Scalable purification of AAV2, AAV4 or AAV5 using ion-exchange chromatography
View Patent ↗The present invention provides methods of purifying adeno-associated virus (AAV) particles. These AAV particles include AAV2, AAV4 and AAV5 particles. The present invention also provides AAV particles purified by the methods of the present invention.
1. A method of purifying adeno-associated virus (AAV) particles comprising:
a) subjecting a culture of wild-type or recombinant AAV particles to high performance liquid chromatography (HPLC), wherein the particles are bound to an ion exchange column equilibrated at a pH of about 6.0 to about 7.2 and at a salt concentration of greater than 100 mM, and wherein the AAV particles are eluted with about 100-600 mM salt in order to obtain purified AAV particles, wherein the culture is not subjected to iodixanol gradient separation;
b) concentrating the eluted AAV particles; and
c) washing the concentrated AAV particles.
2. The method of claim 1 , wherein the ion exchange column is a strong anionic exchange column.
3. The method of claim 1 , wherein the ion exchange column is a weak anionic exchange column.
4. The method of claim 1 , wherein the ion exchange column is a strong cationic exchange column.
5. The method of claim 1 , wherein the ion exchange column is a weak cationic exchange column.
6. The method of claim 1 , wherein the ion exchange column is a weak ion exchange column having a surface coated with a resin selected from the group consisting of polyethyleneimine that is capable of surface ionization up to a pH of about 9, a styrene-divinylbenzene copolymer containing sulfonic acid groups, and a dextran matrix functionalized by diethylaminoethyl.
7. The method of claim 1 , wherein the column is equilibrated with NaCl.
8. The method of claim 1 , wherein the AAV particles are eluted with KCl.
9. A method of purifying adeno-associated virus (AAV) particles comprising:
a. treating a culture of wild-type or recombinant AAV particles with a detergent;
b. subjecting the culture of (a) to high performance liquid chromatography (HPLC), wherein the particles are bound to an ion exchange column equilibrated at a pH of about 6.0 to about 7.2 and at a salt concentration of greater than 100 mM and wherein the AAV particles are eluted with about 150-500 mM salt in order to obtain purified AAV particles, wherein the culture is not subjected to iodixanol gradient separation;
c. concentrating the eluted AAV particles; and
d. washing the concentrated AAV particles.
10. The method of claim 9 , wherein the ion exchange column is a strong anionic exchange column.
11. The method of claim 9 , wherein the ion exchange column is a weak anionic exchange column.
12. The method of claim 9 , wherein the ion exchange column is a strong cationic exchange column.
13. The method of claim 9 , wherein the ion exchange column is a weak cationic exchange column.
14. The method of claim 9 , wherein the ion exchange column is a weak anionic exchange column having a surface coated with a resin selected from the group consisting of polyethyleneimine that is capable of surface ionization up to a pH of about 9, a styrene-divinylbenzene copolymer containing sulfonic acid groups, and a dextran matrix functionalized by diethylaminoethyl.
15. The method of claim 9 , wherein the AAV particles are AAV2 particles, wherein the detergent is octylglucopyranoside (OCG) and wherein the concentration of octylglucopyranoside is at least about 1.5%.
16. The method of claim 9 , wherein the AAV particles are AAV2 particles and wherein the ion exchange column is equilibrated at a pH of about 6.2 to about 7.2.
17. The method of claim 9 , wherein the AAV particles are AAV2 particles and wherein the AAV2 particles are eluted with about 100-400 mM salt.
18. The method of claim 9 , wherein the AAV particles are AAV2 particles, wherein the detergent is OCG and wherein the concentration of OCG is at least about 0.1% and further comprising benzonase.
19. The method of claim 9 , wherein the benzonase is at about 20 μml.
20. The method of claim 19 , wherein the ion exchange column is equilibrated at a pH of about 6.2 to about 7.2.
21. The method of claim 20 , wherein the AAV2 particles are eluted with 100-400 mM salt.
22. The method of claim 9 , wherein the AAV particles are AAV4 particles, wherein the detergent is OCG and wherein the concentration of OCG is about 0.5% and further comprising benzonase.
23. The method of claim 22 , wherein the concentration of benzonase is at least about 20 μ/ml.
24. The method of claim 9 , wherein the AAV particles are AAV4 particles and wherein the ion exchange column is equilibrated at a pH of about 6.7 to about 7.2.
25. The method of claim 9 , wherein the AAV particles are AAV4 particles and wherein the AAV4 particles are eluted with about 200-600 mM salt.
26. The method of claim 9 , wherein the AAV particles are AAV5 particles, wherein the detergent is OCG and wherein the concentration of OCG is about 1.5%.
27. The method of claim 9 , wherein the AAV particles are AAV5 particles and wherein the ion exchange column is equilibrated at a pH of about 6.0 to about 7.2.
28. The method of claim 9 , wherein the AAV particles are AAV5 particles and wherein the AAV5 particles are eluted with about 100-400 mM salt.
29. The method of claim 9 , wherein the AAV particles are adeno-associated virus 2 (AAV2) particles comprising:
a. treating a culture of wild-type or recombinant AAV2 particles with at least about 1.5% octylglucopyranoside (OCG) and;
b. subjecting the culture of (a) to high performance liquid chromatography (HPLC), wherein the particles are bound to an ion exchange column equilibrated at a pH of about 6.5 to about 7.2 and at a salt concentration of greater than 150 mM and wherein the AAV2 particles are eluted with about 100-400 mM salt in order to obtain purified AAV2 particles.
30. The method of claim 29 , wherein the ion exchange column is a strong anionic exchange column.
31. The method of claim 29 , wherein the ion exchange column is a weak anionic exchange column.
32. The method of claim 30 , wherein the strong anionic exchange column has a surface coated with quaternized polyethyleneimine with a surface ionization over a pH range of about 1 to about 14.
33. The method of claim 31 , wherein the weak anionic exchange column has a surface coated with a resin selected from the group consisting of polyethyleneimine that is capable of surface ionization up to a pH of about 9, a styrene-divinylbenzene copolymer containing sulfonic acid groups, and a dextran matrix functionalized by diethylaminoethyl.
34. The method of claim 9 , wherein the AAV particles are adeno-associated virus 2 (AAV2) particles comprising:
a. treating a culture of wild-type or recombinant AAV2 particles with at least about 0.1% octylglucopyranoside (OCG) and about 20 μ/ml benzonase and;
b. subjecting the culture of (a) to high performance liquid chromatography (HPLC), wherein the particles are bound to an ion exchange column equilibrated at a pH of about 6.5 to about 7.2 and at a salt concentration of greater than 150 mM and wherein the AAV2 particles are eluted with about 100-400 mM salt in order to obtain purified AAV2 particles.
35. The method of claim 34 , wherein the ion exchange column is a strong anionic exchange column.
36. The method of claim 34 , wherein the ion exchange column is a weak anionic exchange column.
37. The method of claim 35 , wherein the strong anionic exchange column has a surface coated with quaternized polyethyleneimine with a surface ionization over a pH range of about 1 to about 14.
38. The method of claim 36 , wherein the weak anionic exchange column has a surface coated with a resin selected from the group consisting of polyethyleneimine that is capable of surface ionization up to a pH of about 9, a styrene-divinylbenzene copolymer containing sulfonic acid groups, and a dextran matrix functionalized by diethylaminoethyl.
39. The method of claim 9 , wherein the AAV particles are adeno-associated virus 4(AAV4) particles comprising:
a. treating a culture of wild-type or recombinant AAV4 particles with at least about 0.5% octylglucopyranoside (OCG) and 20 μ/ml benzonase;
b. subjecting the culture of (a) to high performance liquid chromatography (HPLC), wherein the particles are bound to an ion exchange column equilibrated at a pH of about 6.7 to about 7.2 and at a salt concentration of greater than 150 mM, and wherein the AAV 4 particles are eluted with about 200-600 mM salt in order to obtain purified AAV 4 particles.
40. The method of claim 39 , wherein the ion exchange column is a strong anionic exchange column.
41. The method of claim 39 , wherein the ion exchange column is a weak anionic exchange column.
42. The method of claim 40 , wherein the strong anionic exchange column has a surface coated with quaternized polyethyleneimine with a surface ionization over a pH range of about 1 to about 14.
43. The method of claim 41 , wherein the weak anionic exchange column has a surface coated with a resin selected from the group consisting of polyethyleneimine that is capable of surface ionization up to a pH of about 9, a styrene-divinylbenzene copolymer containing sulfonic acid groups, and a dextran matrix functionalized by diethylaminoethyl.
44. The method of claim 9 , wherein the AAV particles are adeno-associated virus 5(AAV5) particles, comprising:
a. treating a culture of wild-type or recombinant AAV4 particles with at least about 1.5% octylglucopyranoside (OCG) and;
b. subjecting the culture of (a) to high performance liquid chromatography (HPLC), wherein the particles are bound to an ion exchange column equilibrated at a pH of about 6.0 to about 7.2 and at a salt concentration of greater than 150 mM, and wherein the AAV 5 particles are eluted with about 100-400 mM salt in order to obtain purified AAV 5 particles.
45. The method of claim 44 , wherein the ion exchange column is a strong anionic exchange column.
46. The method of claim 44 , wherein the ion exchange column is a weak anionic exchange column.
47. The method of claim 45 , wherein the strong anionic exchange column has a surface coated with quaternized polyethyleneimine with a surface ionization over a pH range of about 1 to about 14.
48. The method of claim 46 , wherein the weak anionic exchange column has a surface coated with a resin selected from the group consisting of polyethyleneimine that is capable of surface ionization up to a pH of about 9, a styrene-divinylbenzene copolymer containing sulfonic acid groups, and a dextran matrix functionalized by diethylaminoethyl.
49. A method of purifying AAV2 particles comprising:
a. treating a culture of wild-type or recombinant AAV2 particles with at least about 0.5% deoxycholate (DOC) and;
b. subjecting the culture of (a) to high performance liquid chromatography (HPLC), wherein the particles are bound to an HS column equilibrated at a pH of about 5.5 to about 6.5 and at a salt concentration of greater than 150 mM and wherein the AAV particles are eluted with about 100-400 mM salt in order to obtain purified AAV2 particles, wherein the culture is not subjected to iodixanol gradient seperation.
50. A method of purifying AAV2 particles comprising:
a. treating a culture of wild-type or recombinant AAV2 particles with at least about 0.5% deoxycholate (DOC) and;
b. subjecting the culture of (a) to high performance liquid chromatography (HPLC), wherein the particles are bound to a CM column equilibrated at a pH of about 5.5 to about 7.5 and at a salt concentration of greater than 150 mM and wherein the AAV particles are eluted with about 100-400 mM salt in order to obtain purified AAV particles, wherein the culture is not subjected to iodixanol gradient separation.
51. A method of purifying AAV2 particles comprising:
a. treating a culture of wild-type or recombinant AAV2 particles with at least about 0.5% deoxycholate (DOC) and;
b. subjecting the culture of (a) to high performance liquid chromatography (HPLC), wherein the particles are bound to an HQ column equilibrated at a pH of about 6.5 to about 7.5 and at a salt concentration of greater than 150 mM and wherein the AAV particles are eluted with about 100-300 mM salt in order to obtain purified AAV particles, wherein the culture is not subjected to iodixanol gradient separation.
52. A method of purifying AAV4 particles comprising:
a. treating a culture of wild-type or recombinant AAV4 particles; with at least about 0.5% octylglucopyranoside (OCG) and benzonase; and
b. subjecting the culture of (a) to high performance liquid chromatography (HPLC), wherein the particles are bound to an HQ column equilibrated at a pH of about 6.5 to about 7.5 and at a salt concentration of greater than 150 mM, and wherein the AAV 4 particles are eluted with about 200-600 mM salt in order to obtain purified AAV 4 particles, wherein the culture is not subjected to iodixanol gradient separation.
53. A method of purifying adeno-associated virus (AAV) particles comprising:
a. subjecting a clarified viral lysate of wild-type or recombinant AAV particles to high performance liquid chromatography (HPLC), wherein the particles are bound to an ion exchange column equilibrated at a pH of about 6.0 to about 7.2 and at a salt concentration of greater than 100 mM, and wherein the AAV particles are eluted with about 100-600 mM salt in order to obtain purified AAV particles;
b. concentrating the eluted AAV particles; and
c. washing the concentrated AAV particles.
54. The method of claim 1 , wherein the ion exchange column is a strong anionic exchange column having a surface coated with quaternized polyethyleneimine with a surface ionization over a pH range of about 1 to about 14.
55. The method of claim 9 , wherein the ion exchange column is a strong anionic exchange column having a surface coated with quaternized polyethyleneimine with a surface ionization over a pH range of about 1 to about 14.