Methods for producing preparations of recombinant AAV virions substantially free of empty capsids
Methods for separating AAV empty capsids from mixtures of AAV vector particles and AAV empty capsids are described. The methods use column chromatography techniques and provide for commercially viable levels of recombinant AAV virions.
1. A method for purifying AAV vector particles from an AAV preparation comprising AAV vector particles and AAV empty capsids, to provide an AAV product substantially free of AAV empty capsids, said method comprising:
(a) applying an AAV preparation comprising AAV vector particles and AAV empty capsids to an anion exchange chromatography column under conditions whereby said AAV vector particles and said AAV empty capsids bind the column;
(b) adding a salt buffer to the column from (a) under conditions whereby AAV empty capsids are eluted and AAV vector particles remain bound to the column;
(c) adding a salt buffer with a greater salt concentration relative to the buffer added in (b) to the column from (b) under conditions whereby AAV vector particles are eluted;
(d) collecting eluted fractions from (c) and selecting fractions that consist essentially of AAV vector particles based on the ratio of UV absorption at 260 nm/280 nm to provide an AAV product substantially free of AAV empty capsids.
2. The method of claim 1 , further comprising:
(e) applying the AAV preparation from (d) to a second anion exchange chromatography column under conditions whereby said AAV vector particles and said AAV empty capsids, if present, bind the column;
(f) adding a salt buffer to the column from (e) under conditions whereby AAV empty capsids are eluted and AAV vector particles remain bound to the column;
(g) adding a salt buffer with a greater salt concentration relative to the buffer added in (f) to the column from (f) under conditions whereby AAV vector particles are eluted;
(h) collecting eluted fractions from (g) that comprise AAV vector particles to provide an AAV product substantially free of AAV empty capsids.
3. The method of claim 1 , further comprising:
(e) applying the AAV preparation from (d) to a cation exchange chromatography column under conditions whereby said AAV vector particles and said AAV empty capsids bind the column;
(f) adding a salt buffer to the column from (e) under conditions whereby AAV vector particles are eluted and AAV empty capsids remain bound to the column; and
(g) collecting eluted fractions from (0 that comprise AAV vector particles to provide an AAV product substantially free of AAV empty capsids.
4. The method of claim 1 , wherein AAV vector particles are present in said AAV product in an amount of at least 75%.
5. The method of claim 1 , wherein AAV vector particles are present in said AAV product in an amount of at least 85%.
6. The method of claim 1 , wherein AAV vector particles are present in said AAV product in an amount of at least 90%.
7. The method of claim 1 , wherein said AAV vector particles are from AAV-2.
8. The method of claim 1 , wherein said AAV vector particles are from AAV-5.