C‐terminal lysine variants in fully human monoclonal antibodies: Investigation of test methods and possible causes

TLDR

C‑terminal lysine variation is a common post‑translational modification in monoclonal antibodies and its level is sensitive to the production process. The study aims to evaluate commonly used analytical methods for estimating the relative percentages of C‑terminal lysine variants. The authors compared IEF, cIEF, ion‑exchange chromatography, and LC‑MS to assess their ability to approximate lysine variant percentages. They found that lysine levels differ markedly between B‑cell hybridoma and CHO transfectoma production, change with minor process tweaks, that LC‑MS after trypsin digestion yields reproducible relative percentages, and that carboxypeptidase B or a similar basic carboxypeptidase likely generates the modification.

Abstract

The C-terminal lysine variation is commonly observed in biopharmaceutical monoclonal antibodies. This modification can be important since it is found to be sensitive to the production process. The methods commonly used to probe this charge variation, including IEF, cIEF, ion-exchange chromatography, and LC-MS, were evaluated for their ability to effectively approximate relative percentages of lysine variants. A monoclonal antibody produced in a B cell hybridoma versus a CHO cell transfectoma was examined and it was determined that the relative amount of incorporated C-terminal lysine can vary greatly between these two production schemes. Another case study is shown whereby a different monoclonal antibody is subject to some minor process changes and the extent of lysine variation also exhibits a significant difference. During these studies the different methods for determining the extent of variation were evaluated and it was determined that LC-MS after trypsin digestion provides reproducible relative percentage information and has significant advantages over other methods. The final section of this work investigates the possible origins of this modification and evidence is shown that carboxypeptidase B or another basic carboxypeptidase causes this variation.