Amide Hydrogen–Deuterium Exchange: A Fast Tool for Screening Protein Stabilities in Chromatography

Please direct correspondence to Erik Fernandez at [email protected]. Protein unfolding and aggregation can be serious considerations when designing laboratory and preparative chromatographic purification steps. This problem has been studied most thoroughly within the contexts of reversed-phase chromatography and hydrophobic interaction chromatography. However, there are currently no robust methods for resin selection capable of predicting adsorbed-phase protein stability as a function of amino acid sequence, secondary or tertiary structure, or resin characteristics. Conventional experimental techniques such as circular dichroism and intrinsic fluorescence cannot be applied easily to adsorbed protein on conventional chromatographic matrices because of the opacity of the base matrix. Amide hydrogen–deuterium exchange is a protein-labeling technique sensitive to changes in tertiary structure that has been employed extensively to study conformational changes in a variety of protein-folding studies in solution. Here, we present a methodology for using amide hydrogen–deuterium exchange with chromatographic materials as a means to identify buffer and resin conditions that are destabilizing to specific proteins. The approach is illustrated with hen egg white lysozyme and bovine -lactalbumin adsorbed to Phenyl Sepharose 6 FF high substitution resin under strong binding conditions. Labeling patterns directly demonstrates the strong differences in stability during binding that can lead to irreversible adsorption or low recoveries.