title: Probing of C-terminal lysine variation in a recombinant monoclonal antibody production using Chinese hamster ovary cells with chemically defined media.
abstract: C-terminal lysine (C-K) variants are commonly observed in therapeutic monoclonal antibodies and recombinant proteins. Heterogeneity of C-K residues is believed to result from varying degree of proteolysis by endogenous carboxypeptidase(s) during cell culture production. The achievement of batch-to-batch culture performance and product quality reproducibility is a key cell culture development criterion. Understanding the operational parameters affecting C-K levels provides valuable insight into the cell culture process. A CHO cell line X expressing a recombinant antibody was selected as the model cell line due to the exhibited sensitivity of its C-K level to the process conditions. A weak cation exchange chromatography (WCX) method with or without carboxypeptidase B (CpB) treatment was developed to monitor the C-K level for in-process samples. The effects of operating conditions (i.e., temperature and culture duration) and media trace elements (copper and zinc) on C-K variants were studied. The dominant effect on C-K level was identified as the trace elements concentration. Specifically, increased C-K levels were observed with increase of copper concentration and decrease of zinc concentration in chemically defined medium. Further, a hypothesis for C-K processing with intracellular and extracellular carboxypeptidase activity was proposed, based on preliminary intracellular carboxypeptidase Western blot results and the extracellular HCCF holding study.
authors.affiliation: Oceanside Pharma Technical Development, Genentech, Inc., Oceanside, California 92056, USA. luo.jun@gene.com
authors.forename: Jun
authors.initials: J
authors.lastname: Luo
authors.forename: Jian
authors.initials: J
authors.lastname: Zhang
authors.forename: Diya
authors.initials: D
authors.lastname: Ren
authors.forename: Wen-Lin
authors.initials: WL
authors.lastname: Tsai
authors.forename: Feng
authors.initials: F
authors.lastname: Li
authors.forename: Ashraf
authors.initials: A
authors.lastname: Amanullah
authors.forename: Terry
authors.initials: T
authors.lastname: Hudson
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry; D061249:Batch Cell Culture Techniques; D019149:Bioreactors; D015153:Blotting, Western; D016466:CHO Cells; D043424:Carboxypeptidase B / Q000378:metabolism; D003300:Copper / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media / Q000737:chemistry / Q000378:metabolism*; D008239:Lysine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry; D015032:Zinc / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D003470:Culture Media; D011994:Recombinant Proteins; D003300:Copper; D043424:Carboxypeptidase B; D015032:Zinc; D008239:Lysine