Hydrogen/deuterium
exchange with mass spectrometry (HDX-MS) is
a widely used technique to probe protein structural dynamics, track
conformational changes, and map protein–protein interactions.
Most HDX-MS studies employ a bottom-up approach utilizing the acid
active protease pepsin to digest the protein of interest, often utilizing
immobilized protease in a column format. The extent of proteolytic
cleavage will greatly influence data quality and presents a major
source of variation in HDX-MS studies. Here, we present a simple cocktail
of commonly available peptides that are substrates of pepsin and can
serve as a rapid check of pepsin column activity. The peptide-based
assay requires no system modifications and provides an immediate readout
to check and benchmark pepsin activity across different HDX-MS platforms.
Hydrogen exchange-mass spectrometry (HX-MS) is widely recognized for its potential utility for establishing the equivalence of the higher-order structures of proteins, particularly in comparability and similarity contexts. However, recent progress in the statistical analysis of HX-MS data has instead placed an emphasis on significance testing to identify regions of proteins where there are significant differences in HX between two or more protein states. In the cases involving assessment of similarity or equivalence of the higher-order structure of different protein samples (e.g., biosimilars), significance testing of HX-MS data is unsuitable. To meet this need, we have adapted the univariate two one-sided test (TOST) equivalence testing method for HX-MS data. Equivalence acceptance criteria were determined using maximum deviations from randomized resampling of truly equivalent samples to define hybrid equivalence criteria (maximum deviation of true equivalents, MDTE). Application of the TOST-MDTE test on differential HX-MS measurements of wild-type and mutated maltose-binding proteins demonstrates that the equivalence testing method was fit-for-purpose. Three infliximab biosimilars (Remsima, Renflexis, and Inflectra) were found to be equivalent to their Remicade reference product based on differential HX-MS measurements, while 5% deglycosylated NIST mAb was not statistically equivalent to the unmodified NIST mAb reference.
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