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.

FX knockout CHO hosts can express desired ratios of fucosylated or afucosylated antibodies with high titers and comparable product quality

“…Guide RNA (gRNA) sequences were designed flanking the 5′ and 3′ regions of the FX gene and used to knockout the FX gene as described (Hu et al, ), see supplementary materials for details.…”

Industrial bioprocessing perspectives on managing therapeutic protein charge variant profiles

“…Cell line development has also been particularly effective at controlling C‐terminal Lys. Investigations into the carboxypeptidase activity, the enzyme that cleaves the C‐terminal Lys, allows for targeted modulation by cell line engineering (Dick, Qiu, Mahon, Adamo, & Cheng, ; Hu et al, ), and deletion of C‐terminal amino residues has also successfully controlled the basic charge variants (Hu et al, ; Jiang et al, ). While cell line development may effectively control protein modifications caused by endogenous mechanisms, many charge variant modifications occur due to extracellular interactions in a molecule‐dependent manner that may be difficult to predict and control.…”

Enhanced Genome Editing Tools For Multi‐Gene Deletion Knock‐Out Approaches Using Paired CRISPR sgRNAs in CHO Cells

“…However, this approach raises several potentially problematic issues including altered transcripts or splicing variants, which could compensate or change gene function, as well as the accumulation of incomplete protein or of (frame‐shifted) nonsense protein sequences . Thus, we believe that to study a protein's role in a chosen system, it is necessary to remove the protein, which can only be ensured if the entire gene and thus its expression is completely eliminated as has been done by Hu et al to investigate C‐terminal lysine cleavage in antibody‐producing CHO cells …”