Multi-level structural and functional characterization of therapeutic glycoproteins by mass spectrometry

Therapeutic proteins have been successfully developed for advancing medical treatments. They are usually large molecules produced by host cells and have a high degree of complexity compared to synthetic small molecule-based therapeutics. The complexity is mainly attributed to the heterogenic nature of post-translational modifications (PTMs). Glycosylation is one of the main drivers of protein heterogeneity. Since each modification may potentially impact the safety and efficacy, analyticalmethods for the structural and functional characterization of protein-based therapeutics are highly demanded. This thesis presents novel mass spectrometry (MS)-based methods for the analysis of therapeutic glycoproteins. It covers aspects of glycobioinformatics and sample preparation for improved bottom-up glycoproteomic analysis. Further, the profiling of complex intact glycoproteins by matrix-assisted laser desorption ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) MS is demonstrated. Finally, Fc gamma receptor III affinity chromatography combined with online MS detection is presented for novel proteoform-resolved structure-function insights of monoclonal antibodies.