Rapid Conformational Analysis of Protein Drugs in Formulation by Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS)

Hydrogen Deuterium Exchange coupled to Mass Spectrometry (HDX-MS) has become an established method for analysis of protein higher-order structure. Here, we use HDX-MS methodology based on manual Solid-Phase Extraction (SPE) to allow fast and simplified conformational analysis of proteins under pharmaceutically relevant formulation conditions. Of significant practical utility, the methodology allows global HDX-MS analyses to be performed without refrigeration or external cooling of the setup. In Mode 1, we used DMSO-containing solvents for SPE, allowing the HDX-MS analysis to be performed at acceptable back exchange levels (<30%) without the need for cooling any components of the setup. In mode 2, SPE and chromatography were performed using fast isocratic elution at 0 °C resulting in a back exchange of 10-30%. Real-world applicability was demonstrated by HDX-MS analyses of interferon-β-1a in formulation, using an internal HDX reference peptide (P7I) to control for any sample-to-sample variations in back exchange. Advantages of the methodology include low sample use, optimized excipient removal using multiple solvents, and fast data acquisition. Our results indicate that the SPE-HDX-MS system can provide a reliable approach for fast conformation analysis of proteins in their intended formulations and could facilitate an increased use of HDX-MS in pharmaceutical development research.