Abstract
C-terminal lysine variants are commonly observed in monoclonal antibodies (mAbs) and found sensitive to process conditions, especially specific components in culture medium. The potential roles of media arginine (Arg) and lysine (Lys) in mAb heavy chain C-terminal lysine processing were investigated by monitoring the lysine variant levels under various Arg and Lys concentrations. Both Arg and Lys were found to significantly affect lysine variant level. Specifically, lysine variant level increased from 18.7 to 31.8 % when Arg and Lys concentrations were increased from 2 to 10 mM. Since heterogeneity of C-terminal lysine residues is due to the varying degree of proteolysis by basic carboxypeptidases (Cps), enzyme (basic Cps) level, pH conditions, and product (Arg and Lys) inhibition, which potentially affect the enzymatic reaction, were investigated under various Arg and Lys conditions. Enzyme level and pH conditions were found not to account for the different lysine variant levels, which was evident from the minimal variation in transcription level and intracellular pH. On the other hand, product inhibition effect of Arg and Lys on basic Cps was evident from the notable intracellular and extracellular Arg and Lys concentrations comparable with Ki values (inhibition constant) of basic Cps and further confirmed by cell-free assays. Additionally, a kinetic study of lysine variant level during the cell culture process enabled further characterization of the C-terminal lysine processing.
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Acknowledgments
This work was supported by the National Natural Science Foundation of China (No. 21406066, 21206040), the National High Technology Research and Development Program of China (863 Program) (No. 2012AA02A303).
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Xintao Zhang and Hongping Tang contributed equally to this work.
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Zhang, X., Tang, H., Sun, YT. et al. Elucidating the effects of arginine and lysine on a monoclonal antibody C-terminal lysine variation in CHO cell cultures. Appl Microbiol Biotechnol 99, 6643–6652 (2015). https://doi.org/10.1007/s00253-015-6617-y
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DOI: https://doi.org/10.1007/s00253-015-6617-y