Genetic Engineering and Biotechnology News and Waters recently conducted a webinar on how hydrogen deuterium exchange mass spectrometry (HDX-MS) can improve biotherapeutic drug discovery and development processes.
Our first speaker, Prof. Ganesh Anand — a leading expert on HDX-MS at the National University of Singapore, and a Waters Center of Innovation partner — presented his recent work using HDX-MS to identify lead fragment compounds through protein-ligand binding studies, and shared exciting new research to uncover novel sites within macromolecular assemblies, including viruses.
Our second speaker, Ms. Kai Zhang from the Lilly Biotechnology Center in San Diego, presented her work on epitope and paratope mapping studies of mAbs using HDX-MS, and its ability to advance proteins through biopharmaceutical drug discovery. If you missed the webinar, you can watch it on demand.
We had a great turnout and the speakers received many great questions. Below are some of the highlights from the Q & A session.
What are the application areas of HDX-MS?
Applications of HDX-MS span diverse areas ranging from epitope mapping, structural comparability (innovator vs. biosimilar) and stability studies, formulation studies, protein-ligand (drug) binding studies, lead compound optimization (fragment-based drug design), protein folding/refolding, effects of PTMs, protein characterization, protein structural dynamics, protein-protein interactions studies, and structural biology.
Why don’t you see exchange at side chain positions?
Side chain hydrogens tend to exchange much more quickly, but the off-exchange/back-exchange reaction when they are analyzed by LC-MS is also so quick that the forward exchange is not easily measured. For more details on basic principles of HDX-MS, you can read Engen et.al, Mass Spectrometry Reviews, 2006, 25, 158–170 or visit www.waters.com/hdx.
What kind of reproducibility do you get with HDX-MS?
A study conducted by Houde et.al. from an analysis of multiple peptides and replicate measurements showed that the error or uncertainty of 0.14 Deuterons and a 98% confidence limit of 0.5 Deuterons as a significant difference threshold. A publication by John Engen’s group at Northeastern University described “five levels of replication in HDX-MS experiments: biological, manipulation, labeling, analysis and processing. There is a hierarchy to these replicate levels with biological replication being the highest order and processing replication being lowest order.”
Why do epitope mapping?
Epitope mapping is a key step in locating and characterizing the sites of interaction on the antigen, and is essential for understanding the biological activities and mechanism. It is an important component in the development of effective antibody therapeutics. Mapping the antigen epitope is vital in defining antibody specificity, predicting cross reactivity, assay development, rational vaccine design, and in understanding the fundamental aspects of protein-protein interactions. Finally, it is used to protect intellectual property and for regulatory filing purposes.
What are the benefits of using HDX-MS for epitope mapping?
Epitope mapping has traditionally been done by alanine scanning mutagenesis, protease-protection experiments, NMR, or x-ray crystallography—all of which can be labor intensive, with each approach having unique limitations. HDX-MS is a reliable and robust method for epitope mapping of antibody-antigen interactions in their native solution state and physiological condition. The biggest advantage of the HDX-MS method is the applicability. Unlike the NMR method, there is no size limitation and unlike the x-ray crystallography method, no crystallization is required. Method development in HDX-MS is easier, faster, requires less material, and can be done on more complex proteins.
To learn more on epitope mapping see the following publications:
- The use of hydrogen/deuterium exchange mass spectrometry in epitope mapping (Chemistry Today)
- Hydrogen/Deuterium Exchange Mass Spectrometry for Probing Higher Order Structure of Protein Therapeutics: Methodology and Applications (Drug Discov. Today)
Can you use single point HDX-MS at the protein/peptide level instead of a high-throughput binding assay during fragment-based drug design studies?
Yes, you can use a single point HDX-MS at the protein/peptide level to broadly screen ligands and eliminate those ligands that show no shifts.
Waters has had the honor of leading the commercial development of technologies to perform HDX-MS, and remains the only vendor offering an end-to-end integrated system for HDX-MS studies. Please visit www.waters.com/hdxToolkit to learn more about the power and utility of HDX-MS for higher order structure studies.