Claims for Patent: 9,062,106

Summary for Patent: 9,062,106

Title:	Methods for controlling the galactosylation profile of recombinantly-expressed proteins
Abstract:	The present invention relates to methods for modulating the glycosylation profile of recombinantly-expressed proteins. In particular, the present invention relates to methods of controlling the galactosylation profile of recombinantly-expressed proteins by supplementing production medium, e.g., a hydrolysate-based or a chemically defined medium, with manganese and/or D-galactose.
Inventor(s):	Bengea; Cornelia (Auburn, MA), Rives; Lisa M. (Natick, MA), Hossler; Patrick (Westborough, MA)
Assignee:	AbbVie Inc. (North Chicago, IL)
Application Number:	13/457,020
Patent Litigation and PTAB cases:	See patent lawsuits and PTAB cases for patent 9,062,106
Patent Claims:	1. A method for increasing the galactosylation level of a recombinantly expressed adalimumab, comprising supplementing a media used in the expression of the recombinantly expressed adalimumab with a sufficient amount of a manganese supplement to achieve a manganese concentration in the media of 0.2-100 .mu.M and a sufficient amount of a galactose supplement to achieve a galactose concentration in the media of 1-100 mM, thereby increasing the galactosylation level of the recombinantly expressed adalimumab, wherein the galactosylation level of a recombinantly expressed adalimumab is increased as compared to the galactosylation level of adalimumab recombinantly expressed in media which is not supplemented with said manganese supplement and said galactose supplement. 2. The method of claim 1, wherein the manganese supplement is a biologically acceptable manganese salt. 3. The method of claim 2, wherein the biologically acceptable manganese salt is manganese (II) chloride. 4. The method of claim 1, wherein the galactose supplement is a biologically acceptable galactose containing compound. 5. The method of claim 4, wherein the biologically acceptable galactose containing compound is D-(+)-galactose. 6. The method of claim 1, wherein the media is supplemented with a sufficient amount of the manganese supplement to achieve a manganese concentration in the media selected from the group consisting of 0.2, 0.5, 1.0, 10, 20, 25, 40, 50, 60, 75, 80, and 100 .mu.M. 7. The method of claim 1, wherein the media is supplemented with a sufficient amount of the galactose supplement to achieve a galactose concentration in the media selected from the group consisting of 1, 4, 5, 10, 15, 20, 30, 40, 60, and 100 mM. 8. The method of claim 1, wherein the media is supplemented with a sufficient amount of the manganese supplement and the galactose supplement to achieve a manganese (Mn) and a galactose (Gal) concentration in the media selected from the group consisting of 0.2/1, 0.2/4, 0.2/30, 0.5/1, 0.5/4, 0.5/30, 10/10, 10/20, 10/40, 20/10, 20/20, 20/40, 25/15, 40/10, 40/20, 40/40, 40/100, 50/30, 60/20, 60/40, 60/100, 80/20, 80/40, 80/100, 100/20, 100/40, and 100/100 Mn (.mu.M)/Gal (mM). 9. The method of claim 1, wherein the method further comprises culturing in the media a mammalian cell expressing adalimumab, wherein the media is selected from the group consisting of chemically defined (CD) cell culture media and hydrolysate-based media. 10. The method of claim 9, wherein the culturing is done in a suspension culture. 11. The method of claim 9, wherein the mammalian cell has been adapted for growth in a CD cell culture media. 12. The method of claim 9, wherein the mammalian cell is a CHO cell. 13. The method of claim 9, wherein the mammalian cell is an NS0 cell. 14. The method of claim 9, wherein the mammalian cell is cultured for at least 4 days. 15. The method of claim 9, wherein the mammalian cell is cultured for up to 12 days. 16. The method of claim 9, further comprising recovering said adalimumab from the media. 17. The method of claim 16, further comprising purifying said adalimumab from the media. 18. The method of claim 17, further comprising quantifying the levels of galactose-containing fucosylated biantennary oligosaccharides (NA1F and NA2F) and/or agalactosyl fucosylated biantennary oligosaccharides (NGA2F and NGA2F-GlcNAc) present on said adalimumab. 19. The method of claim 9, wherein the said culturing is done as a fed-batch process. 20. The method of claim 9, wherein said culturing is done in a bioreactor. 21. The method of claim 1, wherein manganese and galactose are present in the media at a concentration sufficient to produce a composition comprising adalimumab in which at least 10% of the total N-linked oligosaccharides present on said adalimumab are of a galactose-containing fucosylated biantennary oligosaccharide (NA1F+NA2F) form. 22. The method of claim 21, wherein at least 15% of the total N-linked oligosaccharides present on said adalimumab are of a galactose-containing fucosylated biantennary oligosaccharide (NA1F+NA2F) form. 23. The method of claim 21, wherein at least 20% of the total N-linked oligosaccharides present on said adalimumab are of a galactose-containing fucosylated biantennary oligosaccharide (NA1F+NA2F) form. 24. The method of claim 1, wherein the media is supplemented with a sufficient amount of the manganese supplement to achieve a manganese concentration in the media of 0.2-40 .mu.M. 25. The method of claim 1, wherein the media is supplemented with a sufficient amount of the manganese supplement to achieve a manganese concentration in the media of 40-100 .mu.M. 26. The method of claim 1, wherein the media is supplemented with a sufficient amount of the manganese supplement to achieve a manganese concentration in the media of 40-60 .mu.M. 27. The method of claim 1, wherein the media is supplemented with a sufficient amount of the manganese supplement to achieve a manganese concentration in the media of 50-80 .mu.M. 28. The method of claim 1, wherein the media is supplemented with a sufficient amount of the manganese supplement to achieve a manganese concentration in the media of 75-100 .mu.M. 29. The method of claim 1, wherein the media is supplemented with a sufficient amount of the galactose supplement to achieve a galactose concentration in the media of 10-100 mM. 30. The method of claim 1, wherein the media is supplemented with a sufficient amount of the galactose supplement to achieve a galactose concentration in the media of 1-60 mM. 31. The method of claim 1, wherein the media is supplemented with a sufficient amount of the galactose supplement to achieve a galactose concentration in the media of 60-100 mM. 32. The method of claim 1, wherein the media is supplemented with a sufficient amount of the galactose supplement to achieve a galactose concentration in the media of 10-60 mM. 33. The method of claim 1, wherein the media is selected from the group consisting of production media and feed media. 34. A method for increasing the galactosylation level of a recombinantly expressed adalimumab, comprising supplementing a media used in the expression of the recombinantly expressed adalimumab with a sufficient amount of a manganese supplement to achieve a manganese concentration in the media of 0.2-100 .mu.M and a sufficient amount of a galactose supplement to achieve a galactose concentration in the media of 1-100 mM and culturing a cell expressing adalimumab in said media, thereby increasing the galactosylation level of the recombinantly expressed adalimumab, wherein the galactosylation level of a recombinantly expressed adalimumab is increased as compared to the galactosylation level of adalimumab recombinantly expressed in media which is not supplemented with said manganese supplement and said galactose supplement. 35. The method of claim 34, wherein the manganese supplement is a biologically acceptable manganese salt. 36. The method of claim 35, wherein the biologically acceptable manganese salt is manganese (II) chloride. 37. The method of claim 34, wherein the galactose supplement is a biologically acceptable galactose containing compound. 38. The method of claim 37, wherein the biologically acceptable galactose containing compound is D-(+)-galactose. 39. The method of claim 34, wherein the media is supplemented with a sufficient amount of the manganese supplement to achieve a manganese concentration in the media selected from the group consisting of 0.2, 0.5, 1.0, 10, 20, 25, 40, 50, 60, 75, 80, and 100 .mu.M. 40. The method of claim 34, wherein the media is supplemented with a sufficient amount of the galactose supplement to achieve a galactose concentration in the media selected from the group consisting of 1, 4, 5, 10, 15, 20, 30, 40, 60, and 100 mM. 41. The method of claim 34, wherein the media is supplemented with a sufficient amount of the manganese supplement and the galactose supplement to achieve a manganese (Mn) and a galactose (Gal) concentration in the media selected from the group consisting of 0.2/1, 0.2/4, 0.2/30, 0.5/1, 0.5/4, 0.5/30, 10/10, 10/20, 10/40, 20/10, 20/20, 20/40, 25/15, 40/10, 40/20, 40/40, 40/100, 50/30, 60/20, 60/40, 60/100, 80/20, 80/40, 80/100, 100/20, 100/40, and 100/100 Mn (.mu.M)/Gal (mM). 42. The method of claim 34, wherein said cell is a mammalian cell and wherein the media is selected from the group consisting of chemically defined (CD) cell culture media and hydrolysate-based media. 43. The method of claim 42, wherein the mammalian cell has been adapted for growth in a CD cell culture media. 44. The method of claim 42, wherein the mammalian cell is a CHO cell. 45. The method of claim 42, wherein the mammalian cell is an NS0 cell. 46. The method of claim 42, wherein the mammalian cell is cultured for at least 4 days. 47. The method of claim 42, wherein the mammalian cell is cultured for up to 12 days. 48. The method of claim 34, wherein manganese and galactose are present in the media at a concentration sufficient to produce a composition comprising adalimumab in which at least 10% of the total N-linked oligosaccharides present on said adalimumab are of a galactose-containing fucosylated biantennary oligosaccharide (NA1F+NA2F) form. 49. The method of claim 48, wherein at least 15% of the total N-linked oligosaccharides present on said adalimumab are of a galactose-containing fucosylated biantennary oligosaccharide (NA1F+NA2F) form. 50. The method of claim 48, wherein 20% of the total N-linked oligosaccharides present on said adalimumab are of a galactose-containing fucosylated biantennary oligosaccharide (NA1F+NA2F) form. 51. The method of claim 34, further comprising recovering said adalimumab from the media. 52. The method of claim 51, further comprising purifying said adalimumab from the media. 53. The method of claim 52, further comprising quantifying the levels of galactose-containing fucosylated biantennary oligosaccharides (NA1F and NA2F) and/or agalactosyl fucosylated biantennary oligosaccharides (NGA2F and NGA2F-GlcNAc) present on said adalimumab. 54. The method of claim 34, wherein the media is supplemented with a sufficient amount of the manganese supplement to achieve a manganese concentration in the media of 0.2-40 .mu.M. 55. The method of claim 34, wherein the media is supplemented with a sufficient amount of the manganese supplement to achieve a manganese concentration in the media of 40-100 .mu.M. 56. The method of claim 34, wherein the media is supplemented with a sufficient amount of the manganese supplement to achieve a manganese concentration in the media of 40-60 .mu.M. 57. The method of claim 34, wherein the media is supplemented with a sufficient amount of the manganese supplement to achieve a manganese concentration in the media of 50-80 .mu.M. 58. The method of claim 34, wherein the media is supplemented with a sufficient amount of the manganese supplement to achieve a manganese concentration in the media of 75-100 .mu.M. 59. The method of claim 34, wherein the media is supplemented with a sufficient amount of the galactose supplement to achieve a galactose concentration in the media of 10-100 mM. 60. The method of claim 34, wherein the media is supplemented with a sufficient amount of the galactose supplement to achieve a galactose concentration in the media of 1-60 mM. 61. The method of claim 34, wherein the media is supplemented with a sufficient amount of the galactose supplement to achieve a galactose concentration in the media of 60-100 mM. 62. The method of claim 34, wherein the media is supplemented with a sufficient amount of the galactose supplement to achieve a galactose concentration in the media of 10-60 mM. 63. The method of claim 34, wherein the said culturing is done as a fed-batch process. 64. The method of claim 34, wherein the media is selected from the group consisting of production media and feed media. 65. The method of claim 34, wherein said culturing is done in a bioreactor.

Details for Patent 9,062,106

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Abbvie Inc.	HUMIRA	adalimumab	Injection	125057	12/31/2002		2031-04-27
Abbvie Inc.	HUMIRA	adalimumab	Injection	125057	02/21/2008		2031-04-27
Abbvie Inc.	HUMIRA	adalimumab	Injection	125057	04/24/2013		2031-04-27