Taurine supplemented cell culture medium and methods of use

Patent metadata

Field	Value
Publication number	US-11312936-B2
Application number	US-201615749037-A
Country	US
Kind code	B2
Filing date	Aug 3, 2016
Priority date	Aug 4, 2015
Publication date	Apr 26, 2022
Grant date	Apr 26, 2022

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. Taurine can be added to 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 or maintaining the cells in a defined cell culture medium during a growth phase, and (b) supplementing the defined cell culture medium with about 0.09 mM to about 0.9 mM ornithine and about 0.1 mM to about 10 mM L-taurine, and expressing the recombinant protein of interest during 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 the L-taurine of (b) is provided from about 1 to about 5 times during production phase.

3. The method of claim 1 , wherein the L-taurine of (b) is provided on each day for the duration of the production phase.

4. The method of claim 1 , further comprising supplementing the defined cell culture medium with about 0.1 mM to about 10 mM L-taurine during the growth phase of (a).

5. The method of claim 1 , wherein the eukaryotic cell is selected from the group consisting of a mammalian cell, avian cell, insect cell, and yeast cell.

6. The method of claim 5 , wherein the cell is selected from the group consisting of a retinal cell, kidney cell lymphocyte epidermal cell, stem cell, tumor cell, and a cell line derived from an aforementioned cell.

7. The method of claim 5 , wherein the cell is a CHO, COS, Vero, CV-1, HEK293, MCDK, HaK, BHK2, HeLa, HepG2, WI38, MRC 5, Colo25, HB 8065, HL-60, Jurkat, Daudi, A431, U937, 3T3, L cell, C127, SP2/0, NS-0, or MMT cell.

8. The method of claim 1 , wherein the recombinant protein of interest is an antigen binding protein.

9. The method of claim 1 , wherein the recombinant protein of interest comprises an Fc domain.

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 selected from the group consisting of an anti-Programmed Cell Death 1 (PD1) antibody, an anti-Programmed Cell Death Ligand-1 (PDL-1) antibody, an anti-Angiopoetin-2 (ANG2) antibody, an anti-Angiopoetin-Like 3 (AngPtl3) antibody, an anti-platelet derived growth factor receptor (PDGFR) antibody, an anti-Prolactin Receptor (PRLR) antibody, an anti-tumor necrosis factor (TNF) antibody, an anti-epidermal growth factor receptor (EGFR) antibody, an anti-Proprotein Convertase Subtilisin Kexin-9 (PCSK9) antibody, an anti-Growth And Differentiation Factor-8 (GDF8) antibody, an anti-Glucagon Receptor (GCGR) antibody, an anti-vascular endothelial growth factor (VEGF) antibody, an anti-interleukin 1 receptor (IL1R) antibody, an anti-interleukin 4 receptor (IL4R) antibody, an anti-interleukin 6 receptor (IL6R) antibody, an anti-interleukin 1 (IL1) antibody, an anti-interleukin 2 (IL2) antibody, an anti-interleukin 3 (IL3) antibody, an anti-interleukin 4 (IL4) antibody, an anti-interleukin 5 (IL5) antibody, an anti-interleukin 6 (IL6) antibody, an anti-interleukin 7 (IL7) antibody, an anti-Respiratory syncytial virus (RSV) antibody, an anti-nerve growth factor (NGF) antibody, an anti-Cluster of differentiation 3 (CD3) antibody, an anti-Cluster of differentiation 20 (CD20) antibody, an anti-Cluster of differentiation 19 (CD19) antibody, an anti-Cluster of differentiation 28 (CD28) antibody, an anti-Cluster of differentiation 48 (CD48) antibody, an anti-CD3/anti-CD20 bispecific antibody, an anti-CD3/anti-Mucin 16 (MUC16) bispecific antibody, and an anti-CD3/anti-Prostate-specific membrane antigen (PSMA) bispecific antibody.

12. The method of claim 10 , wherein the recombinant protein of interest is selected from the group consisting of alirocumab, sarilumab, fasinumab, nesvacumab, dupilumab, trevogrumab, evinacumab, and rinucumab.

13. A method for producing a recombinant protein of interest comprising the steps of:
(a) introducing into a cell or cells a nucleic acid comprising a nucleotide sequence encoding the recombinant protein of interest thereby generating cells expressing the recombinant protein of interest;
(b) isolating the cell(s) expressing the recombinant protein of interest;
(c) culturing the selected cell(s) in a cell culture medium comprising about 0.09 mM to about 0.9 mM ornithine and about 0.1 mM to about 10 mM L-taurine, 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
(d) producing the recombinant protein of interest in the cell, wherein the protein of interest is secreted into the medium.

14. The method of claim 13 , wherein the cells are capable of about 8% or greater increase in yield 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.

15. The method of claim 13 , wherein the cell is a CHO cell, HEK293 cell or BHK cell.

16. The method of claim 13 , wherein the recombinant protein of interest is an antigen-binding protein.

17. The method of claim 13 , wherein the recombinant protein of interest comprises an Fc domain.

18. The method of claim 13 , wherein the recombinant protein of interest is selected from the group consisting of an Fc-fusion protein, a receptor-Fc-fusion protein, an antibody, and an antibody fragment.

19. The method of claim 18 , wherein the recombinant protein of interest is selected from the group consisting of an anti-PD1 antibody, an anti-PDL-1 antibody, an anti-ANG2 antibody, an anti-AngPtl3 antibody, an anti-PDGFR antibody, an anti-PRLR antibody, an anti-TNF antibody, an anti-EGFR antibody, an anti-PCSK9 antibody, an anti-GDF8 antibody, an anti-GCGR antibody, an anti-VEGF antibody, an anti-IL1R antibody, an anti-IL4R antibody, an anti-IL6R antibody, an anti-IL1 antibody, an anti-IL2 antibody, an anti-IL3 antibody, an anti-IL4 antibody, an anti-IL5 antibody, an anti-IL6 antibody, an anti-IL7 antibody, an anti-RSV antibody, an anti-NGF antibody, an anti-CD3 antibody, an anti-CD20 antibody, an anti-CD19 antibody, an anti-CD28 antibody, an anti-CD48 antibody, an anti-CD3/anti-CD20 bispecific antibody, an anti-CD3/anti-MUC16 bispecific antibody, and an anti-CD3/anti-PSMA bispecific antibody.

20. The method of claim 18 , wherein the recombinant protein of interest is selected from the group consisting of alirocumab, sarilumab, fasinumab, nesvacumab, dupilumab, trevogrumab, evinacumab, and rinucumab.

21. A method for producing a protein of interest in a taurine-supplemented culture medium, comprising the steps of:
(a) introducing into a cell a nucleic acid comprising a sequence encoding a protein of interest thereby generating cells expressing the protein of interest;
(b) isolating cells expressing the protein of interest;
(c) culturing the cells expressing the protein of interest in a cell culture medium;
(d) supplementing the cell culture medium with L-taurine in an amount of about 0.1 mM to about 10 mM to produce an L-taurine-supplemented cell culture medium, wherein the taurine-supplemented cell culture medium is also supplemented with about 0.09 mM to about 0.9 mM ornithine;
(e) culturing the cel

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