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.

Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation

“…12 To achieve superior batch-to-batch consistency, the C-terminal lysine content of antibody products could be controlled by recombinantly removing the lysine before cell line development, or possibly by modifying cell culture trace element concentrations. 36,37 Taken together, our studies show that the C-terminal lysinecontaining heavy chain of IgG represents a pro-form in which complement activation via the classical pathway and CDC capability is down regulated. The C-terminal lysine thus acts as a molecular switch that requires clipping to activate the antibodies' full cytotoxic potential.…”

Tailoring recombinant protein quality by rational media design

“…A study concluded that copper concentration in the cell culture medium was the most significant parameter affecting the C-terminal Lys variants of a monoclonal antibody rather than zinc according to previous publications 53 . Further investigations revealed the important role of copper/zinc ratio in both intracellular and extracellular C-terminal Lys processing, with…”

“…Many authors have published extensive reviews, depicting the current state of the art and strategies, which focus on the cell line 1,7,16,45 and the cell culture parameters 18,51 . Furthermore, through the concentration adjustment of selected media components, and in some cases by supplementing the medium with specific co-factors, it is possible to adjust the glycosylation profile 52 , the charge variants 53 , the aggregation level 46,54 , and the abundance of LMW species 55 . Figure 1.1 outlines the three main strategies allowing to affect cell culture process performance and to tailor the quality attributes of therapeutic molecules.…”

“…C-terminal lysine (Lys) and N-terminal glutamine (Gln) charge variants are common for monoclonal antibodies 41,53,61,158 . Even though the mechanism of C-terminal Lys processing has yet to be fully understood, it was suggested that carboxypeptidases cleave the C-terminal Lys residues in a post-translational modification in cultured cells, yielding three antibody species of either 0, 1, or 2 C-terminal Lys residues 41 and lower isoelectric point.…”

“…high copper and low zinc levels favoring basic variants and as such C-terminal Lys 53 . However, large amounts of copper (1 µM) in the culture medium might be undesirable because it can increase Pro amidation 162 .…”