Analyzing Charge Variant Profiles of Monoclonal Antibodies Conjugated to Cytotoxic Agents

This study investigates the charge variant profiles of monoclonal antibodies (mAbs) conjugated to cytotoxic agents, focusing on their implications for therapeutic efficacy and safety. Antibody-drug conjugates (ADCs) are a promising class of targeted therapies, particularly in oncology. The research centers on three specific mAbs: mAB1 and mAB2, targeting EphA2, and mAB3, targeting CD19. Using imaged capillary isoelectric focusing (icIEF), we characterized the charge variants of these mAbs in both their unconjugated state and after conjugation to maytansine and tomaymycin derivatives using non-cleavable linkers. Our findings indicate that mAB1 and mAB2 exhibit greater charge heterogeneity compared to mAB3, with distinct isoelectric points (pI) reflecting their structural diversity. Specifically, mAB1 displayed two charge variants with pI values of 9.00 and 8.95, while mAB3 showed a predominant variant at pI 8.50. Conjugation increased charge heterogeneity and acidity across the ADCs, particularly evident in mAB1 conjugates, which demonstrated a broader pI range. The icIEF method proved effective, showing high repeatability for intra-day and inter-day analyses. These results highlight the critical role of charge variant characterization in ensuring the quality and consistency of ADCs. They underscore how variations in charge profiles can influence mAb pharmacokinetics and therapeutic outcomes, offering insights for developing more effective and safer antibody-drug conjugates in clinical applications.