Methods of producing anti-TNF-alpha antibodies in mammalian cell culture
View Patent ↗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.
1. A method of producing an anti-TNFα antibody in a mammalian cell culture, said method comprising:
a) culturing Chinese Hamster Ovary (CHO) cells comprising a nucleic acid encoding the antibody, or a fragment thereof, in a cell culture growth medium to form the mammalian cell culture; and
b) culturing the CHO cells in a cell culture production medium, wherein glucose is added to the mammalian cell culture to maintain a glucose concentration of at least 2 g/L,
such that the anti-TNFα antibody is produced,
wherein the anti-TNFα antibody comprises a light chain variable region (LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO:3, a CDR2 domain comprising the amino acid sequence of SEQ ID NO:5, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, and comprises a heavy chain variable region (HCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO:4, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO:8.
2. The method of claim 1 , wherein the CHO cells are cultured in a cell culture growth medium at a temperature ranging from 32° to 38° C.
3. The method of claim 1 , wherein the CHO cells are cultured in a cell production medium at a temperature ranging from 32° to 38° C.
4. The method of claim 1 , wherein the CHO cells are cultured in a cell growth phase at a temperature of about 35° C.
5. The method of claim 1 , wherein the cell culture growth media comprises methotrexate.
6. The method of claim 1 , wherein the cell culture production media comprises methotrexate.
7. The method of claim 1 , wherein the cell culture growth media comprises at least one hydrolysate.
8. The method of claim 1 , wherein the cell culture production media comprises at least one hydrolysate.
9. The method of claim 1 , wherein the culturing of the CHO cells is performed in a large-scale cell culture.
10. The method of claim 1 , wherein the mammalian cell culture further comprises a cell protectant.
11. The method of claim 1 , wherein the glucose concentration is maintained from 2 g/L to 5 g/L.
12. A method of producing an anti-TNFα antibody in a mammalian cell culture, said method comprising:
a) culturing Chinese Hamster Ovary (CHO) cells comprising a nucleic acid encoding the antibody, or a fragment thereof, in a cell culture growth medium to form the mammalian cell culture; and
b) culturing the CHO cells in a cell culture production medium, wherein glucose is added to the mammalian cell culture to maintain a glucose concentration of at least 2 g/L,
such that the anti-TNFα antibody is produced,
wherein the anti-TNFα antibody comprises a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:1, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2.
13. The method of claim 12 , wherein the CHO cells are cultured in a cell culture growth medium at a temperature ranging from 32° C. to 38° C.
14. The method of claim 12 , wherein the CHO cells are cultured in a cell production medium at a temperature ranging from 32° to 38° C.
15. The method of claim 12 , wherein the CHO cells are cultured in a cell growth phase at a temperature of about 35° C.
16. The method of claim 12 , wherein the cell culture growth media comprises methotrexate.
17. The method of claim 12 , wherein the cell culture production media comprises methotrexate.
18. The method of claim 12 , wherein the cell culture growth media comprises at least one hydrolysate.
19. The method of claim 12 , wherein the cell culture production media comprises at least one hydrolysate.
20. The method of claim 12 , wherein the culturing of the CHO cells is performed in a large-scale cell culture.
21. The method of claim 12 , wherein the mammalian cell culture further comprises a cell protectant.
22. The method of claim 12 , wherein the glucose concentration is maintained from 2 g/L to 5 g/L.
23. A method of producing adalimumab in a mammalian cell culture, said method comprising:
a) culturing Chinese Hamster Ovary (CHO) cells comprising a nucleic acid encoding adalimumab, or a fragment thereof, in a cell culture growth medium to form the mammalian cell culture; and
b) culturing the CHO cells in a cell culture production medium, wherein glucose is added to the mammalian cell culture to maintain a glucose concentration of at least 2 g/L,
such that adalimumab is produced.
24. The method of claim 23 , wherein the CHO cells are cultured in a cell culture growth medium at a temperature ranging from 32° C. to 38° C.
25. The method of claim 23 , wherein the CHO cells are cultured in a cell production medium at a temperature ranging from 32° to 38° C.
26. The method of claim 23 , wherein the cell growth phase is at a temperature of about 35° C.
27. The method of claim 23 , wherein the cell culture growth media comprises methotrexate.
28. The method of claim 23 , wherein the cell culture production media comprises methotrexate.
29. The method of claim 23 , wherein the cell culture growth media comprises at least one hydrolysate.
30. The method of claim 23 , wherein the cell culture production media comprises at least one hydrolysate.
31. The method of claim 23 , wherein the culturing of the CHO cells is performed in a large-scale cell culture.
32. The method of claim 23 , wherein the mammalian cell culture further comprises a cell protectant.
33. The method of claim 23 , wherein the glucose concentration is maintained from 2 g/L to 5 g/L.
34. A method of producing an anti-TNFα antibody, or an antigen-binding fragment thereof, in a mammalian cell culture, said method comprising culturing Chinese Hamster Ovary (CHO) cells expressing the anti-TNFα antibody, or the antigen-binding fragment thereof, in a cell culture production medium, wherein glucose is added to the cell culture production medium to maintain a glucose concentration of at least 2 g/L, such that the anti-TNFα antibody, or the antigen-binding fragment thereof, is produced,
wherein the anti-TNFα antibody, or the antigen-binding fragment thereof, comprises a light chain variable region (LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, and comprises a heavy chain variable region (HCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 8.
35. The method of claim 34 , wherein the glucose concentration is maintained from 2 g/L to 5 g/L.
36. The method of claim 34 , wherein the anti-TNFα antibody, or antigen-binding fragment thereof, comprises a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:1, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2.
37. The method of claim 36 , wherein the glucose concentration is maintained from 2 g/L to 5 g/L.
38. The method of claim 34 , wherein the anti-TNFα antibody is adalimumab, or an antigen-binding fragment thereof.
39. The method of claim 38 , wherein the glucose concentration is maintained from 2 g/L to 5 g/L.