Characterization of monoclonal antibody size variants containing extra light chains

Abstract

Size exclusion chromatography (SEC) is the most commonly used method to separate and quantify monoclonal antibody (mAb) size variants. MAb-A is an IgG1 subtype humanized monoclonal antibody recombinantly produced in Chinese hamster ovary (CHO) cells. SEC analysis of MAb-A resolved a peak, named Peak 1, which elutes between monomer and dimer peaks. MAb-A lots produced from different clones and production scales all have 0.2–0.3% of SEC Peak 1. Electron spray ionization—time of flight mass spectrometry (ESI-TOF MS), microfluidics capillary electrophoresis and sodium dodecyl sulfate-PAGE (SDS PAGE) results demonstrated that SEC Peak 1 contains two structural variants: MAb-A with one extra light chain (2H3L) and MAb-A with two extra light chains (2H4L). The C-terminal Cys of the extra light chain in Peak 1 variants is either a free thiol, capped by glutathione, cysteine, or another light chain. Both electrophoresis and LC/MS analyses of non-reduced and reduced samples suggested that the extra light chains are linked to the MAb-A light chain through disulfide bonds. Isolated SEC Peak 1 fraction had a potency of 50% relative to MAb-A reference material. The 50% potency loss may result from the reduced accessibility to the antigen-binding site caused by the extra light chain(s)’ steric hindrance.

Keywords: :

• monoclonal antibody
• antibody size variant
• triple light chain antibody variant
• light chain dimer
• antibody high molecular weight species
• Size Exclusion Chromatography

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank Julie Nishihara for helping on the SDS PAGE analysis, Betty Chan for the helping on the Agilent 2100 Bioanalyzer analysis, Melissa Alvarez for setting up the LC/MS/MS analysis, Charity Bechtel for performing SEC-MALS analysis, Aaron Miller for potency data analysis, Angela Meier for performing free thiol quantification by Elman’s reagent and Parbir Grewal for NEM treated peptide map sample preparation. We also thank John Stults and Reed Harris for their support.