Control of Misincorporation of Serine for Asparagine During Antibody Production Using CHO Cells

被引:53
作者
Khetan, Anurag [1 ]
Huang, Yao-ming [1 ]
Dolnikova, Jana [1 ]
Pederson, Nels E. [1 ]
Wen, Dingyi [1 ]
Yusuf-Makagiansar, Helena [1 ]
Chen, Paul [1 ]
Ryll, Thomas [1 ]
机构
[1] Biogen Idec Inc, Cell Culture Dev, Cambridge Ctr 14, Cambridge, MA 02142 USA
关键词
misincorporation; substitution; cell culture; ESCHERICHIA-COLI; AMINO-ACID; RECOMBINANT INTERLEUKIN-2; PROTEIN; EXPRESSION; MISTRANSLATION; NORLEUCINE; IDENTIFICATION; ARGININE; CODONS;
D O I
10.1002/bit.22771
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
A recombinant monoclonal antibody produced by Chinese hamster ovary (CHO) cell fed-batch culture was found to have amino acid sequence misincorporation upon analysis by intact mass and peptide mapping mass spectrometry. A detailed analysis revealed multiple sites for asparagine were being randomly substituted by serine, pointing to mistranslation as the likely source. Results from time-course analysis of cell culture suggest that misincorporation was occurring midway through the fed-batch process and was correlated to asparagine reduction to below detectable levels in the culture. Separate shake flask experiments were carried out that confirmed starvation of asparagine and not excess of serine in the medium as the root cause of the phenomenon. Reduction in serine concentration under asparagine starvation conditions helped reduce extent of misincorporation. Supplementation with glutamine also helped reduce extent of misincorporation. Maintenance of asparagine at low levels in 2 L bench-scale culture via controlled supplementation of asparagine-containing feed eliminated the occurrence of misincorporation. This strategy was implemented in a clinical manufacturing process and scaled up successfully to the 200 and 2,000 L bioreactor scales. Biotechnol. Bioeng. 2010; 107: 116-123. (C) 2010 Wiley Periodicals, Inc.
引用
收藏
页码:116 / 123
页数:8
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