Method of producing recombinant high molecular weight vWF in cell culture

Patent metadata

Field	Value
Publication number	US-10100099-B2
Application number	US-201715591022-A
Country	US
Kind code	B2
Filing date	May 9, 2017
Priority date	Jul 8, 2010
Publication date	Oct 16, 2018
Grant date	Oct 16, 2018

Abstract

Among other aspects, the present invention relates to cell culture conditions for producing high molecular weight vWF, in particular, highly multimericWF with a high specific activity and ADAMTS13 with a high specific activity. The cell culture conditions of the present invention can include, for example, a cell culture medium with an increased copper concentration and/or cell culture supernatant with a low ammonium (NH 4 + ) concentration. The present invention also provides methods for cultivating cells in the cell culture conditions to express high molecular weight vWF and rA13 having high specific activities.

First claim

What is claimed is:

1. A method for producing a recombinant ADAMTS13 (rA13) composition, the method comprising:
culturing one or more mammalian cells comprising a nucleic acid encoding a rA13 protein in a cell culture media containing from 1 μg/L to 6 μg/L copper, such that rA13 is expressed and excreted from the mammalian cells into a culture supernatant; and
recovering at least a portion of the culture supernatant,
wherein the cell density is maintained at less than 4.0×10 6 cells per mL during the step of culturing the one or more mammalian cells; and
wherein at least 1500 Units FRETS-VWF73 activity per liter cell culture media per day is present in the recovered culture supernatant.

2. The method of claim 1 , wherein the cell culture media is a protein free culture media.

3. The method of claim 1 , wherein the cell culture media is a chemically defined culture media.

4. The method of claim 1 , wherein the cell culture media contains from 2.0 μg/L to 4.0 μg/L copper.

5. The method of claim 1 , wherein the cell culture media contains 4 μg/L copper.

6. The method of claim 1 , wherein the cell culture media contains 2 μg/L copper.

7. The method of claim 1 , wherein the mammalian cells are CHO cells.

8. The method of claim 4 , wherein the mammalian cells are CHO cells.

9. The method of claim 5 , wherein the mammalian cells are CHO cells.

10. The method of claim 6 , wherein the mammalian cells are CHO cells.

11. The method of claim 1 , wherein culturing the one or more mammalian cells comprises continuous cultivation of the mammalian cells.

12. The method of claim 11 , wherein the continuous cultivation of mammalian cells is performed in chemostatic mode.

13. The method of claim 11 , wherein the continuous cultivation of mammalian cells is performed in perfusion mode.

14. The method of claim 1 , wherein the one or more mammalian cells is cultured in at least 100 L of the cell culture media.

15. The method of claim 1 , wherein the cell density is maintained at between 2.0×10 6 cells per mL and 4.0×10 6 per mL during the step of culturing the one or more mammalian cells.

16. The method of claim 4 , wherein the cell density is maintained at between 2.0×10 6 cells per mL and 4.0×10 6 per mL during the step of culturing the one or more mammalian cells.

17. The method of claim 1 , wherein the cell culture media contains from 5 mg/L to 10 mg/L nicotinamide.

18. The method of claim 4 , wherein the cell culture media contains from 5 mg/L to 10 mg/L nicotinamide.

19. The method of claim 15 , wherein the cell culture media contains from 5 mg/L to 10 mg/L nicotinamide.

20. The method of claim 1 , wherein the cell culture media contains about 7 mg/L nicotinamide.

21. The method of claim 4 , wherein the cell culture media contains about 7 mg/L nicotinamide.

22. The method of claim 5 , wherein the cell culture media contains about 7 mg/L nicotinamide.

23. The method of claim 6 , wherein the cell culture media contains about 7 mg/L nicotinamide.

Assignees

• Baxalta Inc
• Baxalta GmbH

Inventors

• Grillberger Leopold
• Reiter Manfred
• Fleischanderl Daniel
• Bramberger Gregor

Classifications

• C12Y304/24087

  ADAMTS13 endopeptidase (3.4.24.87) · CPC title

• C12P21/00

  Preparation of peptides or proteins (single cell protein C12N1/00) · CPC title

• C12N2511/00

  Cells for large scale production · CPC title

• C12N2501/998

  Proteins not provided for elsewhere · CPC title

• C12N2500/24

  Iron; Fe chelators; Transferrin · CPC title