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

Jeffrey W. Hudgens, Elyssia S. Gallagher, Ioannis Karageorgos, Kyle W. Anderson, James J. Filliben, Richard Y.C. Huang, Guodong Chen, George M. Bou-Assaf, Alfonso Espada, Michael J. Chalmers, Eduardo Harguindey, Hui Min Zhang, Benjamin T. Walters, Jennifer Zhang, John Venable, Caitlin Steckler, Inhee Park, Ansgar Brock, Xiaojun Lu, Ratnesh PandeyArun Chandramohan, Ganesh Srinivasan Anand, Sasidhar N. Nirudodhi, Justin B. Sperry, Jason C. Rouse, James A. Carroll, Kasper D. Rand, Ulrike Leurs, David D. Weis, Mohammed A. Al-Naqshabandi, Tyler S. Hageman, Daniel Deredge, Patrick L. Wintrode, Malvina Papanastasiou, John D. Lambris, Sheng Li, Sarah Urata

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16 Scopus citations

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 sLab»≤ (0.15 ± 0.01) Da (1σ). All laboratories achieved sLab»≤ 0.4 Da. For immersions of protein at THDX = (3.6 to 25) °C and for D2O exchange times of tHDX = (30 s to 4 h) the reproducibility of back-exchange corrected, deuterium uptake measurements for the 15 laboratories is σreproducibility15 Laboratories(tHDX) = (9.0 ± 0.9) % (1σ). A nine laboratory cohort that immersed samples at THDX = 25 °C exhibited reproducibility of σreproducibility25C cohort(tHDX) = (6.5 ± 0.6) % for back-exchange corrected, deuterium uptake measurements.

Original languageEnglish (US)
Pages (from-to)7336-7345
Number of pages10
JournalAnalytical Chemistry
Volume91
Issue number11
DOIs
StatePublished - Jun 4 2019

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry

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