PRODUCTION OF PROTEINS IN GLUTAMINE-FREE CELL CULTURE MEDIA

• US 20160024217A1
• Filed: 03/26/2015
• Published: 01/28/2016
• Est. Priority Date: 08/11/2009
• Status: Active Grant

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: GAWLITZEK, Martin, LUO, Shun, PETRAGLIA, Christina Teresa
• Granted Patent: US 9,714,293 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/70575 NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154 NGF C07K14/48, TNF C07K14/525
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 16/22 against growth factors ; against growth regulators
• CPC Class Codes: C07K 16/2878 against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• CPC Class Codes: C07K 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C12N 2500/32 Amino acids
• CPC Class Codes: C12N 2500/33 other than alpha-amino carboxylic acids, e.g. beta-amino acids, taurine
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 5/0018 Culture media for cell or tissue culture media for specific animal cell type C12N5/06
• CPC Class Codes: C12N 5/0043 Medium free of human- or animal-derived components

Abstract

The present invention relates generally to glutamine-free cell culture media supplemented with asparagine. The invention further concerns the production of recombinant proteins, such as antibodies, in asparagine-supplemented glutamine-free mammalian cell culture.

Claims

1. A process for producing a polypeptide in a host cell expressing said polypeptide, comprising culturing the host cell in a production phase of the culture in a glutamine-free production culture medium containing asparagine.

2. The process of claim 1 wherein the asparagine is added at a concentration in the range of 2.5 mM to 15 mM.

3. The process of claim 1 wherein the asparagine is added at a concentration in the range of 7.5 mM to 10 mM.

4. The process of claim 1 wherein the asparagine is added at a concentration of 10 mM.

6. The method of claim 1 wherein said host cell is an eukaryotic host cell.

7. The method of claim 1 wherein said eukaryotic host cell is a mammalian host cell.

8. The method of claim 7 wherein said mammalian host cell is a Chinese Hamster Ovary (CHO) cell.

9. The process of claim 8 wherein the mammalian host cell is a dhfr⁻

CHO cell.

10. The process of claim 1 wherein the production medium is serum-free.

11. The process of claim 1 wherein the production culture medium comprises one or more ingredients selected from the group consisting ofan energy source;

2) essential amino acids;

3) vitamins;

4) free fatty acids; and

5) trace elements.

12. The process of claim 11 wherein the production culture medium additionally comprises one or more ingredients selected from the group consisting of:

1) hormones and other growth factors;

2) salts and buffers; and

3) nucleosides.

13. The process of claim 1 wherein the production phase is a batch or fed batch culture phase.

14. The process of claim 1 further comprising the step of isolating said polypeptide.

15. The process of claim 14 further comprising determining one or more of cell viability, culture longevity, specific productivity and final recombinant protein titer following isolation.

16. The process of claim 15 wherein at least one of the cell viability, culture longevity, specific productivity and final recombinant protein titer is increased relative to the same polypeptide produced in a glutamine-containing production medium of the same composition.

17. The process of claim 1 wherein the polypeptide is a mammalian glycoprotein.

18. The process of claim 1 wherein the polypeptide is selected from the group consisting of antibodies, antibody fragments, and immunoadhesins.

19. The process of claim 18 wherein said antibody fragment is selected from the group consisting of Fab, Fab′

, F(ab′

)2, scFv, (scFv)2, dAb, complementarity determining region (CDR) fragments, linear antibodies, single-chain antibody molecules, minibodies, diabodies, and multispecific antibodies formed from antibody fragments.

20. The process of claim 18 wherein the antibody or antibody fragment is chimeric, humanized or human.

21. The method of claim 18 wherein said antibody or antibody fragment is a therapeutic antibody or a biologically functional fragment thereof.

22. The method of claim 21 wherein said therapeutic antibody is selected from the group consisting of anti-HER2 antibodies;

anti-CD20 antibodies;

anti-IL-8 antibodies;

anti-VEGF antibodies;

anti-CD40 antibodies;

anti-CD11a antibodies;

anti-CD18 antibodies;

anti-IgE antibodies;

anti-Apo-2 receptor antibodies;

anti-Tissue Factor (TF) antibodies;

anti-human α

4β

7 integrin antibodies;

anti-EGFR antibodies;

anti-CD3 antibodies;

anti-CD25 antibodies;

anti-CD4 antibodies;

anti-CD52 antibodies;

anti-Fc receptor antibodies;

anti-carcinoembryonic antigen (CEA) antibodies;

antibodies directed against breast epithelial cells;

antibodies that bind to colon carcinoma cells;

anti-CD38 antibodies;

anti-CD33 antibodies;

anti-CD22 antibodies;

anti-EpCAM antibodies;

anti-GpIIb/IIIa antibodies;

anti-RSV antibodies;

anti-CMV antibodies;

anti-HIV antibodies;

anti-hepatitis antibodies;

anti-CA 125 antibodies;

anti-α

vβ

3 antibodies;

anti-human renal cell carcinoma antibodies;

anti-human 17-1A antibodies;

anti-human colorectal tumor antibodies;

anti-human melanoma antibody R24 directed against GD3 ganglioside;

anti-human squamous-cell carcinoma; and

anti-human leukocyte antigen (HLA) antibodies, and anti-HLA DR antibodies.

23. The method of claim 21 wherein said therapeutic antibody is an antibody binding to a HER receptor, VEGF, IgE, CD20, CD11a, CD40, BR3 or DR5.

38. The method of claim 1 wherein said polypeptide is a therapeutic polypeptide.

39. The method of claim 38 wherein said therapeutic polypeptide is selected from the group consisting of a growth hormone, including human growth hormone and bovine growth hormone;

growth hormone releasing factor;

parathyroid hormone;

thyroid stimulating hormone;

lipoproteins;

alpha-1-antitrypsin;

insulin A-chain;

insulin B-chain;

proinsulin;

follicle stimulating hormone;

calcitonin;

luteinizing hormone;

glucagon;

clotting factors such as factor VIIIC, factor IX, tissue factor, and von Willebrands factor;

anti-clotting factors such as Protein C;

atrial natriuretic factor;

lung surfactant;

a plasminogen activator, such as urokinase or human urine or tissue-type plasminogen activator (t-PA);

bombesin;

thrombin;

hemopoietic growth factor;

tumor necrosis factor-alpha and -beta;

enkephalinase;

RANTES (regulated on activation normally T-cell expressed and secreted);

human macrophage inflammatory protein (MIP-1-alpha);

a serum albumin such as human serum albumin;

Muellerian-inhibiting substance;

relaxin A-chain;

relaxin B-chain;

prorelaxin;

mouse gonadotropin-associated peptide;

a microbial protein, such as beta-lactamase;

DNase;

IgE;

a cytotoxic T-lymphocyte associated antigen (CTLA), such as CTLA-4;

inhibin;

activin;

vascular endothelial growth factor (VEGF);

receptors for hormones or growth factors;

Protein A or D;

rheumatoid factors;

a neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF-β

;

platelet-derived growth factor (PDGF);

fibroblast growth factor such as aFGF and bFGF;

epidermal growth factor (EGF);

transforming growth factor (TGF) such as TGF-alpha and TGF-beta, including TGF-β

1, TGF-β

32, TGF-β

33, TGF-β

34, or TGF-β

35;

insulin-like growth factor-I and -II (IGF-I and IGF-II);

des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins;

CD proteins such as CD3, CD4, CD8, CD19, CD20, CD34, and CD40;

erythropoietin;

osteoinductive factors;

immunotoxins;

a bone morphogenetic protein (BMP);

an interferon such as interferon-alpha, -beta, and -gamma;

colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF;

interleukins (ILs), e.g., IL-1 to IL-10;

superoxide dismutase;

T-cell receptors;

surface membrane proteins;

decay accelerating factor;

viral antigen such as, for example, a portion of the AIDS envelope;

transport proteins;

homing receptors;

addressins;

regulatory proteins;

integrins such as CD11a, CD11b, CD11c, CD18, an ICAM, VLA-4 and VCAM;

a tumor associated antigen such as HER2, HER3 or HER4 receptor; and

fragments of said polypeptides.

5. (canceled)

24-37. -37. (canceled)

40. A ready-to-use glutamine-free cell culture medium for the production of a polypeptide in a production phase.