Getting to the core of protein pharmaceuticals--Comprehensive structure analysis by mass spectrometry.

Protein pharmaceuticals are the fastest growing class of novel therapeutic agents, and have been a major research and development focus in the (bio)pharmaceutical industry. Due to their large size and structural diversity, biopharmaceuticals represent a formidable challenge regarding analysis and characterization compared to traditional small molecule drugs. Any changes to the primary, secondary, tertiary or quaternary structure of a protein can potentially impact its function, efficacy and safety. The analysis and characterization of (structural) protein heterogeneity is therefore of utmost importance. Mass spectrometry has evolved as a powerful tool for the characterization of both primary and higher order structures of protein pharmaceuticals. Furthermore, the chemical and physical stability of protein drugs, as well as their pharmacokinetics are nowadays routinely determined by mass spectrometry. Here we review current techniques in primary, secondary and tertiary structure analysis of proteins by mass spectrometry. An overview of established top-down and bottom-up protein analyses will be given, and in particular the use of advanced technologies such as hydrogen/deuterium exchange mass spectrometry (HDX-MS) for higher-order structure analysis will be discussed. Modification and degradation pathways of protein drugs and their detection by mass spectrometry will be described, as well as the growing use of mass spectrometry to assist protein design and biopharmaceutical development.