Advances in mass spectrometry-based approaches for characterizing monoclonal antibodies: resolving structural complexity and analytical challenges
Abhik Mojumdar, Hee-Jin Yoo, Duck‐Hyun Kim, Ji‐Won Park, Sujin Park, Eunji Jeon, Sunhee Choi, Jung Hoon Choi, Moonhee Park, Geul Bang, Kun Cho
Abstract
Abstract Mass spectrometry (MS)-based intact mass analysis and structural characterization of biotherapeutic proteins such as monoclonal antibodies (mAbs) are a crucial characterization approach from upstream drug development to downstream product analysis. Due to various endogenous modifications leading to the structural heterogeneity and several N -linked glycan species resulting in macro-heterogeneity, it is challenging to characterize the mAbs. Hence, it is essential to understand the micro-heterogeneity of such proteins with high level of complexity which may vary in charge, size, or hydrophobicity. The development of high-throughput native separation techniques hyphenated with MS with high sensitivity and excellent mass accuracy has improved the top/middle down analysis, intact mass detection, subunit analysis, enhanced sequence coverage, and accurate localization of site-specific modifications. In this review, we have focused on the critical inroads taken for the improvement in MS-based techniques to resolve the challenges related to analysis of mAbs. Various MS-based techniques and their role in high-order structural analysis and the progress in software development have been explained, and further, the challenges remaining have been discussed. Graphical Abstract