Dataset Information

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.

SUBMITTER: Hudgens JW

PROVIDER: S-EPMC6745711 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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

Hudgens Jeffrey W JW Gallagher Elyssia S ES Karageorgos Ioannis I Anderson Kyle W KW Filliben James J JJ Huang Richard Y-C RY Chen Guodong G Bou-Assaf George M GM Espada Alfonso A Chalmers Michael J MJ Harguindey Eduardo E Zhang Hui-Min HM Walters Benjamin T BT Zhang Jennifer J Venable John J Steckler Caitlin C Park Inhee I Brock Ansgar A Lu Xiaojun X Pandey Ratnesh R Chandramohan Arun A Anand Ganesh Srinivasan GS Nirudodhi Sasidhar N SN Sperry Justin B JB Rouse Jason C JC Carroll James A JA Rand Kasper D KD Leurs Ulrike U Weis David D DD Al-Naqshabandi Mohammed A MA Hageman Tyler S TS Deredge Daniel D Wintrode Patrick L PL Papanastasiou Malvina M Lambris John D JD Li Sheng S Urata Sarah S

Analytical chemistry 20190514 11

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 ...[more]

PMID: 31045344

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Project description:Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a powerful technique to interrogate protein structure and dynamics. With the ability to study almost any protein without a size limit, including intrinsically disordered ones, HDX-MS has shown fast growing importance as a complement to structural elucidation techniques. Current experiments compare two or more related conditions (sequences, interaction partners, excipients, conformational states, etc.) to determine statistically significant differences at a number of fixed time points and highlight areas of changed structural dynamics in the protein. The work presented here builds on the fundamental research performed in the early days of the technique and re-examines exchange rate calculations with the aim of establishing HDX-MS as an absolute and quantitative, rather than relative and qualitative, measurement. We performed millisecond HDX-MS experiments on a mixture of three unstructured peptides (angiotensin, bradykinin, and atrial natriuretic peptide amide rat) and compared experimental deuterium uptake curves with theoretical ones predicted using established exchange rate calculations. With poly-dl-alanine (PDLA) commonly used as a reference,, we find that experimental rates are sometimes faster than theoretically possible, while they agree much better, and are never faster, with the fully unstructured trialanine peptide (3-Ala). Molecular dynamics (MD) simulations confirm the high helical propensity of the longer and partially structured PDLA peptides, which need as few as 15 residues to form a stable helix and are therefore not suitable as an unstructured reference. Reanalysis of previously published data by Weis et al. at 100 mM NaCl however still shows a discrepancy with predictions based on 3-Ala in the absence of salt, highlighting the need for a better understanding of salt effects on exchange rates. Such currently unquantifiable salt effects prevent us from proposing a comprehensive, universal calibration framework at the moment. Nevertheless, an accurate recalibration of intrinsic exchange rate calculations is crucial to enable kinetic modeling of the exchange process and to ultimately allow HDX-MS to move toward a direct link with atomistic structural models.

Dataset Information

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

Publications

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

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