MS-hyphenated separations to characterize proteoforms of antibodies at the intact protein level

This thesis addresses the challenge of characterizing antibodies, which exhibit significant structural heterogeneity and post-translational modifications. The main objective is to develop mass spectrometry (MS)–based analytical strategies for comprehensive characterization of antibody proteoforms, including glycosylation, charge variants, and conjugates. Intact protein analysis was employed to retain structural information that may be lost in peptide-level workflows. To this end, the work focuses on low-flow, MS-compatible separation techniques capable of detecting low-abundance proteoforms with high sensitivity and separation efficiency.New methods were developed to characterize intact monoclonal antibody (mAb) glycoforms using an optimized, polymer-based, monolithic stationary phase with hydrophilic interaction liquid chromatography (HILIC)-MS. Charge variant analysis, traditionally relying on optical detection, was extended through the development of an MS-compatible capillary zone electrophoresis method that enables sensitive separation and identification of acidic and basic variants. For immunoconjugates, an aqueous native size exclusion chromatography–MS approach was established to evaluate payload, while preserving linker stability, and its performance was compared with commonly used analytical techniques. Because mAbs are large and complex biomolecules, multi-attribute analysis is critical for comprehensive structural insight. A salt-based ion-exchange chromatography (IEC) method was developed to separate charge variants, combined with online desalting and fractionation for downstream characterization. An orthogonal workflow integrating IEC and HILIC further enhanced the ability to associate charge variants with corresponding glycoforms, including low-abundance species. Finally, integrated workflows combining intact, subunit, and peptide-level analyses were explored to improve analytical depth and efficiency, and future perspectives for MS-based antibody characterization in biopharmaceutical and endogenous contexts are discussed.