Advanced mass spectrometry workflows for analyzing disulfide bonds in biologics

Proteins are an important class of biologics. Their higher-order structures and therefore their functions are fundamentally determined by the correct formation of disulfide bonds (DSBs), making DSB analysis a central part of their development and production. Mass spectrometry-based bottom-up approaches are most widely used and are further classified according to different methods applied for DSB cleavage. Despite the importance of DSB analysis and the wide range of available methodologies, it is often a challenging and time consuming task. However, due to the current increase in biosimilar development in which animal and clinical trials can be reduced by extensive analytical comparability studies, increased efforts are being made to simplify DSB analysis. As an example of these developments, a matrix-assisted laser desorption/ionization time-of-flight (TOF)/TOF workflow for the automated profiling and identification of DSBs is presented. Furthermore, mass spectrometry based methodologies, which do not identify DSBs directly but measure their influence on the higher-order structure, are also considered.