Serum-free cell culture medium

Patent metadata

Field	Value
Publication number	US-11332771-B2
Application number	US-202016742695-A
Country	US
Kind code	B2
Filing date	Jan 14, 2020
Priority date	Mar 15, 2013
Publication date	May 17, 2022
Grant date	May 17, 2022

Abstract

The specification describes an improved serum-free animal cell culture medium, which can used for the production of a protein of interest. Ornithine, or a combination of ornithine and putrescine can be added to serum-free media or chemically defined media to improve viable cell density, to reduce cell doubling time, and to increase the production of a protein of interest.

First claim

What is claimed is:

1. A method for producing aflibercept comprising:
(a) culturing a CHO cell expressing aflibercept in a cell culture medium comprising 0.6±0.09 mM ornithine and 0.714±0.11 mM putrescine, wherein said medium is serum-free and hydrolysate free; and
(b) expressing aflibercept in the cell, wherein the aflibercept is secreted into the cell culture medium;
wherein the aflibercept is produced at an average 7-day titer that is at least 7% greater than the average 7-day titer produced by a CHO cell expressing aflibercept in a cell culture medium that contains no more than 2.5 μM putrescine and no ornithine.

2. The method of claim 1 , wherein the aflibercept is produced at an average 7-day titer that is at least 14% greater than the average 7-day titer produced by a CHO cell expressing aflibercept in a cell culture medium that contains no more than 2.5 μM putrescine and no ornithine.

3. The method of claim 1 , wherein the aflibercept is produced at an average 7-day titer that is at least 80% greater than the average 7-day titer produced by a CHO cell expressing aflibercept in a cell culture medium that contains no more than 2.5 μM putrescine and no ornithine.

4. The method of claim 1 , wherein the aflibercept is produced at an average 7-day titer that is at least 2-fold greater than the average 7-day titer produced by a CHO cell expressing aflibercept in a cell culture medium that contains no more than 2.5 putrescine and no ornithine.

5. The method of claim 1 , wherein the aflibercept is produced at an average 7-day titer that is at least 3-fold greater than the average 7-day titer produced by a CHO cell expressing aflibercept in a cell culture medium that contains no more than 2.5 putrescine and no ornithine.

6. The method of claim 1 , wherein the CHO cell is a CHO cell derivative.

7. The method of claim 1 , wherein the CHO cell is a CHO-K1 cell.

8. The method of claim 1 , wherein the CHO cell is a CHO DUX B-11 cell, a Veggie-CHO cell, a GS-CHO cell, an S-CHO cell, or a CHO lec mutant cell.

9. The method of claim 1 , wherein the cell culture medium is chemically defined.

10. The method of claim 1 , wherein the cell culture medium comprises a mixture of amino acids or salts thereof.

11. The method of claim 10 , wherein the mixture of amino acids or salts thereof are at a concentration of ≥40 mM±6 mM in the cell culture medium.

12. The method of claim 11 , wherein the mixture of amino acids or salts thereof comprises one or more of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine.

13. The method of claim 11 , wherein the mixture of amino acids or salts thereof comprises alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

14. The method of claim 1 , wherein the cell culture medium comprises one or more fatty acids.

15. The method of claim 14 , wherein the one or more fatty acids are selected from the group consisting of linoleic acid, thioctic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, arachidonic acid, lauric acid, behenic acid, decanoic acid, dodecanoic acid, hexanoic acid, lignoceric acid, myristic acid and octanoic acid.

16. The method of claim 1 , wherein the cell culture medium comprises a mixture of nucleosides.

17. The method of claim 16 , wherein the mixture of nucleosides comprises one or more of adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine.

18. The method of claim 16 , wherein the mixture of nucleosides comprises adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine.

19. The method of claim 1 , wherein the cell culture medium comprises one or more divalent cations.

20. The method of claim 19 , wherein the divalent cation is Ca 2+ , Mg 2+ , or both.

21. The method of claim 1 , further comprising adding one or more point-of-use additions to the cell culture medium.

22. The method of claim 21 , wherein said one or more point-of-use additions are selected from the group consisting of NaHCO 3 , glutamine, insulin, glucose, CuSO 4 , ZnSO 4 , FeCl 3 , NiSO 4 , Na 4 EDTA, Na 3 Citrate and combinations thereof.

23. The method of claim 21 , wherein each of NaHCO 3 , glutamine, insulin, glucose, CuSO 4 , ZnSO 4 , FeCl 3 , NiSO 4 , Na 4 EDTA, and Na 3 Citrate are added to the medium as point-of-use additions.

24. The method of claim 21 , wherein said one or more point-of-use additions are selected from the group consisting of CuSO 4 , ZnSO 4 , FeCl 3 , NiSO 4 , and combinations thereof.

25. The method of claim 21 , wherein said one or more point-of-use additions comprises glutamine.

26. The method of claim 21 , wherein said one or more point-of-use additions comprises insulin.

27. The method of claim 21 , wherein said one or more point-of-use additions comprises glucose.

28. The method of claim 21 , wherein said one or more point-of-use additions are selected from the group consisting of Na 4 EDTA, Na 3 Citrate and a combination thereof.

29. The method of claim 21 , wherein said one or more point-of-use additions comprises NaHCO 3 .

Assignees

• Regeneron Pharma

Inventors

• Oshodi Shadia Abike
• Johnson Amy S
• Lawrence Shawn M

Classifications

• C12N2500/34

  Sugars · CPC title

• C12N2500/24

  Iron; Fe chelators; Transferrin · CPC title

• C12N2500/22

  Zinc; Zn chelators · CPC title

• C12N5/0682

  Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells (oocytes C12N5/0609) · CPC title

• C12N5/0037 Primary

  Serum-free medium, which may still contain naturally-sourced components · CPC title