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1. (WO2019006390) AAV VECTOR COLUMN PURIFICATION METHODS
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WHAT IS CLAIMED IS:

1. A method for purifying recombinant adeno-associated viral (rAAV) vector particles said method comprising the steps of:

(a) harvesting cells and/or cell culture supernatant comprising rAAV vector particles to produce a harvest;

(b) optionally concentrating said harvest produced in step (a) to produce a concentrated harvest;

(c) lysing said harvest produced in step (a) or said concentrated harvest produced in step (b) to produce a lysate;

(d) treating the lysate produced in step (c) to reduce contaminating nucleic acid in the lysate thereby producing a nucleic acid reduced lysate;

(e) optionally filtering said nucleic acid reduced lysate produced in step (d) to produce a clarified lysate, and optionally diluting said clarified lysate to produce a diluted clarified lysate;

(f) subjecting said nucleic acid reduced lysate in in step (d), clarified lysate in step (e) or diluted clarified lysate produced in step (e) to cation exchange column chromatography to produce a column eluate comprised of rAAV vector particles thereby separating rAAV vector particles from protein impurities or other production/process related impurities, and optionally diluting said column eluate to produce a diluted column eluate;

(g) subjecting said column eluate or said diluted column eluate produced in step (f) to anion exchange chromatography to produce a second column eluate comprised of rAAV vector particles thereby separating rAAV vector particles from protein impurities or production/ process related impurities, and optionally concentrating said second column eluate to produce a concentrated second column eluate;

(h) subjecting said second column eluate or said concentrated second column eluate produced in step (g) to size exclusion column chromatography (SEC) to produce a third column eluate comprised of rAAV vector particles thereby separating rAAV vector particles from protein impurities or production/ process related impurities, and optionally concentrating said third column eluate to produce a concentrated third column eluate; and

(i) filtering said third column eluate or said concentrated third column eluate produced in step (h) thereby producing purified rAAV vector particles.

2. A method for purifying recombinant adeno-associated viral (rAAV) vector particles said method comprising the steps of:

(a) harvesting cells and/or cell culture supernatant comprising rAAV vector particles to produce a harvest;

(b) optionally concentrating said harvest produced in step (a) to produce a concentrated harvest;

(c) lysing said harvest produced in step (a) or said concentrated harvest produced in step (b) to produce a lysate;

(d) treating the lysate produced in step (c) to reduce contaminating nucleic acid in the lysate thereby producing a nucleic acid reduced lysate;

(e) optionally filtering said nucleic acid reduced lysate produced in step (d) to produce a clarified lysate, and optionally diluting said clarified lysate to produce a diluted clarified lysate;

(f) subjecting said nucleic acid reduced lysate in in step (d), clarified lysate in step (e) or diluted clarified lysate produced in step (e) to cation exchange column chromatography to produce a column eluate comprised of rAAV vector particles thereby separating rAAV vector particles from protein impurities or other production/ process related impurities, and optionally concentrating said column eluate to produce a concentrated column eluate;

(g) subjecting said column eluate or said concentrated column eluate produced in step (f) to size exclusion column chromatography (SEC) to produce a second column eluate comprised of rAAV vector particles thereby separating rAAV vector particles from protein impurities or other production/ process related impurities, and optionally diluting said second column eluate to produce a concentrated second column eluate;

(h) subjecting said second column eluate or said diluted second column eluate produced in step (g) to anion exchange chromatography to produce a third column eluate comprised of rAAV vector particles thereby separating rAAV vector particles from protein impurities production/ process related impurities,, and optionally diluting said third column eluate to produce a diluted third column eluate; and

(i) filtering said third column eluate or said concentrated third column eluate produced in step (h) thereby producing purified rAAV vector particles.

3. A method for purifying recombinant adeno-associated viral (rAAV) vector particles said method comprising the steps of:

(a) harvesting cells and/or cell culture supernatant comprising rAAV vector particles to produce a harvest;

(b) optionally concentrating said harvest produced in step (a) to produce a concentrated harvest;

(c) lysing said harvest produced in step (a) or said concentrated harvest produced in step (b) to produce a lysate;

(d) treating the lysate produced in step (c) to reduce contaminating nucleic acid in the lysate thereby producing a nucleic acid reduced lysate;

(e) optionally filtering said nucleic acid reduced lysate produced in step (d) to produce a clarified lysate, and optionally diluting said clarified lysate to produce a diluted clarified lysate;

(f) subjecting said nucleic acid reduced lysate in in step (d), clarified lysate in step (e) or diluted clarified lysate produced in step (e) to cation exchange column chromatography to produce a column eluate comprised of rAAV vector particles thereby separating rAAV vector particles from protein impurities or other production/process related impurities, and optionally diluting said column eluate to produce a diluted column eluate;

(g) subjecting said column eluate or said diluted column eluate produced in step (f) to anion exchange chromatography to produce a second column eluate comprised of rAAV vector particles thereby separating rAAV vector particles from production/process related impurities, and optionally concentrating said second column eluate to produce a concentrated second column eluate;

(h) filtering said second column eluate or said concentrated second column eluate produced in step (g) thereby producing purified rAAV vector particles.

4. A method for purifying recombinant adeno-associated viral (rAAV) vector particles said method comprising the steps of:

(a) harvesting cells and/or cell culture supernatant comprising rAAV vector particles to produce a harvest;

(b) optionally concentrating said harvest produced in step (a) to produce a concentrated harvest;

(c) lysing said harvest produced in step (a) or said concentrated harvest produced in step (b) to produce a lysate;

(d) treating the lysate produced in step (c) to reduce contaminating nucleic acid in the lysate thereby producing a nucleic acid reduced lysate;

(e) optionally filtering said nucleic acid reduced lysate produced in step (d) to produce a clarified lysate, and optionally diluting said clarified lysate to produce a diluted clarified lysate;

(f) subjecting said nucleic acid reduced lysate in step (d), or clarified lysate or diluted clarified lysate produced in step (e) to AAV affinity column chromatography to produce a column eluate comprised of rAAV vector particles thereby separating rAAV vector particles from protein impurities or other production/ process related impurities, and optionally diluting said column eluate to produce a diluted column eluate;

(g) subjecting said column eluate or said diluted column eluate produced in step (f) to anion exchange chromatography to produce a second column eluate comprised of rAAV vector particles thereby separating rAAV vector particles from protein impurities or other production/ process related impurities, and optionally concentrating said second column eluate to produce a concentrated second column eluate;

(h) optionally subjecting said second column eluate or said concentrated second column eluate produced in step (g) to size exclusion column chromatography (SEC) to produce a third column eluate comprised of rAAV vector particles thereby separating rAAV vector particles from protein impurities or other production/ process related impurities, and optionally concentrating said third column eluate to produce a concentrated third column eluate; and

(i) filtering said second column eluate or said diluted second column eluate produced in step (g), or filtering said third column eluate or said concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles.

5. A method for purifying recombinant adeno-associated viral (rAAV) vector particles said method comprising the steps of:

(a) harvesting cells and/or cell culture supernatant comprising rAAV vector particles to produce a harvest;

(b) optionally concentrating said harvest produced in step (a) to produce a concentrated harvest;

(c) lysing said harvest produced in step (a) or said concentrated harvest produced in step (b) to produce a lysate;

(d) treating the lysate produced in step (c) to reduce contaminating nucleic acid in the lysate thereby producing a nucleic acid reduced lysate;

(e) optionally filtering said nucleic acid reduced lysate produced in step (d) to produce a clarified lysate, and optionally diluting said clarified lysate to produce a diluted clarified lysate;

(f) subjecting said nucleic acid reduced lysate in step (d), or clarified lysate or diluted clarified lysate produced in step (e) to AAV affinity column chromatography to produce a column eluate comprised of rAAV vector particles thereby separating rAAV vector particles from protein impurities or other production/ process related impurities, and optionally concentrating said column eluate to produce a concentrated column eluate;

(g) subjecting said column eluate or said concentrated column eluate produced in step (f) to size exclusion column chromatography (SEC) to produce a second column eluate comprised of rAAV vector particles thereby separating rAAV vector particles from protein impurities or other production/ process related impurities, and optionally diluting said second column eluate to produce a diluted second column eluate;

(h) optionally subjecting said second column eluate or said diluted second column eluate produced in step (g) to anion exchange chromatography to produce a third column eluate comprised of rAAV vector particles thereby separating rAAV vector particles from protein impurities or other production/ process related impurities, and optionally diluting said third column eluate to produce a diluted third column eluate; and

(i) filtering said second column eluate or said diluted second column eluate produced in step (g), or filtering said third column eluate or said concentrated third column eluate produced in step (h), thereby producing purified rAAV vector particles.

6. A method according to any of claims 1-5, wherein said concentrating of step (b) and/or step (f) and/or step (g) and/or step (h) is via ultrafiltration/diafiltration, optionally by tangential flow filtration (TFF).

7. A method according to any of claims 1-6, wherein said concentrating of step (b) reduces the volume of said harvested cells and cell culture supernatant by about 2-20 fold.

8. A method according to any of claims 1-7, wherein said concentrating of step (f) and/or step (g) and/or step (h) reduces the volume of said column eluate by about 5-20 fold.

9. A method according to any of claims 1-8, wherein said lysing of said harvest produced in step (a) or said concentrated harvest produced in step (b) is by physical or chemical means.

10. A method according to claim 9, wherein the physical means comprises microfluidization or homogenization.

11. A method according to claim 9, wherein the chemical means comprises a detergent, optionally a non-ionic detergent such as triton X-100, optionally at a concentration of between about 0.1 and 1.0%, inclusive.

12. A method according to any of claims 1-11, wherein step (d) comprises treating with a nuclease thereby reducing contaminating nucleic acid.

13. A method according to claim 12, wherein the nuclease comprises benzonase.

14. A method according to any of claims 1-13, wherein said filtering said clarified lysate or said diluted clarified lysate of step (e) is via a filter having a pore diameter of between about 0.1 and 10.0 microns, inclusive.

15. A method according to any of claims 1-14, wherein said diluting of said clarified lysate of step (e) is with an aqueous buffered phosphate, acetate or Tris solution.

16. A method according to any of claims 1-15, wherein said diluting of said column eluate of step (f) or said second column eluate of step (g) is with an aqueous buffered phosphate, acetate or Tris solution.

17. A method according to claim 15 or 16, wherein said aqueous buffered phosphate or acetate solution has a pH of between about 4.0 and 7.4, inclusive.

18. A method according to claim 15 or 16, wherein said aqueous buffered Tris solution has a pH of greater than 7.5, optionally between about 8.0 and 9.0, inclusive.

19. A method according to any of claims 1-18, wherein said rAAV vector particles resulting from step (i) are formulated with a surfactant to produce an AAV vector formulation.

20. A method according to any of claims 1-19, wherein said anion exchange column chromatography of step (f), (g) and/or (h) comprises polyethylene glycol (PEG) modulated column chromatography.

21. A method according to claim 20, wherein said anion exchange column chromatography of step (g) and/or (h) comprises washing said column with a PEG solution prior to elution of said rAAV vector particles from the column.

22. A method according to claim 20 or 21, wherein the PEG has an average molecular weight in a range of about 1,000 to 80,000 g/mol, inclusive.

23. A method according to any of claims 20-22, wherein the PEG is at a concentration of about 4% to about 10%, inclusive.

24. A method according to any of claims 1-23, wherein said anion exchange column of step (g) and/or (h) comprises washing said column with an aqueous surfactant solution prior to elution of said rAAV vector particles from the column.

25. A method according to any of claims 1-24, wherein said cation exchange column of step (f) comprises washing said column with a surfactant solution prior to elution of said rAAV vector particles from the column.

26. A method according to any of claims 21-25, wherein said PEG solution and/or said surfactant solution comprises an aqueous Tris-Cl/NaCl buffer, an aqueous phosphate/NaCl buffer or an aqueous acetate/NaCl buffer.

27. A method according to claim 26, wherein said NaCl buffer comprises a range of between about 20-300 mM NaCl, inclusive, or between about 50-250 mM NaCl, inclusive.

28. A method according to any of claims 24-26, wherein said surfactant comprises a cationic or anionic surfactant.

29. A method according to any of claims 24-28, wherein said surfactant comprises a twelve carbon chained surfactant.

30. A method according to any of claims 24-29, wherein said surfactant comprises

Dodecyltrimethylammonium chloride (DTAC) or Sarkosyl.

31. A method according to any of claims 1-30, wherein said rAAV vector particles are eluted from said anion exchange column of step (f), (g) and/or (h) with an aqueous Tris-Cl/NaCl buffer.

32. A method according to claim 31, wherein said Tris-Cl/NaCl buffer comprises 100-400 mM NaCl, inclusive, optionally at a pH in a range of about 7.5 to about 9.0, inclusive.

33. A method according to any of claims 1-32, wherein said anion exchange column of step (f), (g) and/or (h) is washed with an aqueous Tris-Cl/NaCl buffer.

34. A method according to claim 33, wherein said NaCl in said aqueous Tris-Cl/NaCl buffer is in a range of about 75-125 mM NaCl, inclusive.

35. A method according to any of claims 31-33, wherein said aqueous Tris-Cl/NaCl buffer is at a pH from about 7.5 to about 9.0, inclusive.

36. A method according to any of claims 1-39, wherein said anion exchange column of step (f), (g) and/or (h) is washed one or more times to reduce the amount of AAV empty capsids in the second or third column eluate.

37. A method according to claim 33 or 36, wherein said anion exchange column wash removes AAV empty capsids from the column prior to rAAV removal and/or instead of rAAV, thereby reducing the amount of AAV empty capsids in the second or third column eluate.

38. A method according to claim 33 or 36, wherein said anion exchange column wash removes at least about 50% of the total AAV empty capsids from the column prior to rAAV removal and/or instead of rAAV, thereby reducing the amount of AAV empty capsids in the second or third column eluate by about 50%.

39. A method according to any of claims 33, or 36-38, wherein said NaCl in said aqueous Tris-Cl/NaCl buffer is in a range of about 110-120 mM NaCl, inclusive.

40. A method according to any of claims 33-40, wherein ratios and/or amounts of said rAAV vector particles and AAV empty capsids eluted are controlled by said wash buffer.

41. A method according to any of claims 1-41, wherein said rAAV vector particles are eluted from said cation exchange column of step (f) in an aqueous phosphate/NaCl buffer or an aqueous acetate/NaCl buffer.

42. A method according to claim 41, wherein said phosphate/NaCl buffer or aqueous acetate/NaCl buffer is in a range of about 125-500 mM NaCl, inclusive.

43. A method according to claim 41, wherein said phosphate/NaCl buffer or aqueous acetate/NaCl buffer has a pH in a range of about 5.5 to about 7.5, inclusive.

44. A method according to any of claims 1-43, wherein said anion exchange column of step (f), (g) and/or (h) comprises a quarternary ammonium functional group such as quaternized poly thyleneimine .

45. A method according to any of claims 1-44, wherein said size exclusion column (SEC) of step (g) and/or (h) has a separation/fractionation range (Molecular weight) from about 10,000 to about 600,000, inclusive.

46. A method according to any of claims 1-45, wherein said cation exchange column of step (f) comprises a sulfonic acid or functional group such as sulphopropyl.

47. A method according to any of claims 3-46, wherein the AAV affinity column comprises a protein or ligand that binds to AAV capsid protein.

48. A method according to claim 47, wherein the protein comprises an antibody that binds to AAV capsid protein.

49. A method according to claim 48, wherein the antibody that binds to AAV capsid protein comprises a single-chain Llama antibody (Camelid).

50. A method according to any of claims 1-49, wherein the method excludes a step of cesium chloride gradient ultracentrifugation.

51. A method according to any of claims 1-50, wherein said rAAV vector particles comprise a transgene that encodes a nucleic acid selected from the group consisting of a siRNA, an antisense molecule, miRNA a ribozyme and a shRNA.

52. A method according to any of claims 1-50, wherein said rAAV vector particles comprise a transgene that encodes a gene product selected from the group consisting of insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietins, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor a (TGFa), platelet-derived growth factor (PDGF), insulin growth factors I and II (IGF-I and IGF-II), TGFp, activins, inhibins, bone morphogenic protein (BMP), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT4/5, ciliary neurotrophic factor (CNTF), glial cell line derived neurotrophic factor (GDNF), neurturin, agrin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrins, noggin, sonic hedgehog and tyrosine hydroxylase.

53. A method according to any of claims 1-50, wherein said rAAV vector particles comprise a transgene that encodes a gene product selected from the group consisting of thrombopoietin (TPO), interleukins (IL1 through IL-17), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factors a and β, interferons α, β, and γ, stem cell factor, flk-2/flt3 ligand, IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cell receptors, class I and class II MHC molecules.

54. A method according to any of claims 1-50, wherein said rAAV vector particles comprise a transgene encoding a protein useful for correction of in born errors of metabolism selected from the group consisting of carbamoyl synthetase I, ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase, fumarylacetacetate hydrolase, phenylalanine hydroxylase, alpha- 1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, factor V, factor VIII, factor IX, cystathione beta-synthase, branched chain ketoacid decarboxylase, albumin, isovaleryl-coA dehydrogenase, propionyl CoA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, RPE65, H-protein, T-protein, a cystic fibrosis transmembrane regulator (CFTR) sequence, and a dystrophin cDNA sequence.

55. A method according to any of claims 1-54, wherein said rAAV vector particles comprise a transgene that encodes Factor VIII or Factor IX.

56. A method according to any of claims 1-55, wherein the method recovers approximately 50-90% of the total rAAV vector particles from the harvest produced in step (a) or said concentrated harvest produced in step (b).

57. A method according to any of claims 1-56, wherein the method produces rAAV vector particles having a greater purity than rAAV vector particles produced or purified by a single AAV affinity column purification.

58. A method according to any of claims 1-57, wherein steps (c) and (d) are performed substantially concurrently.

59. A method according to any of claims 1-58, wherein after step (c) but prior to step (f) NaCl is adjusted to be in a range of about 100-400 mM NaCl, inclusive, or in a range of about 140-300 mM NaCl, inclusive.

60. A method according to any of claims 1-59, wherein said rAAV vector particles are derived from an AAV selected from the group consisting of AAVl, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, RhlO and Rh74.

61. A method according to any of claims 1-60, wherein said rAAV vector particles comprise a capsid sequence having 70% or more identity to an AAVl, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, RhlO, Rh74, SEQ ID NO: l or SEQ ID NO:2 capsid sequence.

62. A method according to any of claims 1-61, wherein said rAAV vector particles comprise an ITR sequence having 70% or more identity to an AAVl, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, RhlO, or Rh74 ITR sequence.

63. A method according to any of claims 1-62, wherein said cells comprise suspension or adherent cells.

64. A method according to any of claims 1-63, wherein said cells comprise mammalian cells.

65. A method according to any of claims 1-64, wherein said cells comprise HEK-293 cells.

66. A method according to any of claims 1-65, wherein the method is performed according to any one or more column, condition, concentration, molarity, volume, capacity, flow rate, pressure, material, temperature, pH, or step as set forth in any of Examples 1-3.

67. A method according to any of claims 1-66, wherein the cell lysis and/or preparation prior to column purification is performed according to any one or more condition, concentration, molarity, volume, capacity, flow rate, pressure, material, temperature, pH, or step as set forth in Example 4.