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HDX-MS for Biopharmaceutical Analysis


This project applies hydrogen/deuterium exchange mass spectrometry (HDX-MS) for determining the dynamical structure of therapeutic antibodies, glycoproteins, and proteins; trans-membrane protein receptors; and ligand-receptor interactions. Topics of particular interest include protein-ligand interactions, structure-function relationships between glycan structure and glycoprotein folding, and comparability measurements among innovator drugs and biosimilar drug candidates. This project aims to validate HDX-MS measurement technology by conducting inter-laboratory proficiency testing (aka round robins) and to improve measurement accuracy through parallel HDX-MS (including analyses that employ electron transfer dissociation) and HDX-NMR studies of selected proteins and glycoproteins


Intended Impact

This project is designed to improve, test, and validate hydrogen/deuterium exchange mass spectrometry (HDX-MS) metrology for the determination of dynamical properties of therapeutic proteins and glycoproteins.


  • Explore structure-function relationships between glycan structure and protein folding energies
  • Develop HDX-MS for assessments of dynamical comparability among innovator and candidate biosimilar drugs
  • Measure the reproducibility of proteolytic fragmentation HDX-MS by conducting interlaboratory comparisons on a Fab protein
  • Improve HDX-MS technology for measurements of trans-membrane protein drug targets
  • Measure interactions of biological drugs with surfaces and aggregates
  • Enhance resolution of the HDX-MS method to the single amide level

Technical Approach

The research project uses HDX-MS to measure the D-for-H exchange rates of the amide groups along the backbone of a protein in D2O solution. These rates indicate the protection factors of the amide groups. The protection factors are characteristic of higher order structural features of proteins (e.g., α-helixes, β-sheets …) that are stabilized through hydrogen bonding, disulfide bonds, electrostatic interactions, and hydrophobic forces. Since protection factors can change as the protein binds with ligands or undergoes folding, HDX-MS can provide sensitive diagnostic data evidencing structural differences. HDX-MS is a rapidly evolving metrology. Numerous improvements in automation, instrument resolution and accuracy, and new, more powerful software are being introduced.

Major Accomplishments:

  • Ongoing: NIST HDX-MS Interlaboratory Comparison Study that will determine the Reproducibility of HDX-MS. (Enrollment in this study is closed.)
  • In April 2013 the automated HDX-MS laboratory opened.
  • In NOV 2014 the HDX-MS Interlaboratory Comparison Project sent Fab protein to biopharmaceutical, university, government, and private foundations with the objective of determining the reproducibility of HDX-MS. This project is ongoing.
  • Demonstrated that HDX-MS can detect dynamical differences among glycoforms of the same glycoprotein.
  • Developed a method that measures D-uptake by glycans within glycoproteins.
  • Used HDX-MS and mutagenesis to find the epitope of Proliferating Cell Nuclear Antigen (PCNA) and a previously unknown ligand, TIP. This ligand offers a new vector for attacking solid tumor cancers.


HDX-MS lab

The NIST hydrogen-deuterium exchange mass spectrometry (HDX-MS) laboratory comprises a dual-arm robot (foreground), rapid ultra-high pressure liquid chromatograph (red-orange box), and electrospray ionization tandem mass spectrometer. The mass spectrometer is equipped with an electron transfer dissociation stage that can improve amide resolution and enable top-down HDX-MS studies. This facility resides at the NIST & University of Maryland joint Institute for Bioscience & Biotechnology Research (IBBR).



  • "Cholesterol Hydroxylase CYP46A1: Mapping of the Allosteric Site for Efavirenz, a Drug that Stimulates Enzyme Activity" K. W. Anderson, N. Mast, J. W. Hudgens, J. B. Lin, I. V. Turko, and I. A. Pikuleva, Journal of Biological Chemistry, 291, 11876-11886 (2016). DOI:10.1074/jbc.M116.723577
  • "Mapping Protein–Ligand Interactions with Proteolytic Fragmentation, Hydrogen/Deuterium Exchange-Mass Spectrometry" E. S. Gallagher and Jeffrey W. Hudgens, Methods in Enzymology,566, 357-404. (2016). DOI: 10.1016/bs.mie.2015.08.010
  • "The Effects of Desialylation on Human α1-Acid Glycoprotein-Ligand Interactions" R. Y.-C. Huang and J. W. Hudgens, Biochemistry, 52, 7127-7136 (2013). DOI: 10.1021/bi4011094
  • "A Novel mechanism for regulating the activity of proliferating cell nuclear antigen by a small protein" Z. Li, R. Y.-C. Huang, D. C. Yopp, T. H. Hileman, T. J. Santangelo, J. Hurwitz, J. W. Hudgens, and Z. Kelman, Nucleac Acids Research42(9), 5776-6789  (2014). DOI: 10.1093/nar/gku239 
  • Book Chapter: "Method Validation and Standards in Hydrogen Exchange Mass Spectrometry" J. W. Hudgens, R. Y.-C. Huang and E. D'Ambro, in Hydrogen Exchange Mass Spectrometry of Proteins: Fundamentals, Techniques and Applications; David Weis, ed. (Wiley, 2016).
  •  Book Chapter: "Emerging Technologies to Assess the Higher-Order Structure of Monoclonal Antibodies" J.P. Marino, R.G. Brinson, J.W. Hudgens, J.E. Ladner, D.T. Gallagher, E. S. Gallagher, L.W. Arbogast, and R.Y.-C. Huang In Emerging Technologies To Assess the Higher Order Structure of Monoclonal Antibodies. In State-of-the-Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization Volume 3. Defining the Next Generation of Analytical and Biophysical Techniques, Schiel, J.; Borisov, O.; Davis, D., eds. American Chemical Society: 2015; Vol. 1202, pp 17-43. DOI:10.1021/bk-2015-1202.ch002
Heat maps showing percent deuterium uptake by the PCNA homotrimer 30 and 60 seconds after imersion in D2O, as determined by HDX-MS.

End Date:


Lead Organizational Unit:



Customers: Customers comprise the pharmaceutical industry and research institutions that use HDX-MS.


  • Amgen
  • Biogen Idec
  • Bristol-Myers-Squibb
  • Genentech
  • Genomics Institute of the Novartis Research Foundation
  • Investigacion Básica Lilly, S.A.
  • Janssen Research & Development, LLC
  • Medimmune
  • National University of Singapore
  • The Scripps Research Institute
  • University of California, San Diego
  • University of Copenhagen
  • University of Kansas
  • University of Maryland, School of Pharmacy
  • University of Pennsylvania School of Medicine



Dr. Jeffrey W. Hudgens
Tel. 240-314-6485
Institute for Bioscience and Biotechnology Research
National Institute of Standards and Technology
9600 Gudelsky Drive, Rockville, MD 20850