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1. (WO2016196621) MANGANESE SUPPLEMENTATION FOR CONTROL OF GLYCOSYLATION IN MAMMALIAN CELL CULTURE PROCESS
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WHAT IS CLAIMED IS:

1. A method for achieving a predetermined glycosylation profile of an anti-a4-integrin

antibody comprising providing manganese to a cell culture at a concentration that falls within a target manganese concentration range, wherein the cell culture comprises host cells producing the anti-a4-integrin antibody.

2. The method of claim 1, comprising supplementing the cell culture with manganese if the manganese concentration in the cell culture is below the target manganese concentration range.

3. A method for achieving a predetermined glycosylation profile of an anti-a4-integrin

antibody comprising (i) determining a manganese concentration in a component of a cell culture medium, (ii) if the manganese concentration is below a target manganese concentration range, supplementing the cell culture medium with the component to achieve a manganese concentration within the target manganese concentration range, and (iii) culturing a recombinant host cell producing an anti-a4-integrin antibody in the cell culture medium comprising the cell culture medium component.

4. A method for achieving a predetermined glycosylation profile of an anti-a4-integrin

antibody comprising (i) determining a manganese concentration in a component of a cell culture medium, (ii) if the manganese concentration is below a target manganese concentration range, adding manganese to the component of the cell culture medium to achieve a manganese concentration within the target manganese concentration range, (iii) producing a cell culture medium using the component of cell culture medium with the target manganese concentration, and (iv) culturing a recombinant host cell producing an anti-a4-integrin antibody in the cell culture medium comprising the cell culture medium component with the target manganese concentration.

5. A method for optimizing a cell culture medium for the production of an anti-a4-integrin antibody comprising (i) determining the amount of manganese in a cell culture medium or a component used to produce a cell culture medium, and (ii) if the amount of manganese is below a target range, supplementing the cell culture medium or the component of the cell culture medium with manganese to achieve an amount of manganese within the target range, wherein the target range is sufficient to produce anti-a4-integrin antibody with a predetermined glycosylation profile.

6. A method for optimizing a cell culture medium for the production of an anti-a4-integrin antibody comprising (i) determining the amount of manganese in a cell culture medium or a component used to produce a cell culture medium, and (ii) if the amount of manganese is above a target range, removing manganese from the cell culture medium or the component of the cell culture medium to achieve an amount of manganese within the target range, wherein the target range is sufficient to produce anti-a4-integrin antibody with a predetermined glycosylation profile.

7. The method of any one of claims 1-6, wherein the predetermined glycosylation profile of the anti-a4-integrin antibody comprises 13 to 32 % galactosylation.

8. The method of claim 7, wherein the predetermined glycosylation profile of the anti-a4- integrin antibody comprises 18 to 31 % galactosylation.

9. The method of claim 8, wherein the predetermined glycosylation profile of the anti-a4- integrin antibody comprises about 24% galactosylation.

10. The method of any one of claims 1-9, wherein the predetermined glycosylation profile of the anti-a4-integrin antibody comprises 0.7 to 3.6 % sialylation.

11. The method of claim 10, wherein the predetermined glycosylation profile of the anti-a4- integrin antibody comprises 1.0 to 2.2 % sialylation.

12. The method of claim 11, wherein the predetermined glycosylation profile of the anti-a4- integrin antibody comprises about 1.6 % sialylation.

13. The method of any one of claims 1-12, wherein the target manganese concentration range in the cell culture is 0.025 μΜ to 10 μΜ.

14. The method of claim 13, wherein the target manganese concentration range in the cell culture is 0.1 μΜ to 2.5 μΜ.

15. The method of claim 14, wherein the target manganese concentration range in the cell culture is 0.2 μΜ to 2 μΜ.

16. The method of any of claims 1-15, wherein the target manganese concentration range in the cell culture is at day 0 between 0.002 μΜ and 0.4 μΜ and at day 13 between 0.04 μΜ and 1 μΜ.

17. The method of any one of claims 1-16, wherein the target manganese concentration is maintained through a feedback loop.

18. The method of claim 17, wherein the manganese concentration is constantly monitored and maintained within the target manganese concentration range.

19. The method of any one of claims 1-5 or 7-18, wherein the target manganese concentration is achieved with a single dose of manganese.

20. The method of any one of claims 1-19, wherein the anti-a4-integrin antibody is produced by a eukaryotic host cell.

21. The method of claim 20, wherein the eukaryotic host cell is a mammalian host cell.

22. The method of any one of claims 1-21, wherein the anti-a4-integrin antibody is produced at a manufacturing scale.

23. The method of any one of claims 1-22, wherein the manganese concentration alters the levels of the isoform variants of the anti-a4-integrin antibody.

24. The method of claim 23, wherein the anti-a4-integrin antibody is natalizumab.

25. A method for achieving a predetermined glycosylation profile of an interferon beta- la polypeptide comprising providing manganese to a cell culture at a concentration that falls within a target manganese concentration range, wherein the cell culture comprises host cells producing the interferon beta-la polypeptide.

26. The method of claim 25, comprising supplementing the cell culture with manganese if the manganese concentration in the cell culture is below the target manganese concentration range.

27. A method for achieving a predetermined glycosylation profile of an interferon beta-la polypeptide comprising (i) determining a manganese concentration in a component of a cell culture medium, (ii) if the manganese concentration is below a target manganese concentration range, supplementing the cell culture medium with the component to achieve a manganese concentration within the target manganese concentration range, and (iii) culturing a recombinant host cell producing an interferon beta- la polypeptide in the cell culture medium comprising the cell culture medium component.

28. A method for achieving a predetermined glycosylation profile of an interferon beta-la polypeptide comprising (i) determining a manganese concentration in a component of a cell culture medium, (ii) if the manganese concentration is below a target manganese concentration range, adding manganese to the component of the cell culture medium to achieve a manganese concentration within the target manganese concentration range, (iii) producing a cell culture medium using the component of cell culture medium with the target manganese concentration, and (iv) culturing a recombinant host cell producing an interferon beta-la polypeptide in the cell culture medium comprising the cell culture medium component with the target manganese concentration.

29. A method for optimizing a cell culture medium for the production of an interferon beta-la polypeptide comprising (i) determining the amount of manganese in a cell culture medium or a component used to produce a cell culture medium, and (ii) if the amount of manganese is below a target range, supplementing the cell culture medium or the component of the cell culture medium with manganese to achieve an amount of manganese within the target range, wherein the target range is sufficient to produce interferon beta- la polypeptide with a predetermined glycosylation profile.

30. A method for optimizing a cell culture medium for the production of an interferon beta-la polypeptide comprising (i) determining the amount of manganese in a cell culture medium or a component used to produce a cell culture medium, and (ii) if the amount of manganese is above a target range, removing manganese from the cell culture medium or the component of the cell culture medium to achieve an amount of manganese within the target range, wherein the target range is sufficient to produce interferon beta- la polypeptide with a predetermined glycosylation profile.

31. The method of any one of claims 25-30, wherein the predetermined glycosylation profile of the interferon beta-la polypeptide comprises 91 to 100% sialylation.

32. The method of claim 31, wherein the predetermined glycosylation profile of the

interferon beta- la polypeptide comprises 91 to 97% sialylation.

33. The method of claim 32, wherein the predetermined glycosylation profile of the

interferon beta-la polypeptide comprises 95% sialylation.

34. The method of any one of claims 25-33, wherein the predetermined glycosylation profile of the interferon beta-la polypeptide comprises 55 to 85% biantennary glycoproteins.

35. The method of claim 34, wherein the predetermined glycosylation profile of the

interferon beta- la polypeptide comprises 66 to 73% biantennary glycoproteins.

36. The method of claim 35, wherein the predetermined glycosylation profile of the interferon beta- la polypeptide comprises 70% biantennary glycoproteins.

37. The method of any one of claims 25-36, wherein the predetermined glycosylation profile of the interferon beta-la polypeptide comprises 12 to 46% triantennary glycoproteins.

38. The method of claim 37, wherein the predetermined glycosylation profile of the

interferon beta-la polypeptide comprises 26 to 35% triantennary glycoproteins.

39. The method of claim 38, wherein the predetermined glycosylation profile of the

interferon beta-la polypeptide comprises 30 % triantennary glycoproteins.

40. The method of any one of claims 25-39, wherein the predetermined glycosylation profile of the interferon beta-la polypeptide comprises a biantennary glycoprotein to triantennary glycoprotein ratio of about 1.8 to 4.6.

41. The method of claim 40, wherein the biantennary glycoprotein to triantennary

glycoprotein ratio is about 2.0 to 2.5.

42. The method of claim 41, wherein the biantennary glycoprotein to triantennary

glycoprotein ratio is about 2.3.

43. The method of any one of claims 25-42, wherein the target manganese concentration range in the cell culture is 0.1 μΜ to 5 μΜ.

44. The method of claim 43, wherein the target manganese concentration range in the cell culture is 0.2 μΜ to 4.8 μΜ.

45. The method of claim 44, wherein the target manganese concentration range in the cell culture is 0.3 μΜ to 4.8 μΜ.

46. The method of any one of claims 25-45, wherein the target manganese concentration is maintained through a feedback loop.

47. The method of claim 46, wherein the manganese concentration is constantly monitored and maintained within the target manganese concentration range.

48. The method of any one of claims 25-29 or 31-47, wherein the target manganese

concentration is achieved with a single dose of manganese.

49. The method of any one of claims 25-48, wherein the interferon beta- la polypeptide is produced by a eukaryotic host cell.

50. The method of claim 49, wherein the eukaryotic host cell is a mammalian host cell.

51. The method of any one of claims 25-50, wherein the interferon beta- la polypeptide is produced at a manufacturing scale.

52. The method of any one of claims 25-51, wherein the manganese concentration alters the levels of the isoform variants of the interferon beta-la polypeptide.

53. The method of claim 52, wherein the interferon beta- la polypeptide has the sequence of SEQ ID NCv l .

54. A method for achieving a predetermined glycosylation profile of a rFVIIIFc comprising providing manganese to a cell culture at a concentration that falls within a target manganese concentration range, wherein the cell culture comprises host cells producing the rFVIIIFc.

55. The method of claim 54, comprising supplementing the cell culture with manganese if the manganese concentration in the cell culture is below the target manganese concentration range.

56. A method for achieving a predetermined glycosylation profile of a rFVIIIFc comprising (i) determining a manganese concentration in a component of a cell culture medium, (ii) if the manganese concentration is below a target manganese concentration range, supplementing the cell culture medium with the component to achieve a manganese concentration within the target manganese concentration range, and (iii) culturing a recombinant host cell producing a rFVIIIFc in the cell culture medium comprising the cell culture medium component.

57. A method for achieving a predetermined glycosylation profile of a rFVIIIFc comprising (i) determining a manganese concentration in a component of a cell culture medium, (ii) if the manganese concentration is below a target manganese concentration range, adding manganese to the component of the cell culture medium to achieve a manganese concentration within the target manganese concentration range, (iii) producing a cell culture medium using the component of cell culture medium with the target manganese concentration, and (iv) culturing a recombinant host cell producing a rFVIIIFc in the cell culture medium comprising the cell culture medium component with the target manganese concentration.

58. A method for optimizing a cell culture medium for the production of a rFVIIIFc

comprising (i) determining the amount of manganese in a cell culture medium or a component used to produce a cell culture medium, and (ii) if the amount of manganese is below a target range, supplementing the cell culture medium or the component of the cell culture medium with manganese to achieve an amount of manganese within the target range, wherein the target range is sufficient to produce rFVIIIFc with a predetermined glycosylation profile.

59. A method for optimizing a cell culture medium for the production of a rFVIIIFc

comprising (i) determining the amount of manganese in a cell culture medium or a component used to produce a cell culture medium, and (ii) if the amount of manganese is above a target range, removing manganese from the cell culture medium or the component of the cell culture medium to achieve an amount of manganese within the

target range, wherein the target range is sufficient to produce rFVIIIFc with a

predetermined glycosylation profile.

60. The method of any one of claims 54-59, wherein the predetermined glycosylation profile of the rFVIIIFc comprises 15 to 32% G0+Man6.

61. The method of claim 60, wherein the predetermined glycosylation profile of the rFVIIIFc comprises 16 to 29% G0+Man6.

62. The method of claim 61, wherein the predetermined glycosylation profile of the rFVIIIFc comprises 17 to 27% G0+Man6.

63. The method of any one of claims 54-62, wherein the predetermined glycosylation profile of the rFVIIIFc comprises 19 to 30% Gl+Man7.

64. The method of claim 63, wherein the predetermined glycosylation profile of the rFVIIIFc comprises 22 to 27% Gl+Man7.

65. The method of claim 64, wherein the predetermined glycosylation profile of the rFVIIIFc comprises 24 to 26% Gl+Man7.

66. The method of any one of claims 54-65, wherein the predetermined glycosylation profile of the rFVIIIFc comprises 6 to 17% G2+Man9.

67. The method of claim 66, wherein the predetermined glycosylation profile of the rFVIIIFc comprises 7 to 16% G2+Man9.

68. The method of claim 67, wherein the predetermined glycosylation profile of the rFVIIIFc comprises 10 to 15 % G2+Man9.

69. The method of any one of claims 54-68, wherein the target manganese concentration range in the cell culture is 30 nM to 1800 nM.

70. The method of claim 69, wherein the target manganese concentration range in the cell culture is 50 nM to 300 nM.

71. The method of claim 70, wherein the target manganese concentration range in the cell culture is 75 nM to 200 nM.

72. The method of any one of claims 54-71, wherein the target manganese concentration is maintained through a feedback loop.

73. The method of claim 72, wherein the manganese concentration is constantly monitored and maintained within the target manganese concentration range.

74. The method of any one of claims 54-58 or 60-73, wherein the target manganese

concentration is achieved with a single dose of manganese.

75. The method of any one of claims 54-74, wherein the rFVIIIFc is produced by a

eukaryotic host cell.

76. The method of claim 75, wherein the eukaryotic host cell is a mammalian host cell.

77. The method of any one of claims 54-76, wherein the rFVIIIFc is produced at a

manufacturing scale.