Cell culture process
A method for producing recombinant polypeptides, including antibodies, having targeted levels of carboxyl terminal (C-terminal) lysine or arginine residues is presented. The method involves culturing cells expressing said polypeptides in cell culture medium having sufficient levels of lysine, arginine, and/or agents that change intracellular pH. Culturing the cells under such conditions does not affect cell growth, cell viability, or titer.
1. A method of manufacturing a preparation of a recombinant antibody, comprising:
culturing a cell in a medium comprising 2 g/L lysine to 8 g/L lysine under conditions in which the cell expresses a recombinant antibody;
isolating the recombinant antibody, thereby producing a preparation of the recombinant antibody; and
formulating the preparation into a drug product if the preparation meets a target value of C-terminal variants of the recombinant antibody, wherein the C-terminal variants differ in amino acid sequence only by the presence or absence of a lysine at their carboxyl termini.
2. The method of claim 1 , further comprising providing the target value of C-terminal variants of the recombinant antibody.
3. The method of claim 1 , wherein the medium comprises 3 g/L lysine to 7 g/L lysine.
4. The method of claim 1 , wherein the medium comprises 4 g/L lysine to 6 g/L lysine.
5. The method of claim 1 , wherein the medium comprises 5 g/L lysine.
6. The method of claim 1 , wherein the method further comprises measuring a level of one or more C-terminal variants of the recombinant antibody in the preparation.
7. The method of claim 1 , wherein the C-terminal variants of the recombinant antibody comprise one or more of a K1 lysine variant of the recombinant antibody and a K2 lysine variant of the recombinant antibody.
8. The method of claim 7 , wherein the level of one or more of K1 lysine variant and K2 lysine variant in the preparation is increased relative to a preparation of the recombinant antibody produced using a medium not comprising 2 g/L lysine to 8 g/L lysine.
9. The method of claim 7 , wherein the method further comprises providing a target value of one or more of K1 lysine variants of the recombinant antibody and K2 lysine variants of the recombinant antibody.
10. The method of claim 9 , wherein the target value of K1 lysine variants of the recombinant antibody is 12% to 25% of the recombinant antibody in the preparation.
11. The method of claim 9 , wherein the target value of K1 lysine variants of the recombinant antibody is 15% to 20% of the recombinant antibody in the preparation.
12. The method of claim 9 , wherein the target value of K2 lysine variants of the recombinant antibody is 2% to 6% of the recombinant antibody in the preparation.
13. The method of claim 9 , wherein the target value of K2 lysine variants of the recombinant antibody is 3% to 5% of the recombinant antibody in the preparation.
14. The method of claim 1 , wherein the cell is a CHO cell.
15. The method of claim 1 , wherein the antibody is adalimumab.
16. The method of claim 1 , wherein the medium has a pH of 6.8 to 7.0.
17. The method of claim 1 , wherein the medium has a pH of 6.9.
18. The method of claim 1 , wherein the cell is cultured at a temperature of 34° C. to 37° C.
19. The method of claim 1 , wherein the cell is cultured at a temperature of 36° C.
20. A method of manufacturing a preparation of a recombinant antibody, comprising:
culturing a cell in a medium comprising 2 g/L arginine to 8 g/L arginine under conditions in which the cell expresses a recombinant antibody;
isolating the recombinant antibody, thereby producing a preparation of the recombinant antibody; and
formulating the preparation into a drug product if the preparation meets a target value of C-terminal variants of the recombinant antibody, wherein the C-terminal variants differ in amino acid sequence only by the presence or absence of a lysine at their carboxyl termini.
21. The method of claim 20 , further comprising providing the target value of C-terminal variants of the recombinant antibody.
22. The method of claim 20 , wherein the medium comprises 3 g/L arginine to 7 g/L arginine.
23. The method of claim 20 , wherein the medium comprises 4 g/L arginine to 6 g/L arginine.
24. The method of claim 20 , wherein the medium comprises 5 g/L arginine.
25. The method of claim 20 , wherein the method further comprises measuring a level of one or more C-terminal variants of the recombinant antibody in the preparation.
26. The method of claim 20 , wherein the C-terminal variants of the recombinant antibody comprise one or more of a K1 lysine variant of the recombinant antibody and a K2 lysine variant of the recombinant antibody.
27. The method of claim 26 , wherein the level of one or more of K1 lysine variant and K2 lysine variant in the preparation is increased relative to a preparation of the recombinant antibody produced using a medium not comprising 2 g/L arginine to 8 g/L arginine.
28. The method of claim 26 , wherein the method further comprises providing a target value of one or more of K1 lysine variants of the recombinant antibody and K2 lysine variants of the recombinant antibody.
29. The method of claim 28 , wherein the target value of K1 lysine variants of the recombinant antibody is 12% to 25% of the recombinant antibody in the preparation.
30. The method of claim 28 , wherein the target value of K1 lysine variants of the recombinant antibody is 15% to 20% of the recombinant antibody in the preparation.
31. The method of claim 28 , wherein the target value of K2 lysine variants of the recombinant antibody is 2% to 6% of the recombinant antibody in the preparation.
32. The method of claim 28 , wherein the target value of K2 lysine variants of the recombinant antibody is 3% to 5% of the recombinant antibody in the preparation.
33. The method of claim 20 , wherein the cell is a CHO cell.
34. The method of claim 20 , wherein the antibody is adalimumab.
35. The method of claim 20 , wherein the medium has a pH of 6.8 to 7.0.
36. The method of claim 20 , wherein the medium has a pH of 6.9.
37. The method of claim 20 , wherein the cell is cultured at a temperature of 34° C. to 37° C.
38. The method of claim 20 , wherein the cell is cultured at a temperature of 36° C.