Chapter 12 - Biophysical mass spectrometry for biopharmaceutical process development: focus on hydrogen/deuterium exchange

Abstract

Mass spectrometry is an indispensable method to study biopharmaceuticals. It has been widely used to investigate protein primary structure but can also be instrumental in understanding higher-order structure. Among the tools to assess higher-order protein structure and structural dynamics, solution-phase hydrogen/deuterium exchange monitored by mass spectrometry has emerged as a powerful technique to study protein-protein interactions, folding/unfolding pathways, conformational changes induced by ligand binding, etc. On incubation in deuterated buffer, the labile protein backbone amide hydrogens exchange for deuterium if they are readily exposed to the solvent and are not engaged in secondary-structure hydrogen bonding. Quench of the exchange reaction and subsequent proteolytic digestion generates dozens of peptide fragments for which mass increase as a function of incubation period is monitored by mass spectrometry. Comparison of deuterium incorporation between two states of the protein provides insight into conformational stability and solvent accessibility which are in turn dictated by the secondary, tertiary, and/or quaternary structure of the protein. In this chapter, we briefly discuss direct mass measurement and covalent labeling techniques as other ways of assessing protein higher-order structure. We provide an overview of the molecular mechanism of H/D exchange and highlight the technique developments at every stage of the experiment. We also describe several applications for current and future use in the biopharmaceutical environment.