Elucidating the effects of arginine and lysine on a monoclonal antibody C-terminal lysine variation in CHO cell cultures

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.

Culture temperature modulates monoclonal antibody charge variation distribution in Chinese hamster ovary cell cultures

“…In our previous study, carboxypeptidase B and carboxypeptidase H have been confirmed as two major basic carboxypeptidases responsible for C-terminal lysine cleavage in CHO cells (Zhang et al 2015). GAPDH, which was used as an internal control, was reported not to be affected by culture temperature (Mason et al 2014).…”

Cell culture media for recombinant protein expression in Chinese hamster ovary (CHO) cells: History, key components, and optimization strategies

“…High concentrations of lysine and histidine have also been shown to slightly reduce cellular growth rate in the exponential phase while improving cell viability in the late stage of a production process . However, high concentration of lysine may have an inhibitory effect on basic carboxypeptidases, resulting in an increase in C‐terminal lysine variants . Therefore, the concentration of lysine and histidine may be changed based on specific product requirements.…”

“…However, unlike lysine, high arginine concentrations can inhibit cell growth and reduce viability . Additionally, a rise in arginine concentration has been shown to double the amount of C‐terminal lysine variant and thereby increase the heterogeneity of an antibody product …”

“…77 However, high concentration of lysine may have an inhibitory effect on basic carboxypeptidases, resulting in an increase in C-terminal lysine variants. 78 Therefore, the concentration of lysine and histidine may be changed based on specific product requirements.…”

“…77 Additionally, a rise in arginine concentration has been shown to double the amount of C-terminal lysine variant and thereby increase the heterogeneity of an antibody product. 83 Asparagine and aspartic acid are important nonessential amino acids in cell culture media, playing a central role in key metabolic pathways. CHO cells tend to consume asparagine and aspartate at relatively high rates compared to other amino acids, especially during exponential growth phase.…”