Rapid way to obtain high expression clones of mammalian cells using a methylcellulose and immunoprecipitation screening method

• US 20050118652A1
• Filed: 08/27/2004
• Published: 06/02/2005
• Est. Priority Date: 08/29/2003
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Ly, Celia, Lee, Chichang, Moore, Gordon
• Granted Patent: US 9,128,090 B2
• Time in Patent Office: Days
• US Class Current: 435/7.200
• CPC Class Codes: C07K 16/241 Tumor Necrosis Factors
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: G01N 33/56966 Animal cells

Abstract

The invention provides a genetic screening method for identifying a transfected cell expressing the polypeptide of interest. The methods allows for high throughput screening of recombinant cells for elevated levels of expression of the polypeptide of interest. The invention also provides capture media, formulations and methods of making and using thereof.

Claims

1. A method for selecting high expression cell clones expressing at least one polypeptide of interest, comprising selecting at least one high expression cell clone from cells, cultured in a semi-solid culture medium and expressing said polypeptide of interest, wherein said cells are contacted with a capture molecule that interacts with said at least one polypeptide of interest such that said interaction indicates higher relative expression of said polypeptide for each cell or group of cells.

2. A method according to claim 1, wherein said interaction is precipitation of said polypeptide and said capture molecule.

3. A method according to claim 2, wherein said capture molecule is an antibody or a polypeptide binding fragment thereof.

4. A method according to claim 2, wherein said interaction is by a detectable label.

5. A method according to claim 4, wherein said detectable label is selected from a chemical label, a biological label, a fluorescent label or a radioactive label.

6. A method according to claim 1, wherein the semi-solid culture medium comprises a gelatinization agent selected from cellulose or agar.

7. A method according to claim 6, wherein said cellulose is methylcellulose.

8. A method according to claim 1, wherein said cells are eukaryotic cells.

9. A method according to claim 8, wherein said eukaryotic cells are selected from mammalian cells, yeast cells or insect cells.

10. A method according to claim 8, wherein said mammalian cells are selected from COS-1, COS-7, HEK293, HK21, CHO, BSC-1, HepG2, 653, SP2/0, 293, NSO, DG44 CHO, CHO K1, HeLa, myeloma, or lymphoma cells, or any derivative, immortalized or transformed cells thereof.

11. A method according to claim 1, wherein said cells are prokaryotic cells.

12. A method according to claim 11, wherein said prokaryotic cells are bacterial cells or blue-green algae cells.

13. A method according to claim 1, wherein said at least one polypeptide of interest is a soluble polypeptide.

14. A method according to claim 1, wherein said at least one polypeptide of interest is an immunoglobulin or at least one portion thereof.

15. A method according to claim 1, wherein the capture molecule is selected from at least one portion of a ligand or a receptor of, or an antibody against, said polypeptide of interest.

16. A method according to claim 15, wherein said capture molecule is an antibody against said at least one polypeptide of interest.

17. A method according to claim 1, wherein the cells are myeloma cells, said at least one polypeptide of interest is an immunoglobulin, the capture molecule is an antibody against the immunoglobulin, and the semi-solid culture medium is methylcellulose based.

18. A method according to claim 1, wherein the final concentration of the capture molecule is 0.2-2.0 mg/ml.

19. A method according to claim 18, wherein the final concentration of the capture molecule is 1.0 mg/ml.

20. A high expression cell clone, produced by a method according to claim 1.

21. A high expression cell clone according to claim 20, wherein the cells of said high expression cell clone are eukaryotic cells.

22. A method according to claim 21, wherein said eukaryotic cells are selected from mammalian cells, yeast cells or insect cells.

23. A high expression cell clone according to claim 22, wherein said mammalian cells are selected from COS-1, COS-7, HEK293, HK21, CHO, BSC-1, HepG2, 653, SP2/0, 293, NSO, DG44 CHO, CHO K1, HeLa, myeloma, or lymphoma cells, or any derivative, immortalized or transformed cells thereof.

24. A high expression cell clone according to claim 20, wherein said cells of said high expression cell clone are prokaryotic cells.

25. A high expression cell clone according to claim 24, wherein said prokaryotic cells are bacterial cells or blue-green algae cells.

26. A high expression cell clone according to claim 20, wherein said at least one polypeptide of interest is a soluble polypeptide.

27. A high expression cell clone according to claim 20, wherein said at least one polypeptide of interest is an immunoglobulin or at least one portion thereof.

29. A semi-solid culture medium according to claim 20, wherein said gelatinization agent is selected from cellulose or agar.

30. A semi-solid culture medium according to claim 21, wherein said cellulose is methylcellulose.

31. A method according to claim 2, wherein said precipitation is detectable by diffusion of light directed through said cells.

32. A method according to claim 31, wherein said high expression cell clones are detectable by higher relative size of a halo effect produced by said diffusion of light through cells of said high expression cell clones, relative to the cells having expression of said protein of interest that is lower than said high expression cell lines.

33. A method according to claim 1, wherein said at least one polypeptide of interest is selected from at least one of a growth factor, a cytokine, a blood protein, a neurotransmitter, pharmacologically active peptide, or any portion or derivative thereof.

34. A method according to claim 1, wherein said at least one polypeptide of interest is an antagonist of at least one selected from growth factors, cytokines, blood proteins, neurotransmitters, pharmacologically active peptides, or portions or derivatives thereof.

35. A method according to claim 34, wherein said antagonist is selected from at least one of an antibody, an antibody fusion, an antibody fragment, or any portion or derivative thereof.

28. A semi-solid culture medium to be used to identify a high expression cell clone expressing a polypeptide of interest, said medium comprising a cell growth culture medium and a gelatinization agent.