Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS)

Unique higher-order structures and conformational dynamics are critical for the function and safety of antibody drugs and require fast and reliable analytical methods for their comprehensive characterization. Among the many different separation-based or spectroscopy-based strategies, hydrogen-deuterium exchange mass spectrometry (HDX-MS) is one of the most robust and promising analytical methods for the study of protein conformation and dynamics. At Creative Proteomics, we combine hydrogen/deuterium exchange (HDX) with mass spectrometry (MS) for the study of conformational dynamics, higher-order structures, and ligand-receptor interactions of antibody drugs. Through automation, enhanced MS instrument performance, and data analysis software, our powerful HDX-MS technology provides unique solutions to challenging problems encountered in antibody therapeutic discovery and development.

Introduction of HDX-MS

HDX-MS is one of the key analytical techniques for epitope mapping in protein therapeutics discovery and for comparability studies in product development. The combination of HDX, a "gentle" structure-dependent labeling technique, and the high sensitivity of MS detection provides significant analytical power. In a typical HDX-MS experiment, protein samples are labeled with deuterium at different time points, with or without binding partners. The exchange is quenched by lowering the pH to 2.5 and the deuterated samples are kept at 0 °C to limit back-exchange. After protease digestion, peptides are separated by HPLC/UPLC, and detected by MS (global or "top-dow" HDX-MS analysis) or subjected to proteolytic digestion prior to LC-MS ("bottom-up" or local HDX-MS analysis). By comparison of HDX-MS data before and after the reaction, different HDX spectra in peptides can indicate potential interaction surfaces in protein-small molecule or protein-protein interactions.

Fig. 1 Basic principle and methodology of HDX-MS. (Deng, B. et al., 2016)

Our HDX-MS-Based Antibody Drugs Characterization Services

Creative Proteomics utilizes advanced HDX-MS technology and data analysis tools for comprehensive characterization of antibody drugs. Its applications are focused on two main research areas:

Characterization of Antibody Conformation

• Higher order structure characterization of antibody drugs
• Dynamic changes in conformation due to post-translational modifications
• Any dynamic changes due to drug binding

Probing Protein-Protein Interaction Interfaces

Noncovalent binding events, such as protein-protein interactions and protein-ligand interactions, are involved in many drug action mechanisms. Understanding antigen-antibody interactions and identifying the epitopes are of crucial importance in drug discovery for the design of effective antibody therapeutics.

Epitope Mapping

Epitope mapping is the identification and molecular characterization of the epitope region in a particular antibody–antigen complex. By leveraging the fast and sensitive nature of HDX-MS, we can identify the precise regions on the antibody that interact with specific targets, providing valuable information for the development of therapeutic antibodies.

Protein Aggregation Characterization

By monitoring changes in the deuterium exchange kinetics, we can detect and analyze protein aggregation events, facilitating the formulation and optimization of antibody drugs.

Fig. 2 Scheme for epitope mapping by hydrogen/deuterium exchange mass spectrometry (HDX-MS). (Wei, H. et al., 2014)

Advantages of Our HDX-MS Technology

• High-resolution HDX-MS technology that reveals protein conformational dynamics over a wide range of time scales, providing detailed information about protein folding, surface accessibility, and even conformational changes upon binding to a target.
• Multiple antibody variants or batches can be compared to assess the consistency and stability of their efficacy and quality over time.
• Customized HDX-MS detection methods and strategies are available based on the characteristics of the samples to be tested.
• Professional data analysis, comprehensive analysis reports and provide timely delivery of results.

Creative Proteomics provides advanced HDX-MS technology and related analytical services to help our customers obtain detailed information on the structure, stability and potential interactions of antibody drugs. Contact us to learn more about our service and we will be happy to serve you.

References

1. Deng, B.; et al. Hydrogen deuterium exchange mass spectrometry in biopharmaceutical discovery and development-A review. Analytica chimica acta. 2016, 940: 8-20.
2. Wei, H.; et al. Hydrogen/deuterium exchange mass spectrometry for probing higher order structure of protein therapeutics: methodology and applications. Drug discovery today. 2014, 19(1): 95-102.