Methods and compositions for the purification of adeno-associated virus

Abstract

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.

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)