title: Control of misincorporation of serine for asparagine during antibody production using CHO cells.
abstract: 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.
authors.affiliation: Biogen Idec, Inc., Cambridge, Massachusetts 02142, USA. anurag.khetan@biogenidec.com
authors.forename: Anurag
authors.initials: A
authors.lastname: Khetan
authors.forename: Yao-ming
authors.initials: YM
authors.lastname: Huang
authors.forename: Jana
authors.initials: J
authors.lastname: Dolnikova
authors.forename: Nels E
authors.initials: NE
authors.lastname: Pederson
authors.forename: Dingyi
authors.initials: D
authors.lastname: Wen
authors.forename: Helena
authors.initials: H
authors.lastname: Yusuf-Makagiansar
authors.forename: Paul
authors.initials: P
authors.lastname: Chen
authors.forename: Thomas
authors.initials: T
authors.lastname: Ryll
mesh_terms: D019943:Amino Acid Substitution / Q000502:physiology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism*; D001216:Asparagine / Q000737:chemistry* / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D015202:Protein Engineering / Q000379:methods*; D012694:Serine / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D012694:Serine; D001216:Asparagine