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 dimethyl sulphoxide-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 HDX-MS can provide a reliable approach for fast conformation analysis of proteins in their intended formulations, which could facilitate an increased use of the technique in pharmaceutical development research.
Keywords: LC-MS; analytical biochemistry; analytical chemistry; biopharmaceuticals characterization; formulation; mass spectrometry; physical stability; protein folding/refolding; protein formulation; protein structure.
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