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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
  • Validate HDX-MS trueness through inter-laboratory proficiency testing through inter-laboratory testing
  • 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 across epitopes

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:

  • 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.
  • 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).



  • "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/Deuterium Exchange Mass Spectrometry" J. W. Hudgens, R. Y.-C. Huang and E. D'Ambro, in Hydrogen-Deuterium Exchange Mass Spectrometry: Fundamentals, Techniques and Applications; David Weis, ed. (Wiley, 2014).
  •  Book Chapter: "Ch. 18: Current and Emerging Technologies to Characterize the Higher-Order Structure of Monoclonal Antibodies" J.P. Marino, R.G. Brinson, J.E. Ladner, D.T. Gallagher, L.W. Arbogast, R. Y.-C. Huang , and J.W. Hudgens in Current, State of the Art, and Emerging Technolgies for the Analysis of Monoclonal Antibodies (ACS, 2014), John Schiel, ed.
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.




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