CELL CULTURE IMPROVEMENTS

• US 20140234905A1
• Filed: 03/26/2014
• Published: 08/21/2014
• Est. Priority Date: 09/13/2006
• Status: Active Grant

• Current Assignee: Abbvie Incorporated
• Original Assignee: Abbvie Incorporated
• Inventors: Pla, Itzcoatl A., Matuck, Joseph G., Fann, John C., Schulz, Christof, Roy, Nichole A., Bruton, David F., McIntire, James, Chang, Yu-Hsiang David, Seewoester, Thomas
• Granted Patent: US 8,911,964 B2
• Time in Patent Office: Days
• US Class Current: 435/69.6
• CPC Class Codes: C07K 16/241 Tumor Necrosis Factors
• CPC Class Codes: C07K 16/244 Interleukins [IL]
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C12N 2500/24 Iron; Fe chelators; Transferrin
• CPC Class Codes: C12N 2500/32 Amino acids
• CPC Class Codes: C12N 2500/34 Sugars
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2500/60 Buffer, e.g. pH regulation, osmotic pressure
• CPC Class Codes: C12N 2500/74 from fungi, e.g. yeasts
• CPC Class Codes: C12N 2500/76 from plants
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/33 Insulin together with transferrin C12N2500/25; Insulin-like growth factors C12N2501/105
• CPC Class Codes: C12N 5/0037 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 5/0043 Medium free of human- or animal-derived components

Abstract

The invention describes improved methods and compositions for producing a recombinant protein, e.g., an antibody, in mammalian cell culture. In addition, the invention provides improved cell culture media, including improved production media, feed solutions, and combination feeds, which may be used to improve protein productivity in mammalian cell culture.

Claims

1-20. -20. (canceled)

21. A fed-batch production method of making a human anti-TNFα

antibody which comprises (1) a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO;

1 and (2) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO;

2, said method comprising culturing Chinese Hamster Ovary (CHO) cells comprising a nucleic acid encoding said anti-TNFα

antibody in a cell culture production medium, wherein the glucose concentration in said medium is monitored and glucose is added to said medium if the glucose concentration in said medium decreases to below 2 g/L, or the glucose concentration in said medium is monitored and glucose is added to said medium to maintain the glucose concentration in said medium at a concentration of at least 2 g/L, such that said anti-TNFα

antibody is produced at a titer of at least 2 g/L in said cell culture production medium.

22. The method of claim 21, wherein said anti-TNFα

antibody is produced at a titer of at least 2.5 g/L in said cell culture production medium.

23. The method of claim 21, wherein said anti-TNFα

antibody is produced at a titer of at least 4 g/L in said cell culture production medium.

24. The method of claim 21, wherein said culturing of said CHO cells is performed in a large scale culture volume of greater than 10 L.

25. The method of claim 22, wherein said culture is performed in a large scale culture volume of greater than 10 L.

26. The method of claim 23, wherein said culture is performed in a large scale culture volume of greater than 10 L.

27. The method of claim 24, wherein said culturing lasts 9-15 days.

28. The method of claim 24, wherein said cell culture production medium is a serum-free medium and comprises at least one non-animal-based hydrolysate.

29. The method of claim 24, wherein said cell culture production medium is a biochemically defined cell culture medium.

30. The method of claim 29, wherein said biochemically defined cell culture medium comprises a recombinant growth factor, an osmolarity regulator, a pH buffer agent, glutamine, methotrexate, and a cell protectant.

31. The method of claim 29, wherein said biochemically defined cell culture medium comprises galactose and manganese.

32. The method of claim 21, wherein said CHO cells are cultured at a temperature ranging from 32°

C. to 38°

C.

33. The method of claim 32, wherein said temperature is maintained as a constant temperature throughout said production method, said temperature being selected from a temperature range of 32°

C. to 38°

C.

34. A fed-batch production method of making adalimumab, comprising culturing Chinese Hamster Ovary (CHO) cells comprising a nucleic acid encoding adalimumab in a cell culture production medium, wherein glucose concentration in said medium is monitored and glucose is added to said medium if the glucose concentration in said medium decreases to below 2 g/L, or the glucose concentration in said medium is monitored and glucose is added to said medium to maintain the glucose concentration in said medium at a concentration of at least 2 g/L, such that adalimumab is produced at a titer of at least 2 g/L in said cell culture production medium.

35. The method of claim 34, wherein adalimumab is produced at a titer of at least 2.5 g/L in said cell culture production medium.

36. The method of claim 34, wherein adalimumab is produced at a titer of at least 4 g/L in said cell culture production medium.

37. The method of claim 34, wherein said culturing of said CHO cells is performed in a large scale culture volume of greater than 10 L.

38. The method of claim 37, wherein said culturing lasts 9-15 days.

39. The method of claim 37, wherein during said culturing, said CHO cells reach a cell density of at least 2×

106 cells/ml in said cell culture production medium.

40. The method of claim 37, wherein said culture production medium is a serum-free medium and comprises at least one non-animal-based hydrolysate selected from a yeast-based hydrolysate, a soy-based hydrolysate, or a combination thereof.

41. The method of claim 37, wherein said cell culture production medium is a biochemically defined cell culture medium.

42. The method of claim 41, wherein said biochemically defined cell culture medium comprises a recombinant growth factor, an osmolarity regulator, a pH buffer agent, glutamine, methotrexate, and a cell protectant.

43. The method of claim 41, wherein said biochemically defined cell culture medium comprises galactose and manganese.

44. The method of claim 37, wherein said CHO cells are cultured at a temperature ranging from 32°

C. to 38°

C.

45. The method of claim 44, wherein said temperature is maintained as a constant temperature throughout said production method, said temperature being selected from a temperature range of 32°

C. to 38°

C.

46. The method of claim 35, wherein said culturing of said CHO cells is performed in a large scale culture volume of greater than 10 L.

47. The method of claim 46, wherein said culturing lasts 9-15 days, and wherein said culture production medium is a serum-free medium and comprises at least one non-animal-based hydrolysate, or said culture production medium is a biochemically defined cell culture medium.

48. An improved fed-batch large scale cell culture process for producing an anti-TNFα

antibody which comprises (1) a light chain variable region comprising the amino acid sequence of SEQ ID NO;

1 and (2) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO;

2, said method comprising culturing Chinese Hamster Ovary (CHO) cells comprising a nucleic acid encoding the anti-TNFα

antibody in a cell culture production medium at a temperature between 32°

C. to 38°

C. wherein glucose concentration in said medium is monitored and glucose is added to said medium if the glucose concentration in said medium decreases to below 2 g/L, or the glucose concentration in said medium is monitored and glucose is added to said medium to maintain the glucose concentration in said medium at a concentration of at least 2 g/L, such that said anti TNFα

-antibody is produced at a titer of at least 2 g/L in said cell culture production medium.

49. The method of claim 48, wherein the antibody is adalimumab and said improved batch process produces a greater than 50% increase in yield of said adalimumab.

50. The method of claim 48, wherein said improved batch process produces a 50% increase in the titer at 50% viability as compared to an adalimumab production process that is conducted at variable production method temperatures.