Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) Technology

Unique higher-order structures (HOS) and conformational dynamics are critical to the function and safety of protein therapeutics, requiring fast and reliable analytical methods to fully characterize them. At CD Formulation, we combine hydrogen-deuterium exchange (HDX) with mass spectrometry (MS) and integrate them into our advanced protein characterization platform to study conformational changes of proteins upon binding to different ligands, at different pH or temperature conditions. Our goal is to provide unique solutions to challenging problems encountered in protein therapeutic discovery and development.

What is HDE-MS Technology?

HDX-MS is a powerful biophysical technique used to study the structural dynamics of proteins and protein complexes. The technique exploits the labile nature of protons present on protein backbone amides and involves monitoring the exchange of hydrogen atoms with deuterium atoms when a protein sample is exposed to a deuterated solvent. Amide hydrogens on the biomolecule backbone exchange with deuterium in solution at different rates, which are somewhat conformation-dependent and can be used to infer information about protein structure and conformation. Coupled with MS, it can be used to measure the rate of deuterium uptake. The technique has few limitations in terms of protein size, flexibility or reaction conditions and can be performed in solution at different pH and temperature under controlled redox conditions.

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

Our Services Related to HDX-MS

Thanks to decades of experience in supporting protein/peptide biopharmaceutical development and manufacturing using HDX-MS technology, our team of highly qualified experts offers a range of HDX-MS-related services to accelerate the implementation and success of your projects.

Our scientists will work with you to continuously optimize HDX-MS programs to support all stages of protein/peptide drug development and manufacturing - from early protein/peptide drug discovery to late-stage drug development and post-release testing under GMP.

Utilizing cutting-edge HDX-MS technology, we support the following protein/peptide characterization plans, including but not limited to:

Protein Conformation Analysis

The correct conformation of a protein is a key factor in ensuring its normal function. Our scientists use HDX-MS technology to analyze the key structural features and dynamic properties of proteins, including tertiary and quaternary structures.

Antibody Epitope Mapping

Epitope mapping is a critical step in the development of therapeutic antibodies and vaccines, especially during the lead-generation phase of drug discovery. Both the FDA and EMEA recommend epitope mapping as a tool to uncover information about specific binding sites in therapeutic antibodies. Our scientists use HDX-MS to perform epitope mapping to support the development of drug candidates and to assess immune responses elicited by vaccines or other therapies.

Protein Aggregation Characterization

The stability of aggregation affects the rate of hydrogen-deuterium exchange. Aggregates are more stable and the exchange rate of hydrogen atoms is usually slower. Our scientists compare the hydrogen-deuterium exchange patterns of monomers and aggregates to identify conformational changes, including changes in α-helices and β-folds, so as to study the kinetics of protein aggregation under different conditions, such as pH, salt concentration, temperature changes, etc.

In addition to the key plans mentioned above, we also use HDX-MS technology to support the following studies, including but not limited to:

• Protein-protein interaction studies
• Exploring the structure-function relationship between glycan structure and protein folding energy.
• Evaluating the dynamic comparability between innovator drugs and biosimilar candidates.

Our Procedure for HDX-MS Analysis

At CD Formulation, we offer the following two HDX-MS protocols, including:

Local/Bottom-up HDX-MS

The most common strategy for this technology involves in-solution labeling and enzymatic digestion of proteins into peptides, which are then analyzed using mass spectrometry. This ensures complete sequence coverage and captures specific region information from the protein.

Typical workflow includes:

• Labeling: Incubate the protein sample in D₂O buffer to allow H/D exchange (10 seconds to several hours).
• Quenching: Reduce pH and temperature to prevent further exchange or back exchange.
• Digestion: Digest the protein with a suitable protease into shorter peptides for analysis.
• LC-MS analysis: The mass spectrometer analyzes the peptides, producing a set of LC-MS chromatograms corresponding to the peptide groups at different labeling times.
• Data processing: Use data analysis software to determine the uptake/incorporation of deuterium in the digested peptides and plot graphs at multiple time points.

Fig. 2 Workflow for local or bottom-up HDX-MS. (CD Formulation)

Native / Up-bottom HDX-MS

This protocol is an alternative to bottom-up HDX-MS.

In intact/up-down HDX-MS, proteins are introduced to the mass spectrometer after deuterium exchange without any enzymatic digestion. This technique allows the measurement of deuterium uptake at the intact level.

Advantages of Our HDX-MS Technology

• High efficiency: HDX-MS experiments can usually be completed in weeks, while structure determination by X-ray crystallography or NMR may take months.
• High sensitivity and accuracy: Allows accurate characterization of proteins with different molecular weights from ~10 kDa to ~2MDa.
• High resolution: HDX-MS technology can reveal protein conformational dynamics over a wide range of time scales, providing detailed information about protein folding, surface accessibility, and even conformational changes when binding to targets.
• Low sample volume required: HDX-MS requires only 0.1 mg of protein, far less than X-ray crystallography or NMR.
• No size limit: Unlike NMR, there is no theoretical size limit for proteins analyzed by HDX-MS, which makes it ideal for studying large biomolecular complexes such as membrane proteins.
• No chemical labeling required: HDX-MS does not require additional chemical labels, such as isotope labeling required for NMR.

Custom HDX-MS Services

Why Choose Our HDX-MS Technology?

• Our team of experienced professionals with extensive knowledge in mass spectrometry and structural biology ensures high-quality services and accurate results.
• We have accumulated decades of expertise and successful project experience using HDX-MS technology to support protein/peptide biopharmaceutical development.
• We utilize state-of-the-art HDX-MS instrumentation that allows for high sensitivity and resolution, leading to better-quality data.
• Our services offer a wide range of applications, from studying protein conformational dynamics to understanding protein-ligand interactions and protein-protein interactions.
• We provide customized experimental designs to meet the specific needs of your research project, ensuring the collection of relevant and meaningful data.
• We not only provide raw data but also comprehensive analysis and interpretation support to help you understand the meaning of your results.
• We have a strong track record of success, with many satisfied customers benefiting from our HDX-MS services.

Publication

CD Formulation aims to provide a powerful analytical tool for protein/peptide HOS and conformational dynamics characterization. Please feel free to contact us if you are interested in our services. Learn how our HDX-MS technology can support the smooth implementation of your protein/peptide biopharmaceutical program.

How It Works

STEP 1

Submit a service request describing your specific research or development need.

STEP 2

We'll email you to provide your quote and confirm order details if applicable.

STEP 3

Execute the project with real-time communication, and deliver the final report promptly.