered through an in-line 0.2 pM Sartorius Sartopore® filter loaded onto a 2000 mL (range: 1846 to 2154 mL) Poros™ 50 HQ Anion Exchange column (13 +/- 1 cm high x 14 cm wide) at a flow rate of 130 cm/hr and a residence time of 6.0 min/CV. Elution was via 37.5 CV of a Sodium Acetate gradient at 0 to 75% Buffer B. All other steps of the AEX process (Table 23) were performed at a flow rate of 390 cm/hr and a residence time of 2.0 min/CV (Table 23).

[0417] Table 23. AEX process parameters

[0418] * process steps performed with upward flow, all other steps performed with downward flow.

[0419] AAV3B empty capsids were recovered in the unbound load fraction, empty capsid wash and fronting shoulder of the elution peak. Enriched full/intermediate capsids were eluted from the column in the center of the elution peak.

[0420] Twenty 0.5 CV elution fractions were collected from 32% to 50% buffer B in vessels pre-charged with 0.066 CV of 250 mM sodium citrate, pH 3.5. Each fraction was analyzed by SEC to determine A260/A280 ratios and qPCR to quantitate viral genome titer. The SEC A260/A280 ranged from 0.68 (fraction 1 ) to 1 .36 (fractions 9-11 ). The vg titer ranged from 9.66E+10 VG/mL (fraction 1 ) to 3.90E+13 VG/mL (fraction 9) (Table 24). [0421] Pooling started with the first fraction that had a SEC A260/A280 ratio > 0.99. All consecutive fractions that would account for >2% of the total vg of the theoretical pool were pooled. The percent vg of the fraction contribution to the theoretical pool is equal to (fraction titer x fraction volume)/(summed theoretical pool titer x summer theoretical pool volume) x

100. A total of 10 fractions (fractions 5 through 14) were pooled. The SEC A260/A280 of the pooled fractions ranged from 1 .04 (fraction 5) to 1 .36 (fractions 9-11 ). The vg titer ranged from 3.67E+12 VG/mL (fraction 14) to 3.90E+13 VG/mL (fraction 9) (Table 24). The pH of the AEX pool was adjusted to pH 7.69 with a target range from 7.5 to 7.7. [0422] Table 24. Analysis of fractions

[0423] The affinity pool , AEX load and AEX pool were analyzed by SEC to determine A260/A280 ratios, vp/mL, % HMMS and % monomer and by qPCR to quantitate viral genome titer, and by AUC to determine percentages of full, intermediate, and empty capsids.

[0424] Table 25. Quality attributes of Affinity pool and AEX pool

[0425] The AEX pool also had a 2.57E+13 vp/mL, 4.7% HMMS and 95% monomer.

[0426] These data demonstrate that the improved AEX purification process resulted in efficient separation of empty capsids from full capsids and produced an AEX eluate with a step yield of greater than 60% and an A260/A280 of greater than 1 .30.

Example 9: AEX Process run at 2000 L scale

[0427] HEK 293 cells were grown in a 2000 L suspension culture and transfected with 3 plasmids to produce rAAV3B vector per standard methods known in the art. HEK 293 cells were harvested, lysed, flocculated, and the resulting lysate was filtered. rAAV3B vector was purified from the clarified lysate by affinity chromatography. An affinity column was equilibrated, loaded with clarified lysate, washed, and the purified rAAV3B vector was eluted. The rAAV3B vector comprised a vector genome with a transgene encoding the amino acid of SEQ ID NO:1 (copper transporting ATPase 2 with deletion of metal binding sites 1-4).

[0428] The affinity pool was neutralized (target pH: 7.6; range: 7.4 to 7.8) using 6.3% v/v (target: 5% v/v) of 1 M Tris Base. The affinity pool was then spiked with 2.51% v/v (target: 2.5% v/v, range: 2.3% to 2.7% v/v) of 1 M MgCh to yield a final MgCh target concentration of 25 mM (range: 23 to 27 mM).

[0429] Before loading on the AEX column to separate remaining impurities and empty capsids from full capsids, the neutralized and spiked affinity eluate was diluted 4.6 fold (target: 5 fold, range: 4 to 7 fold) by weight with a dilution buffer (190 mM Histidine, 190 mM Tris, 9 mM Sodium Citrate, 11 mM MgCh, 18 mM NaCI, 0.50% P188 with a pH of 8.8 (range: 8.7 to 9.0) to obtain a conductivity of 6.4 mS/cm (target: 6.4 mS/cm, range: 6.0 to 6.8 mS/cm) and a pH of 8.8 (target: 8.8, range: 8.6 to 9.0). The load capacity target was 5.0E+13 VG/mL with a range of 4.0E+13 to 1.3E+14 VG/mL. The actual load challenge was 5.64E+14 VG/mL.

[0430] The diluted affinity pool was pre-filtered through an in-line 0.2 pM Sartorius Sartopore® 2, 20” filter and loaded onto a 2000 mL (range: 1846 to 2154 mL) Poros™ 50 HQ Anion Exchange column (13 +/- 1 cm high x 14 cm wide) at a target flow rate of 130 cm/hr (range: 117-143 cm/hr) and a residence time of 6.5 min/CV. The skid was set up under pressure control for all phases to lower the flow rates, if needed, to maintain the maximum pressure at < 4.5 bar-g. The column was equilibrated with two buffers prior to product loading. A third equilibrium was performed after product loading, followed by an empty capsid wash with a bis-tris propane solution. The third equilibrium was repeated after the empty capsid wash, before the product was eluted to remove the bis-tris propane from the column.

[0431] All steps of the AEX process, other than the load and 17.5% ethanol storage, were performed at a target flow rate of 390 cm/hr (range 351 -429) and a residence time of

2.2 min/CV (Table 26). The 17.5% ethanol storage was performed at a target flow rate of 390 cm/hr (range: 351-429 cm/hr) and a residence time of 3.23 min/CV.

[0432] Table 26. AEX process parameters

[0433] * process steps performed with upward flow, al other steps performed with downward flow.

[0434] The product was eluted over a sodium acetate gradient (0-75% high conductivity elution buffer after starting with low conductivity buffer); elution was performed over a 37.5 CV elution gradient, with the AAV3B empty capsids eluting in the unbound and fronting shoulder of the elution peak. Enriched full/intermediate capsids eluted from the column in the center of the elution peak. Collection started when the UV A260/A280 ratio was 1 .02 (target: >0.99) and the minimum UV280 > 10mAU/mm path length (5 mm path length) was 0.1509. [0435] Collection ended when a 3.0 CV minimum volume was collected and either the UV280 was <7.5% of the peak UV280, or the UV A260/A280 ratio was <0.99. In this run, collection ended when the UV280 was 0.0336 and the A260/A280 was 1 .08. The peak UV280 was 0.4818 and the peak UV260/280 was 1.26 (FIG. 10).The method also allows for ending collection when a volume of > 5.5 CV is collected, but the UV conditions are not met. Collection ended at approximately 32% to 52% buffer B. [0436] After eluate collection, the pH was adjusted to a target pH of 7.6 (range 7.5-7.7).

If needed the eluate can be titrated with 1 M tris base to increase the pH or 250 mM sodium citrate, pH 3.5 to decrease the pH.

[0437] The Step Yield was determined at multiple steps in the process.

[0438] Table 27. Step Yield.

** Step yield calculation accounts for VG loss due to sampling. [0439] The neutralized affinity and AEX pools were analyzed to determine quality attributes (Table 28).

[0440] Table 28. Quality attributes of Affinity pool and AEX Pool

NMT = not more than

[0441] These data demonstrate that the improved AEX purification process resulted in efficient separation of empty capsids from full capsids and produced an AEX eluate with a step yield of greater than 60% and an A260/A280 of greater than 1 .30. Specifically, for this Example the improved AEX purification process resulted in efficient separation of empty capsids from full capsids and produced an AEX eluate with greater than 76% full capsids, an A260/A280 of greater than 1 .3 and a step yield of at least 100%.

SEQUENCE LISTING

Claims

We claim:

1 . A method of purifying an rAAV vector by AEX, the method comprising a step of: i) loading a solution comprising the rAAV vector to be purified onto a stationary phase in a column; ii) contacting the stationary phase with an empty capsid wash (ECW) solution; iii) performing gradient elution of material from the stationary phase in the column wherein a percentage of a first gradient elution buffer is varied in a manner inversely proportional to variation in a percentage of a second gradient elution buffer; and iv) collecting at least one fraction of eluate from the column during the gradient elution.

2. The method of purifying an rAAV vector by AEX of claim 1 , wherein the solution comprising a rAAV vector to be purified is an affinity eluate and comprises full capsids and empty capsids.

3. The method of purifying an rAAV vector by AEX of claim 1 or 2, wherein the solution comprising a rAAV vector to be purified is spiked with 1 M MgCI₂ to achieve a final concentration of 23 mM to 27 mM MgCh.

4. The method of purifying an rAAV vector by AEX of any one of claims 1 -3, wherein the solution comprising a rAAV vector to be purified is diluted 4 to 7-fold with a dilution solution comprising 100 mM to 300 mM histidine, 100 mM to 300 mM Tris, 1 mM to 20 mM sodium citrate, 1 mM to 20 mM MgCh, 10 mM to 30 mM NaCI, and 0.1% to 1% P188 and has a pH of 7 to 9.2 and a conductivity of 6.0 mS/cm to 6.8 mS/cm.

5. The method of purifying an rAAV vector by AEX of any one of claims 1-4, wherein the ECW solution removes bound empty capsids from the stationary phase, optionally, wherein the ECW solution removes 10% or more of the bound empty capsids from the stationary phase.

6. The method of purifying an rAAV vector by AEX of any one of claims 1-5, wherein the ECW solution removes 5% or less of the bound full capsids from the stationary phase, optionally wherein the ECW solution does not remove bound full capsids from the stationary phase. The method of purifying an rAAV vector by AEX of any one of claims 1 -6, wherein the ECW solution removes less than 5% of vector genomes (VG) from the stationary phase. The method of purifying an rAAV vector by AEX of any one of claims 1 -7, wherein the stationary phase is contacted with 4 to 6 column volumes (CV) of ECW solution and wherein the ECW solution comprises about 20 mM to about 30 mM bis-tris propane, about 80 mM to 100 mM NaCI and has a pH of about 9.3 to 9.5 and a conductivity of 9.3 mS/cm to 1 1 .6 mS/cm. The method of purifying an rAAV vector by AEX of any one of claims 1 -8, wherein an SEC A260/A280 ratio of the ECW solution after contact with the stationary phase is less than 0.80, less than 0.75, less than 0.70, less than 0.65, less than 0.60, less than 0.55 or less than 0.50. The method of purifying an rAAV vector by AEX of any one of claims 1 -9, wherein a % viral particles (VP) in the ECW solution after contact with the stationary phase is greater than 5%, greater than 10%, greater than 15%, greater than 20%, greater than 25% of total VP in the solution comprising the rAAV capsid. The method of purifying an rAAV vector by AEX of any one of claims 1 -10, wherein a % VG in the ECW solution after contact with the stationary phase is less than 10%, less than 8%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% of total VG in the solution comprising the rAAV capsid. The method of purifying an rAAV vector by AEX of any one of claims 1 -11 , wherein the stationary phase is contacted with an equilibration solution before the stationary phase is contacted with the ECW solution, after the stationary phase is contacted with the ECW solution, or both. The method of purifying an rAAV vector by AEX of claim 12, wherein the equilibration solution comprises a buffer, MgCh and a detergent. The method of purifying an rAAV vector by AEX of claims 12 or 13, wherein the equilibration solution comprises 0.5 mM to 5 mM MgCh, optionally about 2 mM MgCh.

15. The method of purifying an rAAV vector by AEX of any one of claims 12-14, wherein the equilibration solution comprises 50 mM to 150 mM Tris, 0.5 mM to 5 mM MgCh, 0.005% to 0.015% P188 and has a pH of about 8.5 to 9.9.

16. The method of purifying an rAAV vector by AEX of any one of claims 1-15, wherein at the start of performing the gradient elution the percentage of the first gradient elution buffer (optionally buffer A) is 50% to 100% and at the end of performing the gradient elution the percentage of the second gradient elution buffer, (optionally buffer B) is 50% to 100%, wherein the percentage of the second gradient elution buffer increases at a rate of 1 .3% to 2.7% (optionally 2%) per CV; and wherein 37.5 CV of the first gradient elution buffer, the second gradient elution buffer, or a mixture of both, are applied to the stationary phase during the gradient elution.

17. The method of purifying an rAAV vector by AEX of any one of claims 1-16, wherein the first gradient elution buffer (optionally buffer A) comprises about 50 mM to about 150 mM (optionally about 100 mM) Tris, about 0.005% to about 0.015% (optionally about 0.01 %) P188 and has a pH of about pH 8.5 to 9.5 (optionally about 8.9); wherein the second gradient elution buffer (optionally buffer B) comprises about 400 mM to about 600 mM (optionally about 500 mM) sodium acetate or sodium sulfate, about 50 mM to about 150 mM (optionally about 100 mM) Tris, about 0.005% to about 0.015% (optionally about 0.01%) P188 and has a pH of about pH 8.5 to 9.5 (optionally about 8.9).

18. The method of purifying an rAAV vector by AEX of claim 17, wherein a concentration of sodium acetate or sodium sulfate increases continuously during the gradient elution; wherein a rate of increase of the concentration of sodium acetate or sodium sulfate is equivalent to a change in concentration of the sodium acetate or sodium sulfate per total CV; wherein the rate of change in concentration of the sodium acetate or sodium sulfate during the gradient elution is 6.7 mM/CV to 13.3 mM/CV (optionally about 10 mM/CV) or a combination thereof.

19. The method of purifying an rAAV vector by AEX of any one of claims 1-18, wherein the at least one fraction of eluate is collected when the gradient comprises from about 32%

20. The method of purifying an rAAV vector by AEX of any one of claims 1-19, wherein full capsids are eluted from the stationary phase in the center of the elution peak and wherein empty capsids are recovered in a column flow-through, in the unbound and fronting shoulder of the elution peak or both, when performing the gradient elution.

21 . The method of purifying an rAAV vector by AEX of any one of claims 1-20, wherein the at least one fraction of eluate meets predefined pooling criteria comprising i) a first fraction of eluate that has an SEC A260/A280 ratio greater than or equal to 0.99 and ii) all subsequent fractions of eluate that would account for greater than or equal to 2% of a total VG titer of a theoretical pool.

22. The method of purifying an rAAV vector by AEX of claim 21 , wherein the number of fractions of eluate that meet the predefined pooling criteria are combined to form a pooled eluate.

23. The method of purifying an rAAV vector by AEX of claim 22, wherein the pooled eluate is characterized by i) a percentage of full capsids greater than or equal to 50%; ii) an A260/A280 ratio of 1 .16 or greater; iii) a %VG step yield of 60% or greater; iv) or a combination thereof.

24. The method of purifying an rAAV vector by AEX of any one of claims 1-20, wherein the collecting of the at least one fraction of eluate starts when the UV A260/A280 ratio is >0.99 and the minimum UV280 is > 10mAU/mm path length (5 mm path length).

25. The method of purifying an rAAV vector by AEX of claim 24, wherein the collecting of the at least one fraction of eluate ends when i) a minimum volume of 3.0 CVs is collected and either the UV280 is <7.5% of the peak UV280 measurement, or the UV A260/A280 ratio is <0.99 or ii) when a volume of 5.5 CV is collected.

26. The method of purifying an rAAV vector by AEX of any one of claims 1-25, wherein the stationary phase is a polystyrenedivinylbenzene particle with covalently bound quaternized polyethyleneimine.

27. The method of purifying a rAAV vector by AEX of any one of claims 1-26, wherein the rAAV vector comprises an AAV capsid protein from an AAV serotype selected from the group consisting of AAV1 , AAV2, AAV3 (including AAV3A, AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhIO, AAVrh74, AAV12, AAV2i8, NP4, NP22, NP66, AAVDJ, AAVDJ/8, AAVDJ/9, AAVLK03, AAV1 .1 , AAV2.5, AAV6.1 , AAV6.3.1 , AAV9.45, RHM4-1 (SEQ ID N0:5 of WO 2015/013313), RHM15-1 , RHM15-2, RHM15- 3/RHM15-5, RHM15-4, RHM15-6, AAV hu.26, AAV1 .1 , AAV2.5, AAV6.1 , AAV6.3.1 , AAV9,45, AAV2i8, AAV29G, AAV2,8G9, AVV-LK03, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N, AAVHSC1 , AAVHSC2, AAVHSC3, AAVHSC4, AAVHSC5, AAVHSC6, AAVHSC7, AAVHSC8, AAVHSC9, AAVHSC10, AAVHSC11 , AAVHSC12, AAVHSC13, AAVHSC14 and AAVHSC15. The method of purifying a rAAV vector by AEX of any one of claims 1-27, wherein the rAAV vector comprises an AAV3B capsid protein and a transgene comprising a nucleic acid encoding the amino acid sequence of SEQ ID NO:1 . A method of preparing a solution comprising a rAAV vector for purification by AEX, the method comprising a step of: diluting the solution 2 to 10-fold (e.g., 5-fold) with a dilution solution comprising histidine, Tris, P188, sodium citrate, magnesium chloride and sodium chloride to form a diluted solution, wherein the ratio of sodium citrate, magnesium chloride and sodium chloride is at a molar ratio of 1 to 1 .25 to 2; wherein the pH of the diluted solution is 8.6 to 9.0; and wherein the conductivity of the diluted solution is 6.0 mS/cm to 6.8 mS/cm. The method of preparing a solution comprising a rAAV vector for purification by AEX of claim 29, wherein the solution comprising a rAAV vector to be purified is an affinity eluate and comprises full capsids and empty capsids. The method of preparing a solution comprising a rAAV vector for purification by AEX of claim 29 or 30, wherein the solution comprising a rAAV vector to be purified is neutralized prior to the step of diluting by adjusting the pH of the solution to a range of 7.4 to 7.8 (e.g., 7.6). The method of preparing a solution comprising a rAAV vector for purification by AEX of any one of claims 29-31 , wherein the solution comprising a rAAV vector to be purified is spiked with 1 M MgCls to achieve a final concentration of 23 mM to 27 mM (optionally 25 mM) MgCh prior to the step of diluting. The method of preparing a solution comprising a rAAV vector for purification by AEX of any one of claims 29-32, wherein the solution comprising a rAAV vector to be purified is diluted 4 to 7-fold with a dilution solution comprising 100 mM to 300 mM histidine, 100 mM to 300 mM Tris, 1 mM to 20 mM sodium citrate, 1 mM to 20 mM MgCL, 10 mM to 30 mM NaCI, and 0.1 % to 1% P188 and has a pH of 7 to 9.2 and a conductivity of 6.0 mM to 6.8 mS/cm. The method of preparing a solution comprising a rAAV vector for purification by AEX of any one of claims 29-33, wherein the dilution solution comprises about 190 mM histidine, about 190 mM Tris, about 9 mM sodium citrate, about 1 1 mM MgCh and about 18 mM NaCI, and about 0.5% P188, has a pH of 8.6 to 9.0 and a conductivity of 6.0 to 6.8 mS/cm. The method of preparing a solution comprising a rAAV vector for purification by AEX of any one of claims 29-34, wherein the solution comprising a rAAV vector is diluted until the conductivity of the diluted solution reaches a pH range of 6.0 to 6.8 mS/cm (e.g., about 6.4 mS/cm). The method of preparing a solution comprising a rAAV vector for purification by AEX of any one of claims 29-35, wherein the solution comprising a rAAV vector is diluted until the pH of the diluted solution reaches a conductivity range of 8.6 to 9.0 (e.g., about 8.8). The method of preparing a solution comprising a rAAV vector for purification by AEX any one of claims 29-36, wherein the solution comprising the rAAV vector to be purified has pH of 7.4 to 7.8 and the diluted solution has a pH of 8.6 to 9.0 (e.g., 8.8). The method of preparing a solution comprising a rAAV vector for purification by AEX of any one of claims 29-37 wherein the conductivity of the solution comprising the rAAV vector to be purified is adjusted to 6.4 mS/cm. The method of preparing a solution comprising a rAAV vector for purification by AEX of any one of claims 29-38, further comprising the step of filtering the solution comprising a rAAV vector through a filter to produce a diluted and filtered solution. The method of preparing a solution comprising a rAAV vector for purification by AEX of any one of claims 29-39, further comprising a step of loading the diluted solution onto a stationary phase in a column.

41 . The method of preparing a solution comprising a rAAV vector for purification by AEX of claim 40, wherein the diluted solution (optionally the load) comprises about 1.0E+13 VG/mL resin to about 5.0E+14 VG/mL resin (optionally about 5.0E+13 VG/mL resin, about 2.5 VG/mL resin).

42. The method of preparing a solution comprising a rAAV vector for purification by AEX of claim 40 or 41 , wherein the diluted solution comprises about 1 .0E+17 to 1 .0E+18 (optionally about 2.10E+17) total VG.

43. The method of preparing a solution comprising a rAAV vector for purification by AEX of any one of claims 40-42, wherein greater than 95% of VG in the diluted solution binds the stationary phase in the column.

44. The method of preparing a solution comprising a rAAV vector for purification by AEX of any one of claims 40-43, wherein a column flow-through has a SEC A260/A280 ratio of less than 0.70

45. The method of preparing a solution comprising a rAAV vector for purification by AEX of any one of claims 29-44, wherein the stationary phase is a polystyrenedivinylbenzene particle with covalently bound quaternized polyethyleneimine.

46. The method of preparing a solution comprising a rAAV vector for purification by AEX of purifying a rAAV vector by AEX of any one of claims 29-45, wherein the rAAV vector comprises an AAV capsid protein from an AAV serotype selected from the group consisting of AAV1 , AAV2, AAV3 (including AAV3A, AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhIO, AAVrh74, AAV12, AAV2i8, NP4, NP22, NP66, AAVDJ, AAVDJ/8, AAVDJ/9, AAVLK03, AAV1 .1 , AAV2.5, AAV6.1 , AAV6.3.1 , AAV9.45, RHM4-1 (SEQ ID NO:5 of WO 2015/013313), RHM15-1 , RHM15-2, RHM15- 3/RHM15-5, RHM15-4, RHM15-6, AAV hu.26, AAV1 .1 , AAV2.5, AAV6.1 , AAV6.3.1 , AAV9,45, AAV2i8, AAV29G, AAV2,8G9, AVV-LK03, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N, AAVHSC1 , AAVHSC2, AAVHSC3, AAVHSC4, AAVHSC5, AAVHSC6, AAVHSC7, AAVHSC8, AAVHSC9, AAVHSC10, AAVHSC11 , AAVHSC12, AAVHSC13, AAVHSC14 and AAVHSC15.

47. The method of preparing a solution comprising a rAAV vector for purification by AEX of purifying a rAAV vector by AEX of any one of claims 29-46, wherein the rAAV vector comprises an AAV3B capsid protein and a transgene comprising a nucleic acid encoding the amino acid sequence of SEQ ID NO:1 .

48. A solution comprising a rAAV vector purification prepared by the method of any one of claims 29-47.

49. A solution comprising a rAAV vector to be purified, wherein the solution has a pH of 7.4-7.8 and comprises 25 mM MgCh. 50. The solution comprising a rAAV vector of claim 49, wherein the solution is diluted 4 to

7 fold with a dilution solution comprising 100 mM to 300 mM histidine, 100 mM to 300 mM Tris, 1 mM to 20 mM sodium citrate, 1 mM to 20 mM MgCh, 10 mM to 30 mM NaCI, and 0.1 % to 1% P188, and following dilution has a conductivity of 6.0 to 6.8 mS/cm.

51 . The solution comprising a rAAV vector of claim 50, wherein the solution has a pH of 8.6 to 9.0.