ExSAR utilizes proprietary platform amide hydrogen/deuterium exchange mass spectrometry technology (H/D-Ex) to facilitate drug development and target validation. ExSAR’s H/D-Ex platform utilizes automated sample handling and proprietary software to generate rapid and reproducible measures of amide proton exchange rates. Typical applications involve drug binding interface determination and the detection of alterations in protein structure following protein/protein or protein/ligand complex formation. Other H/D-Ex applications focus on the detection of structure alterations in response to environmental modifications (e.g. new formulation, new buffer, etc.) or following genetic mutation (e.g. wild-type vs. inherited mutation).

Discovery and Development of Biologicals – Epitope Mapping

• Antigen-antibody epitope mapping identification.
• Protein-protein or protein-peptide interface mapping.
• Bioequivalence of follow-on or biogeneric biologicals.
  

Drug Discovery

• Ligand binding site identification
• Binding site and pharmacology correlations (e.g. agonist vs. antagonist).
  
• Enabling “fragment assembly” strategies in the absence of ligand-bound X-ray structures.
  

Structural Biology

• Identification of disordered regions in protein constructs that prevent crystallization
  
• Identification of optimum crystallization conditions
  
• Identifying the effect of point mutations on protein stability (e.g. wild-type protein vs. congenital mutations)

Epitope mapping is an essential aspect of the discovery and development of diagnostic and therapeutic antibodies. Epitope mapping can streamline the selection of lead candidate molecules, particularly where epitope similarity or dissimilarity issues are involved. Intellectual property considerations of patentability, with freedom to operate consequences, can hinge upon the epitope itself. Furthermore, regulatory agencies recommend that prior to use in humans and whenever possible, the protein bearing the reactive epitope should be biochemically defined and the antigenic epitope itself determined (PTC/FDA, 94D-0259).


While an X-ray co-crystal of the antigen:antibody complex remains the gold standard of epitope determination, it is technically challenging, tedious and not always feasible due to the difficulty of obtaining high quality well diffracting crystals. In the absence of a crystal complex, hydrogen/deuterium exchange (e.g. DXMS, H/D-Ex, etc.) is the next best available option. ExSAR employs hydrogen/deuterium exchange mass spectrometry as a tool to identify binding interfaces. Differences in the rate of exchange serve to highlight the location of an epitope. Hydrogen/deuterium exchange (H/D-Ex) was utilized to identify the epitope of a potent neutralizing antibody to IL-17, a homodimeric cytokine involved in a number of pro-inflammatory signaling pathways. Illustrated in red in the above figure are areas where exchange was significantly reduced upon complex formation. Pink highlighted regions indicate areas that were moderaty affected upon complex formation. The epitope was subsequently validated by the X-ray crystal structure of the complex.

H/D Exchange Overview | H/D Exchange Science | H/D Exchange Applications | H/D Exchange Partnering Opportunities
Discovery and Development of Biologicals | Drug Discovery | Structural Biology