Statistical Equivalence Testing of Higher-Order Protein Structures with Differential Hydrogen Exchange-Mass Spectrometry (HX-MS)

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.