Novel Strong Cation Exchange Ultra-Performance Liquid Chromatography Coupled to UV and MS Detectors for Robust Characterization of ADC Charge Variants

• Published in: Journal of biomolecular techniques 2020-08, Vol.31 (Suppl), p.S22-S22
• Main Authors: Kliman, Michal, Aistars, Arnie, Matsuda, Yutaka, Mendelsohn, Brian A.
• Format: Article
• Language: English
• Subjects: Poster Abstracts
• ISSN: 1524-0215; 1943-4731

ADCs (antibody-drug conjugates) are powerful new antibody-based drugs that require advanced analytics for full characterization of the antibody charge variants and the drug payload. We set out to develop novel ion exchange UPLC-UV-MS methods that could be used on a small footprint UPLC-UV-MS platform and implemented in core laboratories of academic and industry research facilities. Strong Cation Exchange (SCX) Ultra-Performance Liquid Chromatography (UPLC) was used for highly reproducible and information rich characterization of AJ2, a site-specific ADC with a defined drug antibody ratio (2.0) based on AJICAPâ„¢ technology (Ref 1,2). SCX UPLC-UV and SCX UPLC-MS data is presented for Naked mAb, Linker Modified mAb, Linker and Drug Modified 1.0 and 2.0 DAR ADCs. The key findings of the study are as follows: (1) High separation capability of the SCX UPLC method allowed high resolution characterization of charge variants throughout the ADC manufacturing process. (2) High retention time reproducibility after SCX UPLC column equilibration allowed monitoring of small changes in product quality. (3) SCX UPLC-MS performed with novel MS friendly mobile phase allowed confirmation of linker position, number of attachments, and glycoforms of the ADC heavy chain IdeS cleavage fragments with high mass accuracy to verify linker/drug incorporation. 1) Yamada, K.; Shikida, N.; Shimbo, K.; Ito, Y.; Khedri, Z.; Matsuda, Y.; Mendelsohn, B. A.; Angew. Chem. Int. Ed. 2019, 58, 5592 - 5597. 2) Matsuda, Y.; Robles, V.; Malinao, M-C.; James, S.; Mendelsohn, B. A. Anal. Chem. 2019, in press. DOI: 10.1021/acs.analchem.9b02192