HDX-MS for Biopharmaceutical Analysis

Summary

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

Description

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.

Objectives

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.

Publications

"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 Research, 42(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