Accelerating Characterization of Therapeutic Antibodies: A Comparative Assessment of icIEF-UV/MS and the Traditional Fractionation Workflow.

Accurate characterization of charge variants in biologics is crucial to uphold stringent quality standards ensuring the safety and efficacy of biotherapeutic products and regulatory requirements for the Biologic Licensing Application (BLA) process enabling marketing application. These variants, arising from post-translational modifications (PTMs) during upstream processing and enzymatic/nonenzymatic reactions in downstream processing and storage, can significantly impact therapeutic potency, efficacy, and immunogenicity. Conventional methods for characterizing charge variants typically involve labor-intensive fraction enrichment, consuming time and resources. However, recent technological advancements, exemplified by the Intabio ZT system's innovative platform, enable the seamless and direct integration of imaged capillary isoelectric focusing (icIEF) and UV quantitation with mass spectrometry (MS) PTM identification, facilitating rapid and unbiased characterization. In this study, we conduct a comparative assessment of the charge variant characterization of investigative mAb-1 using icIEF-UV/MS analysis and a traditional fractionation-based workflow. Our results demonstrated that icIEF-UV/MS-based proteoform characterization provided comparable icIEF-UV separation and quantitation and superior direct MS-based peak characterization with shorter time and less sample processing compared to conventional fractionation approaches.