Cell culture processes
View Patent ↗Culturing heterologous protein-secreting mammalian cells, such as CHO or BHK cells, at 35.1-36.5° C. and/or at pH 7.15-7.20 and/or at a dissolved CO 2 concentration of 10% or lower. Preferred heterologous proteins are Factor VIII, ADAMTS-13, furin or Factor VII.
1. A method comprising:
expressing heterologous Factor VIII protein in a mammalian cell in continuous cell culture wherein the continuous cell culture is maintained at a temperature of 36.0±0.5° C. and a cell culture density of from 1.4×10 6 to 2.6×10 6 cells/ml, and wherein the mammalian cell is a CHO cell.
2. The method of claim 1 , wherein the temperature of the continuous cell culture is maintained at 36±0.2° C.
3. The method of claim 1 , wherein the temperature of the continuous cell culture is maintained at 36° C.
4. The method of claim 1 , wherein the temperature of the continuous cell culture is maintained at 36.5° C.
5. The method of claim 1 , wherein the continuous cell culture is maintained at a pH of from 7.10 to 7.25.
6. The method of claim 5 , wherein the pH of the continuous cell culture is maintained at 7.20±0.05.
7. The method of claim 5 , wherein the pH of the continuous cell culture is maintained at 7.20±0.03.
8. The method of claim 5 , wherein the pH of the continuous cell culture is maintained at 7.20±0.01.
9. The method of claim 5 , wherein the pH of the continuous cell culture is maintained at 7.20.
10. The method of claim 5 , wherein the pH of the continuous cell culture is maintained at 7.15±0.05.
11. The method of claim 5 , wherein the pH of the continuous cell culture is maintained at 7.15±0.03.
12. The method of claim 5 , wherein the pH of the continuous cell culture is maintained at 7.15±0.01.
13. The method of claim 5 , wherein the pH of the continuous cell culture is maintained at 7.15.
14. The method of claim 1 , wherein the continuous cell culture is maintained at a dissolved CO 2 concentration of from 1% to 10%.
15. The method of claim 14 , wherein the CO 2 concentration of the continuous cell culture is maintained at from 4.0% to 9.0%.
16. The method of claim 14 , wherein the CO 2 concentration of the continuous cell culture is maintained at from 5.5% to 8.5%.
17. The method of claim 14 , wherein the continuous cell culture is maintained at a pH of from 7.10 to 7.25.
18. The method of claim 17 , wherein the cell culture supernatant of the continuous cell culture is buffered with bicarbonate.
19. The method of claim 14 , wherein the dissolved CO 2 concentration of the continuous cell culture is maintained by sparging the continuous mammalian cell culture with air.
20. The method of claim 1 , wherein the cell density of the continuous cell culture is maintained at from 1.6×10 6 to 2.6×10 6 cells/ml.
21. The method of claim 20 , wherein the cell density of the continuous cell culture is maintained at from 1.8×10 6 to 2.4×10 6 cells/ml.
22. The method of claim 1 , wherein the Factor VIII is coexpressed with von Willebrand Factor.
23. The method of any one of claims 1 , 5 , or 14 , wherein the cell culture supernatant of the continuous cell culture comprises copper (Cu 2+ ) at a concentration of at least 5 ppb.
24. The method of claim 23 , wherein the Cu 2+ concentration of the cell culture supernatant is at least 7 ppb.
25. The method of claim 1 , wherein the heterologous protein is FVIII, and the cell culture density of the continuous cell culture is maintained by:
measuring the cell culture density of the continuous cell culture using an in-line sensor, and
automatically controlling the influx of fresh medium into the continuous cell culture to maintain the cell culture density of from 1.4×10 6 to 2.6×10 6 cells/ml.
26. The method of claim 1 , wherein the continuous cell culture is operated as a chemostat or a turbidostat continuous cell culture.