Identification of a monoclonal antibody clipping variant by cross-validation using capillary electrophoresis-sodium dodecyl sulfate, capillary zone electrophoresis-mass spectrometry and capillary isoelectric focusing-mass spectrometry

Critical quality attributes (CQAs) of recombinant monoclonal antibody therapeutics are constantly mon-itored throughout the life cycle of drug development and manufacturing. In the past few decades, nu-merous analytical techniques have been developed for the characterization of CQAs. In this regard, non -reduced and reduced capillary electrophoresis - sodium dodecyl sulfate (CE-SDS) methods have been widely adopted by the biopharmaceutical industry for the evaluation of size-related heterogeneities in biologics. In this work we demonstrate that, with recent development of capillary electrophoresis - mass spectrometry (CE-MS) technologies, a clipping variant of bevacizumab may be identified directly by both capillary zone electrophoresis - mass spectrometry (CZE-MS) and capillary isoelectric focusing - mass spectrometry (cIEF-MS) approaches, providing a powerful addition to the traditional CE-SDS analysis workflow. In this novel workflow, linear regression between the mobility and molecular weight first re-sults in an approximate size range of this variant. The intact masses of all species in the bevacizumab are then obtained, after deconvolution of all features identified in the CZE-MS analysis. Subsequent CZE-MS analysis of the subunits of bevacizumab leads to the confirmation of a clipped heavy chain. Furthermore, cIEF-MS of the intact bevacizumab confirms the existence of this clipping variant. The cross-validation between CE-SDS, CZE-MS, and cIEF-MS, creates a comprehensive roadmap for monoclonal antibody size variants profiling. These CE-based analytical techniques are complementary to each other, leading to or-thogonal verification for size heterogeneity characterization.(c) 2022 Elsevier B.V. All rights reserved.

Automated summary

This article describes the method of using capillary electrophoresis-mass spectrometry (CE-MS) to characterize the critical quality attributes (CQAs) of recombinant monoclonal antibody therapeutics. By combining the CZE-MS and cIEF-MS methods with the traditional CE-SDS analysis workflow, a comprehensive profile of monoclonal antibody size variants can be obtained.