Taurine supplemented cell culture medium and methods of use

Patent metadata

Field	Value
Publication number	US-12359165-B2
Application number	US-202418672959-A
Country	US
Kind code	B2
Filing date	May 23, 2024
Priority date	Aug 4, 2015
Publication date	Jul 15, 2025
Grant date	Jul 15, 2025

Abstract

The specification describes a composition comprising an improved eukaryotic cell culture medium, which can be used for the production of a protein of interest. TaXULne can be added to the serum-free media or chemically-defined media to increase the production of a protein of interest. Methods for recombinantly expressing high levels of protein using the media compositions are included.

First claim

What is claimed is:

1. A method for culturing recombinant eukaryotic cells for improved production of a recombinant protein of interest, comprising the steps of:
(a) propagating recombinant eukaryotic cells in a defined cell culture medium during a growth phase, wherein said cell culture medium is free of glutamine;
(b) supplementing the defined cell culture medium with about 0.1 mM to about 10 mM L-taurine and one or more of the following: 0 mM to 11.2 mM alanine, 2.4 mM to 11.9 mM arginine, 1.3 mM to 33.3 mM asparagine, 1.5 mM to 93.9 mM aspartic acid, 1.1 mM to 19.9 mM cysteine, 1.4 mM to 47.6 mM glutamic acid, 0 mM to 16.7 mM glycine, 1 mM to 9.5 mM histidine, 1.5 mM to 22.9 mM isoleucine, 1.5 mM to 38.1 mM leucine, 2.7 mM to 24.6 mM lysine, 1.3 mM to 13.4 mM methionine, 1.2 mM to 18.2 mM phenylalanine, 1.7 mM to 26.1 mM proline, 1.9 mM to 57.1 mM serine, 1.7 mM to 33.6 mM threonine, 0.5 mM to 14.7 mM tryptophan, 0.9 mM to 22.2 mM tyrosine, and 1.7 mM to 34.1 mM valine; and
(c) expressing the recombinant protein of interest during a production phase,
wherein the addition of L-taurine increases the titer of the recombinant protein of interest by at least 3% when compared to cells expressing the recombinant protein of interest in a cell culture medium containing less than 0.1 mM L-taurine.

2. The method of claim 1 , wherein said cell culture medium is serum-free.

3. The method of claim 1 , wherein a total amount of amino acids or amino acid salts with which the defined cell culture medium is supplemented does not exceed 115 mM.

4. The method of claim 1 , further comprising the step of supplementing said cell culture medium with ≤16 g/L hydrolysate.

5. The method of claim 1 , further comprising the step of supplementing said cell culture medium with fatty acids.

6. The method of claim 1 , further comprising the step of supplementing said cell culture medium with nucleosides including one or more of the following: adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine.

7. The method of claim 1 , further comprising the step of supplementing said cell culture medium with salts of calcium, magnesium, and phosphate.

8. The method of claim 1 , wherein the taurine supplement is further provided at least three additional times during the production phase.

9. The method of claim 1 , wherein the taurine supplement is provided on each day for the duration of the production phase.

10. The method of claim 1 , wherein the recombinant protein of interest is selected from the group consisting of an Fc-fusion protein, a receptor-Fc-fusion protein, an antibody, an antibody fragment, and a ScFv-Fc fusion protein.

11. The method of claim 10 , wherein the recombinant protein of interest is an anti-vascular endothelial growth factor (VEGF) antibody or an anti-VEGF receptor-Fc-fusion protein.

12. The method of claim 1 , wherein the recombinant protein of interest is aflibercept.

13. The method of claim 1 , wherein the defined cell culture medium is supplemented with about 0.09 mM to about 0.9 mM ornithine.

14. The method of claim 1 , wherein the eukaryotic cells are CHO cells.

15. The method of claim 1 , wherein said cells are maintained at a first temperature of 35° C. to 38° C. during said growth phase and a second temperature of 29° C. to 37° C. during said production phase, wherein the first temperature is higher than the second temperature.

16. The method of claim 1 , wherein the addition of L-taurine reduces the amount of ammonia byproduct when compared to cells expressing the recombinant protein of interest in a cell culture medium containing less than 0.1 mM L-taurine.

17. A method for culturing recombinant eukaryotic cells for improved production of a recombinant protein of interest, comprising the steps of:
(a) propagating cells in a defined cell culture medium during a growth phase, and
(b) supplementing the defined cell culture medium with about 0.1 mM to about 10 mM L-taurine and expressing the recombinant protein of interest during a production phase, wherein the addition of L-taurine increases the titer of the recombinant protein of interest by at least 3% when compared to cells expressing the recombinant protein of interest in a cell culture medium containing less than 0.1 mM L-taurine, and wherein said propagating cells during said growth phase is performed at a temperature of about 35° C. to 38° C., and said expressing a recombinant protein of interest during said production phase is performed at a temperature of about 29° C. to 37° C., wherein the temperature of said growth phase is higher than the temperature of said production phase.

18. The method of claim 17 , wherein the L-taurine is supplemented from about 1 to about 5 times during the production phase.

19. The method of claim 17 , wherein the L-taurine is supplemented on each day for the duration of the production phase.

20. The method of claim 17 , further comprising supplementing the defined cell culture medium with about 0.1 mM to about 10 mM L-taurine during the growth phase.

21. The method of claim 17 , wherein the defined cell culture medium is supplemented with about 0.09 mM to about 0.9 mM ornithine.

22. The method of claim 17 , wherein the cells are CHO cells.

23. The method of claim 17 , wherein the recombinant protein of interest is an antigen-binding protein.

24. The method of claim 17 , wherein the recombinant protein of interest comprises an Fc domain.

25. The method of claim 17 , wherein the recombinant protein of interest is selected from the group consisting of an Fc-fusion protein, a receptor-Fc-fusion protein, an antibody, an antibody fragment, and a ScFv-Fc fusion protein.

26. The method of claim 25 , wherein the recombinant protein of interest is an anti-vascular endothelial growth factor (VEGF) antibody or an anti-VEGF receptor-Fc-fusion protein.

27. The method of claim 17 , wherein the recombinant protein of interest is aflibercept.

28. The method of claim 17 , wherein the cells are capable of about 7% or greater increase in titer of the recombinant protein of interest compared to cells expressing the recombinant protein of interest in a cell culture medium containing less than 0.1 mM L-taurine.

29. The method of claim 17 , wherein the method is capable of increasing the recombinant protein of interest titer by at least 0.1 g/L, at least 0.5 g/L, at least 1 g/L, at least 1.2 g/L, at least 1.4 g/L, at least 1.6 g/L, at least 1.8 g/L, at least 2 g/L, at least 2.2 g/L, at least 2.4 g/L, or at least 2.5 g/L compared to a similar method in a cell culture medium that contains less than 0.1 mM L-taurine.

30. The method of claim 17 , wherein the cells are cultured in the L-taurine-supplemented defined cell culture medium of (b) for at least 6 days and express a higher titer of the recombinant protein of interest compared to cells expressing the protein of interest cultured in the defined cell culture medium for at least 6 days in the absence of L-taurine.

Assignees

• Regeneron Pharma

Inventors

• Johnson Amy S
• Casey Meghan E
• Oshodi Shadia
• Lawrence Shawn

Classifications

• C12N15/85

  for animal cells · CPC title

• C12P21/00

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

• C12N2500/90

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

• C12N2500/46

  Amines, e.g. putrescine · CPC title

• C12N2510/02

  Cells for production · CPC title