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Interlaboratory Comparison of Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) MMeasurements of the Fab fragment of NISTmAb



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


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 fifteen laboratories. Laboratories reported  78,900 centroids for 430 proteolytic peptide sequences of the heavy and light chains of NISTmAb, providing 100 % coverage. 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 〈σ^Lab 〉  0.15 ±0.01 Da (1σ_x ̅ ), and all laboratories achieved 〈σ^Lab 〉  0.4 Da. For immersions of protein at THDX = (3.6 to 25) oC and for D2O exchange times between 30 s and 4 h the reproducibility of back-exchange corrected, deuterium uptake measurements for the 15 laboratories is σ_reproducibility^(15 Labs) (t_HDX ) = (9.0 ±0.9) % (1σ). A smaller 9 laboratory cohort that immersed samples at THDX = 25 oC exhibited reproducibility of σ_reproducibility^(25C cohort) (t_HDX ) = (6.5 ±0.6) % for back-exchange corrected, deuterium uptake measurements.
Analytical Chemistry


hydrogen-deuterium exchange, inerlaboratory comparison, mass spectrometry, peptide, precision, proteomics, reference material, repeatability, reproducibility.


Hudgens, J. , Karageorgos, I. , Gallagher, E. , Anderson, K. , Filliben, J. , Huang, R. , Chen, G. , Chalmers, M. , Walters, B. , Zhang, J. , Venable, J. , Steckler, C. , , I. , Brock, A. , Lu, X. , Pandey, R. , Chandramohan, A. , , G. , Nirudodhi, S. , Sperry, J. , Rouse, J. , Carroll, J. , Rand, K. , Leurs, U. , Weis, D. , Al-Naqshabandi, M. , Hageman, T. , Wintrode, P. , Lambris, J. , Urata, S. , Bou-Assaf, G. and Espada, A. (2019), Interlaboratory Comparison of Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) MMeasurements of the Fab fragment of NISTmAb, Analytical Chemistry, [online], (Accessed May 21, 2022)
Created May 2, 2019, Updated May 4, 2021