Methods of preventing and removing trisulfide bonds

Patent metadata

Field	Value
Publication number	US-9562252-B2
Application number	US-201214117554-A
Country	US
Kind code	B2
Filing date	May 11, 2012
Priority date	May 13, 2011
Publication date	Feb 7, 2017
Grant date	Feb 7, 2017

Abstract

The present invention pertains to methods of preventing and eliminating trisulfide bonds in proteins such as antibodies. In one embodiment, trisulfide bonds in proteins are converted to disulfide bonds as part of chromatographic purification procedures. In another embodiment, the formation of trisulfide bonds in proteins is inhibited by implementation of methods described herein during the cell culture production of such proteins. In another embodiment, monoclonal antibodies are produced by the methods described herein.

First claim

What is claimed is:

1. A method for reducing the formation of trisulfide bonds in a protein comprising culturing cells expressing said protein in the presence of an effective amount of an inhibitor of cysteine degradation, whereby trisulfide linkage formation in said protein is reduced relative to cells cultured in medium without the inhibitor of cysteine degradation, and wherein said inhibitor is methyl pyruvate, ethyl pyruvate, glyceraldehyde, or glyoxylic acid.

2. A method for reducing the formation of trisulfide bonds in a protein during large-scale production comprising culturing cells expressing said protein in the presence of an effective amount of an inhibitor of cysteine degradation, whereby trisulfide linkage formation in said protein is reduced relative to cells cultured in medium without the inhibitor of cysteine degradation, and wherein said inhibitor is methyl pyruvate, ethyl pyruvate, glyceraldehyde, or glyoxylic acid.

3. The method of claim 1 , wherein said cells are mammalian cells.

4. The method of claim 3 , wherein said mammalian cells are CHO (Chinese Hamster Ovary) (including CHO-K1, CHO DG44, and CHO DUXB11), VERO, HeLa, (human cervical carcinoma), CV1 (monkey kidney line) (including COS and COS-7), BHK (baby hamster kidney), MDCK, C127, PC12, HEK-293 (including HEK-293T and HEK-293E), PER C6, NS0, WI38, R1610 (Chinese hamster fibroblast), BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line), SP2/O (mouse myeloma), P3x63-Ag3.653 (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), or RAJI (human lymphocyte) cells.

5. The method of claim 1 , wherein the ratio of the inhibitor to cysteine is about 5:1 to 1:10.

6. The method of claim 1 , wherein said inhibitor is added at a concentration of between about 50 μM and about 500 mM.

7. The method of claim 6 , wherein said inhibitor is added at a concentration of between about 100 μM and about 100 mM.

8. The method of claim 1 , wherein said inhibitor is added at the beginning of the culturing.

9. The method of claim 1 , wherein said inhibitor is added during a feed in a fed-batch culture.

10. The method of claim 1 , wherein said cells are cultured in a bioreactor.

11. The method of claim 1 , wherein said cells are grown in suspension.

12. The method of claim 1 , wherein said protein is an antibody or Fc-fusion protein.

13. The method of claim 1 , wherein said cells are in IMDM or DMEM.

Assignees

• Kshirsagar Rashmi Rohit
• Gilbert Alan
• Biogen Ma Inc

Inventors

• Kshirsagar Rashmi Rohit
• Gilbert Alan

Classifications

• C07K2317/41

  Glycosylation, sialylation, or fucosylation · CPC title

• C07K1/1133

  by redox-reactions involving cystein/cystin side chains · CPC title

• A61K2039/505

  comprising antibodies · CPC title

• A61K39/39591 Primary

  Stabilisation, fragmentation · CPC title

• C12N5/0018

  Culture media for cell or tissue culture (media for specific animal cell type C12N5/06) · CPC title