METHODS FOR CONTROLLING THE GALACTOSYLATION PROFILE OF RECOMBINANTLY-EXPRESSED PROTEINS

• US 20150050693A1
• Filed: 09/22/2014
• Published: 02/19/2015
• Est. Priority Date: 04/27/2011
• Status: Active Grant

• Current Assignee: Abbvie Incorporated
• Original Assignee: Abbvie Incorporated
• Inventors: Bengea, Cornelia, Rives, Lisa M.
• Granted Patent: US 9,090,688 B2
• Time in Patent Office: Days
• US Class Current: 435/69.6
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/241 Tumor Necrosis Factors
• CPC Class Codes: C07K 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/41 Glycosylation, sialylation, or fucosylation
• CPC Class Codes: C12N 2500/20 Transition metals
• CPC Class Codes: C12N 2500/34 Sugars
• CPC Class Codes: C12N 2511/00 Cells for large scale production
• CPC Class Codes: C12N 5/0018 Culture media for cell or tissue culture media for specific animal cell type C12N5/06
• CPC Class Codes: C12P 21/005 Glycopeptides, glycoproteins

Abstract

The present invention relates to methods for modulating the glycosylation profile of recombinantly-expressed proteins. In particular, the present invention relates to methods of controlling the galactosylation profile of recombinantly-expressed proteins by supplementing production medium, e.g., a hydrolysate-based or a chemically defined medium, with manganese and/or D-galactose.

Claims

1. A process for producing adalimumab, comprising culturing a mammalian cell capable of producing adalimumab in a chemically defined cell culture media that has been supplemented with a manganese supplement and a galactose supplement.

2. The process of claim 1, wherein said culturing is done in a suspension culture.

3. The process of claim 1, wherein the media is HyClone CDM4CHO media.

4. The process of claim 1, wherein the mammalian cell is a CHO cell.

5. The process of claim 1, wherein the mammalian cell is cultured for at least 4 days.

6. The process of claim 1, wherein the process is a fed batch process.

7. The process of claim 1, wherein the media is selected from the group consisting of production media and feed media.

8. The process of claim 1, wherein the media is supplemented with a sufficient amount of the manganese supplement to achieve a manganese concentration of at least 0.1 μ

M.

9. The process of claim 1, wherein the media is supplemented with a sufficient amount of the galactose supplement to achieve a galactose concentration of at least 1 mM.

10. The process of claim 1, wherein the media is supplemented with a sufficient amount of the manganese supplement and the galactose supplement to achieve a manganese (Mn) and a galactose (Gal) concentration of at least 0.2/1 Mn (μ

M)/Gal (mM).

11. The process of claim 1, wherein the manganese supplement and the galactose supplement are each present in the media at a concentration sufficient to produce a composition comprising adalimumab in which at least 10% of the total N-linked oligosaccharides present on said adalimumab are of a galactose-containing fucosylated biantennary oligosaccharide (NA1F+NA2F) form.

12. The process of claim 11, wherein at least 15% of the total N-linked oligosaccharides present on said adalimumab are of a galactose-containing fucosylated biantennary oligosaccharide (NA1F+NA2F) form.

13. A process for producing adalimumab, comprising culturing a mammalian cell capable of producing adalimumab in a media that has been supplemented with a manganese supplement and a galactose supplement.

14. The process of claim 13, wherein said culturing is done in a suspension culture.

15. The process of claim 13, wherein the media comprises a hydrolysate.

16. The process of claim 13, wherein the mammalian cell is a CHO cell.

17. The process of claim 13, wherein the mammalian cell is cultured for at least 4 days.

18. The process of claim 13, wherein the process is a fed-batch process.

19. The process of claim 15, wherein the media is selected from the group consisting of production media and feed media.

20. The process of claim 13, wherein the manganese supplement is a biologically acceptable manganese salt.

21. The process of claim 20, wherein the biologically acceptable manganese salt is manganese (II) chloride.

22. The process of claim 13, wherein the media is supplemented with a sufficient amount of the manganese supplement to achieve a manganese concentration of at least 0.1 μ

M.

23. The process of claim 13, wherein the galactose supplement is a biologically acceptable galactose containing compound.

24. The process of claim 23, wherein the biologically acceptable galactose containing compound is D-(+)-galactose.

25. The process of claim 13, wherein the media is supplemented with a sufficient amount of the galactose supplement to achieve a galactose concentration of at least 1 mM.

26. The process of claim 13, wherein the media is supplemented with a sufficient amount of the manganese supplement and the galactose supplement to achieve a manganese (Mn) and a galactose (Gal) concentration of at least 0.2/1 Mn (μ

M)/Gal (mM).

27. The process of claim 13, wherein the manganese supplement is present in the media at a concentration sufficient to produce a composition comprising adalimumab in which at least 10% of the total N-linked oligosaccharides present on said adalimumab are of a galactose-containing fucosylated biantennary oligosaccharide (NA1F+NA2F) form.

28. The process of claim 27, wherein at least 15% of the total N-linked oligosaccharides present on said adalimumab are of a galactose-containing fucosylated biantennary oligosaccharide (NA1F+NA2F) form.