Application of Native Ion Exchange Mass Spectrometry to Intact and Subunit Analysis of Site-Specific Antibody-Drug Conjugates Produced by AJICAP First Generation Technology.

Abstract

Antibody-drug conjugates (ADCs) are at the forefront of the next generation of oncology biopharmaceuticals. Conventional ADCs involve stochastic conjugation of the antibody to a cytotoxic drug, creating a highly heterogeneous product. The resulting stochastic distribution often leads to a narrow therapeutic index and makes it difficult to analyze the composition of heterogeneous ADCs. With the goal of overcoming these issues, we developed a site-specific conjugation technology, named AJICAP, for production of low heterogeneity ADCs. For analysis of these site-specific ADCs, we report herein strong cation exchange chromatography coupled with UV and mass spectrometry detection (SCX-UV-MS). Retention time reproducibility after SCX column equilibration enabled monitoring of important changes in product quality. SCX-UV-MS performed with MS-compatible mobile phases was conducted for intact native ADC analysis, allowing drug-antibody ratio characterization and charge variant characterization in single analysis. Furthermore, subunit analysis of the site-specific ADCs by native SCX-UV-MS confirmed the Fc site selectivity of ADCs generated by AJICAP conjugation.