METHODS FOR MODULATING PROTEIN GLYCOSYLATION PROFILES OF RECOMBINANT PROTEIN THERAPEUTICS USING MONOSACCHARIDES AND OLIGOSACCHARIDES

• US 20140271632A1
• Filed: 03/13/2014
• Published: 09/18/2014
• Est. Priority Date: 03/14/2013
• Status: Active Grant

• Current Assignee: Abbvie Incorporated
• Original Assignee: Abbvie Incorporated
• Inventors: Correia, Ivan, Hossler, Patrick, McDermott, Sean, Racicot, Christopher
• Granted Patent: US 9,499,614 B2
• Time in Patent Office: Days
• US Class Current: 424/133.1
• 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 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/41 Glycosylation, sialylation, or fucosylation
• CPC Class Codes: C07K 2317/60 characterized by non-natural combinations of immunoglobulin fragments
• CPC Class Codes: C07K 2317/732 Antibody-dependent cellular cytotoxicity [ADCC]
• CPC Class Codes: C12N 2500/34 Sugars
• 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 the field of protein production, and in particular to methods and compositions for modulating glycosylation of proteins expressed in host cells.

Claims

1. A method of producing a composition comprising a protein with a modulated glycosylation profile, said method comprising:

culturing a host cell expressing said protein in cell culture media supplemented with a monosaccharide and/or an oligosaccharide, thereby producing said composition comprising said protein with a modulated glycosylation profile as compared to a control,

wherein said control is a composition comprising a protein produced by culturing a host cell expressing said protein in cell culture media which is not supplemented with a monosaccharide and/or an oligosaccharide.

2. The method of claim 1, further comprising purifying said composition comprising said protein with a modulated glycosylation profile.

3. The method of claim 1, wherein the protein is an antibody or antigen-binding portion thereof.

4. The method of claim 3, wherein the antibody is an anti-TNFα

antibody.

5. The method of claim 4, wherein the anti-TNFα

antibody is adalimumab, or antigen binding fragment thereof.

6. The method of claim 1, wherein the protein is a dual variable domain immunoglobulin (DVD-Ig).

7. The method of claim 1, wherein the protein is selected from the group consisting of a TVD-IG, a half-body and a RAB.

8. The method of claim 1, wherein the monosaccharide is tagatose.

9. The method of claim 1, wherein the oligosaccharide is sucrose.

10. The method of claim 1, wherein the cell culture media is supplemented with a sufficient amount of the monosaccharide to achieve a monosaccharide concentration selected from the group consisting of about 1 mM, about 5 mM, about 7 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM and about 100 mM.

11. The method of claim 10, wherein the monosaccharide concentration is 30 mM.

12. The method of claim 10 or 11, wherein the monosaccharide is tagatose.

13. The method of claim 1 wherein the cell culture media is supplemented with a sufficient amount of the oligosaccharide to achieve an oligosaccharide concentration selected from the group consisting of about 1 mM, about 5 mM, about 7 mM, about 10 mM, about 20 mM, about 30 mM. about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM and about 100 mM.

14. The method of claim 13, wherein the oligosaccharide concentration is 30 mM.

15. The method of claim 13 or 14, wherein the oligosaccharide is sucrose.

16. The method of claim 1, wherein the modulated glycosylation profile of the protein comprises modulation of a fucosylation level and/or a mannosylated N-glycan oligosaccharide level in said protein.

17. The method of claim 16, wherein the modulation of the fucosylation level comprises a decrease in the fucosylation level in said protein.

18. The method of claim 17, wherein the decrease in the fucosylation level comprises a decrease in the level of NGA2F, NA1F-GlcNAc, NA1F and/or NA2F in said protein.

19. The method of claim 18, wherein the decrease in the level of NGA2F, NA1F-GlcNAc, NA1F and/or NA2F is a decrease of about 0.1%, 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65%.

20. The method of claim 16, wherein the modulation of the fucosylation level comprises an increase in the fucosylation level in said protein.

21. The method of claim 20, wherein increase in the fucosylation level comprises an increase in the level of NGA2F-GlcNAc, NA1F-GlcNAc, NA1F and/or NA2F in said protein.

22. The method of claim 20, wherein the increase in the level of NGA2F-GlcNAc, NA1F-GlcNAc, NA1F or NA2F is an increase of about 0.1%, 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15% or 20%.

23. The method of claim 17, wherein the overall decrease in the fucosylation level comprises an increase or a decrease in the level of NGA2F, NGA2F-GlcNAc, NA1F-GlcNAc, NA1F and/or NA2F in said protein.

24. The method of claim 23, wherein in the overall decrease in the fucosylation level is a decrease of about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%.

25. The method of claim 16, wherein the modulation of the mannosylated N-glycan level comprises an increase in the mannosylation level of said protein.

26. The method of claim 25, wherein the increase in the mannosylation level is an increase of about 0.1%, 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%.

27. The method of claim 25, wherein the increase in the mannosylation level comprises an increase in the level of a high mannose N-glycan oligosaccharide selected from the group consisting of Man 5 glycan, Man 6 glycan, Man 7 glycan and Man 8 glycan.

28. The method of claim 27, wherein the levels of Man 5 glycan, Man 6 glycan, Man 7 glycan and/or Man 8 glycan are increased by about 0.1%, 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40, 45% or 50%.

29. The method of claim 1, wherein said host cell is a CHO cell.

37. A pharmaceutical composition comprising the composition produced by the methods of any one of claim 1, 30 or 32 and a pharmaceutically acceptable carrier.

38. A composition comprising a therapeutic protein with a modulated glycosylation profile produced by the methods of any one of claim 1, 30 or 32.

39. The composition of claim 38, wherein the therapeutic protein is an antibody.

30. A method of producing a composition comprising an antibody, or antigen binding fragment thereof, with a modulated glycosylation profile, said method comprising:

culturing a host cell expressing said antibody, or antigen binding fragment thereof, in cell culture media supplemented with sucrose and/or tagatose, thereby producing said composition comprising said antibody, or antigen binding fragment thereof, with an increased level of mannosylated N-glycans and a decreased level of fucosylated N-glycans as compared to a control,

wherein said control is a composition comprising an antibody, or antigen binding fragment thereof, produced by culturing a host cell expressing said antibody, or antigen binding fragment thereof, in cell culture media which is not supplemented with tagatose and/or glucose.

31. The method of claim 30, wherein the antibody is adalimumab, or antigen binding fragment thereof.

32. A method of producing a composition comprising an antibody, or antigen binding fragment thereof, with a modulated glycosylation profile, said method comprising:

culturing a host cell expressing said antibody, or antigen binding fragment thereof, in cell culture media supplemented with sucrose and/or tagatose, thereby producing said composition comprising said antibody, or antigen binding fragment thereof, with a 1-50% increase in the level of mannosylated N-glycans and a 1-50% decrease in the level of fucosylated N-glycans as compared to a control,

wherein said control is a composition comprising an antibody, or antigen binding fragment thereof, produced by culturing a host cell expressing said antibody, or antigen binding fragment thereof, in cell culture media which is not supplemented with tagatose and/or glucose.

33. The method of claim 32, wherein the antibody is adalimumab, or antigen binding fragment thereof.

34. A composition comprising a cell culture media comprising a monosaccharide and/or an oligosaccharide.

35. The composition of claim 34, wherein the monosaccharide is tagatose.

36. The composition of claim 34, wherein the oligosaccharide is sucrose.

40. A composition comprising a therapeutic protein, wherein said protein comprises a 1-50% increase in the level of mannosylated N-glycans and a 1-50% decrease in the level of fucosylated N-glycans, as compared to a control,

wherein said control is a composition comprising a protein produced by culturing a host cell expressing said protein in cell culture media which is not supplemented with a monosaccharide and/or an oligosaccharide

41. The composition of claim 40, wherein the therapeutic protein is selected from the group consisting of an antibody, an antigen-binding portion thereof, DVD-Ig, TVD-Ig, RAB and half-body.

42. A composition of claim 40, wherein the therapeutic protein is an antibody.

43. A method of producing a composition comprising an antibody, or antigen binding fragment thereof, with a modulated glycosylation profile, said method comprising:

culturing a host cell expressing said antibody, or antigen binding fragment thereof, in cell culture media supplemented with sucrose and/or tagatose, thereby producing said composition comprising said antibody, or antigen binding fragment thereof, with a 1-30% increase in antibody-dependent cellular cytotoxicity (ADCC) response as compared to a control,

wherein said control is a composition comprising an antibody, or antigen binding fragment thereof, produced by culturing a host cell expressing said antibody, or antigen binding fragment thereof, in cell culture media which is not supplemented with tagatose and/or glucose.

44. The method of claim 43, wherein the antibody is adalimumab, or antigen binding fragment thereof.