Cell culture processes

Patent metadata

Field	Value
Publication number	US-9359629-B2
Application number	US-34180708-A
Country	US
Kind code	B2
Filing date	Dec 22, 2008
Priority date	Dec 27, 2007
Publication date	Jun 7, 2016
Grant date	Jun 7, 2016

Abstract

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.

First claim

The invention claimed is:

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.

Assignees

• Giovagnoli Andre
• Roy Sylvain
• Ducros Veronique
• Charlot Virginie
• Stauffer Yves-Olivier
• Baxalta Inc
• Baxalta GmbH

Inventors

• Giovagnoli Andre
• Roy Sylvain
• Ducros Veronique
• Charlot Virginie
• Stauffer Yves-Olivier

Classifications

• C12N5/06 Primary

  Animal cells or tissues; Human cells or tissues (preservation of excised living parts A01N1/10) · CPC title

• C12N9/6454

  Dibasic site splicing serine proteases, e.g. kexin (3.4.21.61); furin (3.4.21.75) and other proprotein convertases · CPC title

• C12P21/02 Primary

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C12N9/6437 Primary

  Coagulation factor VIIa (3.4.21.21) · CPC title

• C12Y304/21021

  Coagulation factor VIIa (3.4.21.21) · CPC title