Interlaboratory Comparison of Hydrogen–Deuterium Exchange Mass Spectrometry Measurements of the Fab Fragment of NISTmAb

Abstract

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is an established, powerful tool for investigating protein-ligand interactions, protein folding, and protein dynamics. However, HDX-MS is still an emergent tool for quality control of biopharmaceuticals and for establishing dynamic similarity between a biosimilar and an innovator therapeutic. Because industry will conduct quality control and similarity measurements over a product lifetime and in multiple locations, an understanding of HDX-MS reproducibility is critical. To determine the reproducibility of continuous-labeling, bottom-up HDX-MS measurements, the present interlaboratory comparison project evaluated deuterium uptake data from the Fab fragment of NISTmAb reference material (PDB: 5K8A ) from 15 laboratories. Laboratories reported ∼89 800 centroid measurements for 430 proteolytic peptide sequences of the Fab fragment (∼78 900 centroids), giving ∼100% coverage, and ∼10 900 centroid measurements for 77 peptide sequences of the Fc fragment. Nearly half of peptide sequences are unique to the reporting laboratory, and only two sequences are reported by all laboratories. The majority of the laboratories (87%) exhibited centroid mass laboratory repeatability precisions of ⟨ s^Lab⟩ ≤ (0.15 ± 0.01) Da (1σ_x̅). All laboratories achieved ⟨s^Lab⟩ ≤ 0.4 Da. For immersions of protein at T_HDX = (3.6 to 25) °C and for D₂O exchange times of t_HDX = (30 s to 4 h) the reproducibility of back-exchange corrected, deuterium uptake measurements for the 15 laboratories is σ_{reproducibility}^{15 Laboratories}( t_HDX) = (9.0 ± 0.9) % (1σ). A nine laboratory cohort that immersed samples at T_HDX = 25 °C exhibited reproducibility of σ_{reproducibility}^{25C cohort}( t_HDX) = (6.5 ± 0.6) % for back-exchange corrected, deuterium uptake measurements.