Complementary Structural Information for Stressed Antibodies from Hydrogen-Deuterium Exchange and Covalent Labeling Mass Spectrometry

Identifying changes in the higher-order structure (HOS) of therapeutic monoclonal antibodies upon storage, stress, or mishandling is important for ensuring efficacy and avoiding adverse effects. Here, we demonstrate diethylpyrocarbonate (DEPC)-based covalent labeling (CL) mass spectrometry (MS) and hydrogen-deuterium exchange (HDX)/MS can be used together to provide site-specific information about subtle conformational changes that are undetectable by traditional techniques. Using heat-stressed rituximab as a model protein, we demonstrate that CL/MS is more sensitive than HDX/MS to subtle HOS structural changes under low stress conditions (e.g., 45 and 55 degrees C for 4 h). At higher heat stress (65 degrees C for 4 h), we find CL/MS and HDX/MS provide complementary information, as CL/MS reports on changes in side chain orientation while HDX/MS reveals changes in backbone dynamics. More interestingly, we demonstrate that the two techniques work synergistically to identify likely aggregation sites in the heat-stressed protein. In particular, the C-H3 and C-L domains experience decreases in deuterium uptake after heat stress, while only the C-H3 domain experiences decreases in DEPC labeling extent as well, suggesting the C-H3 domain is a likely site of aggregation and the C-L domain only undergoes a decrease in backbone dynamics. The combination of DEPC-CL/MS and HDX/MS provides valuable structural information, and the two techniques should be employed together when investigating the HOS of protein therapeutics.

Automated summary

Identifying changes in the higher-order structure (HOS) of therapeutic monoclonal antibodies is crucial for ensuring efficacy and preventing adverse effects. This study demonstrates that DEPC-CL/MS and HDX/MS can be used together to provide valuable structural information about subtle conformational changes that traditional techniques may not detect. The combination of the two techniques proves to be effective in identifying likely aggregation sites and complementary in reporting changes in side chain orientation and backbone dynamics.