title: Carboxypeptidase D is the only enzyme responsible for antibody C-terminal lysine cleavage in Chinese hamster ovary (CHO) cells.
abstract: Heterogeneity of C-terminal lysine levels often observed in therapeutic monoclonal antibodies is believed to result from the proteolysis by endogenous carboxypeptidase(s) during cell culture production. Identifying the responsible carboxypeptidase(s) for C-terminal lysine cleavage in CHO cells would provide valuable insights for antibody production cell culture processes development and optimization. In this study, five carboxypeptidases, CpD, CpM, CpN, CpB, and CpE, were studied for message RNA (mRNA) expression by qRT-PCR analysis in two most commonly used blank hosts (DUXB-11 derived DHFR-deficient DP12 host and DHFR-positive CHOK1 host), used for therapeutic antibody production, as well an antibody-expressing cell line derived from each host. Our results showed that CpD had the highest mRNA expression. When CpD mRNA levels were reduced by RNAi (RNA interference) technology, C-terminal lysine levels increased, whereas there was no obvious change in C-terminal lysine levels when a different carboxypeptidase mRNA level was knocked down suggesting that carboxypeptidase D is the main contributor for C-terminal lysine processing. Most importantly, when CpD expression was knocked out by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology, C-terminal lysine cleavage was completely abolished in CpD knockout cells based on mass spectrometry analysis, demonstrating that CpD is the only endogenous carboxypeptidase that cleaves antibody heavy chain C-terminal lysine in CHO cells. Hence, our work showed for the first time that the cleavage of antibody heavy chain C-terminal lysine is solely mediated by the carboxypeptidase D in CHO cells and our finding provides one solution to eliminating C-terminal lysine heterogeneity for therapeutic antibody production by knocking out CpD gene expression. Biotechnol. Bioeng. 2016;113: 2100-2106. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Department of Early Stage Cell Culture, Genentech Inc., 1 DNA Way, South San Francisco, California 94080.
authors.forename: Zhilan
authors.initials: Z
authors.lastname: Hu
authors.affiliation: Department of Early Stage Cell Culture, Genentech Inc., 1 DNA Way, South San Francisco, California 94080.
authors.forename: Henry
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Molecular Biology, Genentech Inc., 1 DNA Way, South San Francisco, California.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Haley
authors.affiliation: Department of Analytical Development and Quality Control, Genentech Inc., 1 DNA Way, South San Francisco, California.
authors.forename: Frank
authors.initials: F
authors.lastname: Macchi
authors.affiliation: Department of Analytical Development and Quality Control, Genentech Inc., 1 DNA Way, South San Francisco, California.
authors.forename: Feng
authors.initials: F
authors.lastname: Yang
authors.affiliation: Department of Early Stage Cell Culture, Genentech Inc., 1 DNA Way, South San Francisco, California 94080.
authors.forename: Shahram
authors.initials: S
authors.lastname: Misaghi
authors.affiliation: Department of Early Stage Cell Culture, Genentech Inc., 1 DNA Way, South San Francisco, California 94080.
authors.forename: Joseph
authors.initials: J
authors.lastname: Elich
authors.affiliation: Department of Analytical Development and Quality Control, Genentech Inc., 1 DNA Way, South San Francisco, California.
authors.forename: Renee
authors.initials: R
authors.lastname: Yang
authors.affiliation: Department of Analytical Development and Quality Control, Genentech Inc., 1 DNA Way, South San Francisco, California.
authors.forename: Yun
authors.initials: Y
authors.lastname: Tang
authors.affiliation: Department of Early Stage Cell Culture, Genentech Inc., 1 DNA Way, South San Francisco, California 94080.
authors.forename: John C
authors.initials: JC
authors.lastname: Joly
authors.affiliation: Department of Early Stage Cell Culture, Genentech Inc., 1 DNA Way, South San Francisco, California 94080.
authors.forename: Bradley R
authors.initials: BR
authors.lastname: Snedecor
authors.affiliation: Department of Early Stage Cell Culture, Genentech Inc., 1 DNA Way, South San Francisco, California 94080. shen.amy@gene.com.
authors.forename: Amy
authors.initials: A
authors.lastname: Shen
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics*; D016466:CHO Cells / Q000201:enzymology; D002268:Carboxypeptidases / Q000235:genetics / Q000378:metabolism*; D003412:Cricetulus; D055785:Gene Knockdown Techniques / Q000379:methods*; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*
chemical_list: C429108:107-1A4 monoclonal antibody; D000911:Antibodies, Monoclonal; D002268:Carboxypeptidases; C078874:carboxypeptidase D; D008239:Lysine
keywords: C-terminal lysine; CHO cell; CRISPR (clustered regularly interspaced short palindromic repeats) knockout; RNAi (RNA interference); carboxypeptidase D (CpD); charge variants; shRNA (small interference RNA)