METHODS AND COMPOSITIONS FOR THE PURIFICATION OF ADENO-ASSOCIATED VIRUS

Provided herein are methods and compositions for the separation of an adeno-associated virus (AAV) particle from a mixture of the AAV and at least one contaminant using anion exchange chromatography. These methods and compositions allow for improved purification of complete AAV particles from contaminants such as AAV particles that lack a complete genome (e.g., empty capsids) and AAV degradation products.

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Description
RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application Ser. Nos. 63/263,305, filed Oct. 29, 2021, the entire disclosure of which is hereby incorporated herein by reference.

SEQUENCE LISTING

The content of the electronically submitted sequence listing in ASCII format (Name: “HMW-048_SL_ST26”; Size: 125,560 bytes; and Date of Creation: Oct. 27, 2022) is incorporated herein by reference in its entirety.

BACKGROUND

Gene therapy using adeno-associated virus (AAV) vectors has the potential to treat a wide variety of human disorders. Commercial therapeutic use of AAV vectors requires the large-scale manufacture of highly purified preparations of these vectors. A major challenge in the large-scale manufacture of AAV vectors is the separation of intact AAV particles from process-related contaminants, such as incomplete AAV particles, e.g., AAV particles that lack a complete genome (e.g., empty capsids).

Accordingly, there is a need in the art for novel AAV manufacturing methods that can provide for the consistent large-scale purification of intact AAV particles.

SUMMARY

The present disclosure provides methods for the separation of an adeno-associated virus (AAV) particle from a mixture of the AAV and at least one contaminant. The methods generally comprise contacting a mixture of an AAV particle and at least one contaminant with an anion exchange chromatography (AEX) medium such that the AAV particle binds to the AEX medium, and washing the AEX medium with a first wash solution under conditions such that the AAV particle remains bound to the AEX medium and the at least one contaminant does not bind the AEX medium. The present disclosure provides in certain embodiments, that the mixture and the first wash solution comprise an acetate. In certain embodiments, the mixture further comprises magnesium chloride (MgCl2). In certain embodiments, the first wash solution further comprises urea. The present disclosure also provides methods further comprising washing the AEX medium with a second wash solution under conditions such that the AAV particle remains bound to the AEX medium and the at least one contaminant does not bind to the AEX medium, wherein the second wash solution is a low conductivity wash solution that has a conductivity of less than about 3 mS/cm. The low conductivity wash is performed prior to elution. Also provided herein are compositions comprising an AEX medium and a mixture comprising MgCl2 and an acetate, and compositions comprising an AEX medium and a wash solution comprising urea and an acetate. The methods and compositions provided by the present disclosure allow for the improved purification of intact AAV particles from contaminants such as AAV particles that lack a complete genome (e.g., empty capsids) and AAV degradation products. Consequently, the methods disclosed herein, and the compositions disclosed herein for use in such methods, result in compositions having a high purity of intact AAV particles. Without wishing to be bound by any particular theory, the improved purification of intact AAV particles is at least partly achieved through “weak partitioning”, a process by which a stronger binding species displaces or “weakly partitions” a weaker binding species from the chromatography column by inhibiting the binding of the weaker binding species to the column.

Accordingly, in one aspect, the present disclosure provides a method for the separation of an adeno-associated virus (AAV) particle from a mixture of the AAV particle and at least one contaminant, the method comprising: contacting the mixture with an anion exchange chromatography (AEX) medium under conditions such that a subset of empty AAV particles does not bind to the AEX medium and the full AAV particle binds to the AEX medium, wherein the mixture comprises magnesium chloride and an acetate; and washing the AEX medium with a first wash solution under conditions such that the AAV particle remains bound to the AEX medium and the at least one contaminant does not bind the AEX medium, thereby separating the AAV particle from the at least one contaminant.

In certain embodiments, the first wash solution comprises an acetate. In certain embodiments, the first wash solution comprises urea.

In another aspect, the present disclosure provides a method for the separation of an adeno-associated virus (AAV) particle from a mixture of the AAV particle and at least one contaminant, the method comprising: contacting the mixture with an anion exchange chromatography (AEX) medium under conditions such that the AAV particle binds to the AEX medium; and washing the AEX medium with a first wash solution comprising urea and an acetate under conditions such that the AAV particle remains bound to the AEX medium and the at least one contaminant does not bind to the AEX medium, thereby separating the AAV particle from the at least one contaminant. In another aspect, the method comprises contacting the mixture of the AAV particle and at least one contaminant with an AEX medium under conditions such that a subset of empty AAV particles does not bind to the AEX medium and the full AAV particle binds to the AEX medium, and washing the AEX medium with a first wash solution comprising urea and an acetate under conditions such that the AAV particle remains bound to the AEX medium and the at least one contaminant does not bind the AEX medium, thereby separating the AAV particle from the at least one contaminant.

In certain embodiments, the method further comprises washing the anion exchange chromatography medium with a second wash solution.

In another aspect, the present disclosure provides a method for the separation of an adeno-associated virus (AAV) particle from a mixture of the AAV particle and at least one contaminant, the method comprising: providing an AEX medium that has been contacted with the mixture, wherein the mixture comprises magnesium chloride and an acetate, and wherein the AEX medium comprises the AAV particle bound thereto and has been washed with a first wash solution such that the AAV particle remained bound to the AEX medium and the at least one contaminant did not bind to the AEX medium; and washing the AEX medium with a second wash solution under conditions such that the AAV particle remains bound to the AEX medium and the at least one contaminant does not bind to the AEX medium, thereby separating the AAV particle from the at least one contaminant.

In certain embodiments, the first wash solution comprises an acetate. In certain embodiments, the first wash solution comprises urea.

In another aspect, the present disclosure provides a method for the separation of an adeno-associated virus (AAV) particle from a mixture of the AAV particle and at least one contaminant, the method comprising: providing an AEX medium that has been contacted with the mixture, wherein the AEX medium comprises the AAV particle bound thereto and has been washed with a first wash solution comprising urea and an acetate such that the AAV particle remained bound to the AEX medium and the at least one contaminant did not bind to the AEX medium; and washing the AEX medium with a second wash solution under conditions such that the AAV particle remains bound to the AEX medium and the at least one contaminant does not bind to the AEX medium, thereby separating the AAV particle from the at least one contaminant.

In certain embodiments, the at least one contaminant is selected from the group consisting of an AAV particle lacking a complete genome, an AAV degradation product, a host cell protein, a host cell fragment, and any combination thereof. In certain embodiments, the at least one contaminant is an AAV particle that lacks a complete genome.

In certain embodiments, the AEX medium has an average pore size of at least about 100 nm. In certain embodiments, the AEX medium has an average pore size of at least about 500 nm. In certain embodiments, the AEX medium comprises a quaternary amine. In certain embodiments, the AEX medium comprises a quaternary polyethyleneimine group.

In certain embodiments, the mixture comprises a partially purified AAV composition. In certain embodiments, the mixture comprises an eluate of an affinity chromatography column.

In certain embodiments, the mixture comprises about 10 mM to about 40 mM of an acetate. In certain embodiments, the mixture comprises about 10 mM to about 40 mM ammonium acetate. In certain embodiments, the mixture comprises about 28 mM ammonium acetate.

In certain embodiments, the mixture comprises about 2 mM to about 6 mM magnesium chloride. In certain embodiments, the mixture comprises about 2 mM magnesium chloride.

In certain embodiments, the mixture comprises about 0.001% (w/v) to about 0.05% (w/v) Poloxamer 188. In certain embodiments, the mixture comprises about 0.01% (w/v) Poloxamer 188.

In certain embodiments, the pH of the mixture is about 9 to about 10.5. In certain embodiments, the pH of the mixture is about 9.3.

In certain embodiments, the first wash solution comprises about 0.1 M to about 4 M urea. In certain embodiments, the first wash solution comprises about 2 M urea.

In certain embodiments, the first wash solution comprises an acetate selected from the group consisting of ammonium acetate, potassium acetate, sodium acetate, and cesium acetate. In certain embodiments, the first wash solution comprises ammonium acetate. In certain embodiments, the first wash solution comprises about 10 mM to about 40 mM of the acetate. In certain embodiments, the first wash solution comprises about 10 mM to about 40 mM ammonium acetate. In certain embodiments, the first wash solution comprises about 28 mM ammonium acetate.

In certain embodiments, the first wash solution comprises about 2 mM to about 6 mM magnesium chloride. In certain embodiments, the first wash solution comprises about 2 mM magnesium chloride.

In certain embodiments, the first wash solution comprises about 0.001% (w/v) to about 0.05% (w/v) Poloxamer 188. In certain embodiments, the first wash solution comprises about 0.01% (w/v) Poloxamer 188.

In certain embodiments, the pH of the first wash solution is about 9 to about 10.5. In certain embodiments, the pH of the first wash solution is about 9.3.

In certain embodiments, the first wash solution has a conductivity of about 1 mS/cm to about 3 mS/cm.

In certain embodiments, the second wash solution comprises about 0.1 mM to about 15 mM ammonium acetate. In certain embodiments, the second wash solution comprises about 10 mM ammonium acetate.

In certain embodiments, the second wash solution comprises about 0.001% (w/v) to about 0.05% (w/v) Poloxamer 188. In certain embodiments, the second wash solution comprises about 0.01% (w/v) Poloxamer 188.

In certain embodiments, the pH of the second wash solution is about 9 to about 10.5. In certain embodiments, the pH of the second wash solution is about 9.3.

In certain embodiments, the second wash solution has a conductivity of less than about 3 mS/cm. In certain embodiments, the second wash solution has a conductivity of about 1 mS/cm to about 3 mS/cm. In certain embodiments, the second wash solution has a conductivity of about 1 mS/cm.

In certain embodiments, the method further comprises eluting the AAV particle from the AEX medium. In certain embodiments, the AAV particle is eluted from the AEX medium with an eluant using a step gradient. In certain embodiments, the AAV particle is eluted from the AEX medium with an eluant using a linear gradient.

In certain embodiments, the eluant comprises a salt at a concentration of about 10 mM to about 1 M. In certain embodiments, the salt is an acetate salt. In certain embodiments, the acetate salt is selected from the group consisting of ammonium acetate, potassium acetate, sodium acetate, and cesium acetate. In certain embodiments, the acetate salt is ammonium acetate. In certain embodiments, the acetate salt is sodium acetate.

In certain embodiments, the eluant comprises about 10 mM to about 1 M ammonium acetate. In certain embodiments, the eluant comprises about 10 mM to about 150 mM ammonium acetate. In certain embodiments, the eluant comprises about 100 mM to about 300 mM ammonium acetate.

In certain embodiments, the eluant comprises about 0.001% (w/v) to about 0.05% (w/v) Poloxamer 188. In certain embodiments, the eluant comprises about 0.01% (w/v) Poloxamer 188.

In certain embodiments, the pH of the eluant is about 9 to about 10.5. In certain embodiments, the pH of the eluant is about 9.3.

In certain embodiments, the eluant further comprises about 50 mM ethanolamine.

In certain embodiments, the eluant has a conductivity of about 8.5 mS/cm to about 30 mS/cm. In certain embodiments, the eluant has a conductivity of about 8.5 mS/cm to about 10.5 mS/cm. In certain embodiments, the eluant has a conductivity of about 10 mS/cm to about 11.5 mS/cm. In certain embodiments, the eluant has a conductivity of about 14 mS/cm to about 17.5 mS/cm. In certain embodiments, the eluant has a conductivity of about 18.5 mS/cm. In certain embodiments, the eluant has a conductivity of about 19 mS/cm. In certain embodiments, the eluant has a conductivity of about 26 mS/cm.

In certain embodiments, the method results in an eluate comprising less than about 15% AAV particles that lack a complete genome. In certain embodiments, the method results in an eluate comprising less than about 10% AAV particles that lack a complete genome.

In certain embodiments, the method further comprises formulating the eluted AAV particle in a formulation buffer suitable for administration to a human subject.

In certain embodiments, the AAV is a recombinant AAV (rAAV) comprising an rAAV genome comprising a transgene. In certain embodiments, the transgene encodes a polypeptide. In certain embodiments, the transgene encodes an miRNA, shRNA, siRNA, antisense RNA, gRNA, antagomir, miRNA sponge, RNA aptazyme, RNA aptamer, lncRNA, ribozyme, or mRNA. In certain embodiments, the transgene encodes a protein selected from the group consisting of iduronate-2-sulfatase (I2S), frataxin (FXN), glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), cyclin-dependent kinase-like 5 (CDKL5/STK9), galactose-1 phosphate uridyltransferase, phenylalanine hydroxylase (PAH), branched-chain alpha-keto acid dehydrogenase, fumarylacetoacetate hydrolase, methylmalonyl-CoA mutase, medium-chain acyl-CoA dehydrogenase, ornithine transcarbamylase (OTC), argininosuccinic acid synthetase (AS S1), low density lipoprotein receptor (LDLR) protein, UDP-glucuronosyltransferase, adenosine deaminase, hypoxanthine guanine phosphoribosyltransferase, biotinidase, alpha-galactosidase A, copper-transporting ATPase 2 (ATP7B), beta-glucocerebrosidase, 70 kDa peroxisomal membrane protein (PMP70), and arylsulfatase A (ARSA). In certain embodiments, the transgene encodes an antibody or a fragment thereof selected from the group consisting of: muromonab-cd3, efalizumab, tositumomab, daclizumab, nebacumab, catumaxomab, edrecolomab, abciximab, rituximab, basiliximab, palivizumab, infliximab, trastuzumab, adalimumab, ibritumomab tiuxetan, omalizumab, cetuximab, bevacizumab, natalizumab, panitumumab, ranibizumab, eculizumab, certolizumab, ustekinumab, canakinumab, golimumab, ofatumumab, tocilizumab, denosumab, belimumab, ipilimumab, brentuximab vedotin, pertuzumab, raxibacumab, obinutuzumab, alemtuzumab, siltuximab, ramucirumab, vedolizumab, blinatumomab, nivolumab, pembrolizumab, idarucizumab, necitumumab, dinutuximab, secukinumab, mepolizumab, alirocumab, evolocumab, daratumumab, elotuzumab, ixekizumab, reslizumab, olaratumab, bezlotoxumab, atezolizumab, obiltoxaximab, inotuzumab ozogamicin, brodalumab, guselkumab, dupilumab, sarilumab, avelumab, ocrelizumab, emicizumab, benralizumab, gemtuzumab ozogamicin, durvalumab, burosumab, erenumab, galcanezumab, lanadelumab, mogamulizumab, tildrakizumab, cemiplimab, fremanezumab, ravulizumab, emapalumab, ibalizumab, moxetumomab, caplacizumab, romosozumab, risankizumab, polatuzumab, eptinezumab, leronlimab, sacituzumab, brolucizumab, isatuximab, and teprotumumab. In certain embodiments, the transgene encodes a protein which is not selected from the group consisting of phenylalanine hydroxylase (PAH), iduronate-2-sulfatase (I2S), arylsulfatase A (ARSA), and an anti-complement component 5 antibody.

In certain embodiments, the transgene encodes a protein selected from the group consisting of frataxin (FXN), glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), cyclin-dependent kinase-like 5 (CDKL5/STK9), galactose-1 phosphate uridyltransferase, branched-chain alpha-keto acid dehydrogenase, fumarylacetoacetate hydrolase, methylmalonyl-CoA mutase, medium-chain acyl-CoA dehydrogenase, ornithine transcarbamylase (OTC), argininosuccinic acid synthetase (ASS1), low density lipoprotein receptor (LDLR) protein, UDP-glucuronosyltransferase, adenosine deaminase, hypoxanthine guanine phosphoribosyltransferase, biotinidase, alpha-galactosidase A, copper-transporting ATPase 2 (ATP7B), beta-glucocerebrosidase, and 70 kDa peroxisomal membrane protein (PMP70).

In certain embodiments, the rAAV genome further comprises a transcriptional regulatory element operably linked to the transgene. In certain embodiments, the transcriptional regulatory element comprises a promoter element and/or an intron element.

In certain embodiments, the rAAV genome further comprises a polyadenylation sequence. In certain embodiments, the polyadenylation sequence is 3′ to the transgene.

In certain embodiments, the rAAV genome comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 50, 51, 52, 53, or 54

In certain embodiments, the rAAV genome further comprises a 5′ inverted terminal repeat (5′ ITR) nucleotide sequence 5′ of the transgene, and a 3′ inverted terminal repeat (3′ ITR) nucleotide sequence 3′ of the transgene. In certain embodiments, the 5′ ITR nucleotide sequence is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 39, 41, or 42, and/or the 3′ ITR nucleotide sequence is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 40, 43, or 44.

In certain embodiments, the rAAV genome comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 55, 56, 57, 58, or 59.

In certain embodiments, the rAAV comprises an AAV capsid comprising an AAV capsid protein. In certain embodiments, the AAV capsid protein is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAVS, AAV6, AAV7, AAV8, AAV9, AAV-DJ, AAV-LK03, NP59, VOY101, VOY201, VOY701, VOY801, VOY1101, AAVPHP.N, AAVPHP.A, AAVPHP.B, PHP.B2, PHP.B3, G2A3, G2B4, G2B5, and PHP.S.

In certain embodiments, the AAV capsid protein does not comprise an amino acid sequence that is at least 85% identical to the amino acid sequence of amino acids 203-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17, wherein: the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO: 16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G or Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C; or, the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G. In certain embodiments, the AAV capsid protein does not comprise an amino acid sequence that is at least 85% identical to the amino acid sequence of amino acids 203-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17, wherein: (a) the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G, and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G; (b) the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H, the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; (c) the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; (d) the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A, and the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; or (e) the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C. In certain embodiments, the AAV capsid protein does not comprise the amino acid sequence of amino acids 203-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17.

In certain embodiments, the AAV capsid protein does not comprise an amino acid sequence that is at least 85% identical to the amino acid sequence of amino acids 138-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17, wherein: the amino acid in the capsid protein corresponding to amino acid 151 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 160 of SEQ ID NO: 16 is D; the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO: 16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G or Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C; or, the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G. In certain embodiments, the AAV capsid protein does not comprise an amino acid sequence that is at least 85% identical to the amino acid sequence of amino acids 138-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17, wherein: (a) the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G, and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G; (b) the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H, the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; (c) the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; (d) the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A, and the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; or (e) the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C. In certain embodiments, the AAV capsid protein does not comprise the amino acid sequence of amino acids 138-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 16, or 17.

In certain embodiments, the AAV capsid protein does not comprise an amino acid sequence that is at least 85% identical to the amino acid sequence of amino acids 1-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17, wherein: the amino acid in the capsid protein corresponding to amino acid 2 of SEQ ID NO: 16 is T; the amino acid in the capsid protein corresponding to amino acid 65 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 68 of SEQ ID NO: 16 is V; the amino acid in the capsid protein corresponding to amino acid 77 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 119 of SEQ ID NO: 16 is L; the amino acid in the capsid protein corresponding to amino acid 151 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 160 of SEQ ID NO: 16 is D; the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO: 16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G or Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C; or, the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G. In certain embodiments, the AAV capsid protein does not comprise an amino acid sequence that is at least 85% identical to the amino acid sequence of amino acids 1-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17, wherein: (a) the amino acid in the capsid protein corresponding to amino acid 2 of SEQ ID NO: 16 is T, and the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q; (b) the amino acid in the capsid protein corresponding to amino acid 65 of SEQ ID NO: 16 is I, and the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is Y; (c) the amino acid in the capsid protein corresponding to amino acid 77 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K; (d) the amino acid in the capsid protein corresponding to amino acid 119 of SEQ ID NO: 16 is L, and the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S; (e) the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G, and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G; (f) the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H, the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; (g) the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; (h) the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A, and the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; or (i) the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C. In certain embodiments, the AAV capsid protein does not comprise the amino acid sequence of amino acids 1-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17.

In certain embodiments, the AAV capsid protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of amino acids 203-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO: 16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G or Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C; or, the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G. In certain embodiments: (a) the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G, and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G; (b) the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H, the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; (c) the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; (d) the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A, and the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; or (e) the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C. In certain embodiments, the AAV capsid protein comprises the amino acid sequence of amino acids 203-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17.

In certain embodiments, the AAV capsid protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of amino acids 138-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 151 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 160 of SEQ ID NO: 16 is D; the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO: 16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G or Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C; or, the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G. In certain embodiments: (a) the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G, and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G; (b) the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H, the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; (c) the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; (d) the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A, and the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; or (e) the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C. In certain embodiments, the AAV capsid protein comprises the amino acid sequence of amino acids 138-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 16, or 17.

In certain embodiments, the AAV capsid protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of amino acids 1-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 2 of SEQ ID NO: 16 is T; the amino acid in the capsid protein corresponding to amino acid 65 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 68 of SEQ ID NO: 16 is V; the amino acid in the capsid protein corresponding to amino acid 77 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 119 of SEQ ID NO: 16 is L; the amino acid in the capsid protein corresponding to amino acid 151 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 160 of SEQ ID NO: 16 is D; the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO: 16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G or Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C; or, the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G. In certain embodiments: (a) the amino acid in the capsid protein corresponding to amino acid 2 of SEQ ID NO: 16 is T, and the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q; (b) the amino acid in the capsid protein corresponding to amino acid 65 of SEQ ID NO: 16 is I, and the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is Y; (c) the amino acid in the capsid protein corresponding to amino acid 77 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K; (d) the amino acid in the capsid protein corresponding to amino acid 119 of SEQ ID NO: 16 is L, and the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S; (e) the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G, and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G; (f) the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H, the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; (g) the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; (h) the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A, and the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; or (i) the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C. In certain embodiments, the AAV capsid protein comprises the amino acid sequence of amino acids 1-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17.

In another aspect, the present disclosure provides a composition comprising an AEX medium and a mixture comprising magnesium chloride (MgCl2) and an acetate.

In certain embodiments, the mixture comprises an eluate of an affinity chromatography column.

In certain embodiments, the mixture comprises about 10 mM to about 200 mM of an acetate. In certain embodiments, the mixture comprises about 10 mM to about 155 mM of an acetate. In certain embodiments, the mixture comprises about 10 mM to about 200 mM of ammonium acetate. In certain embodiments, the mixture comprises about 10 mM to about 155 mM of ammonium acetate. In certain embodiments the mixture comprises about 153 mM ammonium acetate. In certain embodiments, the mixture comprises about 10 mM to about 40 mM of an acetate. In certain embodiments, the mixture comprises about 10 mM to about 40 mM ammonium acetate. In certain embodiments, the mixture comprises about 28 mM ammonium acetate.

In certain embodiments, the mixture comprises about 2 mM to about 6 mM magnesium chloride. In certain embodiments, the mixture comprises about 2 mM magnesium chloride.

In certain embodiments, the mixture comprises about 0.001% (w/v) to about 0.05% (w/v) Poloxamer 188. In certain embodiments, the mixture comprises about 0.01% (w/v) Poloxamer 188.

In certain embodiments, the pH of the mixture is about 9 to about 10.5. In certain embodiments, the pH of the mixture is about 9.3.

In another aspect, the present disclosure provides a composition comprising an AEX medium and a first wash solution, wherein the first wash solution comprises urea and an acetate.

In certain embodiments, the AEX medium has an average pore size of at least about 100 nm. In certain embodiments, the AEX medium has an average pore size of at least about 500 nm.

In certain embodiments, the AEX medium comprises a quaternary amine. In certain embodiments, the AEX medium comprises a quaternary polyethyleneimine group.

In certain embodiments, the first wash solution comprises about 0.1 M to about 4 M urea. In certain embodiments, the first wash solution comprises about 2 M urea.

In certain embodiments, the first wash solution comprises about 10 mM to about 200 mM of an acetate. In certain embodiments, the first wash solution comprises about 10 mM to about 155 mM of an acetate. In certain embodiments, the first wash solution comprises about 10 mM to about 200 mM of ammonium acetate. In certain embodiments, the first wash solution comprises about 10 mM to about 155 mM of ammonium acetate. In certain embodiments the first wash solution comprises about 153 mM ammonium acetate. In certain embodiments, the first wash solution comprises about 10 mM to about 32 mM of the acetate. In certain embodiments, the first wash solution comprises about 10 mM to about 32 mM ammonium acetate. In certain embodiments, the first wash solution comprises about 28 mM ammonium acetate.

In certain embodiments, the first wash solution comprises about 2 mM to about 5.7 mM magnesium chloride. In certain embodiments, the first wash solution comprises about 2 mM magnesium chloride.

In certain embodiments, the first wash solution comprises about 0.001% (w/v) to about 0.05% (w/v) Poloxamer 188. In certain embodiments, the first wash solution comprises about 0.01% (w/v) Poloxamer 188.

In certain embodiments, the pH of the first wash solution is about 9 to about 10.5. In certain embodiments, the pH of the first wash solution is about 9.3.

In certain embodiments, the first wash solution has a conductivity of about 3 mS/cm.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A is a plot showing the chromatographic overlay of the elution profiles obtained from an AEX process that included Bis-Tris Propane and sodium chloride (BTP/NaCl) in the load composition and wash steps, with or without 5.7 mM MgCl2 in the load composition. FIG. 1B is a plot showing the chromatographic overlay of the elution profiles obtained from an AEX process to separate intact AAV particles from empty capsids that included ammonium acetate (AmAc) in the load composition and wash steps, and an AEX process that included Bis-Tris Propane and sodium chloride (BTP/NaCl) in the load composition and wash steps. FIG. 1C is a plot showing the chromatographic overlay of the elution profiles obtained from an AEX process that included AmAc in the load composition and wash steps, with 5.7 mM MgCl2, or without MgCl2 in the load composition. FIG. 1D is a plot showing the chromatographic profiles of samples collected from the flowthrough and wash (FT/Wash) steps of AEX processes that included ammonium acetate in the load composition and wash steps, with or without 5.7 mM MgCl2 in the load composition. In FIGS. 1A-1D, the A260 (dashed lines) and A280 (solid lines) profiles are shown; the γ-axis absorbance units are in mAU, and the x-axis represents run volume in ml. In general, an A260 value higher than A280 value indicates an enrichment of intact AAV, and an A280 value higher than A260 value indicates an enrichment of empty AAV.

FIG. 2 shows a flow diagram of an AEX process with and without magnesium chloride.

FIG. 3 is a plot showing the chromatographic overlay of elution profiles obtained from an AEX process that included magnesium chloride in both the load composition and the first wash solution, and an AEX process run that did not include magnesium chloride in the load composition and first wash solution. Profiles and axis units are identical to those described for FIGS. 1A-1D.

FIG. 4 is a flow diagram of an AEX process with wash prior to elution at 1 mS/cm conductivity and an AEX process with wash prior to elution at 3 mS/cm conductivity.

FIG. 5 is a plot showing the chromatographic overlay of the step gradient elution profiles obtained from an AEX process that included a 1 mS/cm conductivity wash prior to elution, and an AEX process that included a 3 mS/cm conductivity wash prior to elution. Profiles and axis units are identical to those described for FIGS. 1A-1D.

FIG. 6 is a flow diagram of an AEX process run with and without the use of a wash step with a wash solution comprising urea.

FIGS. 7A-7B are plots showing the chromatographic overlay of wash (FIG. 7A) and elution (FIG. 7B) profiles obtained from an AEX process that did not employ the use of a wash solution comprising urea and an AEX process that employed the use of a wash solution comprising urea. Profiles and axis units are identical to those described for FIGS. 1A-1D.

FIGS. 8A-8B are a series of elution profiles obtained from AEX process runs performed using AEX media having the indicated average pore sizes for representative AAV comprising a self-complementary vector genome (FIG. 8A) or single-stranded vector genome (FIG. 8B).

FIGS. 9A-9B are plots showing the full chromatogram (FIG. 9A) and elution profile (FIG. 9B) obtained from AEX with linear gradient elution for a representative AAV8 vector. In FIGS. 9A-9B, the A260 (black lines) and A280 (grey lines) profiles are shown; the γ-axis absorbance units are in mAU, and the x-axis represents run volume in ml.

FIGS. 10A-10C are plots showing the full chromatogram (FIG. 10A) and elution profiles (FIGS. 10B-C) obtained from AEX with isocratic elution for a representative AAV8 vector. The elution profile is shown for the AEX process performed with the 13 mS/cm load condition relative to a 3 mS/cm load condition (FIG. 10C). In FIGS. 10A-10B, the A260 (black lines) and A280 (grey lines) profiles are shown; the γ-axis absorbance units are in mAU, and the x-axis represents run volume in ml. In FIG. 10C the A280 profiles are shown; the γ-axis absorbance units are in mAU, and the x-axis represents run volume in ml.

FIGS. 11A-11B are plots showing the full chromatogram (FIG. 11A) and elution profile (FIG. 11B) obtained from AEX with linear gradient elution for a representative AAV9 vector. In FIGS. 11A-11B, the A260 (dashed lines) and A280 (solid lines) profiles are shown; the y-axis absorbance units are in mAU, and the x-axis represents run volume in ml.

FIGS. 12A-12B are plots showing the full chromatogram (FIG. 12A) and elution profile (FIG. 12B) obtained from AEX with step gradient elution for a representative AAV9 vector. In FIGS. 12A-12B, the A260 (dashed lines) and A280 (solid lines) profiles are shown; the y-axis absorbance units are in mAU, and the x-axis represents run volume in ml.

FIGS. 13A-13B are plots showing the full chromatogram (FIG. 13A) and elution profile (FIG. 13B) obtained from AEX with linear gradient elution for a representative AAV2 vector. In FIGS. 13A-13B, the A260 (black lines) and A280 (grey lines) profiles are shown; the y-axis absorbance units are in mAU, and the x-axis represents run volume in ml.

FIGS. 14A-14C are plots showing the full chromatogram (FIG. 14A) and elution profiles (FIGS. 14B-C) obtained from AEX with isocratic elution for a representative AAV2 vector. The elution profile shows the A260 (dashed lines) and A280 (solid lines) profiles for the AEX process performed with the 13 mS/cm load condition (FIG. 14C). In FIGS. 14A-14C, the A260 (black lines) and A280 (grey lines) profiles are shown; the γ-axis absorbance units are in mAU, and the x-axis represents run volume in ml.

 DE TAILED DESCRIPTION

The present disclosure provides methods for the separation of an adeno-associated virus (AAV) particle from a mixture of the AAV and at least one contaminant. The methods and compositions provided by the present disclosure allow for the improved purification of intact AAV particles from contaminants such as AAV particles that lack a complete genome (e.g., empty capsids) and AAV degradation products. Consequently, the methods disclosed herein, and the compositions disclosed herein for use in such methods, result in compositions having a high purity of intact AAV particles.

I. Definitions

As used herein, the term “recombinant adeno-associated virus” or “rAAV” refers to an adeno-associated virus (AAV) comprising a genome lacking functional rep and cap genes.

As used herein, the term “cap gene” refers to a nucleic acid sequence that encodes an AAV capsid protein.

As used herein, the term “rAAV genome” refers to a nucleic acid molecule comprising the genome sequence of an rAAV. The skilled artisan will appreciate that where an rAAV genome comprises a transgene, the rAAV genome can be in the sense or antisense orientation relative to the direction of transcription of the transgene.

As used herein, the term “editing genome” refers to a recombinant AAV genome that is capable of integrating an editing element (e.g., one or more nucleotides or an internucleotide bond) via homologous recombination into a target locus to correct a genetic defect in a target gene. The skilled artisan will appreciate that the portion of an editing genome comprising the 5′ homology arm, editing element, and 3′ homology arm can be in the sense or antisense orientation relative to the target locus.

As used herein, the term “editing element” refers to the portion of an editing genome that when integrated at a target locus modifies the target locus. An editing element can mediate insertion, deletion, or substitution of one or more nucleotides at the target locus. As used herein, the term “target locus” refers to a region of a chromosome or an internucleotide bond (e.g., a region or an internucleotide bond of a target gene) that is modified by an editing element.

As used herein, the term “homology arm” refers to a portion of an editing genome positioned 5′ or 3′ of an editing element that is substantially identical to the genome flanking a target locus.

As used herein, the “percentage identity” between two nucleotide sequences or between two amino acid sequences is calculated by multiplying the number of matches between the pair of aligned sequences by 100, and dividing by the length of the aligned region, including internal gaps. Identity scoring only counts perfect matches and does not consider the degree of similarity of amino acids to one another. Note that only internal gaps are included in the length, not gaps at the sequence ends.

As used herein, the term “coding sequence” refers to the portion of a complementary DNA (cDNA) that encodes a polypeptide, starting at the start codon and ending at the stop codon. A gene may have one or more coding sequences due to alternative splicing, alternative translation initiation, and variation within the population. A coding sequence may be wild-type or a non-naturally occurring variant (e.g., a codon optimized variant).

As used herein, the term “transcriptional regulatory element” or “TRE” refers to a cis-acting nucleotide sequence, for example, a DNA sequence, that regulates (e.g., controls, increases, or reduces) transcription of an operably linked nucleotide sequence by an RNA polymerase to form an RNA molecule. A TRE relies on one or more trans-acting molecules, such as transcription factors, to regulate transcription. Thus, one TRE may regulate transcription in different ways when it is in contact with different trans-acting molecules, for example, when it is in different types of cells. A TRE may comprise one or more promoter elements and/or enhancer elements. A skilled artisan would appreciate that the promoter and enhancer elements in a gene may be close in location, and the term “promoter” may refer to a sequence comprising a promoter element and an enhancer element. Thus, the term “promoter” does not exclude an enhancer element in the sequence. The promoter and enhancer elements do not need to be derived from the same gene or species, and the sequence of each promoter or enhancer element may be either identical or substantially identical to the corresponding endogenous sequence in the genome.

As used herein, the term “operably linked” is used to describe the connection between a TRE and a coding sequence to be transcribed. Typically, gene expression is placed under the control of a TRE comprising one or more promoter and/or enhancer elements. The coding sequence is “operably linked” to the TRE if the transcription of the coding sequence is controlled or influenced by the TRE. The promoter and enhancer elements of the TRE may be in any orientation and/or distance from the coding sequence, as long as the desired transcriptional activity is obtained. In certain embodiments, the TRE is upstream from the coding sequence.

As used herein, the term “polyadenylation sequence” refers to a DNA sequence that when transcribed into RNA constitutes a polyadenylation signal sequence. The polyadenylation sequence can be native or exogenous. The exogenous polyadenylation sequence can be a mammalian or a viral polyadenylation sequence (e.g., an SV40 polyadenylation sequence).

As used herein, “exogenous polyadenylation sequence” refers to a polyadenylation sequence not identical or substantially identical to the endogenous polyadenylation sequence of a transgene. In certain embodiments, an exogenous polyadenylation sequence is a polyadenylation sequence of a gene different from the transgene, but within the same species (e.g., human). In certain embodiments, an exogenous polyadenylation sequence is a polyadenylation sequence of a different organism (e.g., a virus).

As used herein, the term “contaminant” refers to any material present at any stage of a method disclosed herein that is not the desired intact AAV particle. Contaminants include, without limitation, viral and cellular proteins or nucleic acids, or byproducts thereof, that arise in the production process of the desired intact AAV particle, or any undesired AAV particle or byproduct thereof, including, for example, an AAV particle that lacks a complete vector genome (also referred to herein as an “empty capsid”). A contaminant also includes any host cell protein, host cell nucleic acids, or host cell fragment that results from any stage of an AAV production process. The terms “host cell protein,” “host cell nucleic acid,” and “host cell fragment” are used herein to refer to any unwanted protein, nucleic acid, or cell fragment that originates from the cell used to produce an AAV particle.

As used herein, the term “AAV degradation product” refers to a product or intermediate thereof that arises from the degradation (e.g., physical, chemical, or enzymatic degradation) of an AAV particle.

As used herein, the term “about,” when in reference to a value or parameter herein, includes a variability of ±5% of the value or parameter. For example, when referring to a pH value, “about” refers to a range that includes the value 5% below the referenced value, and the value 5% above the referenced value. Thus, a pH of about 10 refers to a pH that encompasses a pH of 9.5 to a pH of 10.5, inclusive.

II. Anion Exchange Chromatography Methods and Compositions

One challenge in the downstream processing of AAV is the efficient separation of intact AAV from contaminants, including process-related impurities such as AAV particles that lack a complete genome (e.g., empty capsids) and AAV degradation products. In one aspect, the present disclosure provides methods for the separation of an adeno-associated virus (AAV) particle from a mixture of the AAV particle and at least one contaminant, using an anion exchange chromatography (AEX) process. In general, an AEX process comprises the following steps in sequential order: preparation of the load composition comprising a mixture of the AAV and at least one contaminant, application of the load composition to the AEX medium, washing of the AEX medium, and elution of the AAV. The skilled artisan will appreciate that depending on the desired purpose and outcome, the AEX process can comprise additional intermediate steps, and the skilled artisan will be able to determine the optimal AEX process for the desired purpose and outcome.

The present disclosure relates to improved methods for the separation of intact AAV from contaminants, such as AAV particles that lack a complete genome (e.g., empty capsids). In one aspect, such methods comprise the application of a mixture comprising the AAV and contaminants to an AEX medium, wherein the mixture further comprises MgCl2 and an acetate, followed by subsequent washing of the AEX medium having AAV bound thereto. In another aspect, such methods comprise the application of a mixture comprising the AAV and contaminants to an AEX medium, followed by subsequent washing of the AEX medium having AAV bound thereto with a wash solution that comprises urea and an acetate.

In certain embodiments, methods of the present disclosure include a low conductivity wash step prior to elution, and the low conductivity wash is believed to be important for allowing for the efficient step gradient (isocratic) elution of AAV particles. Further, it has been found that the presence of MgCl2 and/or urea in an AEX process to purify AAV particles, contributes to achieving compositions having a high purity of intact AAV particles. Without being bound by any theory, it is believed that the presence of MgCl2 diminishes the binding of empty capsids to the AEX media, and that the chaotropic nature of urea disrupts the structure of empty capsids and aids in dissociating the empty capsids from the column. Together, this facilitates the desorption of empty capsids from the AEX media, leading to compositions having high purity of intact AAV particles. In particular, this facilitates the inhibition of binding of empty capsids and the desorption of empty capsids, leading to compositions having high purity of intact AAV particles. Methods of the present disclosure achieve unexpected separation efficiencies, resulting in final eluates that comprise less than about 15% AAV particles that lack a complete genome (e.g., empty capsids), and in some cases, less than about 10% AAV particles that lack a complete genome.

In one aspect, the present disclosure provides a method for the separation of an adeno-associated virus (AAV) particle from a mixture of the AAV particle and at least one contaminant, comprising: contacting the mixture with an anion exchange chromatography (AEX) medium under conditions such that the AAV particle binds to the AEX medium, wherein the mixture comprises magnesium chloride (MgCl2) and an acetate; and washing the AEX medium with a first wash solution under conditions such that the AAV particle remains bound to the AEX medium and the at least one contaminant does not bind to the AEX medium, thereby separating the AAV particle from the at least one contaminant. In another aspect, the method comprises contacting the mixture of the AAV particle and at least one contaminant with an AEX medium under conditions such that a subset of empty AAV particles does not bind to the AEX medium and the full AAV particle binds to the AEX medium, wherein the mixture comprises magnesium chloride (MgCl2) and an acetate; and washing the AEX medium with a first wash solution comprising urea and an acetate under conditions such that the AAV particle remains bound to the AEX medium and the at least one contaminant does not bind the AEX medium, thereby separating the AAV particle from the at least one contaminant.

In another aspect, the present disclosure provides a method for the separation of an adeno-associated virus (AAV) particle from a mixture of the AAV particle and at least one contaminant, comprising: contacting the mixture with an anion exchange chromatography (AEX) medium under conditions such that the AAV particle binds to the AEX medium; and washing the AEX medium with a first wash solution comprising urea and an acetate under conditions such that the AAV particle remains bound to the AEX medium and the at least one contaminant does not bind to the AEX medium, thereby separating the AAV particle from the at least one contaminant. In another aspect, the method comprises contacting the mixture of the AAV particle and at least one contaminant with an AEX medium under conditions such that a subset of empty AAV particles does not bind to the AEX medium and the full AAV particle binds to the AEX medium; and washing the AEX medium with a first wash solution comprising urea and an acetate under conditions such that the AAV particle remains bound to the AEX medium and the at least one contaminant does not bind the AEX medium, thereby separating the AAV particle from the at least one contaminant. In certain embodiments, the method further comprises washing the AEX medium with a second wash solution.

In certain embodiments, the methods further comprise washing the AEX medium with a second wash solution.

In another aspect, the present disclosure provides a method for the separation of an adeno-associated virus (AAV) particle from a mixture of the AAV particle and at least one contaminant, comprising: providing an AEX medium that has been contacted with the mixture, wherein the mixture comprises magnesium chloride (MgCl2) and an acetate, and wherein the AEX medium comprises the AAV particle bound thereto and has been washed with a first wash solution such that the AAV particle remained bound to the AEX medium and the at least one contaminant did not bind to the AEX medium; and washing the AEX medium with a second wash solution under conditions such that the AAV particle remains bound to the AEX medium and the at least one contaminant does not bind to the AEX medium, thereby separating the AAV particle from the at least one contaminant.

In another aspect, the present disclosure provides a method for the separation of an adeno-associated virus (AAV) particle from a mixture of the AAV particle and at least one contaminant, comprising: providing an AEX medium that has been contacted with the mixture, wherein the AEX medium comprises the AAV particle bound thereto and has been washed with a first wash solution comprising urea and an acetate such that the AAV particle remained bound to the AEX medium and the at least one contaminant did not bind to the AEX medium; and washing the AEX medium with a second wash solution under conditions such that the AAV particle remains bound to the AEX medium and the at least one contaminant does not bind to the AEX medium, thereby separating the AAV particle from the at least one contaminant.

In certain embodiments, the methods of the present disclosure further comprise eluting the desired AAV from the AEX medium. In certain embodiments, the methods of the present disclosure further comprise eluting the desired AAV from the AEX medium, wherein the elution step is performed sequentially after the AEX medium has been washed with a low conductivity wash solution (e.g., a second wash solution described herein). In certain embodiments, the AEX medium is washed with a low conductivity wash solution (e.g., a second wash solution described herein) prior to eluting the AAV particle from the AEX medium. In certain embodiments, the methods of the present disclosure further comprise formulating the eluted AAV particle in a formulation buffer. In certain embodiments, the formulation buffer is suitable for administration to a human subject.

In another aspect, the present disclosure provides a composition comprising an AEX medium and a mixture comprising magnesium chloride and an acetate.

In another aspect, the present disclosure provides a composition comprising an AEX medium and a first wash solution, wherein the first wash solution comprises urea and an acetate.

Anion Exchange Chromatography Medium

The methods generally comprise contacting the mixture with an anion exchange chromatography (AEX) medium under conditions such that the AAV particle binds to the AEX medium, and washing the AEX medium comprising the AAV particle bound thereto with a first wash solution. In another aspect, the methods comprise contacting the mixture with an AEX medium under conditions such that a subset of empty AAV particles does not bind to the AEX medium and the full AAV particle binds to the AEX medium, and washing the AEX medium comprising the AAV particle bound thereto with a first wash solution. The methods disclosed herein separate the AAV particle from the at least one contaminant.

The contaminant can be any material present at any stage of a method disclosed herein that is not the desired AAV particle. Contaminants include, without limitation, viral and cellular proteins or nucleic acids, or byproducts thereof, that arise in the production process of the AAV particle. Contaminants also include any undesired AAV particles or byproducts thereof. Undesired AAV particles include, without limitation, AAV particles that lack a complete vector genome, for example, AAV particles that lack a complete vector genome (e.g., empty capsids). In certain embodiments, a method of the present disclosure is useful in separating desired AAV particles from undesired material such as, without limitation, viral and cellular proteins or nucleic acids, or byproducts thereof, e.g., host cell proteins and host cell fragments, AAV degradation products, AAV particles that lack a complete vector genome, and any combination thereof.

In particular, a method of the present disclosure provides improved separation of desired AAV particles from AAV particles that lack a complete genome (e.g. empty capsids), resulting in an eluate that comprises a higher percentage of desired AAV particles. In certain embodiments, the eluate comprising the desired AAV particles, comprises less than about 40%, e.g., less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5% AAV particles that lack a complete genome. In certain embodiments, a method of the present disclosure results in an eluate comprising the desired AAV particles, wherein the eluate comprises less than about 15% AAV particles that lack a complete genome (e.g., empty capsids). In certain embodiments, a method of the present disclosure results in an eluate comprising the desired AAV particles, wherein the eluate comprises less than about 10% AAV particles that lack a complete genome.

Methods of the present disclosure employ an AEX medium. In certain embodiments, the AEX medium includes an insoluble matrix or solid support (e.g., beads) capable of having a surface ionization. In certain embodiments, the AEX medium comprises acrylamides, agarose-based materials, cellulose, methacrylates, polystyrene divinyl benzene, or silica-based materials.

In certain embodiments, the AEX medium comprises a weak anion exchange resin comprising a solid support having a surface coated with a weak anion exchanger. In certain embodiments, the AEX medium comprises a strong anion exchange resin comprising a solid support having a surface coated with a weak anion exchanger. Examples of AEX media suitable for use in the methods described herein include, without limitation, those that comprise a quaternary amine, a quaternary polyethyleneimine, a quaternary ammonium, or a diethylaminoethanol (DEAE) ligand. In certain embodiments, a method of the present disclosure employs the use of an AEX medium comprising a quaternary amine. In certain embodiments, a method of the present disclosure employs the use of an AEX medium comprising a quaternary polyethyleneimine. In certain embodiments, the anion exchange chromatography medium is from the CEVIMULTUS™ series of anion exchange media, e.g., CEVIMULTUS™ QA (BlAseparations), or the POROS™ series of anion exchange media, e.g., POROS™ HQ, POROS™ XQ, POROS™ PI (ThermoFisher Scientific). Other examples of anion exchange chromatography media include, without limitation, Nuvia HP-Q, SOURCE™ 15Q (Cytiva), Q-SepharoseXL (Cytiva), NATRIX® Q, HITRAP® Q (Cytiva), and Capto™ Q (Cytiva).

Suitable AEX media for use in the methods of the present disclosure have an average pore size of at least about 100 nm. In certain embodiments, the AEX medium has an average pore size of about 100 nm or greater, e.g., at least about 150 nm, at least about 200 nm, at least about 250 nm, at least about 300 nm, at least about 350 nm, at least about 400 nm, at least about 450 nm, at least about 500 nm. In certain embodiments, the AEX medium has an average pore size of about 500 nm or greater.

In certain embodiments, the present disclosure provides a composition comprising an AEX medium and a mixture comprising magnesium chloride and an acetate. Various components of the mixture are further described herein. In certain embodiments, the present disclosure provides a composition comprising an AEX medium and a first wash solution, wherein the first wash solution comprises urea and an acetate. Various components of the first wash solution are further described herein.

Mixtures

Methods of the present disclosure comprise contacting a mixture of the AAV particle and at least one contaminant with an AEX medium under conditions such that the AAV particle binds to the AEX medium. In another aspect, the methods comprise contacting the mixture of the AAV particle and at least one contaminant with an AEX medium under conditions such that a subset of empty AAV particles does not bind to the AEX medium and the full AAV particle binds to the AEX medium.

In certain embodiments, the mixture comprises a partially purified AAV composition. For example, the mixture can comprise a partially purified AAV composition that has undergone an affinity chromatography step. In certain embodiments, the mixture comprises an eluate from an affinity chromatography step. Such an affinity chromatography step generally comprises separating AAV from a mixture that contains other non-AAV contaminants such as viral and cellular proteins or nucleic acids, or byproducts thereof. Briefly, affinity chromatography can utilize a solid support having an AAV-specific binding protein bound thereto that can capture AAV particles from the mixture. Affinity chromatography comprises the use of affinity chromatography media which may be in the form of a resin packed into a column. In certain embodiments, the mixture comprises an eluate of an affinity chromatography column. In certain embodiments, the mixture comprises a diluted eluate of an affinity chromatography column.

In certain embodiments, the mixture comprises one or more salts. The salt can encompass a monovalent cation or a bivalent cation. In certain embodiments, the mixture comprises a salt that encompasses a monovalent cation. It is understood by those of skill in the art, that salts that encompass a monovalent cation dissociate into the monovalent cation and a monovalent anion. For example, ammonium acetate dissociates into an ammonium cation and an acetate ion. Examples of salts that encompass a monovalent cation include, without limitation, ammonium acetate, potassium acetate, sodium acetate, cesium chloride, lithium chloride, potassium chloride, sodium chloride, and the like. In certain embodiments, the mixture comprises about 10 mM to about 200 mM of a salt that encompasses a monovalent cation. For example, the mixture comprises about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM, about 120 mM, about 125 mM, about 130 mM, about 135 mM, about 140 mM, about 145 mM, about 150 mM, about 155 mM, about 160 mM, about 165 mM, about 170 mM, about 175 mM, about 180 mM, about 185 mM, about 190 mM, about 195 mM, or about 200 mM of a salt that encompasses a monovalent cation. In certain embodiments, the mixture comprises about 10 mM to about 155 mM of a salt that encompasses a monovalent cation. In certain embodiments, the mixture comprises about 1 mM to about 50 mM of a salt that encompasses a monovalent cation. For example, the mixture comprises about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM of a salt that encompasses a monovalent cation. In certain embodiments, the mixture comprises about 10 mM to about 32 mM of a salt that encompasses a monovalent cation.

In certain embodiments, the mixture comprises an acetate. In certain embodiments, the mixture comprises about 10 mM to about 200 mM of an acetate or a salt equivalent thereof. For example, the mixture comprises about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM, about 120 mM, about 125 mM, about 130 mM, about 135 mM, about 140 mM, about 145 mM, about 150 mM, about 155 mM, about 160 mM, about 165 mM, about 170 mM, about 175 mM, about 180 mM, about 185 mM, about 190 mM, about 195 mM, or about 200 mM of an acetate or a salt equivalent thereof. In certain embodiments, the mixture comprises about 10 mM to about 155 mM of an acetate or a salt equivalent thereof. In certain embodiments, the mixture comprises about 153 mM of an acetate or a salt equivalent thereof. In certain embodiments, the mixture comprises about 1 mM to about 50 mM of an acetate or a salt equivalent thereof. In certain embodiments, the mixture comprises about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM of an acetate or a salt equivalent thereof. In certain embodiments, the mixture comprises about 10 mM to about 32 mM of an acetate or a salt equivalent thereof. In certain embodiments, the mixture comprises about 28 mM of an acetate or a salt equivalent thereof. In certain embodiments, the mixture comprises about 29 mM of an acetate or a salt equivalent thereof.

Examples of suitable acetates are known to those of skill in the art, and include, without limitation, ammonium acetate, sodium acetate, and potassium acetate. In certain embodiments, the mixture comprises ammonium acetate. In certain embodiments, the mixture comprises about 10 mM to about 200 mM ammonium acetate. For example, the mixture comprises about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM, about 120 mM, about 125 mM, about 130 mM, about 135 mM, about 140 mM, about 145 mM, about 150 mM, about 155 mM, about 160 mM, about 165 mM, about 170 mM, about 175 mM, about 180 mM, about 185 mM, about 190 mM, about 195 mM, or about 200 mM ammonium acetate. In certain embodiments, the mixture comprises about 10 mM to about 155 mM ammonium acetate. In certain embodiments, the mixture comprises about 153 ammonium acetate. In certain embodiments, the mixture comprises about 1 mM to about 50 mM ammonium acetate. In certain embodiments, the mixture comprises about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM ammonium acetate. In certain embodiments, the mixture comprises about 10 mM to about 32 mM ammonium acetate. In certain embodiments, the mixture comprises about 28 mM ammonium acetate. In certain embodiments, the mixture comprises about 29 mM ammonium acetate.

In certain embodiments, the mixture comprises a salt that encompasses a divalent cation. Examples of salts that encompass a divalent cation include, without limitation, magnesium chloride, manganese chloride, copper chloride, zinc chloride, and the like. In certain embodiments, the mixture comprises magnesium chloride. In certain embodiments, the mixture comprises about 1 mM to about 10 mM magnesium chloride. In certain embodiments, the mixture comprises 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, or about 10 mM magnesium chloride. In certain embodiments, the mixture comprises about 2 mM to about 6 mM magnesium chloride. In certain embodiments, the mixture comprises about 2 mM magnesium chloride. In certain embodiments, the mixture comprises about 5.7 mM magnesium chloride.

In certain embodiments, the mixture comprises a salt that encompasses a monovalent cation and magnesium chloride. In certain embodiments, the mixture comprises about 10 mM to about 200 mM salt that encompasses a monovalent cation, and about 1 mM to about 10 mM magnesium chloride. In certain embodiments, the mixture comprises about 10 mM to about 200 mM salt that encompasses a monovalent cation, and about 2 mM to 6 mM magnesium chloride. In certain embodiments, the mixture comprises about 10 mM to about 200 mM salt that encompasses a monovalent cation, and about 2 mM magnesium chloride. In certain embodiments, the mixture comprises about 1 mM to about 50 mM salt that encompasses a monovalent cation, and about 1 mM to about 10 mM magnesium chloride. In certain embodiments, the mixture comprises about 1 mM to about 50 mM salt that encompasses a monovalent cation, and about 2 mM to 6 mM magnesium chloride. In certain embodiments, the mixture comprises about 1 mM to about 50 mM salt that encompasses a monovalent cation, and about 2 mM magnesium chloride. In certain embodiments, the mixture comprises about 1 mM to about 50 mM salt that encompasses a monovalent cation, and about 5.7 mM magnesium chloride.

In certain embodiments, the mixture comprises an acetate or a salt equivalent thereof (e.g., ammonium acetate) and magnesium chloride. In certain embodiments, the mixture comprises about 10 mM to about 200 mM of an acetate or a salt equivalent thereof (e.g., ammonium acetate), and about 1 mM to about 10 mM magnesium chloride. In certain embodiments, the mixture comprises about 10 mM to about 200 mM of an acetate or a salt equivalent thereof (e.g., ammonium acetate), and about 2 mM to 6 mM magnesium chloride. In certain embodiments, the mixture comprises about 10 mM to about 200 mM of an acetate or a salt equivalent thereof (e.g., ammonium acetate), and about 2 mM magnesium chloride. In certain embodiments, the mixture comprises about 10 mM to about 155 mM of an acetate or a salt equivalent thereof (e.g., ammonium acetate), and about 2 mM magnesium chloride. In certain embodiments, the mixture comprises about 153 mM ammonium acetate and about 2 mM magnesium chloride. In certain embodiments, the mixture comprises about 1 mM to about 50 mM of an acetate or a salt equivalent thereof (e.g., ammonium acetate), and about 1 mM to about 10 mM magnesium chloride. In certain embodiments, the mixture comprises about 1 mM to about 50 mM of an acetate or a salt equivalent thereof (e.g., ammonium acetate), and about 2 mM to 6 mM magnesium chloride. In certain embodiments, the mixture comprises about 1 mM to about 50 mM of an acetate or a salt equivalent thereof (e.g., ammonium acetate), and about 2 mM magnesium chloride. In certain embodiments, the mixture comprises about 1 mM to about 50 mM of an acetate or a salt equivalent thereof (e.g., ammonium acetate), and about 5.7 mM magnesium chloride. In certain embodiments, the mixture comprises about 10 mM to about 32 mM of an acetate or a salt equivalent thereof (e.g., ammonium acetate), and about 1 mM to about 10 mM magnesium chloride. In certain embodiments, the mixture comprises about 10 mM to about 32 mM of an acetate or a salt equivalent thereof (e.g., ammonium acetate), and about 2 mM to 6 mM magnesium chloride. In certain embodiments, the mixture comprises about 10 mM to about 32 mM of an acetate or a salt equivalent thereof (e.g., ammonium acetate), and about 2 mM magnesium chloride. In certain embodiments, the mixture comprises about 10 mM to about 32 mM of an acetate or a salt equivalent thereof (e.g., ammonium acetate), and about 5.7 mM magnesium chloride. In certain embodiments, the mixture comprises about 28 mM of an acetate or a salt equivalent thereof (e.g., ammonium acetate) and about 2 mM magnesium chloride. In certain embodiments, the mixture comprises about 29 mM of an acetate or a salt equivalent thereof (e.g., ammonium acetate) and about 2 mM magnesium chloride. In certain embodiments, the mixture comprises about 28 mM of an acetate or a salt equivalent thereof (e.g., ammonium acetate) and about 5.7 mM magnesium chloride. In certain embodiments, the mixture comprises about 29 mM of an acetate or a salt equivalent thereof (e.g., ammonium acetate) and about 5.7 mM magnesium chloride. In certain embodiments, the mixture comprises about 28 mM ammonium acetate and about 2 mM magnesium chloride. In certain embodiments, the mixture comprises about 29 mM ammonium acetate and about 2 mM magnesium chloride. In certain embodiments, the mixture comprises about 28 mM ammonium acetate and about 5.7 mM magnesium chloride. In certain embodiments, the mixture comprises about 29 mM ammonium acetate and about 5.7 mM magnesium chloride.

In certain embodiments, the mixture comprises a surfactant. In certain embodiments, the surfactant is a non-ionic surfactant. Examples of non-ionic surfactants include, without limitation, Brij™-35, Brij™-58, NP-40, octyl-beta-glucoside, octylthioglucoside (OTG), poloxamer 188 (P-188), poloxamer 407, polysorbate 20, polysorbate 80, Triton X-100, or Triton X-114. In certain embodiments, the mixture comprises about 0.001% (w/v) to about 0.05% (w/v) of a non-ionic surfactant. In certain embodiments, the mixture comprises about 0.001% (w/v), about 0.005% (w/v), about 0.01% (w/v), about 0.015% (w/v), about 0.02% (w/v), about 0.03% (w/v), about 0.03 5% (w/v), about 0.04% (w/v), about 0.045% (w/v), or about 0.05% (w/v) of a non-ionic surfactant. In certain embodiments, the mixture comprises about 0.01% (w/v) of a non-ionic surfactant. In certain embodiments, the mixture comprises poloxamer 188. In certain embodiments, the mixture comprises about 0.001% (w/v) to about 0.05% (w/v) poloxamer 188. In certain embodiments, the mixture comprises about 0.001% (w/v), about 0.005% (w/v), about 0.01% (w/v), about 0.015% (w/v), about 0.02% (w/v), about 0.03% (w/v), about 0.035% (w/v), about 0.04% (w/v), about 0.045% (w/v), or about 0.05% (w/v) poloxamer 188. In certain embodiments, the mixture comprises about 0.01% (w/v) poloxamer 188.

In certain embodiments, the pH of the mixture is about 9 to about 10.5. In certain embodiments, the pH of the mixture is about 9, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, about 10, about 10.1, about 10.2, about 10.3, about 10.4, or about 10.5. In certain embodiments, the pH of the mixture is about 9.3.

In certain embodiments, the mixture has a pH of about 9.2, a conductivity of about 3 mS/cm, and comprises about 28 mM ammonium acetate, about 2 mM magnesium chloride, and about 0.01% (w/v) poloxamer 188. In certain embodiments, the mixture has a pH of about 9.3, a conductivity of about 3 mS/cm, and comprises about 28 mM ammonium acetate, about 2 mM magnesium chloride, and about 0.01% (w/v) poloxamer 188. In certain embodiments, the mixture has a pH of about 9.3, a conductivity of about 3 mS/cm, and comprises about 29 mM ammonium acetate, about 2 mM magnesium chloride, and about 0.01% (w/v) poloxamer 188.

Wash Solutions and Methods

After contacting a mixture with the AEX medium, the AEX medium is washed with one or more wash solutions to remove contaminants. In certain embodiments, the AEX medium is washed with a first wash solution (e.g., in a first wash step). In certain embodiments, the AEX medium is washed with a first wash solution and/or a second wash solution (e.g., in a second wash step). In certain embodiments, the AEX medium is washed with a first wash solution and a second wash solution.

In certain embodiments, a method of the present disclosure comprises, after application of the mixture to an AEX medium, washing the AEX medium with a first wash solution. The first wash solution comprises urea. It has been found that a first wash using a first wash solution comprising urea provides enhanced separation of the desired AAV from unwanted contaminants (e.g., empty capsids). Without being bound by theory, it is believed that the chaotropic nature of urea disrupts the structure of the contaminants including empty capsids, dissociating them from the AEX medium, resulting in their removal prior to elution. In certain embodiments, the first wash solution comprises about 0.1 M to about 4 M urea. In certain embodiments, the first wash solution comprises about 0.1 M, about 0.2 M, about 0.3 M, about 0.4 M, about 0.5 M, about 0.6 M, about 0.7 M, about 0.8 M, about 0.9 M, about 1.0 M, about 1.1 M, about 1.2 M, about 1.3 M, about 1.4 M, about 1.5 M, about 1.6 M, about 1.7 M, about 1.8 M, about 1.9 M, about 2.0 M, about 2.1 M, about 2.2 M, about 2.3 M, about 2.4 M, about 2.5 M, about 2.6 M, about 2.7 M, about 2.8 M, about 2.9 M, about 3.0 M, about 3.1 M, about 3.2 M, about 3.3 M, about 3.4 M, about 3.5 M, about 3.6 M, about 3.7 M, about 3.8 M, about 3.9 M, about 4.0 M urea. In certain embodiments, the first wash solution comprises about 2 M urea.

In certain embodiments, the first wash solution comprises urea and an acetate. In certain embodiments, the first wash solution comprises about 10 mM to about 200 mM of an acetate or a salt equivalent thereof. For example, the first wash solution comprises about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM, about 120 mM, about 125 mM, about 130 mM, about 135 mM, about 140 mM, about 145 mM, about 150 mM, about 155 mM, about 160 mM, about 165 mM, about 170 mM, about 175 mM, about 180 mM, about 185 mM, about 190 mM, about 195 mM, or about 200 mM of an acetate or a salt equivalent thereof. In certain embodiments, the first wash solution comprises about 10 mM to about 155 mM of an acetate or a salt equivalent thereof. In certain embodiments, the first wash solution comprises about 153 mM of an acetate or a salt equivalent thereof. In certain embodiments, the first wash solution comprises about 1 mM to about 50 mM of an acetate or a salt equivalent thereof. In certain embodiments, the first wash solution comprises about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM of an acetate or a salt equivalent thereof. In certain embodiments, the first wash solution comprises about 10 mM to about 40 mM of an acetate or a salt equivalent thereof. In certain embodiments, the first wash solution comprises about 10 mM to about 32 mM of an acetate or a salt equivalent thereof. In certain embodiments, the first wash solution comprises about 28 mM of an acetate or a salt equivalent thereof. In certain embodiments, the first wash solution comprises about 29 mM of an acetate or a salt equivalent thereof.

Examples of suitable acetates are known to those of skill in the art, and include, without limitation, ammonium acetate, sodium acetate, and potassium acetate. In certain embodiments, the first wash solution comprises ammonium acetate. In certain embodiments, the first wash solution comprises about 1 mM to about 50 mM ammonium acetate. In certain embodiments, the first wash solution comprises about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM ammonium acetate. In certain embodiments, the first wash solution comprises about 10 mM to about 40 mM ammonium acetate. In certain embodiments, the first wash solution comprises about 10 mM to about 32 mM ammonium acetate. In certain embodiments, the first wash solution comprises about 28 mM ammonium acetate. In certain embodiments, the first wash solution comprises about 29 mM ammonium acetate.

In certain embodiments, the first wash solution comprises a salt that encompasses a divalent cation. Examples of salts that encompass a divalent cation include, without limitation, magnesium chloride, manganese chloride, copper chloride, zinc chloride, and the like. In certain embodiments, the first wash solution comprises magnesium chloride. In certain embodiments, the first wash solution comprises about 10 mM to about 200 mM of ammonium acetate. For example, the first wash solution comprises about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM, about 120 mM, about 125 mM, about 130 mM, about 135 mM, about 140 mM, about 145 mM, about 150 mM, about 155 mM, about 160 mM, about 165 mM, about 170 mM, about 175 mM, about 180 mM, about 185 mM, about 190 mM, about 195 mM, or about 200 mM ammonium acetate. In certain embodiments, the first wash solution comprises about 10 mM to about 155 mM ammonium acetate. In certain embodiments, the first wash solution comprises about 153 mM ammonium acetate. In certain embodiments, the first wash solution comprises about 1 mM to about 10 mM magnesium chloride. In certain embodiments, the first wash solution comprises 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, or about 10 mM magnesium chloride. In certain embodiments, the first wash solution comprises about 2 mM to about 6 mM magnesium chloride. In certain embodiments, the first wash solution comprises about 2 mM magnesium chloride. In certain embodiments, the first wash solution comprises about 5.7 mM magnesium chloride.

In certain embodiments, the first wash solution comprises a surfactant. In certain embodiments, the surfactant is a non-ionic surfactant. Examples of non-ionic surfactants include, without limitation, Brij™-35, Brij™-58, NP-40, octyl-beta-glucoside, octylthioglucoside (OTG), poloxamer 188 (P-188), poloxamer 407, polysorbate 20, polysorbate 80, Triton X-100, or Triton X-114. In certain embodiments, the first wash solution comprises about 0.001% (w/v) to about 0.05% (w/v) of a non-ionic surfactant. In certain embodiments, the first wash solution comprises about 0.001% (w/v), about 0.005% (w/v), about 0.01% (w/v), about 0.015% (w/v), about 0.02% (w/v), about 0.03% (w/v), about 0.035% (w/v), about 0.04% (w/v), about 0.045% (w/v), or about 0.05% (w/v) of a non-ionic surfactant. In certain embodiments, the first wash solution comprises about 0.01% (w/v) of a non-ionic surfactant. In certain embodiments, the first wash solution comprises poloxamer 188. In certain embodiments, the first wash solution comprises about 0.001% (w/v) to about 0.05% (w/v) poloxamer 188. In certain embodiments, the first wash solution comprises about 0.001% (w/v), about 0.005% (w/v), about 0.01% (w/v), about 0.015% (w/v), about 0.02% (w/v), about 0.03% (w/v), about 0.035% (w/v), about 0.04% (w/v), about 0.045% (w/v), or about 0.05% (w/v) poloxamer 188. In certain embodiments, the first wash solution comprises about 0.01% (w/v) poloxamer 188.

In certain embodiments, the pH of the first wash solution is about 9 to about 10.5. In certain embodiments, the pH of the first wash solution is about 9, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, about 10, about 10.1, about 10.2, about 10.3, about 10.4, or about 10.5. In certain embodiments, the pH of the first wash solution is about 9.3.

In certain embodiments, the first wash solution has a conductivity of about 1 mS/cm to about 20 mS/cm. In certain embodiments, the first wash solution has a conductivity of about 1 mS/cm, about 2 mS/cm, about 3 mS/cm, about 4 mS/cm, about 5 mS/cm, about 6 mS/cm, about 7 mS/cm, about 8 mS/cm, about 9 mS/cm, about 10 mS/cm, about 11 mS/cm, about 12 mS/cm, about 13 mS/cm, about 14 mS/cm, about 15 mS/cm, about 16 mS/cm, about 17 mS/cm, about 18 mS/cm, about 19 mS/cm, or about 20 mS/cm. In certain embodiments, the first wash solution has a conductivity of about 13 mS/cm. In certain embodiments, the first wash solution has a conductivity of about 1 mS/cm to about 3 mS/cm. In certain embodiments, the first wash solution has a conductivity of about 1 mS/cm, about 1.1 mS/cm, about 1.2 mS/cm, about 1.3 mS/cm, about 1.4 mS/cm, about 1.5 mS/cm, about 1.6 mS/cm, about 1.7 mS/cm, about 1.8 mS/cm, about 1.9 mS/cm, about 2 mS/cm, about 2.1 mS/cm, about 2.2 mS/cm, about 2.3 mS/cm, about 2.4 mS/cm, about 2.5 mS/cm, about 2.6 mS/cm, about 2.7 mS/cm, about 2.8 mS/cm, about 2.9 mS/cm, or about 3 mS/cm. In certain embodiments, the first wash solution has a conductivity of about 3 mS/cm.

In certain embodiments, the first wash solution has a pH of about 9.2, a conductivity of about 3 mS/cm, and comprises about 28 mM ammonium acetate, about 2 mM magnesium chloride, and 0.01% (w/v) poloxamer 188. In certain embodiments, the first wash solution has a pH of about 9.3, a conductivity of about 3 mS/cm, and comprises about 28 mM ammonium acetate, about 2 mM magnesium chloride, and 0.01% (w/v) poloxamer 188. In certain embodiments, the first wash solution has a pH of about 9.3, a conductivity of about 3 mS/cm, and comprises about 29 mM ammonium acetate, about 2 mM magnesium chloride, and 0.01% (w/v) poloxamer 188. In certain embodiments, the first wash solution has a pH of about 9.3, a conductivity of about 13 mS/cm, and comprises about 153 mM ammonium acetate, about 2 mM magnesium chloride, and 0.01% (w/v) poloxamer 188.

In certain embodiments, the first wash solution has a pH of about 9.2, a conductivity of about 3 mS/cm, and comprises about 28 mM ammonium acetate, up to about 2 M urea, about 2 mM magnesium chloride, and 0.01% (w/v) poloxamer 188. In certain embodiments, the first wash solution has a pH of about 9.3, a conductivity of about 3 mS/cm, and comprises about 28 mM ammonium acetate, up to about 2 M urea, about 2 mM magnesium chloride, and 0.01% (w/v) poloxamer 188. In certain embodiments, the first wash solution has a pH of about 9.3, a conductivity of about 3 mS/cm, and comprises about 29 mM ammonium acetate, up to about 2 M urea, about 2 mM magnesium chloride, and 0.01% (w/v) poloxamer 188.

In certain embodiments, the first wash solution has a pH of about 9.2, a conductivity of about 3 mS/cm, and comprises about 28 mM ammonium acetate, about 2 M urea, about 2 mM magnesium chloride, and 0.01% (w/v) poloxamer 188. In certain embodiments, the first wash solution has a pH of about 9.3, a conductivity of about 3 mS/cm, and comprises about 28 mM ammonium acetate, about 2 M urea, about 2 mM magnesium chloride, and 0.01% (w/v) poloxamer 188. In certain embodiments, the first wash solution has a pH of about 9.3, a conductivity of about 3 mS/cm, and comprises about 29 mM ammonium acetate, about 2 M urea, about 2 mM magnesium chloride, and 0.01% (w/v) poloxamer 188. In certain embodiments, the first wash solution has a pH of about 9.3, a conductivity of about 13 mS/cm, and comprises about 153 mM ammonium acetate, about 2 M urea, about 2 mM magnesium chloride, and 0.01% (w/v) poloxamer 188.

In certain embodiments, a method of the present disclosure comprises, after washing the AEX medium after application of the mixture with a first wash solution, washing the AEX medium with a second wash solution. In certain embodiments, the second wash solution comprises an acetate. In certain embodiments, the second wash solution comprises about 0.1 mM to about 20 mM of an acetate or a salt equivalent thereof. In certain embodiments, the second wash solution comprises about 0.1 mM, 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, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM of an acetate or a salt equivalent thereof. In certain embodiments, the second wash solution comprises about 10 mM of an acetate or a salt equivalent thereof.

Examples of suitable acetates are known to those of skill in the art, and include, without limitation, ammonium acetate, sodium acetate, and potassium acetate. In certain embodiments, the second wash solution comprises ammonium acetate. In certain embodiments, the second wash solution comprises about 0.1 mM to about 20 mM ammonium acetate. In certain embodiments, the second wash solution comprises about 0.1 mM, 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, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM ammonium acetate. In certain embodiments, the second wash solution comprises about 10 mM ammonium acetate.

In certain embodiments, the second wash solution comprises a surfactant. In certain embodiments, the surfactant is a non-ionic surfactant. Examples of non-ionic surfactants include, without limitation, Brij™-35, Brij™-58, NP-40, octyl-beta-glucoside, octylthioglucoside (OTG), poloxamer 188 (P-188), poloxamer 407, polysorbate 20, polysorbate 80, Triton X-100, or Triton X-114. In certain embodiments, the second wash solution comprises about 0.001% (w/v) to about 0.05% (w/v) of a non-ionic surfactant. In certain embodiments, the second wash solution comprises about 0.001% (w/v), about 0.005% (w/v), about 0.01% (w/v), about 0.015% (w/v), about 0.02% (w/v), about 0.03% (w/v), about 0.035% (w/v), about 0.04% (w/v), about 0.045% (w/v), or about 0.05% (w/v) of a non-ionic surfactant. In certain embodiments, the second wash solution comprises about 0.01% (w/v) of a non-ionic surfactant. In certain embodiments, the second wash solution comprises poloxamer 188. In certain embodiments, the second wash solution comprises about 0.001% (w/v) to about 0.05% (w/v) poloxamer 188. In certain embodiments, the second wash solution comprises about 0.001% (w/v), about 0.005% (w/v), about 0.01% (w/v), about 0.015% (w/v), about 0.02% (w/v), about 0.03% (w/v), about 0.035% (w/v), about 0.04% (w/v), about 0.045% (w/v), or about 0.05% (w/v) poloxamer 188. In certain embodiments, the second wash solution comprises about 0.01% (w/v) poloxamer 188.

In certain embodiments, the pH of the second wash solution is about 9 to about 10.5. In certain embodiments, the pH of the second wash solution is about 9, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, about 10, about 10.1, about 10.2, about 10.3, about 10.4, or about 10.5. In certain embodiments, the pH of the second wash solution is about 9.3.

In certain embodiments, the second wash solution has a conductivity of less than about 3 mS/cm. In certain embodiments, the second wash solution has a conductivity of about 1 mS/cm to about 3 mS/cm. In certain embodiments, the second wash solution has a conductivity of about 1 mS/cm, about 1.1 mS/cm, about 1.2 mS/cm, about 1.3 mS/cm, about 1.4 mS/cm, about 1.5 mS/cm, about 1.6 mS/cm, about 1.7 mS/cm, about 1.8 mS/cm, about 1.9 mS/cm, about 2 mS/cm, about 2.1 mS/cm, about 2.2 mS/cm, about 2.3 mS/cm, about 2.4 mS/cm, about 2.5 mS/cm, about 2.6 mS/cm, about 2.7 mS/cm, about 2.8 mS/cm, about 2.9 mS/cm, or about 3 mS/cm. In certain embodiments, the second wash solution has a conductivity of about 1 mS/cm.

In certain embodiments, the second wash solution has a pH of about 9.2, a conductivity of about 1 mS/cm, and comprises about 10 mM ammonium acetate, and 0.01% (w/v) poloxamer 188. In certain embodiments, the second wash solution has a pH of about 9.3, a conductivity of about 1 mS/cm, and comprises about 10 mM ammonium acetate, and 0.01% (w/v) poloxamer 188. In certain embodiments, the second wash solution has a pH of about 9.3, a conductivity of about 3 mS/cm, and comprises about 34 mM ammonium acetate, and 0.01% (w/v) poloxamer 188.

Elution from AEX Medium

Methods of the present disclosure further comprise eluting the AAV from the AEX medium. AAV bound to the AEX medium can be eluted using a gradient elution or a step isocratic elution (also referred to herein as a step elution). The gradient elution may be a linear gradient elution. In certain embodiments, methods of the present disclosure comprise eluting the AAV from the AEX medium under a linear gradient elution. In certain embodiments, methods of the present disclosure comprise eluting the AAV from the AEX medium under a step isocratic elution.

Elution of the AAV bound to the AEX medium is achieved by applying an eluant to the AEX medium. In certain embodiments, for linear gradient elution, elution is performed using a linear gradient volume of an eluant with an increasing conductivity. In certain embodiments, for step elution, elution is performed by sequential addition of an eluant at increasing conductivities.

In certain embodiments, the AEX medium is washed with a second wash solution prior to eluting the AAV from the AEX medium.

In certain embodiments, the eluant comprises a salt. In certain embodiments, the eluant comprises an acetate. In certain embodiments, the eluant comprises about 10 mM to about 1 M of an acetate or a salt equivalent thereof. In certain embodiments, the eluant comprises about 10 mM to about 150 mM of an acetate or a salt equivalent thereof. In certain embodiments, the eluant comprises about 100 mM to about 300 mM of an acetate or a salt equivalent thereof. In certain embodiments, the eluant comprises about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, about 500 mM, about 550 mM, about 600 mM, about 650 mM, about 700 mM, about 750 mM, about 800 mM, about 850 mM, about 900 mM, about 950 mM, or about 1 M of an acetate or a salt equivalent thereof.

Examples of suitable acetates are known to those of skill in the art, and include, without limitation, ammonium acetate, sodium acetate, and potassium acetate. In certain embodiments, the eluant comprises ammonium acetate. In certain embodiments, the eluant comprises about 10 mM to about 1 M ammonium acetate. In certain embodiments, the eluant comprises about 10 mM to about 150 mM ammonium acetate. In certain embodiments, the eluant comprises about 100 mM to about 300 mM ammonium acetate. In certain embodiments, the eluant comprises about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, about 500 mM, about 550 mM, about 600 mM, about 650 mM, about 700 mM, about 750 mM, about 800 mM, about 850 mM, about 900 mM, about 950 mM, or about 1 M ammonium acetate.

In certain embodiments, the eluant comprises a surfactant. In certain embodiments, the surfactant is a non-ionic surfactant. Examples of non-ionic surfactants include, without limitation, Brij™-35, Brij™-58, NP-40, octyl-beta-glucoside, octylthioglucoside (OTG), poloxamer 188 (P-188), poloxamer 407, polysorbate 20, polysorbate 80, Triton X-100, or Triton X-114. In certain embodiments, the eluant comprises about 0.001% (w/v) to about 0.05% (w/v) of a non-ionic surfactant. In certain embodiments, the eluant comprises about 0.001% (w/v), about 0.005% (w/v), about 0.01% (w/v), about 0.015% (w/v), about 0.02% (w/v), about 0.03% (w/v), about 0.035% (w/v), about 0.04% (w/v), about 0.045% (w/v), or about 0.05% (w/v) of a non-ionic surfactant. In certain embodiments, the eluant comprises about 0.01% (w/v) of a non-ionic surfactant. In certain embodiments, the eluant comprises poloxamer 188. In certain embodiments, the eluant comprises about 0.001% (w/v) to about 0.05% (w/v) poloxamer 188. In certain embodiments, the eluant comprises about 0.001% (w/v), about 0.005% (w/v), about 0.01% (w/v), about 0.015% (w/v), about 0.02% (w/v), about 0.03% (w/v), about 0.035% (w/v), about 0.04% (w/v), about 0.045% (w/v), or about 0.05% (w/v) poloxamer 188. In certain embodiments, the eluant comprises about 0.01% (w/v) poloxamer 188.

In certain embodiments, the pH of the eluant is about 9 to about 10.5. In certain embodiments, the pH of the eluant is about 9, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, about 10, about 10.1, about 10.2, about 10.3, about 10.4, or about 10.5. In certain embodiments, the pH of the eluant is about 9.3.

In certain embodiments, the eluant is applied to the AEX medium at a conductivity of about 1 mS/cm to about 40 mS/cm. In certain embodiments, the eluant is applied to the AEX medium at a conductivity of about 8.5 mS/cm to about 30 mS/cm. In certain embodiments, the eluant is applied to the AEX medium at a conductivity of about 8.5 mS/cm to about 10.5 mS/cm. In certain embodiments, the eluant is applied to the AEX medium at a conductivity of about 10 mS/cm to about 11.5 mS/cm. In certain embodiments, the eluant is applied to the AEX medium at a conductivity of about 14 mS/cm to about 17.5 mS/cm. In certain embodiments, the eluant is applied to the AEX medium at a conductivity of about 1 mS/cm, about 2 mS/cm, about 3 mS/cm, about 4 mS/cm, about 5 mS/cm, about 6 mS/cm, about 7 mS/cm, about 8 mS/cm, about 9 mS/cm, about 10 mS/cm, about 11 mS/cm, about 12 mS/cm, about 13 mS/cm, about 14 mS/cm, about 15 mS/cm, about 16 mS/cm, about 17 mS/cm, about 18 mS/cm, about 19 mS/cm, about 20 mS/cm, about 21 mS/cm, about 22 mS/cm, about 23 mS/cm, about 24 mS/cm, about 25 mS/cm, about 26 mS/cm, about 27 mS/cm, about 28 mS/cm, about 29 mS/cm, about 30 mS/cm, about 31 mS/cm, about 32 mS/cm, about 33 mS/cm, about 34 mS/cm, about 35 mS/cm, about 36 mS/cm, about 37 mS/cm, about 38 mS/cm, about 39 mS/cm, or about 40 mS/cm. In certain embodiments, the eluant is applied to the AEX medium at a conductivity of about 6.5 mS/cm. In certain embodiments, the eluant is applied to the AEX medium at a conductivity of about 7 mS/cm. In certain embodiments, the eluant is applied to the AEX medium at a conductivity of about 7.5 mS/cm. In certain embodiments, the eluant is applied to the AEX medium at a conductivity of about 8 mS/cm. In certain embodiments, the eluant is applied to the AEX medium at a conductivity of about 8.5 mS/cm. In certain embodiments, the eluant is applied to the AEX medium at a conductivity of about 9 mS/cm. In certain embodiments, the eluant is applied to the AEX medium at a conductivity of about 9.5 mS/cm. In certain embodiments, the eluant is applied to the AEX medium at a conductivity of about 10 mS/cm. In certain embodiments, the eluant is applied to the AEX medium at a conductivity of about 11 mS/cm. In certain embodiments, the eluant is applied to the AEX medium at a conductivity of about 18.5 mS/cm. In certain embodiments, the eluant is applied to the AEX medium at a conductivity of about 19 mS/cm. In certain embodiments, the eluant is applied to the AEX medium at a conductivity of about 26 mS/cm. In certain embodiments, the eluant is applied to the AEX medium at a conductivity of about 30 mS/cm.

In certain embodiments, the eluant has a pH of about 9.2, and comprises 110 mM ammonium acetate, and 0.01% (w/v) poloxamer 188, and is applied to the AEX medium at a conductivity of about 9.5 mS/cm. In certain embodiments, the eluant has a pH of about 9.3, and comprises 400 mM ammonium acetate, and 0.01% (w/v) poloxamer 188, and is applied to the AEX medium at a conductivity of about 30 mS/cm. In certain embodiments, the eluant has a pH of about 9.3 and comprises 500 mM ammonium acetate, and 0.01% (w/v) poloxamer 188, and is applied to the AEX medium at a conductivity of about 10 mS/cm. In certain embodiments, the eluant has a pH of about 9.3, and comprises about 293 mM ammonium acetate, and 0.01% (w/v) poloxamer 188, and is applied to the AEX medium at a conductivity of about 18.5 mS/cm. In certain embodiments, the eluant has a pH of about 9.3, and comprises about 305 mM ammonium acetate, and 0.01% (w/v) poloxamer 188, and is applied to the AEX medium at a conductivity of about 19 mS/cm. In certain embodiments, the eluant has a pH of about 9.3, and comprises about 89 mM ammonium acetate, and 0.01% (w/v) poloxamer 188, and is applied to the AEX medium at a conductivity of about 6.5 mS/cm. In certain embodiments, the eluant has a pH of about 9.3, and comprises about 89 mM ammonium acetate, and 0.01% (w/v) poloxamer 188, and is applied to the AEX medium at a conductivity of about 7 mS/cm. In certain embodiments, the eluant has a pH of about 9.3, and comprises about 89 mM ammonium acetate, and 0.01% (w/v) poloxamer 188, and is applied to the AEX medium at a conductivity of about 7.5 mS/cm. In certain embodiments, the eluant has a pH of about 9.3, and comprises about 89 mM ammonium acetate, and 0.01% (w/v) poloxamer 188, and is applied to the AEX medium at a conductivity of about 8 mS/cm. In certain embodiments, the eluant has a pH of about 9.3, and comprises about 89 mM ammonium acetate, and 0.01% (w/v) poloxamer 188, and is applied to the AEX medium at a conductivity of about 8.5 mS/cm. In certain embodiments, the eluant has a pH of about 9.3, and comprises about 89 mM ammonium acetate, and 0.01% (w/v) poloxamer 188, and is applied to the AEX medium at a conductivity of about 9 mS/cm. In certain embodiments, the eluant has a pH of about 9.3, and comprises about 89 mM ammonium acetate, and 0.01% (w/v) poloxamer 188, and is applied to the AEX medium at a conductivity of about 9.5 mS/cm. In certain embodiments, the eluant has a pH of about 9.3, and comprises about 89 mM ammonium acetate, and 0.01% (w/v) poloxamer 188, and is applied to the AEX medium at a conductivity of about 10 mS/cm. In certain embodiments, the eluant has a pH of about 9.3, and comprises about 89 mM ammonium acetate, and 0.01% (w/v) poloxamer 188, and is applied to the AEX medium at a conductivity of about 11 mS/cm.

Those of ordinary skill in the art will appreciate that the eluant may comprise additional components that aid in the elution of AAV from the AEX medium. Such components may include additional buffering agents and additives that aid in, for example, dissociation, solubilization, and metal chelation. In certain embodiments, the eluant further comprises ethanolamine. In certain embodiments, the eluant comprises about 1 mM to about 100 mM ethanolamine, e.g., about 1 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM ethanolamine. In certain embodiments, the eluant comprises about 50 mM ethanolamine. In certain embodiments, the eluant further comprises Bis-Tris Propane (BTP). In certain embodiments, the eluant comprises about 1 mM to about 100 mM BTP, e.g., about 1 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM BTP. In certain embodiments, the eluant further comprises glycine. In certain embodiments, the eluant comprises about 1 mM to about 2000 mM glycine, e.g., about 1 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 300 mM, about 400 mM, about 500 mM, about 600 mM, about 700 mM, about 800 mM, about 900 mM, about 1000 mM, about 1100 mM, about 1200 mM, about 1300 mM, about 1400 mM, about 1500 mM, about 1600 mM, about 1700 mM, about 1800 mM, about 1900 mM, about 2000 mM glycine.

III. Recombinant Adeno-Associated Virus

The methods provided by the present disclosure are for the separation of an AAV particle from a mixture of the AAV particle and at least one contaminant. In certain embodiments, the AAV is a recombinant adeno-associated virus (rAAV).

rAAV Genome

In certain embodiments, the methods provided by the present disclosure are for the separation of an rAAV particle from a mixture of the rAAV and at least one contaminant. The rAAV comprises an rAAV genome. In certain embodiments, the rAAV genome comprises a transgene.

In certain embodiments, the transgene comprises one or more sequences encoding an RNA molecule. Suitable RNA molecules include, without limitation, miRNA, shRNA, siRNA, antisense RNA, gRNA, antagomirs, miRNA sponges, RNA aptazymes, RNA aptamers, mRNA, lncRNAs, ribozymes, and synthetic RNAs known in the art.

In certain embodiments, the transgene encodes one or more polypeptides, or a fragment thereof. Such transgenes can comprise the complete coding sequence of a polypeptide, or only a fragment of a coding sequence of a polypeptide. In certain embodiments, the transgene encodes a polypeptide that is useful to treat a disease or disorder in a subject. Suitable polypeptides include, without limitation, β-globin, hemoglobin, tissue plasminogen activator, and coagulation factors; colony stimulating factors (CSF); interleukins, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, etc.; growth factors, such as keratinocyte growth factor (KGF), stem cell factor (SCF), fibroblast growth factor (FGF, such as basic FGF and acidic FGF), hepatocyte growth factor (HGF), insulin-like growth factors (IGFs), bone morphogenetic protein (BMP), epidermal growth factor (EGF), growth differentiation factor-9 (GDF-9), hepatoma derived growth factor (HDGF), myostatin (GDF-8), nerve growth factor (NGF), neurotrophins, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-β), and the like; soluble receptors, such as soluble TNF-a receptors, soluble interleukin receptors (e.g., soluble IL-1 receptors and soluble type II IL-1 receptors), soluble γ/Δ T cell receptors, ligand-binding fragments of a soluble receptor, and the like; enzymes, such as a-glucosidase, imiglucerase, β-glucocerebrosidase, and alglucerase; enzyme activators, such as tissue plasminogen activator; chemokines, such as IP-10, monokine induced by interferon-gamma (Mig), Groa/IL-8, RANTES, MIP-1a, MCP-1, PF-4, and the like; angiogenic agents, such as vascular endothelial growth factors (VEGFs, e.g., VEGF121, VEGF165, VEGF-C, VEGF-2), glioma-derived growth factor, angiogenin, angiogenin-2; and the like; anti-angiogenic agents, such as a soluble VEGF receptor; protein vaccine; neuroactive peptides, such as nerve growth factor (NGF), bradykinin, cholecystokinin, gastrin, secretin, oxytocin, gonadotropin-releasing hormone, beta-endorphin, enkephalin, substance P, somatostatin, prolactin, galanin, growth hormone-releasing hormone, bombesin, dynorphin, warfarin, neurotensin, motilin, thyrotropin, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagons, vasopressin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptide, a sleep peptide, and the like; thrombolytic agents; atrial natriuretic peptide; relaxin; glial fibrillary acidic protein; follicle stimulating hormone (FSH); human alpha-1 antitrypsin; leukemia inhibitory factor (LIF); tissue factors; macrophage activating factors; tumor necrosis factor (TNF); neutrophil chemotactic factor (NCF); tissue inhibitors of metalloproteinases; vasoactive intestinal peptide; angiogenin; angiotropin; fibrin; hirudin; IL-1 receptor antagonists; ciliary neurotrophic factor (CNTF); brain-derived neurotrophic factor (BDNF); neurotrophins 3 and 4/5 (NT-3 and -4/5); glial cell derived neurotrophic factor (GDNF); aromatic amino acid decarboxylase (AADC); Factor VIII, Factor IX, Factor X; dystrophin or mini-dystrophin; lysosomal acid lipase; phenylalanine hydroxylase (PAH); glycogen storage disease-related enzymes, such as glucose-6-phosphatase, acid maltase, glycogen debranching enzyme, muscle glycogen phosphorylase, liver glycogen phosphorylase, muscle phosphofructokinase, phosphorylase kinase, glucose transporter, aldolase A, β-enolase, glycogen synthase; lysosomal enzymes, such as iduronate-2-sulfatase (I2S), and arylsulfatase A; and mitochondrial proteins, such as frataxin.

In certain embodiments, the transgene encodes a protein that may be defective in one or more lysosomal storage diseases. Suitable proteins include, without limitation, a-sialidase, cathepsin A, α-mannosidase, β-mannosidase, glycosylasparaginase, α-fucosidase, α-N-acetylglucosaminidase, β-galactosidase, β-hexosaminidase α-subunit, β-hexosaminidase β-subunit, GM2 activator protein, glucocerebrosidase, Saposin C, Arylsulfatase A, Saposin B, formyl-glycine generating enzyme, β-galactosylceramidase, α-galactosidase A, iduronate sulfatase, α-iduronidase, heparan N-sulfatase, acetyl-CoA transferase, N-acetyl glucosaminidase, β-glucuronidase, N-acetyl glucosamine 6-sulfatase, N-acetylgalactosamine 4-sulfatase, galactose 6-sulfatase, hyaluronidase, α-glucosidase, acid sphingomyelinase, acid ceramidase, acid lipase, cathepsin K, tripeptidyl peptidase, palmitoyl-protein thioesterase, cystinosin, sialin, UDP-N-acetylglucosamine, phosphotransferase γ-subunit, mucolipin-1, LAMP-2, NPC1, CLN3, CLN 6, CLN 8, LYST, MYOV, RAB27A, melanophilin, and AP3 β-subunit. In certain embodiments, suitable proteins include α-sialidase, cathepsin A, α-mannosidase, β-mannosidase, glycosylasparaginase, α-fucosidase, α-N-acetylglucosaminidase, β-galactosidase, β-hexosaminidase a-subunit, β-hexosaminidase β-subunit, GM2 activator protein, glucocerebrosidase, Saposin C, Saposin B, formyl-glycine generating enzyme, β-galactosyl ceramidase, α-galactosidase A, α-iduronidase, heparan N-sulfatase, acetyl-CoA transferase, N-acetyl glucosaminidase, β-glucuronidase, N-acetyl glucosamine 6-sulfatase, N-acetylgalactosamine 4-sulfatase, galactose 6-sulfatase, hyaluronidase, α-glucosidase, acid sphingomyelinase, acid ceramidase, acid lipase, cathepsin K, tripeptidyl peptidase, palmitoyl-protein thioesterase, cystinosin, sialin, UDP-N-acetylglucosamine, phosphotransferase γ-subunit, mucolipin-1, LAMP-2, NPC1, CLN3, CLN 6, CLN 8, LYST, MYOV, RAB27A, melanophilin, and AP3 β-subunit.

In certain embodiments, the transgene encodes a protein selected from the group consisting of iduronate-2-sulfatase (I2S), frataxin (FXN), glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), cyclin-dependent kinase-like 5 (CDKL5/STK9), galactose-1 phosphate uridyltransferase, phenylalanine hydroxylase (PAH), branched-chain alpha-keto acid dehydrogenase, fumarylacetoacetate hydrolase, methylmalonyl-CoA mutase, medium-chain acyl-CoA dehydrogenase, ornithine transcarbamylase (OTC), argininosuccinic acid synthetase (ASS1), low density lipoprotein receptor (LDLR) protein, UDP-glucuronosyltransferase, adenosine deaminase, hypoxanthine guanine phosphoribosyltransferase, biotinidase, alpha-galactosidase A, copper-transporting ATPase 2 (ATP7B), beta-glucocerebrosidase, 70 kDa peroxisomal membrane protein (PMP70), and arylsulfatase A (ARSA). In certain embodiments, the transgene encodes a protein which is not selected from the group consisting of phenylalanine hydroxylase (PAH), iduronate-2-sulfatase (I2S), arylsulfatase A (ARSA), and an anti-complement component 5 antibody.

In certain embodiments, the transgene encodes an antibody or a fragment thereof (e.g., a Fab, scFv, or full-length antibody). Suitable antibodies include, without limitation, muromonab-cd3, efalizumab, tositumomab, daclizumab, nebacumab, catumaxomab, edrecolomab, abciximab, rituximab, basiliximab, palivizumab, infliximab, trastuzumab, adalimumab, ibritumomab tiuxetan, omalizumab, cetuximab, bevacizumab, natalizumab, panitumumab, ranibizumab, eculizumab, certolizumab, ustekinumab, canakinumab, golimumab, ofatumumab, tocilizumab, denosumab, belimumab, ipilimumab, brentuximab vedotin, pertuzumab, raxibacumab, obinutuzumab, alemtuzumab, siltuximab, ramucirumab, vedolizumab, blinatumomab, nivolumab, pembrolizumab, idarucizumab, necitumumab, dinutuximab, secukinumab, mepolizumab, alirocumab, evolocumab, daratumumab, elotuzumab, ixekizumab, reslizumab, olaratumab, bezlotoxumab, atezolizumab, obiltoxaximab, inotuzumab ozogamicin, brodalumab, guselkumab, dupilumab, sarilumab, avelumab, ocrelizumab, emicizumab, benralizumab, gemtuzumab ozogamicin, durvalumab, burosumab, erenumab, galcanezumab, lanadelumab, mogamulizumab, tildrakizumab, cemiplimab, fremanezumab, ravulizumab, emapalumab, ibalizumab, moxetumomab, caplacizumab, romosozumab, risankizumab, polatuzumab, eptinezumab, leronlimab, sacituzumab, brolucizumab, isatuximab, and teprotumumab.

In certain embodiments, the transgene encodes a nuclease. Suitable nucleases include, without limitation, zinc fingers nucleases (ZFN) (see, e.g., Porteus, and Baltimore (2003) Science 300: 763; Miller et al. (2007) Nat. Biotechnol. 25:778-785; Sander et al. (2011) Nature Methods 8:67-69; and Wood et al. (2011) Science 333:307, each of which is hereby incorporated by reference in its entirety), transcription activator-like effectors nucleases (TALEN) (see, e.g., Wood et al. (2011) Science 333:307; Boch et al. (2009) Science 326:1509-1512; Moscou and Bogdanove (2009) Science 326:1501; Christian et al. (2010) Genetics 186:757-761; Miller et al. (2011) Nat. Biotechnol. 29:143-148; Zhang et al. (2011) Nat. Biotechnol. 29:149-153; and Reyon et al. (2012) Nat. Biotechnol. 30(5): 460-465, each of which is hereby incorporated by reference in its entirety), homing endonucleases, meganucleases (see, e.g., U.S. Patent Publication No. US 2014/0121115, which is hereby incorporated by reference in its entirety), and RNA-guided nucleases (see, e.g., Makarova et al. (2018) The CRISPR Journal 1(5): 325-336; and Adli (2018) Nat. Communications 9:1911, each of which is hereby incorporated by reference in its entirety).

In certain embodiments, the transgene encodes an RNA-guided nuclease. Suitable RNA-guided nucleases include, without limitation, Class I and Class II clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleases. Class I is divided into types I, III, and IV, and includes, without limitation, type I (Cas3), type I-A (Cas8a, Cas5), type I-B (Cas8b), type I-C (Cas8c), type 1-D (Cas10d), type I-E (Csel, Cse2), type I-F (Csy1, Csy2, Csy3), type I-U (GSU0054), type III (Cas10), type III-A (Csm2), type III-B (Cmr5), type III-C (Csx10 or Csx11), type III-D (Csx10), and type IV (Csfl). Class II is divided into types II, V, and VI, and includes, without limitation, type II (Cas9), type II-A (Csn2), type II-B (Cas4), type V (Cpf1, C2c1, C2c3), and type VI (Cas13a, Cas13b, Cas13c). RNA-guided nucleases also include naturally-occurring Class II CRISPR nucleases such as Cas9 (Type II) or Cas12a/Cpf1 (Type V), as well as other nucleases derived or obtained therefrom. Exemplary Cas9 nucleases that may be used in the present invention include, but are not limited to, S. pyogenes Cas9 (SpCas9), S. aureus Cas9 (SaCas9), N. meningitidis Cas9 (NmCas9), C. jejuni Cas9 (CjCas9), and Geobacillus Cas9 (GeoCas9).

In certain embodiments, the transgene encodes one or more reporter sequences, which upon expression produce a detectable signal. Such reporter sequences include, without limitation, DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), red fluorescent protein (RFP), chloramphenicol acetyltransferase (CAT), luciferase, membrane bound proteins, including, for example, CD2, CD4, CD8, the influenza hemagglutinin protein, and others well known in the art, to which high affinity antibodies directed thereto exist or can be produced by conventional means, and fusion proteins comprising a membrane bound protein appropriately fused to an antigen tag domain from, among others, hemagglutinin or Myc.

In certain embodiments, the rAAV genome comprises a transcriptional regulatory element (TRE) operably linked to the transgene, to control expression of an RNA or polypeptide encoded by the transgene. In certain embodiments, the TRE comprises a constitutive promoter. In certain embodiments, the TRE can be active in any mammalian cell (e.g., any human cell). In certain embodiments, the TRE is active in a broad range of human cells. Such TREs may comprise constitutive promoter and/or enhancer elements, including any of those described herein, and any of those known to one of skill in the art. In certain embodiments, the TRE comprises an inducible promoter. In certain embodiments, the TRE may be a tissue-specific TRE, i.e., it is active in specific tissue(s) and/or organ(s). A tissue-specific TRE comprises one or more tissue-specific promoter and/or enhancer elements, and optionally one or more constitutive promoter and/or enhancer elements. A skilled artisan would appreciate that tissue-specific promoter and/or enhancer elements can be isolated from genes specifically expressed in the tissue by methods well known in the art.

Suitable promoters include, e.g., cytomegalovirus promoter (CMV) (Stinski et al. (1985) Journal of Virology 55(2): 431-441), CMV early enhancer/chicken β-actin (CBA) promoter/rabbit β-globin intron (CAG) (Miyazaki et al. (1989) Gene 79(2): 269-277), CBSB (Jacobson et al. (2006) Molecular Therapy 13(6): 1074-1084), human elongation factor la promoter (EF1a) (Kim et al. (1990) Gene 91 (2): 217-223), human phosphoglycerate kinase promoter (PGK) (Singer-Sam et al. (1984) Gene 32(3): 409-417), mitochondrial heavy-strand promoter (Loderio et al. (2012) PNAS 109(17): 6513-6518), ubiquitin promoter (Wulff et al. (1990) FEBS Letters 261: 101-105). In certain embodiments, the TRE comprises a cytomegalovirus (CMV) promoter/enhancer (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 18 or 19), an SV40 promoter, a chicken beta actin (CBA) promoter (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 20 or 21), a smCBA promoter (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22), a human elongation factor 1 alpha (EF1a) promoter (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 23), a minute virus of mouse (MVM) intron which comprises transcription factor binding sites (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 24 or 25), a human phosphoglycerate kinase (PGK1) promoter, a human ubiquitin C (Ubc) promoter, a human beta actin promoter, a human neuron-specific enolase (ENO2) promoter, a human beta-glucuronidase (GUSB) promoter, a rabbit beta-globin element (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 26 or 27), a human calmodulin 1 (CALM1) promoter (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28), a human ApoE/C-I hepatic control region (HCR1) (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 29), a human al-antitrypsin (hAAT) promoter (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 30, 31, or 32), an extended HCR1 (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 33), a HS-CRM8 element of an hAAT promoter (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 34), a human transthyretin (TTR) promoter (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 35), and/or a human Methyl-CpG Binding Protein 2 (MeCP2) promoter. Any of the TREs described herein can be combined in any order to drive efficient transcription. For example, an rAAV genome may comprise a TRE comprising a CMV enhancer, a CBA promoter, and the splice acceptor from exon 3 of the rabbit beta-globin gene, collectively called a CAG promoter (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 36). For example, an rAAV genome may comprise a TRE comprising a hybrid of CMV enhancer and CBA promoter followed by a splice donor and splice acceptor, collectively called a CASI promoter region (e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 37). For example, an rAAV genome may comprise a TRE comprising an HCR1 and hAAT promoter (also referred to as an LP1 promoter, e.g., comprising a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 38).

In certain embodiments, the TRE is brain-specific (e.g., neuron-specific, glial cell-specific, astrocyte-specific, oligodendrocyte-specific, microglia-specific and/or central nervous system-specific). Exemplary brain-specific TREs may comprise one or more elements from, without limitation, human glial fibrillary acidic protein (GFAP) promoter, human synapsin 1 (SYN1) promoter, human synapsin 2 (SYN2) promoter, human metallothionein 3 (MT3) promoter, and/or human proteolipid protein 1 (PLP1) promoter. More brain-specific promoter elements are disclosed in WO 2016/100575A1, which is incorporated by reference herein in its entirety.

In certain embodiments, the native promoter for the transgene may be used. The native promoter may be preferred when it is desired that expression of the transgene should mimic the native expression. The native promoter may be used when expression of the transgene must be regulated temporally or developmentally, or in a tissue-specific manner, or in response to specific transcriptional stimuli. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites or Kozak consensus sequences may also be used to mimic the native expression.

In certain embodiments, the rAAV genome comprises an editing genome. Editing genomes can be used to edit the genome of a cell by homologous recombination of the editing genome with a genomic region surrounding a target locus in the cell. In certain embodiments, the editing genome is designed to correct a genetic defect in a gene by homologous recombination. Editing genomes generally comprise: (i) an editing element for editing a target locus in a target gene, (ii) a 5′ homology arm nucleotide sequence 5′ of the editing element having homology to a first genomic region 5′ to the target locus, and (iii) a 3′ homology arm nucleotide sequence 3′ of the editing element having homology to a second genomic region 3′ to the target locus, wherein the portion of the editing genome comprising the 5′ homology arm, editing element, and 3′ homology arm can be in the sense or antisense orientation relative to the target locus. Suitable target genes for editing using an editing genome include, without limitation, phenylalanine hydroxylase (PAH), cystic fibrosis conductance transmembrane regulator (CFTR), beta hemoglobin (HBB), oculocutaneous albinism II (OCA2), Huntingtin (HTT), dystrophia myotonica-protein kinase (DMPK), low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), neurofibromin 1 (NF1), polycystic kidney disease 1 (PKD1), polycystic kidney disease 2 (PKD2), coagulation factor VIII (F8), dystrophin (DMD), phosphate-regulating endopeptidase homologue, X-linked (PHEX), methyl-CpG-binding protein 2 (MECP2), and ubiquitin-specific peptidase 9Y, Y-linked (USP9Y).

In certain embodiments, the rAAV genomes disclosed herein further comprise a transcription terminator (e.g., a polyadenylation sequence). In certain embodiments, the transcription terminator is 3′ to the transgene. The transcription terminator may be any sequence that effectively terminates transcription, and a skilled artisan would appreciate that such sequences can be isolated from any genes that are expressed in the cell in which transcription of the at least a portion of an antibody coding sequence is desired. In certain embodiments, the transcription terminator comprises a polyadenylation sequence. In certain embodiments, the polyadenylation sequence is identical or substantially identical to the endogenous polyadenylation sequence of an immunoglobulin gene. In certain embodiments, the polyadenylation sequence is an exogenous polyadenylation sequence. In certain embodiments, the polyadenylation sequence is an SV40 polyadenylation sequence (e.g., comprising a nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 14, 47, or 48, or a nucleotide sequence complementary thereto). In certain embodiments, the polyadenylation sequence comprises the nucleotide sequence set forth in SEQ ID NO: 14. In certain embodiments, the polyadenylation sequence consists of the nucleotide sequence set forth in SEQ ID NO: 14. In certain embodiments, the polyadenylation sequence is a bovine growth hormone (BGH) polyadenylation sequence (e.g., comprising a nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 49, or a nucleotide sequence complementary thereto). In certain embodiments, the polyadenylation sequence comprises the nucleotide sequence set forth in SEQ ID NO: 49. In certain embodiments, the polyadenylation sequence consists of the nucleotide sequence set forth in SEQ ID NO: 49.

In certain embodiments, the rAAV genome comprises a nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 50, 51, 52, 53, 54, or 64. In certain embodiments, the editing element comprises the nucleotide sequence set forth in SEQ ID NO: 50, 51, 52, 53, 54, or 64. In certain embodiments, the editing element consists of the nucleotide sequence set forth in SEQ ID NO: 50, 51, 52, 53, 54, or 64.

In certain embodiments, the rAAV genomes disclosed herein further comprise a 5′ inverted terminal repeat (5′ ITR) nucleotide sequence 5′ of the TRE, and a 3′ inverted terminal repeat (3′ ITR) nucleotide sequence 3′ of the polyadenylation sequence associated with an antibody light chain coding sequence. ITR sequences from any AAV serotype or variant thereof can be used in the rAAV genomes disclosed herein. The 5′ and 3′ ITR can be from an AAV of the same serotype or from AAVs of different serotypes. Exemplary ITRs for use in the rAAV genomes disclosed herein are set forth in SEQ ID NOs: 39, 40, 41, 42, 43, and 44, herein.

In certain embodiments, the 5′ ITR or 3′ ITR is from AAV2. In certain embodiments, both the 5′ ITR and the 3′ ITR are from AAV2. In certain embodiments, the 5′ ITR nucleotide sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 39, or the 3′ ITR nucleotide sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 40. In certain embodiments, the 5′ ITR nucleotide sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 39, and the 3′ ITR nucleotide sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 40. In certain embodiments, the rAAV genome comprises a 5′ ITR nucleotide sequence having the sequence of SEQ ID NO: 39, and a 3′ ITR nucleotide sequence having the sequence of SEQ ID NO: 40.

In certain embodiments, the 5′ ITR or 3′ ITR are from AAVS. In certain embodiments, both the 5′ ITR and 3′ ITR are from AAVS. In certain embodiments, the 5′ ITR nucleotide sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 42, or the 3′ ITR nucleotide sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 43. In certain embodiments, the 5′ ITR nucleotide sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 42, and the 3′ ITR nucleotide sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 43. In certain embodiments, the rAAV genome comprises a 5′ ITR nucleotide sequence having the sequence of SEQ ID NO: 42, and a 3′ ITR nucleotide sequence having the sequence of SEQ ID NO: 43.

In certain embodiments, the 5′ ITR nucleotide sequence and the 3′ ITR nucleotide sequence are substantially complementary to each other (e.g., are complementary to each other except for mismatch at 1, 2, 3, 4, or 5 nucleotide positions in the 5′ or 3′ ITR).

In certain embodiments, the 5′ ITR or the 3′ ITR is modified to reduce or abolish resolution by Rep protein (“non-resolvable ITR”). In certain embodiments, the non-resolvable ITR comprises an insertion, deletion, or substitution in the nucleotide sequence of the terminal resolution site. Such modification allows formation of a self-complementary, double-stranded DNA genome of the AAV after the rAAV genome is replicated in an infected cell. Exemplary non-resolvable ITR sequences are known in the art (see, e.g., those provided in U.S. Pat. Nos. 7,790,154 and 9,783,824, which are incorporated by reference herein in their entirety). In certain embodiments, the 5′ ITR comprises a nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 41. In certain embodiments, the 5′ ITR consists of a nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 41. In certain embodiments, the 5′ ITR consists of the nucleotide sequence set forth in SEQ ID NO: 41. In certain embodiments, the 3′ ITR comprises a nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 44. In certain embodiments, the 5′ ITR consists of a nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 44. In certain embodiments, the 3′ ITR consists of the nucleotide sequence set forth in SEQ ID NO: 44. In certain embodiments, the 5′ ITR consists of the nucleotide sequence set forth in SEQ ID NO: 41, and the 3′ ITR consists of the nucleotide sequence set forth in SEQ ID NO: 44. In certain embodiments, the 5′ ITR consists of the nucleotide sequence set forth in SEQ ID NO: 41, and the 3′ ITR consists of the nucleotide sequence set forth in SEQ ID NO: 44.

In certain embodiments, the 5′ ITR is flanked by an additional nucleotide sequence derived from a wild-type AAV2 genomic sequence. In certain embodiments, the 5′ ITR is flanked by an additional 46 bp sequence derived from a wild-type AAV2 sequence that is adjacent to a wild-type AAV2 ITR in an AAV2 genome. In certain embodiments, the additional 46 bp sequence is 3′ to the 5′ ITR in the rAAV genome. In certain embodiments, the 46 bp sequence consists of the nucleotide sequence set forth in SEQ ID NO: 45.

In certain embodiments, the 3′ ITR is flanked by an additional nucleotide sequence derived from a wild-type AAV2 genomic sequence. In certain embodiments, the 3′ ITR is flanked by an additional 37 bp sequence derived from a wild-type AAV2 sequence that is adjacent to a wild-type AAV2 ITR in an AAV2 genome. See, e.g., Savy et al., Human Gene Therapy Methods (2017) 28(5): 277-289 (which is hereby incorporated by reference herein in its entirety). In certain embodiments, the additional 37 bp sequence is 5′ to the 3′ ITR in the rAAV genome. In certain embodiments, the 37 bp sequence consists of the nucleotide sequence set forth in SEQ ID NO: 46.

In certain embodiments, the rAAV genome comprises a nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 55, 56, 57, 58, 59, or 63. In certain embodiments, the editing element comprises the nucleotide sequence set forth in SEQ ID NO: 55, 56, 57, 58, 59, or 63. In certain embodiments, the editing element consists of the nucleotide sequence set forth in SEQ ID NO: 55, 56, 57, 58, 59, or 63.

AAV Capsid Protein

In certain embodiments, the rAAV comprises an AAV capsid. In certain embodiments, the AAV capsid comprises an AAV capsid protein. The rAAV can comprise an AAV capsid comprising an AAV capsid protein from any AAV capsid known in the art, including natural AAV isolates and variants thereof.

AAV capsid proteins include VP1, VP2, and VP3 capsid proteins. VP1, VP2, and/or VP3 capsid proteins assemble into a capsid that surrounds the rAAV genome. In certain embodiments, assembly of the capsid proteins is facilitated by the assembly-activating protein (AAP). Capsids of certain AAV serotypes require the role of AAP in transporting the capsid proteins to the nucleolus for assembly. For example, AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV12 require AAP to form capsids, while capsids of AAV4, AAVS, and AAV11 can assemble without AAP. See, e.g., Earley et al. (2017) J. Virol. 91(3): e01980-16.

Different AAV serotypes or variants thereof comprise AAV capsid proteins having different amino acid sequences. Suitable AAV capsid proteins include, without limitation, a capsid protein from AAV1, AAV2 (e.g., comprising the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 60), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 (e.g., comprising the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 61), AAV9 (e.g., comprising the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 62), AAV10, AAV11, AAV12, AAV13, AAV-DJ, AAV-LK03, NP59, VOY101, VOY201, VOY701, VOY801, VOY1101, AAVPHP.N, AAVPHP.A, AAVPHP.B, PHP.B2, PHP.B3, G2A3, G2B4, G2B5, PHP.S, AAVRh32.33, AAVrh74, AAVrh10, AAVHSC1, AAVHSC2, AAVHSC3, AAVHSC4, AAVHSC5, AAVHSC6, AAVHSC7, AAVHSC8, AAVHSC9, AAVHSC10, AAVHSC11, AAVHSC12, AAVHSC13, AAVHSC14, AAVHSC15, AAVHSC16, AAVHSC17, and any variants thereof. In certain embodiments, the AAV capsid protein is not from an AAVHSC. The sequences of the various AAV capsid proteins are disclosed in, e.g., U.S. Patent Publication Nos.: US20140359799, US20150376607, US20150159173, US20170081680, and US20170360962A1, and PCT Publication No. W02020227515, the disclosures of which are incorporated by reference herein in their entireties.

For example, in certain embodiments, the capsid protein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of amino acids 203-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17. In certain embodiments, the capsid protein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of amino acids 203-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17, wherein: the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO: 16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G or Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C; or, the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G, and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H, the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A, and the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C. In certain embodiments, the capsid protein comprises the amino acid sequence of amino acids 203-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17.

For example, in certain embodiments, the capsid protein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of amino acids 138-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17. In certain embodiments, the capsid protein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of amino acids 138-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17, wherein: the amino acid in the capsid protein corresponding to amino acid 151 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 160 of SEQ ID NO: 16 is D; the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO: 16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G or Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C; or, the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G, and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H, the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A, and the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C. In certain embodiments, the capsid protein comprises the amino acid sequence of amino acids 138-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17.

For example, in certain embodiments, the capsid protein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of amino acids 1-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17. In certain embodiments, the capsid protein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of amino acids 1-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17, wherein: the amino acid in the capsid protein corresponding to amino acid 2 of SEQ ID NO: 16 is T; the amino acid in the capsid protein corresponding to amino acid 65 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 68 of SEQ ID NO: 16 is V; the amino acid in the capsid protein corresponding to amino acid 77 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 119 of SEQ ID NO: 16 is L; the amino acid in the capsid protein corresponding to amino acid 151 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 160 of SEQ ID NO: 16 is D; the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO: 16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G or Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C; or, the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 2 of SEQ ID NO: 16 is T, and the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 65 of SEQ ID NO: 16 is I, and the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is Y. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 77 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 119 of SEQ ID NO: 16 is L, and the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G, and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H, the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A, and the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R. In certain embodiments, the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C. In certain embodiments, the capsid protein comprises the amino acid sequence of amino acids 1-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17.

In certain embodiments, the AAV capsid comprises two or more of: (a) a capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 1, 2, 3, 4, 6, 7, 10, 11, 12, 13, 15, 16, or 17; (b) a capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 16, or 17; and (c) a capsid protein comprising the amino acid sequence of amino acids 1-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17. In certain embodiments, the AAV capsid comprises: (a) a capsid protein having an amino acid sequence consisting of amino acids 203-736 of SEQ ID NO: 1, 2, 3, 4, 6, 7, 10, 11, 12, 13, 15, 16, or 17; (b) a capsid protein having an amino acid sequence consisting of amino acids 138-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 16, or 17; and (c) a capsid protein having an amino acid sequence consisting of amino acids 1-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17.

In certain embodiments, the AAV capsid comprises one or more of: (a) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence of amino acids 203-736 of SEQ ID NO: 8; (b) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence of amino acids 138-736 of SEQ ID NO: 8; and (c) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence of amino acids 1-736 of SEQ ID NO: 8. In certain embodiments, the AAV capsid comprises one or more of: (a) a capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 8; (b) a capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 8; and (c) a capsid protein comprising the amino acid sequence of amino acids 1-736 of SEQ ID NO: 8. In certain embodiments, the AAV capsid comprises two or more of: (a) a capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 8; (b) a capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 8; and (c) a capsid protein comprising the amino acid sequence of amino acids 1-736 of SEQ ID NO: 8. In certain embodiments, the AAV capsid comprises: (a) a capsid protein having an amino acid sequence consisting of amino acids 203-736 of SEQ ID NO: 8; (b) a capsid protein having an amino acid sequence consisting of amino acids 138-736 of SEQ ID NO: 8; and (c) a capsid protein having an amino acid sequence consisting of amino acids 1-736 of SEQ ID NO: 8.

In certain embodiments, the AAV capsid comprises one or more of: (a) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence of amino acids 203-736 of SEQ ID NO: 11; (b) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence of amino acids 138-736 of SEQ ID NO: 11; and (c) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence of amino acids 1-736 of SEQ ID NO: 11. In certain embodiments, the AAV capsid comprises one or more of: (a) a capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 11; (b) a capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 11; and (c) a capsid protein comprising the amino acid sequence of amino acids 1-736 of SEQ ID NO: 11. In certain embodiments, the AAV capsid comprises two or more of: (a) a capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 11; (b) a capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 11; and (c) a capsid protein comprising the amino acid sequence of amino acids 1-736 of SEQ ID NO: 11. In certain embodiments, the AAV capsid comprises: (a) a capsid protein having an amino acid sequence consisting of amino acids 203-736 of SEQ ID NO: 11; (b) a capsid protein having an amino acid sequence consisting of amino acids 138-736 of SEQ ID NO: 11; and (c) a capsid protein having an amino acid sequence consisting of amino acids 1-736 of SEQ ID NO: 11.

In certain embodiments, the AAV capsid comprises one or more of: (a) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence of amino acids 203-736 of SEQ ID NO: 13; (b) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence of amino acids 138-736 of SEQ ID NO: 13; and (c) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence of amino acids 1-736 of SEQ ID NO: 13. In certain embodiments, the AAV capsid comprises one or more of: (a) a capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 13; (b) a capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 13; and (c) a capsid protein comprising the amino acid sequence of amino acids 1-736 of SEQ ID NO: 13. In certain embodiments, the AAV capsid comprises two or more of: (a) a capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 13; (b) a capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 13; and (c) a capsid protein comprising the amino acid sequence of amino acids 1-736 of SEQ ID NO: 13. In certain embodiments, the AAV capsid comprises: (a) a capsid protein having an amino acid sequence consisting of amino acids 203-736 of SEQ ID NO: 13; (b) a capsid protein having an amino acid sequence consisting of amino acids 138-736 of SEQ ID NO: 13; and (c) a capsid protein having an amino acid sequence consisting of amino acids 1-736 of SEQ ID NO: 13.

In certain embodiments, the AAV capsid comprises one or more of: (a) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of amino acids 203-736 of SEQ ID NO: 16; (b) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of amino acids 138-736 of SEQ ID NO: 16; and (c) a capsid protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of amino acids 1-736 of SEQ ID NO: 16. In certain embodiments, the AAV capsid comprises one or more of: (a) a capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 16; (b) a capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 16; and (c) a capsid protein comprising the amino acid sequence of amino acids 1-736 of SEQ ID NO: 16. In certain embodiments, the AAV capsid comprises two or more of: (a) a capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 16; (b) a capsid protein comprising the amino acid sequence of amino acids 138-736 of SEQ ID NO: 16; and (c) a capsid protein comprising the amino acid sequence of amino acids 1-736 of SEQ ID NO: 16. In certain embodiments, the AAV capsid comprises: (a) a capsid protein having an amino acid sequence consisting of amino acids 203-736 of SEQ ID NO: 16; (b) a capsid protein having an amino acid sequence consisting of amino acids 138-736 of SEQ ID NO: 16; and (c) a capsid protein having an amino acid sequence consisting of amino acids 1-736 of SEQ ID NO: 16.

EXAMPLES

The following examples are offered by way of illustration, and not by way of limitation.

Example 1: Optimization of AEX Process: MgCl2 and Ammonium Acetate

In order to investigate the impact of magnesium chloride (MgCl2) on the AEX process, the presence of MgCl2 in the mixture (e.g., load composition) that was applied to the AEX medium was tested for its ability to improve the separation of intact AAV particles from AAV particles that lack a complete genome (e.g., empty capsids).

Representative AAV affinity chromatography product was used as starting material and the AEX media used were 0.2 mL Natrix® Q anion exchange chromatography membranes (Millipore Sigma). A load challenge of 1.5E15 capsids/mL membrane volume was used.

A comparison was made between a process that included Bis-Tris Propane and sodium chloride (BTP/NaCl) in the load composition and wash steps, with 5.7 mM MgCl2 or without MgCl2 in the load composition. The product was eluted in a Sodium Chloride gradient (BTP/NaCl) of increasing conductivity. FIG. 1A shows a chromatographic overlay of the gradient elution profiles obtained from a +BTP/NaCl process run with 5.7 mM MgCl2 in the load composition and a +BTP/NaCl process run without MgCl2 in the load composition. The elution profile of the +BTP/NaCl process run with 5.7 mM MgCl2 in the load composition showed one single first peak with significant tailing and no discernable second peak. The +BTP/NaCl process run without MgCl2 in the load composition resulted in about 24% empty capsids in the product peak, compared to about 45% empty capsids in the product peak that resulted from the +BTP/NaCl process run with 5.7 mM MgCl2 in the load composition. This data is summarized in Table 1.

Table 1: Summary of analytical data

Condition % Empty capsids +BTP/NaCl without MgCl2 in the load composition 23.6% +BTP/NaCl with 5.7 mM MgCl2 in the load composition 44.7% +AmAc without MgCl2 in the load composition 16.5% +AmAc with 5.7 mM MgCl2 in the load composition 7.9%

A comparison was made between a process that included ammonium acetate (AmAc) in the load composition and wash steps, with or without MgCl2 in the load composition, and a control process that included Bis-Tris Propane and sodium chloride (BTP/NaCl) in the load composition and wash steps, with or without MgCl2 in the load composition.

FIG. 1B shows a chromatographic overlay of the gradient elution profiles obtained from AEX processes run using load compositions, wash and elution steps that contained AmAc (+AmAc) or BTP/NaCl (+BTP/NaCl). The product was eluted in an Ammonium Acetate gradient (+AmAc) or a Sodium Chloride gradient (+BTP/NaCl) of increasing conductivity. It was found that the +AmAc process run resulted in significantly improved resolution between the first (product) peak and the second peak. As shown in FIG. 1B and Table 1, the full peak area of the +BTP/NaCl process run was about 10% larger than the peak area of the +AmAc run, however, the increased recovery appeared to be at the expense of enrichment. The +AmAc run resulted in about 17% empty capsids in the product peak compared to about 24% empty capsids in the product peak that resulted from the +BTP/NaCl run. Taken together, this shows that the presence of ammonium acetate in the load and elution results in increased peak resolution and increased purity.

Next, a comparison was made between +AmAc process runs with or without MgCl2 in the load composition. FIG. 1C shows a chromatographic overlay of the gradient elution profiles obtained from a +AmAc process run with 5.7 mM MgCl2 in the load composition, and a +AmAc process run without MgCl2 in the load composition. The product was eluted in an Ammonium Acetate gradient (+AmAc) of increasing conductivity As shown in FIG. 1C and Table 1, the +AmAc process run without MgCl2 in the load composition resulted in about 17% empty capsids in the product peak, compared to about 8% empty capsids in the product peak that resulted from the +AmAc process run with MgCl2 in the load composition. Further, the presence of MgCl2 in the load composition resulted in a reduction in the overall area of both first and second peaks. Without being bound by any theory, the decrease in peak area observed may be due to the presence of MgCl2 causing significantly more capsids to flow through the AEX medium (FIG. 1D). The A260/A280 ratios of the flow through and wash step (FT/Wash) for the +AmAc process run with 5.7 mM MgCl2 in the load composition and the +AmAc process run without MgCl2 in the load composition are almost identical at 0.82, suggesting that the presence of MgCl2 in the load composition did not change the population of capsids flowing through the AEX membrane, and that most of the unbound capsids (i.e., capsids in the flow through) were empty capsids.

Based on the foregoing, the addition of MgCl2 to the load composition impacts the AEX process in different ways depending on whether BTP/NaCl or AmAc is used. Adding MgCl2 to the load composition of a +BTP/NaCl process resulted in a negative impact on product enrichment. Adding MgCl2 to the load composition of a +AmAc process resulted in improved purity (Table 1).

To confirm the benefit of the addition of MgCl2 to an AEX process, the presence of 2 mM MgCl2 was tested in a process using a CIMmultus QA (“CIM-QA”; BIA Separations) monolith at a load challenge of 3.92E14 capsids/ml. The presence of 2 mM MgCl2 in the CIM-QA load preparation as well as in the re-equilibration buffer (+MgCl2) was compared to an analogous CIM-QA process in which no MgCl2 was used (−MgCl2). In each case, the load composition had a target pH of 9.3 and a conductivity of 3 mS/cm. FIG. 2 shows a flow diagram of the two processes.

In both conditions, the capsids were eluted with an Ammonium Acetate gradient of increasing conductivity. The conditions used are described in Table 2.

TABLE 2 Summary of conditions for CIMQ processes with and without magnesium chloride CIM-QA Load Dilution CIM-QA Re- Process Buffer Equilibration Buffer 2 mM 29 mM Ammonium Acetate 29 mM Ammonium Acetate Magnesium 0.01% Poloxamer-188 0.01% Poloxamer-188 Chloride 2 mM MgCl2 2 mM MgCl2 pH 9.3 pH 9.3 No Magnesium 34 mM Ammonium Acetate 34 mM Ammonium Acetate Chloride 0.01% Poloxamer-188 0.01% Poloxamer-188 pH 9.3 pH 9.3

Chromatograms for the CIM-QA process runs with and without MgCl2 in the load composition are shown in FIG. 3. FIG. 3 shows a chromatographic overlay of +MgCl2 and −MgCl2 CIM-QA process runs, with the elution peak magnified. As shown in FIG. 3, the CIM-QA process run without MgCl2 in the load composition resulted in about 35% empty capsids in the product peak, compared to about 25% empty capsids in the product peak that resulted from the CIM-QA process run with MgCl2 in the load composition. Without being bound by any theory, it is believed that magnesium chloride may diminish binding of empty capsids to the AEX media.

Example 2: Optimization of AEX Process: Wash Step Prior to Elution

Based on the above findings, the presence of 2 mM MgCl2 was maintained in the load composition and re-equilibration buffer. To investigate the impact of wash buffer conductivity prior to elution in an AEX process on the purity of resultant AAV drug substance, an AEX process carried out with a 3 mS/cm conductivity wash prior to elution was compared to an AEX process carried out with a 1 mS/cm conductivity wash prior to elution. FIG. 4 shows a flow diagram of the two processes. A CIM-QA monolith was challenged at 3.92E14 capsids/ml for each run and various step elution conductivities using AmAc were tested to investigate their impact on the separation of intact and empty capsids.

3 mS/cm Wash Step Prior to Elution

Elution steps at five different conductivities were tested at pH 9.3: 6.5 mS/cm, 7.0 mS/cm, 7.5 mS/cm, 8.5 mS/cm, and 9.5 mS/cm. Elution steps were performed with a 3 mS/cm wash step (second wash step) just prior to elution.

Table 3 summarizes the purification results from the elution peaks from different elution conductivities.

TABLE 3 Summary of purification results Step elution conductivity (mS/cm) % Empty capsids 6.5 Did not assay due to low A260:280 ratio observed 7.0 (in the range of process runs without MgCl2) indicating > 30% empty capsids 7.5 31 8.5 34 9.5 36

The eluates from the step elutions conducted at 6.5 and 7.0 mS/cm were not assayed as their A260:A280 ratio was under 1.21. Such ratios were similar to the −MgCl2 CIM-QA process runs which resulted in >30% empty capsids as determined by analytical ultracentrifugation (AUC) analysis, a method used to quantify macromolecules based on sedimentation coefficients (see, Table 5). The percent empty capsids obtained in the elution peaks at step elutions ranging from 7.5-9.5 mS/cm (pH 9.3) was above 30% (Table 5).

1 mS/cm Wash Step Prior to Elution

Elution steps at six different conductivities with AmAc were tested at pH 9.3: 7.0 mS/cm, 8.0 mS/cm, 9.0 mS/cm, 9.5 mS/cm, 10.0 mS/cm and 11 mS/cm. To investigate if an AEX process comprising a lower conductivity wash step prior to elution would lower the percent empty capsids in the product peak, elution steps were performed with a 1 mS/cm wash step (second wash step) just prior to elution on a CIM-QA monolith challenged at 3.92E14 capsids/ml.

Table 4 summarizes the purification results from the elution peaks from different elution conductivities.

TABLE 4 Summary of purification results Step elution conductivity (mS/cm) % Empty capsids 7.5 14 8.0 12 9.0 14 9.5 16 10.0 28 11.0 32

As shown in Table 4, with a 1 mS/cm wash step prior to elution and 2 mM MgCl2 in the load composition, most elution conductivities tested resulted in <30% empty capsids obtained from the elution peaks.

FIG. 5 shows a chromatographic overlay of the step gradient elution profiles obtained from an AEX process performed with a 1 mS/cm wash step prior to elution as described above, and an AEX process performed with a 3 mS/cm wash step prior to elution as described above. As shown, 14% empty capsids were recovered from an AEX process that included a 1 mS/cm wash prior to elution, compared to 31% empty capsids recovered from an AEX process that included a 3 mS/cm wash prior to elution.

The foregoing data supports an AEX process to separate empty capsids and full capsids, comprising MgCl2 in the load composition, followed by a low conductivity second wash solution (1 mS/cm) prior to a linear gradient elution or step elution.

Example 3: Optimization of AEX Process: Urea Wash Step

In order to investigate the impact of a urea wash step on the AEX process, an addition of a 2M urea wash step was evaluated to determine its impact on the % empty capsids obtained in the product. A comparison was made between a process that includes the 2M urea wash step and a previously established control process that did not include the 2M urea wash step (Table 5). The product in each case was eluted at pH 9.3 with a conductivity of 9.5 mS/cm using AmAc. FIG. 6 and Table 5 shows a flow diagram of the two processes.

TABLE 5 Summary of conditions Control Run 2M urea wash Run No wash buffer used 2M Urea 28 mM Ammonium Acetate 2 mM Magnesium Chloride 0.01% Poloxamer-188 pH 9.3 Conductivity: 3 mS/cm

Affinity chromatography product was used as starting material and a POROS HQ column was used at a load ratio of 7E+14 capsids/ml.

Table 6 summarizes the purification results from the elution peaks comparing the control process and the process that includes a 2M urea wash step.

TABLE 6 Summary of purification results Condition % Empty capsids Control (No Urea Wash) 26 2M Urea Wash 12

As shown in Table 6 and FIGS. 7A and 7B, the addition of a 2M urea wash step significantly improved chromatographic separation and reduced the percent of empty capsids in the product from about 26% to less than 15%. The AEX process that included a 2M urea wash step resulted in recovery of 12% empty capsids under a step elution, compared to 26% empty capsids recovered under a step elution from an AEX process that did not include a 2M urea wash step.

Taken together, it is hypothesized that the addition of the urea wash step enhanced separation by removing additional empty capsids that co-elute with the product in the first peak. Without being bound by theory, it is believed that the chaotropic nature of urea disrupts the structure of empty capsids and aids in dissociating the empty capsids from the column. This may occur by breaking down non-specific hydrogen bond interactions between the empty capsids and the AEX medium, thus facilitating desorption of empty capsids.

Example 4: Optimization of AEX Process: Choice of AEX Media

In order to investigate the effect of AEX chromatography media pore size on peak resolution, single strand (SS) and self complementary (SC) AAV vectors were processed on various AEX media as shown in Table 7.

TABLE 7 AEX Media Used AAV Vector Approximate Average Pore Type Used AEX Media Type Size (nm) SC Fractogel TMAE Hicap (M) 80 Praesto Q 100 Poros HQ50 200 SS Eshmuno Q 80 Poros HQ50 200 Nuvia HP-Q 500

For AAV comprising self-complementary and single strand vector genome, affinity chromatography product was used as starting material. For each resin tried for self-complementary vector, the product was desorbed with an elution gradient of 10-400 mM Ammonium acetate over 60 column volumes at a 6 minute residence time. For each resin tried for single strand vector, the product was desorbed with an elution gradient of 10-1000 mM Ammonium acetate over 60 column volumes at a 3 minute residence time

It was found that increasing AEX media pore size aids in the obtaining of higher quality product from the elution peak (FIGS. 8A and 8B). As shown in FIGS. 8A and 8B, improved separation performance was achieved when purifying AAV comprising single-stranded vector genomes (FIG. 8B) and AAV comprising self-complementary vector genomes (FIG. 8A).

Example 5: Optimization of AEX Process: Evaluation across AAV Capsid Serotypes

In order to investigate the ability of the AEX process to separate empty capsids and full capsids for different AAV capsid serotypes, the AEX process was evaluated for AAV8, AAV9 and AAV2 capsid serotypes.

AAV8 Anion Exchange Chromatography with Linear Gradient Elution

The conditions listed in Table 8 were utilized to evaluate the anion exchange chromatography process with AAV8 vector packaged with a PAH transgene. Neutralized AAV8 affinity product was in 20.5 mM Ammonium Acetate, 2.14 mM Magnesium Chloride, 0.01% Poloxamer-188, pH 9.3 and loaded onto POROS HQ column equilibrated with a pH 9.3 and 3.0 mS/cm ammonium acetate buffer. Following the load, the column was washed with the 3.0 mS/cm equilibration buffer followed by a pH 9.3 and 1.0 mS/cm ammonium acetate buffer. Bound vector was eluted with a 10 to 400 mM ammonium acetate gradient at pH 9.3.

TABLE 8 Conditions for AAV8 Anion Exchange Chromatography with Linear Gradient Elution Residence Time Step Buffer Composition CV (min) Equilibration 28 mM Ammonium 5 1 Acetate, 2 mM Magnesium Chloride, 0.01% Poloxamer- 188, pH 9.3 Sample Neutralized AAV8 N/A 2 Application Affinity Product diluted in 20.5 mM Ammonium Acetate, 2.14 mM Magnesium Chloride, 0.01% Poloxamer-188, pH 9.3 Wash 1 28 mM Ammonium 5 2 Acetate, 2 mM Magnesium Chloride, 0.01% Poloxamer- 188, pH 9.3 Wash 2 10 mM Ammonium 5 2 Acetate, 2 mM Magnesium Chloride, 0.01% Poloxamer- 188, pH 9.3 Linear Gradient 10-400 mM 60 3 Elution Ammonium Acetate, 0.01% Poloxamer- 188, pH 9.3 Elution Hold 400 mM Ammonium 20 3 Acetate, 0.01% Poloxamer-188, pH 9.3

The AAV8 anion exchange purification with a linear gradient elution resulted in one elution peak that was split into three pools. The full chromatogram is shown in FIG. 9A and the elution peak profile is shown in FIG. 9B. Table 9 shows the percentage of empty capsids in the load and elution pools determined via analytical ultracentrifugation. The first pool at the front of the peak contained predominantly empty capsids. The middle of the peak, or the product pool, consisted of 29% empty capsids. The final pool at the tail of the peak contained mostly empty capsids.

TABLE 9 AAV8 Anion Exchange Chromatography with Linear Gradient Elution Fraction % Empty Capsids Load 49% Elution Pool 1 88% Elution Pool 2 29% Elution Pool 3 62%

AAV8 Anion Exchange Chromatography with Isocratic Elution

The conditions listed in Table 10 were utilized to evaluate the anion exchange chromatography process with AAV8 vector packaged with a PAH transgene. The equilibration conditions were selected to promote weak partitioning of the empty capsids into the flowthrough and were identified from the linear gradient results in FIG. 9B. Neutralized AAV8 affinity product was diluted in a pH 9.3 and 13 mS/cm ammonium acetate buffer and loaded onto a POROS HQ column that was equilibrated with the 13 mS/cm buffer. Following the load, the column was washed with 13 mS/cm equilibration buffer followed by a pH 9.3 and 1.0 mS/cm ammonium acetate buffer. Bound vector was eluted over 5 CVs with a pH 9.3 and 19 mS/cm ammonium acetate buffer. The elution condition was identified from the linear gradient results in FIG. 9B.

TABLE 10 Conditions for AAV8 Anion Exchange Chromatography with Isocratic Elution Residence Time Step Buffer Composition CV (min) Equilibration 153 mM Ammonium 5 1 Acetate, 2 mM Magnesium Chloride, 0.01% Poloxamer- 188, pH 9.3 Sample Neutralized AAV8 N/A 2 Application Affinity Product diluted in 153 mM Ammonium Acetate, 2 mM Magnesium Chloride, 0.01% Poloxamer-188, pH 9.3 Wash 1 153 mM Ammonium 5 2 Acetate, 2 mM Magnesium Chloride, 0.01% Poloxamer- 188, pH 9.3 Wash 2 10 mM Ammonium 5 2 Acetate, 2 mM Magnesium Chloride, 0.01% Poloxamer- 188, pH 9.3 Elution 305 mM Ammonium 5 6 Acetate, 0.01% Poloxamer-188, pH 9.3

The AAV8 anion exchange purification with an isocratic elution resulted in a single elution peak consisting of low levels of empty capsids. The full chromatogram is shown in FIG. 10A and the elution peak is shown in FIG. 10B. The flowthrough A280 signal for the AAV8 anion exchange purification performed with the 13 mS/cm load condition relative to a 3 mS/cm load condition is shown in FIG. 10C. The increased A280 signal observed when loading at higher conductivity suggested the presence of empty capsids in the flowthrough. Analytical ultracentrifugation confirmed the presence of mainly empty capsids in the flowthrough and enrichment of full capsids through the anion exchange purification. The analytical ultracentrifugation capsid packaging data are shown in Table 11.

TABLE 11 AAV8 Anion Exchange Chromatography with Isocratic Elution Fraction % Empty Capsids Load 35% Flowthrough 93% Product 17%

AAV9 Anion Exchange Chromatography with Linear Gradient Elution

The conditions listed in Table 12 were utilized to evaluate the anion exchange chromatography process with AAV9 vector packaged with a PAH transgene. The AAV9 vector was loaded onto a POROS HQ50 column under the experimental parameters described in Table 12 below. Thawed affinity product was diluted with and adjusted to 28 mM Ammonium Acetate, 2 mM MgCl2, 0.01% (w/v%) Poloxamer 188 pH 9.3. The conductivity of the solution was adjusted to 3 mS/cm using 0.01% (w/v%) Poloxamer 188, 2 mM MgCl2. The final pH and conductivity of the solution was 9.30 and 3.0 mS/cm.

TABLE 12 Conditions for AAV9 Anion Exchange Chromatography with Linear Gradient Elution Residence Time Step Buffer Composition CV (min) Equilibration 1 28 mM Ammonium Acetate 20 1 0.01% Poloxamer-188 2 mM MgCl2 pH 9.3 Sample Load pH 9.3 N/A 1 application Conductivity: 3 mS/cm First wash 28 mM Ammonium Acetate 10 1 step 0.01% Poloxamer-188 2 mM MgCl2 pH 9.3 Second wash 10 mM Ammonium Acetate 20 1 step 0.01% Poloxamer-188 pH 9.3 Linear gradient 400 mM Ammonium Acetate 60 6 elution 0.01% Poloxamer-188 pH 9.3 0-100% over 60 CV

The AAV9 anion exchange purification with a linear gradient elution resulted in one elution peak that was split into three pools. The full chromatogram is shown in FIG. 11A and the elution peak profile is shown in FIG. 11B. Table 13 shows the percentage of empty capsids in the load and elution pools determined via analytical ultracentrifugation. The first pool at the front of the peak consisted of 45.5% empty capsids. The middle of the peak, or the product pool, consisted of 3.3% empty capsids. The final pool at the tail of the peak contained predominantly empty capsids.

TABLE 13 AAV9 Anion Exchange Chromatography with Linear Gradient Elution Fraction % Empty Capsids Load 45.5% Flowthrough 94.7% Product 3.3%

AAV9 Anion Exchange Chromatography with Step Gradient Elution

The conditions listed in Table 14 were utilized to evaluate the process with AAV9 vector packaged with a PAH transgene. The PAH-AAV9 vector was loaded onto a POROS HQ50 column under the experimental parameters described in Table 14 below. Thawed affinity product was diluted with and adjusted to 28 mM Ammonium Acetate, 2 mM MgCl2, 0.01% (w/v%) Poloxamer 188 pH 9.3. The conductivity of the solution was adjusted to 3 mS/cm using of 0.01% (w/v%) Poloxamer 188, 2 mM MgCl2. The final pH and conductivity of the solution was 9.28 and 3.06 mS/cm.

TABLE 14 Conditions for AAV9 Anion Exchange Chromatography with Step Gradient Elution Residence Time Step Buffer Composition CV (min) Equilibration 1 29 mM Ammonium Acetate 20 1 0.01% Poloxamer-188 2 mM MgCl2 pH 9.3 Sample Load pH 9.3 N/A 1 application Conductivity: 3 mS/cm First wash 29 mM Ammonium Acetate 10 1 step 0.01% Poloxamer-188 2 mM MgCl2 pH 9.3 Second wash 10 mM Ammonium Acetate 20 1 step 0.01% Poloxamer-188 pH 9.3 Step gradient 112 mM Ammonium Acetate  5 6 elution 0.01% Poloxamer-188 pH 9.3

The AAV9 anion exchange purification with a step gradient elution resulted in a single elution peak consisting of low levels of empty capsids. The full chromatogram is shown in FIG. 12A and the elution peak is shown in FIG. 12B. These data show that the product pool consisted of 1.9% empty capsids, confirming the presence of mainly empty capsids in the flowthrough and enrichment of full capsids through the anion exchange purification.

AAV2 Anion Exchange Chromatography with Linear Gradient Elution

The conditions listed in Table 15 were utilized to evaluate the anion exchange chromatography process with AAV2 vector packaged with a PAH transgene. Neutralized AAV2 affinity product was in 20.5 mM Ammonium Acetate, 2.14 mM Magnesium Chloride, 0.01% Poloxamer-188, pH 9.3 and loaded onto POROS HQ column equilibrated with a pH 9.3 and 3.0 mS/cm ammonium acetate buffer. Following the load, the column was washed with the 3.0 mS/cm equilibration buffer followed by a pH 9.3 and 1.0 mS/cm ammonium acetate buffer. Bound vector was eluted with a 10 to 400 mM ammonium acetate gradient at pH 9.3.

TABLE 15 Conditions for AAV2 Anion Exchange Chromatography with Linear Gradient Elution Residence Time Step Buffer Composition CV (min) Equilibration 28 mM Ammonium 5 1 Acetate, 2 mM Magnesium Chloride, 0.01% Poloxamer-188, pH 9.3 Sample Neutralized AAV8 N/A 2 Application Affinity Product diluted in 20.5 mM Ammonium Acetate, 2.14 mM Magnesium Chloride, 0.01% Poloxamer-188, pH 9.3 Wash 1 28 mM Ammonium 5 2 Acetate, 2 mM Magnesium Chloride, 0.01% Poloxamer-188, pH 9.3 Wash 2 10 mM Ammonium 5 2 Acetate, 2 mM Magnesium Chloride, 0.01% Poloxamer-188, pH 9.3 Linear Gradient 10-400 mM 60 3 Elution Ammonium Acetate, 0.01% Poloxamer-188, pH 9.3 Elution Hold 400 mM Ammonium 20 3 Acetate, 0.01% Poloxamer-188, pH 9.3

The AAV2 anion exchange purification with a linear gradient elution resulted in one elution peak that was split into three pools. The full chromatogram is shown in FIG. 13A and the elution peak profile is shown in FIG. 13B. Table 16 shows the percentage of empty capsids in the load and elution pools determined via analytical ultracentrifugation. The first pool at the front of the peak contained predominantly empty capsids. The middle of the peak, or the product pool, consisted of 24% empty capsids.

TABLE 16 AAV2 Anion Exchange Chromatography with Linear Gradient Elution Fraction % Empty Capsids Load 40% Elution Pool 1 82% Elution Pool 2 24% Elution Pool 3 NA

AAV2 Anion Exchange Chromatography with Isocratic Elution

The conditions listed in Table 17 were utilized to evaluate the anion exchange chromatography process with AAV2 vector packaged with a PAH transgene. The equilibration conditions were selected to promote weak partitioning of the empty capsids into the flowthrough and were identified from the linear gradient results in FIG. 13B. Neutralized AAV2 affinity product was diluted in a pH 9.3 and 13 mS/cm ammonium acetate buffer and loaded onto a POROS HQ column that was equilibrated with the 13 mS/cm buffer. Following the load, the column was washed with 13 mS/cm equilibration buffer followed by a pH 9.3 and 1.0 mS/cm ammonium acetate buffer. Bound vector was eluted with a pH 9.3 and 18.5 mS/cm ammonium acetate buffer. The elution condition was identified from the linear gradient results in FIG. 13B.

TABLE 17 Conditions for AAV2 Anion Exchange Chromatography with Isocratic Elution Residence Time Step Buffer Composition CV (min) Equilibration 153 mM Ammonium 5 2 Acetate, 2 mM Magnesium Chloride, 0.01% Poloxamer-188, pH 9.3 Sample Neutralized AAV8 N/A 2 Application Affinity Product diluted in 153 mM Ammonium Acetate, 2 mM Magnesium Chloride, 0.01% Poloxamer-188, pH 9.3 Wash 1 153 mM Ammonium 5 2 Acetate, 2 mM Magnesium Chloride, 0.01% Poloxamer-188, pH 9.3 Wash 2 10 mM Ammonium 5 2 Acetate, 2 mM Magnesium Chloride, 0.01% Poloxamer-188, pH 9.3 Elution 293 mM Ammonium 5 6 Acetate, 0.01% Poloxamer-188, pH 9.3

The AAV2 anion exchange purification with an isocratic elution resulted in a single elution peak consisting of low levels of empty capsids. The full chromatogram is shown in FIG. 14A and the elution peak is shown in FIG. 14B. The flowthrough A280 signal for the AAV8 anion exchange purification performed with the 13 mS/cm load condition is shown in FIG. 14C. Analytical ultracentrifugation confirmed the presence of mainly empty capsids in the flowthrough and enrichment of full capsids through the anion exchange purification. The analytical ultracentrifugation capsid packaging data are shown in Table 18.

TABLE 18 AAV2 Anion Exchange Chromatography with Isocratic Elution Fraction % Empty Capsids Load 31% Flowthrough 78% Product  8%

The foregoing data demonstrate that the AEX process described herein can be applied across different AAV serotypes.

Further embodiments of the invention are set out in the following clauses:

Clause 1. A method for the separation of an adeno-associated virus (AAV) particle from a mixture of the AAV particle and at least one contaminant, the method comprising: contacting the mixture with an anion exchange chromatography (AEX) medium under conditions such that the AAV particle binds to the AEX medium, wherein the mixture comprises magnesium chloride and an acetate; and washing the AEX medium with a first wash solution under conditions such that the AAV particle remains bound to the AEX medium and the at least one contaminant does not bind the AEX medium, thereby separating the AAV particle from the at least one contaminant.

Clause 2. The method of Clause 1, wherein the first wash solution comprises an acetate.

Clause 3. The method of Clause 1 or 2, wherein the first wash solution comprises urea.

Clause 4. A method for the separation of an adeno-associated virus (AAV) particle from a mixture of the AAV particle and at least one contaminant, the method comprising: contacting the mixture with an anion exchange chromatography (AEX) medium under conditions such that the AAV particle binds to the AEX medium; and washing the AEX medium with a first wash solution comprising urea and an acetate under conditions such that the AAV particle remains bound to the AEX medium and the at least one contaminant does not bind to the AEX medium, thereby separating the AAV particle from the at least one contaminant.

Clause 5. The method of any one of Clauses 1-4, further comprising washing the anion exchange chromatography medium with a second wash solution.

Clause 6. A method for the separation of an adeno-associated virus (AAV) particle from a mixture of the AAV particle and at least one contaminant, the method comprising: providing an AEX medium that has been contacted with the mixture, wherein the mixture comprises magnesium chloride and an acetate, and wherein the AEX medium comprises the AAV particle bound thereto and has been washed with a first wash solution such that the AAV particle remained bound to the AEX medium and the at least one contaminant did not bind to the AEX medium; and washing the AEX medium with a second wash solution under conditions such that the AAV particle remains bound to the AEX medium and the at least one contaminant does not bind to the AEX medium, thereby separating the AAV particle from the at least one contaminant.

Clause 7. The method of Clause 6, wherein the first wash solution comprises an acetate.

Clause 8. The method of Clause 6 or 7, wherein the first wash solution comprises urea.

Clause 9. A method for the separation of an adeno-associated virus (AAV) particle from a mixture of the AAV particle and at least one contaminant, the method comprising: providing an AEX medium that has been contacted with the mixture, wherein the AEX medium comprises the AAV particle bound thereto and has been washed with a first wash solution comprising urea and an acetate such that the AAV particle remained bound to the AEX medium and the at least one contaminant did not bind to the AEX medium; and washing the AEX medium with a second wash solution under conditions such that the AAV particle remains bound to the AEX medium and the at least one contaminant does not bind to the AEX medium, thereby separating the AAV particle from the at least one contaminant.

Clause 10. The method of any preceding Clause, wherein the at least one contaminant is selected from the group consisting of an AAV particle lacking a complete genome, an AAV degradation product, a host cell protein, a host cell fragment, and any combination thereof.

Clause 11. The method of any preceding Clause, wherein the at least one contaminant is an AAV particle that lacks a complete genome.

Clause 12. The method of any preceding Clause, wherein the AEX medium has an average pore size of at least about 100 nm.

Clause 13. The method of any preceding Clause, wherein the AEX medium has an average pore size of at least about 500 nm.

Clause 14. The method of any preceding Clause, wherein the AEX medium comprises a quaternary amine.

Clause 15. The method of any preceding Clause, wherein the AEX medium comprises a quaternary polyethyleneimine group.

Clause 16. The method of any preceding Clause, wherein the mixture comprises an eluate of an affinity chromatography column.

Clause 17. The method of any preceding Clause, wherein the mixture comprises about 10 mM to about 40 mM of an acetate.

Clause 18. The method of any preceding Clause, wherein the mixture comprises about 10 mM to about 40 mM ammonium acetate.

Clause 19. The method of any preceding Clause, wherein the mixture comprises about 28 mM ammonium acetate.

Clause 20. The method of any preceding Clause, wherein the mixture comprises about 2 mM to about 6 mM magnesium chloride.

Clause 21. The method of any preceding Clause, wherein the mixture comprises about 2 mM magnesium chloride.

Clause 22. The method of any preceding Clause, wherein the mixture comprises about 0.001% (w/v) to about 0.05% (w/v) Poloxamer 188.

Clause 23. The method of any preceding Clause, wherein the mixture comprises about 0.01% (w/v) Poloxamer 188.

Clause 24. The method of any preceding Clause, wherein the pH of the mixture is about 9 to about 10.5.

Clause 25. The method of any preceding Clause, wherein the pH of the mixture is about 9.3.

Clause 26. The method of any preceding Clause, wherein the first wash solution comprises about 0.1 M to about 4 M urea.

Clause 27. The method of any preceding Clause, wherein the first wash solution comprises about 2 M urea.

Clause 28. The method of any preceding Clause, wherein the first wash solution comprises an acetate selected from the group consisting of ammonium acetate, potassium acetate, sodium acetate, and cesium acetate.

Clause 29. The method of any preceding Clause, wherein the first wash solution comprises ammonium acetate.

Clause 30. The method of any preceding Clause, wherein the first wash solution comprises about 10 mM to about 40 mM of the acetate.

Clause 31. The method of any preceding Clause, wherein the first wash solution comprises about 10 mM to about 40 mM ammonium acetate.

Clause 32. The method of any preceding Clause, wherein the first wash solution comprises about 28 mM ammonium acetate.

Clause 33. The method of any preceding Clause, wherein the first wash solution comprises about 2 mM to about 6 mM magnesium chloride.

Clause 34. The method of any preceding Clause, wherein the first wash solution comprises about 2 mM magnesium chloride.

Clause 35. The method of any preceding Clause, wherein the first wash solution comprises about 0.001% (w/v) to about 0.05% (w/v) Poloxamer 188.

Clause 36. The method of any preceding Clause, wherein the first wash solution comprises about 0.01% (w/v) Poloxamer 188.

Clause 37. The method of any preceding Clause, wherein the pH of the first wash solution is about 9 to about 10.5.

Clause 38. The method of any preceding Clause, wherein the pH of the first wash solution is about 9.3.

Clause 39. The method of any preceding Clause, wherein the first wash solution has a conductivity of about 1 mS/cm to about 3 mS/cm.

Clause 40. The method of any one of Clauses 5-39, wherein the second wash solution comprises about 0.1 mM to about 15 mM ammonium acetate.

Clause 41. The method of any one of Clauses 5-40, wherein the second wash solution comprises about 10 mM ammonium acetate.

Clause 42. The method of any one of Clauses 5-41, wherein the second wash solution comprises about 0.001% (w/v) to about 0.05% (w/v) Poloxamer 188.

Clause 43. The method of any one of Clauses 5-42, wherein the second wash solution comprises about 0.01% (w/v) Poloxamer 188.

Clause 44. The method of any one of Clauses 5-43, wherein the pH of the second wash solution is about 9 to about 10.5.

Clause 45. The method of any one of Clauses 5-44, wherein the pH of the second wash solution is about 9.3.

Clause 46. The method of any one of Clauses 5-45, wherein the second wash solution has a conductivity of less than about 3 mS/cm.

Clause 47. The method of any one of Clauses 5-46, wherein the second wash solution has a conductivity of about 1 mS/cm to about 3 mS/cm.

Clause 48. The method of any one of Clauses 5-47, wherein the second wash solution has a conductivity of about 1 mS/cm.

Clause 49. The method of any preceding Clause, further comprising eluting the AAV particle from the AEX medium.

Clause 50. The method of Clause 49, wherein the AAV particle is eluted from the AEX medium with an eluant using a step gradient.

Clause 51. The method of Clause 49, wherein the AAV particle is eluted from the AEX medium with an eluant using a linear gradient.

Clause 52. The method of Clause 50 or 51, wherein the eluant comprises a salt at a concentration of about 10 mM to about 1 M.

Clause 53. The method of Clause 52, wherein the salt is an acetate salt.

Clause 54. The method of Clause 53, wherein the acetate salt is selected from the group consisting of ammonium acetate, potassium acetate, sodium acetate, and cesium acetate.

Clause 55. The method of Clause 53 or 54, wherein the acetate salt is ammonium acetate.

Clause 56. The method of Clause 53 or 54, wherein the acetate salt is sodium acetate.

Clause 57. The method of any one of Clauses 50-56, wherein the eluant comprises about 10 mM to about 1 M ammonium acetate.

Clause 58. The method of any one of Clauses 50-57, wherein the eluant comprises about 10 mM to about 150 mM ammonium acetate.

Clause 59. The method of any one of Clauses 50-58, wherein the eluant comprises about 100 mM to about 300 mM ammonium acetate.

Clause 60. The method of any one of Clauses 50-59, wherein the eluant comprises about 0.001% (w/v) to about 0.05% (w/v) Poloxamer 188.

Clause 61. The method of any one of Clauses 50-60, wherein the eluant comprises about 0.01% (w/v) Poloxamer 188.

Clause 62. The method of any one of Clauses 50-61, wherein the pH of the eluant is about 9 to about 10.5.

Clause 63. The method of any one of Clauses 50-62, wherein the pH of the eluant is about 9.3.

Clause 64. The method of any one of Clauses 50-63, wherein the eluant further comprises about 50 mM ethanolamine.

Clause 65. The method of any one of Clauses 50-64, wherein the eluant has a conductivity of about 8.5 mS/cm to about 30 mS/cm.

Clause 66. The method of any one of Clauses 50-64, wherein the eluant has a conductivity of about 8.5 mS/cm to about 10.5 mS/cm.

Clause 67. The method of any one of Clauses 50-64, wherein the eluant has a conductivity of about 10 mS/cm to about 11.5 mS/cm.

Clause 68. The method of any one of Clauses 50-64, wherein the eluant has a conductivity of about 14 mS/cm to about 17.5 mS/cm.

Clause 69. The method of any one of Clauses 50-64, wherein the eluant has a conductivity of about 26 mS/cm.

Clause 70. The method of any preceding Clause, wherein the method results in an eluate comprising less than about 15% AAV particles that lack a complete genome.

Clause 71. The method of any preceding Clause, wherein the method results in an eluate comprising less than about 10% AAV particles that lack a complete genome.

Clause 72. The method of any one of Clauses 50-71, further comprising formulating the eluted AAV particle in a formulation buffer suitable for administration to a human subject.

Clause 73. The method of any preceding Clause, wherein the AAV is a recombinant AAV (rAAV) comprising an rAAV genome comprising a transgene.

Clause 74. The method of Clause 73, wherein the transgene encodes a polypeptide.

Clause 75. The method of Clause 73, wherein the transgene encodes an miRNA, shRNA, siRNA, antisense RNA, gRNA, antagomir, miRNA sponge, RNA aptazyme, RNA aptamer, lncRNA, ribozyme, or mRNA.

Clause 76. The method of Clause 73, wherein the transgene encodes a protein selected from the group consisting of iduronate-2-sulfatase (I2S), frataxin (FXN), glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), cyclin-dependent kinase-like 5 (CDKLS/STK9), galactose-1 phosphate uridyltransferase, phenylalanine hydroxylase (PAH), branched-chain alpha-keto acid dehydrogenase, fumarylacetoacetate hydrolase, methylmalonyl-CoA mutase, medium-chain acyl-CoA dehydrogenase, ornithine transcarbamylase (OTC), argininosuccinic acid synthetase (AS S1), low density lipoprotein receptor (LDLR) protein, UDP-glucuronosyltransferase, adenosine deaminase, hypoxanthine guanine phosphoribosyltransferase, biotinidase, alpha-gal actosidase A, copper-transporting ATPase 2 (ATP7B), beta-glucocerebrosidase, 70 kDa peroxisomal membrane protein (PMP70), and aryl sulfatase A (ARSA).

Clause 77. The method of Clause 73, wherein the transgene encodes an antibody or a fragment thereof selected from the group consisting of: muromonab-cd3, efalizumab, tositumomab, daclizumab, nebacumab, catumaxomab, edrecolomab, abciximab, rituximab, basiliximab, palivizumab, infliximab, trastuzumab, adalimumab, ibritumomab tiuxetan, omalizumab, cetuximab, bevacizumab, natalizumab, panitumumab, ranibizumab, eculizumab, certolizumab, ustekinumab, canakinumab, golimumab, ofatumumab, tocilizumab, denosumab, belimumab, ipilimumab, brentuximab vedotin, pertuzumab, raxibacumab, obinutuzumab, alemtuzumab, siltuximab, ramucirumab, vedolizumab, blinatumomab, nivolumab, pembrolizumab, idarucizumab, necitumumab, dinutuximab, secukinumab, mepolizumab, alirocumab, evolocumab, daratumumab, elotuzumab, ixekizumab, reslizumab, olaratumab, bezlotoxumab, atezolizumab, obiltoxaximab, inotuzumab ozogamicin, brodalumab, guselkumab, dupilumab, sarilumab, avelumab, ocrelizumab, emicizumab, benralizumab, gemtuzumab ozogamicin, durvalumab, burosumab, erenumab, galcanezumab, lanadelumab, mogamulizumab, tildrakizumab, cemiplimab, fremanezumab, ravulizumab, emapalumab, ibalizumab, moxetumomab, caplacizumab, romosozumab, risankizumab, polatuzumab, eptinezumab, leronlimab, sacituzumab, brolucizumab, isatuximab, and teprotumumab.

Clause 78. The method of any one of Clauses 73-77, wherein the rAAV genome further comprises a transcriptional regulatory element operably linked to the transgene.

Clause 79. The method of Clause 78, wherein the transcriptional regulatory element comprises a promoter element and/or an intron element.

Clause 80. The method of any one of Clauses 73-79, wherein the rAAV genome further comprises a polyadenylation sequence.

Clause 81. The method of Clause 80, wherein the polyadenylation sequence is 3′ to the transgene.

Clause 82. The method of any one of Clauses 73-81, wherein the rAAV genome comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 50, 51, 52, 53, or 54

Clause 83. The method of any one of Clauses 73-82, wherein the rAAV genome further comprises a 5′ inverted terminal repeat (5′ ITR) nucleotide sequence 5′ of the transgene, and a 3′ inverted terminal repeat (3′ ITR) nucleotide sequence 3′ of the transgene.

Clause 84. The method of Clause 83, wherein the 5′ ITR nucleotide sequence is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 39, 41, or 42, and/or the 3′ ITR nucleotide sequence is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 40, 43, or 44.

Clause 85. The method of any one of Clauses 73-84, wherein the rAAV genome comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 55, 56, 57, 58, or 59.

Clause 86. The method of any one of Clauses 73-85, wherein the rAAV comprises an AAV capsid comprising an AAV capsid protein.

Clause 87. The method of Clause 86, wherein the AAV capsid protein is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAVS, AAV6, AAV7, AAV8, AAV9, AAV-DJ, AAV-LK03, NP59, VOY101, VOY201, VOY701, VOY801, VOY1101, AAVPHP.N, AAVPHP.A, AAVPHP.B, PHP.B2, PHP.B3, G2A3, G2B4, G2B5, and PHP. S.

Clause 88. The method of Clause 86 or 87, wherein the AAV capsid protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of amino acids 203-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17.

Clause 89. The method of Clause 88, wherein: the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO: 16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G or Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C; or, the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G.

Clause 90. The method of Clause 89, wherein:

    • (a) the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G, and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G;
    • (b) the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H, the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M;
    • (c) the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R;
    • (d) the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A, and the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; or
    • (e) the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C.

Clause 91. The method of Clause 89, wherein the AAV capsid protein comprises the amino acid sequence of amino acids 203-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17.

Clause 92. The method of any one of Clauses 86-91, wherein the AAV capsid protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of amino acids 138-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17.

Clause 93. The method of Clause 92, wherein: the amino acid in the capsid protein corresponding to amino acid 151 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 160 of SEQ ID NO: 16 is D; the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO: 16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G or Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C; or, the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G.

Clause 94. The method of Clause 93, wherein:

    • (a) the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G, and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G;
    • (b) the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H, the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M;
    • (c) the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R;
    • (d) the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A, and the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; or
    • (e) the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C.

Clause 95. The method of Clause 93, wherein the AAV capsid protein comprises the amino acid sequence of amino acids 138-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 16, or 17.

Clause 96. The method of any one of Clauses 86-95, wherein the AAV capsid protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of amino acids 1-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17.

Clause 97. The method of Clause 96, wherein: the amino acid in the capsid protein corresponding to amino acid 2 of SEQ ID NO: 16 is T; the amino acid in the capsid protein corresponding to amino acid 65 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 68 of SEQ ID NO: 16 is V; the amino acid in the capsid protein corresponding to amino acid 77 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 119 of SEQ ID NO: 16 is L; the amino acid in the capsid protein corresponding to amino acid 151 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 160 of SEQ ID NO: 16 is D; the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO: 16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G or Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C; or, the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G.

Clause 98. The method of Clause 97, wherein:

    • (a) the amino acid in the capsid protein corresponding to amino acid 2 of SEQ ID NO: 16 is T, and the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q;
    • (b) the amino acid in the capsid protein corresponding to amino acid 65 of SEQ ID NO: 16 is I, and the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is Y;
    • (c) the amino acid in the capsid protein corresponding to amino acid 77 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K;
    • (d) the amino acid in the capsid protein corresponding to amino acid 119 of SEQ ID NO: 16 is L, and the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S;
    • (e) the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G, and the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G;
    • (f) the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H, the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M;
    • (g) the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R;
    • (h) the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A, and the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; or
    • (i) the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I, the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R, and the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C.

Clause 99. The method of Clause 97, wherein the AAV capsid protein comprises the amino acid sequence of amino acids 1-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17.

Clause 100. A composition comprising an AEX medium and a mixture comprising magnesium chloride (MgCl2) and an acetate.

Clause 101. The composition of Clause 100, wherein the mixture comprises an eluate of an affinity chromatography column.

Clause 102. The composition of Clause 100 or 101, wherein the mixture comprises about 10 mM to about 40 mM of an acetate.

Clause 103. The composition of any one of Clauses 100-102, wherein the mixture comprises about 10 mM to about 40 mM ammonium acetate.

Clause 104. The composition of any one of Clauses 100-103, wherein the mixture comprises about 28 mM ammonium acetate.

Clause 105. The composition of any one of Clauses 100-104, wherein the mixture comprises about 2 mM to about 6 mM magnesium chloride.

Clause 106. The composition of any one of Clauses 100-105, wherein the mixture comprises about 2 mM magnesium chloride.

Clause 107. The composition of any one of Clauses 100-106, wherein the mixture comprises about 0.001% (w/v) to about 0.05% (w/v) Poloxamer 188.

Clause 108. The composition of any one of Clauses 100-107, wherein the mixture comprises about 0.01% (w/v) Poloxamer 188.

Clause 109. The composition of any one of Clauses 100-108, wherein the pH of the mixture is about 9 to about 10.5.

Clause 110. The composition of any one of Clauses 100-109, wherein the pH of the mixture is about 9.3.

Clause 111. A composition comprising an AEX medium and a first wash solution, wherein the first wash solution comprises urea and an acetate.

Clause 112. The composition of Clause 111, wherein the AEX medium has an average pore size of at least about 100 nm.

Clause 113. The composition of Clause 111 or 112, wherein the AEX medium has an average pore size of at least about 500 nm.

Clause 114. The composition of any one of Clauses 111-113, wherein the AEX medium comprises a quaternary amine.

Clause 115. The composition of any one of Clauses 111-114, wherein the AEX medium comprises a quaternary polyethyleneimine group.

Clause 116. The composition of any one of Clauses 111-115, wherein the first wash solution comprises about 0.1 M to about 4 M urea.

Clause 117. The composition of any one of Clauses 111-116, wherein the first wash solution comprises about 2 M urea.

Clause 118. The composition of any one of Clauses 111-117, wherein the first wash solution comprises about 10 mM to about 32 mM of the acetate.

Clause 119. The composition of any one of Clauses 111-118, wherein the first wash solution comprises about 10 mM to about 32 mM ammonium acetate.

Clause 120. The composition of any one of Clauses 111-119, wherein the first wash solution comprises about 28 mM ammonium acetate.

Clause 121. The composition of any one of Clauses 111-120, wherein the first wash solution comprises about 2 mM to about 5.7 mM magnesium chloride.

Clause 122. The composition of any one of Clauses 111-121, wherein the first wash solution comprises about 2 mM magnesium chloride.

Clause 123. The composition of any one of Clauses 111-122, wherein the first wash solution comprises about 0.001% (w/v) to about 0.05% (w/v) Poloxamer 188.

Clause 124. The composition of any one of Clauses 111-123, wherein the first wash solution comprises about 0.01% (w/v) Poloxamer 188.

Clause 125. The composition of any one of Clauses 111-124, wherein the pH of the first wash solution is about 9 to about 10.5.

Clause 126. The composition of any one of Clauses 111-125, wherein the pH of the first wash solution is about 9.3.

Clause 127. The composition of any one of Clauses 111-126, wherein the first wash solution has a conductivity of about 3 mS/cm.

The invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

All references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Other embodiments are within the following claims.

Claims

1. A method for the separation of an adeno-associated virus (AAV) particle from a mixture of the AAV particle and at least one contaminant, the method comprising:

contacting the mixture with an anion exchange chromatography (AEX) medium under conditions such that the AAV particle binds to the AEX medium; and
washing the AEX medium with a first wash solution under conditions such that the AAV particle remains bound to the AEX medium and the at least one contaminant does not bind the AEX medium,
thereby separating the AAV particle from the at least one contaminant,
wherein the mixture comprises magnesium chloride and an acetate, or the first wash solution comprises urea and an acetate, and
wherein the AAV particle is a recombinant AAV (rAAV) comprising an rAAV genome comprising a transgene, and an AAV capsid comprising an AAV capsid protein, wherein: (i) the transgene is selected from the group consisting of phenylalanine hydroxylase (PAH), arylsulfatase A (ARSA), iduronate 2-sulfatase (I2S), and an anti-complement component 5 (C5) antibody; or (ii) the AAV capsid protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of amino acids 1-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17; wherein: the amino acid in the capsid protein corresponding to amino acid 2 of SEQ ID NO: 16 is T; the amino acid in the capsid protein corresponding to amino acid 65 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 68 of SEQ ID NO: 16 is V; the amino acid in the capsid protein corresponding to amino acid 77 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 119 of SEQ ID NO: 16 is L; the amino acid in the capsid protein corresponding to amino acid 151 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 160 of SEQ ID NO: 16 is D; the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO: 16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A; the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G or Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C; or, the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G.

2. The method of claim 1, wherein the mixture comprises magnesium chloride and an acetate, and the first wash solution comprises urea and an acetate.

3-4. (canceled)

5. The method of claim 1, further comprising washing the anion exchange chromatography medium with a second wash solution.

6. A method for the separation of an adeno-associated virus (AAV) particle from a mixture of the AAV particle and at least one contaminant, the method comprising:

providing an AEX medium that has been contacted with the mixture, and wherein the AEX medium comprises the AAV particle bound thereto and has been washed with a first wash solution such that the AAV particle remained bound to the AEX medium and the at least one contaminant did not bind to the AEX medium;
wherein the first wash solution comprises urea and an acetate, and
washing the AEX medium with a second wash solution under conditions such that the AAV particle remains bound to the AEX medium and the at least one contaminant does not bind to the AEX medium,
thereby separating the AAV particle from the at least one contaminant,
wherein the AAV particle is a recombinant AAV (rAAV) comprising an rAAV genome comprising a transgene, and an AAV capsid comprising an AAV capsid protein, wherein: (i) the transgene is selected from the group consisting of phenylalanine hydroxylase (PAH), arylsulfatase A (ARSA), iduronate 2-sulfatase (I2S), and an anti-complement component 5 (C5) antibody; or (ii) the AAV capsid protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of amino acids 1-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17; wherein: the amino acid in the capsid protein corresponding to amino acid 2 of SEQ ID NO: 16 is T; the amino acid in the capsid protein corresponding to amino acid 65 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 68 of SEQ ID NO: 16 is V; the amino acid in the capsid protein corresponding to amino acid 77 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 119 of SEQ ID NO: 16 is L; the amino acid in the capsid protein corresponding to amino acid 151 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 160 of SEQ ID NO: 16 is D; the amino acid in the capsid protein corresponding to amino acid 206 of SEQ ID NO: 16 is C; the amino acid in the capsid protein corresponding to amino acid 296 of SEQ ID NO: 16 is H; the amino acid in the capsid protein corresponding to amino acid 312 of SEQ ID NO: 16 is Q; the amino acid in the capsid protein corresponding to amino acid 346 of SEQ ID NO: 16 is A;
the amino acid in the capsid protein corresponding to amino acid 464 of SEQ ID NO: 16 is N; the amino acid in the capsid protein corresponding to amino acid 468 of SEQ ID NO: 16 is S; the amino acid in the capsid protein corresponding to amino acid 501 of SEQ ID NO: 16 is I; the amino acid in the capsid protein corresponding to amino acid 505 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 590 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 626 of SEQ ID NO: 16 is G or Y; the amino acid in the capsid protein corresponding to amino acid 681 of SEQ ID NO: 16 is M; the amino acid in the capsid protein corresponding to amino acid 687 of SEQ ID NO: 16 is R; the amino acid in the capsid protein corresponding to amino acid 690 of SEQ ID NO: 16 is K; the amino acid in the capsid protein corresponding to amino acid 706 of SEQ ID NO: 16 is C; or, the amino acid in the capsid protein corresponding to amino acid 718 of SEQ ID NO: 16 is G.

7-9. (canceled)

10. The method of claim 1, wherein the at least one contaminant is selected from the group consisting of an AAV particle lacking a complete genome, an AAV degradation product, a host cell protein, a host cell fragment, and any combination thereof.

11. (canceled)

12. The method of claim 1, wherein:

the AEX medium has an average pore size of at least about 100 nm or at least about 500 nm;
the AEX medium comprises a quaternary amine; and/or
the AEX medium comprises a quaternary polyethyleneimine group.

13-15. (canceled)

16. The method of claim 1, wherein:

the mixture comprises an eluate of an affinity chromatography column the mixture comprises about 10 mM to about 200 mM of the acetate;
the mixture comprises about 10 mM to about 200 mM ammonium acetate, optionally about 28 mM ammonium acetate;
the mixture comprises about 2 mM to about 6 mM magnesium chloride, optionally about 2 mM magnesium chloride;
the mixture comprises about 0.001% (w/v) to about 0.05% (w/v) Poloxamer 188, optionally about 0.01% (w/v) Poloxamer 188; and/or
the mixture has a pH of about 9 to about 10.5, optionally about 9.3.

17-30. (canceled)

31. The method of claim 1, wherein:

the first wash solution comprises about 0.1 M to about 4 M urea, optionally about 2 M urea;
the first wash solution comprises an acetate selected from the group consisting of ammonium acetate, potassium acetate, sodium acetate, and cesium acetate;
the first wash solution comprises about 10 mM to about 200 mM of the acetate;
the first wash solution comprises about 10 mM to about 200 mM ammonium acetate, optionally about 153 mM ammonium acetate, or about 28 mM ammonium acetate;
the first wash solution comprises about 2 mM to about 6 mM magnesium chloride, optionally about 2 mM magnesium chloride;
the first wash solution comprises about 0.001% (w/v) to about 0.05% (w/v) Poloxamer 188, optionally about 0.01% (w/v) Poloxamer 188;
the first wash solution has a pH of about 9 to about 10.5, optionally about 9.3; and/or
the first wash solution has a conductivity of about 1 mS/cm to about 13 mS/cm, optionally about 13 mS/cm.

32-51. (canceled)

52. The method of claim 5, wherein:

the second wash solution comprises about 0.1 mM to about 15 mM ammonium acetate, optionally about 10 mM ammonium acetate;
the second wash solution comprises about 0.001% (w/v) to about 0.05% (w/v) Poloxamer 188, optionally about 0.01% (w/v) Poloxamer 188;
the second wash solution has a pH of about 9 to about 10.5, optionally about 9.3; and/or
the second wash solution has a conductivity of less than about 3 mS/cm, optionally about 1 mS/cm.

53-60. (canceled)

61. The method of claim 1, further comprising eluting the AAV particle from the AEX medium, optionally wherein the AAV particle is eluted from the AEX medium with an eluant using a step gradient or a linear gradient, optionally wherein:

the eluant comprises a salt at a concentration of about 10 mM to about 1 M, optionally wherein the salt is an acetate salt optionally selected from the group consisting of ammonium acetate, potassium acetate, sodium acetate, and cesium acetate;
the eluant comprises about 10 mM to about 1 M ammonium acetate;
the eluant comprises about 0.001% (w/v) to about 0.05% (w/v) Poloxamer 188, optionally 0.01% (w/v) Poloxamer 188;
the eluant has a pH of about 9 to about 10.5, optionally about 9.3;
the eluant further comprises about 50 mM ethanolamine; and/or
the eluant has a conductivity of about 8.5 mS/cm to about 30 mS/cm, optionally about 18.5 mS/cm, about 19 mS/cm, or about 26 mS/cm.

62-83. (canceled)

84. The method of claim 1, wherein the method results in an eluate comprising less than about 15%, or less than about 10% AAV particles that lack a complete genome.

85. (canceled)

86. The method of claim 61, further comprising formulating the eluted AAV particle in a formulation buffer suitable for administration to a human subject.

87. The method of claim 1, wherein:

the transgene further encodes an miRNA, shRNA, siRNA, antisense RNA, gRNA, antagomir, miRNA sponge, RNA aptazyme, RNA aptamer, lncRNA, ribozyme, or mRNA:,.
the rAAV genome further comprises a transcriptional regulatory element operably linked to the transgene, optionally wherein the transcriptional regulatory element comprises a promoter element and/or an intron element
the rAAV genome further comprises a polyadenylation sequence, optionally wherein the polyadenylation sequence is 3′ to the transgene;
the rAAV genome further comprises a 5′ inverted terminal repeat (5′ ITR) nucleotide sequence 5′ of the transgene, and a 3′ inverted terminal repeat (3′ ITR) nucleotide sequence 3′ of the transgene, optionally wherein the 5′ ITR nucleotide sequence is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 39, 41, or 42, and/or the 3′ ITR nucleotide sequence is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 40, 43, or 44;
the rAAV genome comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59; and/or
the AAV capsid protein comprises the amino acid sequence of amino acids 203-736, amino acids 138-736, and/or amino acids 1-736 of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, or 17.

88-135. (canceled)

Patent History
Publication number: 20230183659
Type: Application
Filed: Oct 28, 2022
Publication Date: Jun 15, 2023
Inventors: Michael MERCALDI (Bedford, MA), Ashish Sharma (Bedford, MA), Thomas Thiers (Bedford, MA), Luke Mustich (Bedford, MA), Shanshan Zhou (Bedford, MA), Zaid Junaid (Bedford, MA), Iraj Ghazi (Bedford, MA), Achyuta Teella (Bedford, MA), Alex Meola (Bedford, MA)
Application Number: 18/050,812
Classifications
International Classification: C12N 7/02 (20060101); C12N 15/864 (20060101); C07K 14/005 (20060101);