The expanding role of therapeutic proteins in medicine has increased the need for analytical tools that can report on higher-order protein structure quickly, with high structural resolution, and minimal sample consumption. Hydrogen exchange mass spectrometry (HX-MS) is gaining acceptance for its potential to meet these needs for structural interrogation in pharmaceutical discovery and development contexts. Here, two examples will be used to illustrate the capabilities of HX-MS: measuring structural comparability of proteins and mapping contact sites in monoclonal antibodies undergoing reversible self-association. In a simulated comparability study, we were able to detect the presence of as little as 5% misfolded protein with HX-MS. Here, we used wild-type maltose-binding protein spiked with small fractions of a single-point mutated form representing the misfolded protein. Although the 5% misfolded sample was indistinguishable from 100% wild-type protein using conventional biophysical methods (differential scanning calorimetry, size-exclusion chromatography, and far UV circular dichroism), statistically significant differences were found in the HX-MS data. No statistically significant differences were found in null experiments where 100% wild-type samples were compared. Using a novel hydrogen exchange process, we have mapped the interfaces of monoclonal antibodies undergoing reversible self-association at high concentration (&gt;50 mg/mL). To maintain the high concentration that promotes reversible self-association, hydrogen exchange was initiated by reconstitution of lyophilized antibody with D2O. Comparison of HX-MS data obtained using the reconstitution method at high and low concentrations revealed significant increases in protection within the complimentarity-determining regions of the light and heavy chains, indicating that these regions form the interfaces during reversible self-association.

Hydrogen exchange mass spectrometry:  An enabling tool for therapeutic protein development

251st National Meeting (2016)

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