Identification of Method-Induced Artifacts and Light Chain C-terminal Extension Sequence Variants in Therapeutic Monoclonal Antibodies by Complementary Analytical Methods

Abstract

Background: Atypical peaks were observed in capillary electrophoresis with sodium dodecyl sulfate (CE-SDS) during the development of therapeutic monoclonal IgG4 antibodies (mAb-X). Based on the previous literature reports, the atypical peak may be caused by various factors such as post-translational modifications (PTMs), method-induced artifacts, sample degradation and sequence variants. Due to the high complexity structure of mAbs and the limitations of CE-SDS, acquiring comprehensive profiling of atypical peaks has historically been challenging. Objective: Here we developed a strategy utilizing complementary analytical methods to identify the cause of atypical peak. Methods: This strategy includes optimizing reduced CE-SDS method to evaluate artifacts induced by the analytical method, excluding potential glycation modifications, and utilizing Liquid Chromatograph Mass Spectrometer (LC-MS) to characterize mAb-X. Results: Our study demonstrates that the atypical peaks of mAb-X are a mixture of method-induced artifacts and variants in the C-terminal extension sequence of the light chain. Conclusion: Strategy for complementary analytical methods tools helps to identify the cause of unknown species and plays a key role in product and process characterization.