Methods of decreasing trisulfide bonds during recombinant production of polypeptides
AssigneeGenentech, Inc.
InventorsMartin GAWLITZEK, Sven MARKERT, Oliver POPP, Masaru Ken SHIRATORI, Thomas TROEBS, Jessica WUU
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Abstract
Provided herein are cell culture media and methods culturing host cells expressing polypeptides to reduce the level of trisulfide bonds in polypeptides produced by the host cells.
Claims49
A method for decreasing trisulfide bond levels in a polypeptide comprising:
- (a) contacting a host cell comprising a nucleic acid encoding the polypeptide with a basal medium, wherein the basal medium comprises one or more of the following components:
- i) between about 2 μM to about 35 μM iron,
- ii) between about 0.11 μM to about 2μM riboflavin (vitamin B2),
- iii) between about 4.5μM to about 80 μM pyridoxine or pyridoxal (vitamin B6),
- iv) between about 3.4 μM to about 23 μM folic acid (vitamin B9),
- v) between about 0.2 μM to about 2.5 μM cyanocobalamin (vitamin B12),
- vi) between about 9 mM and about 10 mM hypotaurine;
- and
- vii) between about 0 and about 1.58 mM methionine;
- (b) culturing the host cell to produce the polypeptide;
- and
- (c) harvesting the polypeptide produced by the host cell.
A method for producing a polypeptide, comprising:
- (a) contacting a host cell comprising a nucleic acid encoding the polypeptide with a basal medium, wherein the basal medium comprises one or more of the following components:
- i) between about 2 μM to about 35 μM iron,
- ii) between about 0.11 μM to about 2μM riboflavin (vitamin B2),
- iii) between about 4.5μM to about 80 μM pyridoxine or pyridoxal (vitamin B6),
- iv) between about 3.4 μM to about 23 μM folic acid (vitamin B9),
- v) between about 0.2 μM to about 2.5 μM cyanocobalamin (vitamin B12),
- vi) between about 9 mM and about 10 mM hypotaurine;
- and
- vii) between about 0 and about 1.58 mM methionine;
- (b) culturing the host cell to produce the polypeptide;
- and
- (c) harvesting the polypeptide produced by the host cell.
A method for decreasing trisulfide bond levels in a polypeptide comprising:
- (a) culturing a host cell comprising a nucleic acid encoding the polypeptide in a cell culture medium, wherein the cell culture medium comprises one or more of the following components:
- i) between about 2 μM to about 35 μM iron,
- ii) between about 0.11 μM to about 2μM riboflavin (vitamin B2),
- iii) between about 4.5μM to about 80 μM pyridoxine or pyridoxal (vitamin B6),
- iv) between about 3.4 μM to about 23 μM folate/folic acid (vitamin B9),
- v) between about 0.2 μM to about 2.5 μM cyanocobalamin (vitamin B12),
- vi) between about 9 mM and about 10 mM hypotaurine;
- and
- vii) between about 0 and about 4.5 mM methionine;
- (b) producing the polypeptide;
- (c) and harvesting the polypeptide produced by the host cell.
A method for decreasing level of trisulfide bonds in a polypeptide produced by a host cell comprising:
- (i) supplementing a culture of said host cell with a reducing agent and a chelating agent prior to harvest;
- (ii) supplementing a pre-harvest cell culture fluid (PHCCF) of said host cell with a chelating agent and a reducing agent;
- or
- (iii) supplementing a harvested cell culture fluid (HCCF) of said host cell with a reducing agent and a chelating agent.
Use of between about 0 and about 4.5 μM methionine in a cell culture medium for decreasing trisulfide bond levels in a polypeptide selected from the group consisting of:
- a CEA-IL2v immuocytokine, a FAP-IL2v immunocytokine, an anti-CEA/anti-CD3 bispecific antibody, an anti-VEGF/anti-angiopoietin bispecific antibody, an anti-Ang2/anti-VEGF bispecific antibody, an anti-C5 antibody, and an anti-CD40 antibody.
Dependencies are inferred from the claim text.