For higher order structure studies, hydrogen deuterium exchange with mass spectrometry – alternatively referred to as H/D exchange, HDX-MS, or HXMS – has become indispensable for biotech companies.
Recent innovations in LC-MS, automation, and informatics technologies have converted HDX-MS from a complex academic exercise to a robust tool for discovery and development of protein drugs.
The HDX-MS Tool Box. Supporting materials to help you demonstrate the value of HDX-MS data within your organization. Get access to overview Powerpoint slides, key literature citations and summary of their key points and information about Waters solutions. |
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This video demonstrates how LC-MS technologies, including HDX-MS, can help biosimilar developers thoroughly understand the structure and variation of the innovator product to set specifications for their biosimilar. |
Professor John Engen, a leading expert on HDX-MS, presents “How hydrogen exchange mass spectrometry can help investigate protein higher order structure” at HOS 2015. |
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Dr Ganesh Anand, of the National University of Singapore, and a Waters Center of Innovation Program honoree, describes how he uses HDX-MS to structurally analyze biological molecules. |
In this webinar recording, Ganesh Anand, Associate Professor, Ph.D., National University of Singapore and Kai Zhang, Consultant Biologist, Lilly Biotechnology Center, discuss their experiences using HDX-MS. |
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Quality Assistance, an analytical CRO based in Belgium, talks about how they will be using the HDX-MS solution to conduct advanced characterization for their customers. |
Rachel Garlish, PhD, of UCB, talks about how they rely on HDX-MS to better understand the binding of molecules to select proteins and therefore optimize molecules for future drugs. |
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Jing Fang, Senior Scientist at Waters Corporation, presents "Contribution of HDX-MS to Evaluate the Conformational Similarity of Biosimilars to Reference Products." Originally presented at PAMS 2015. |
Learn more about the ACQUITY UPLC M-Class System with HDX Technology and high-resolution mass spectrometry. |
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Read the app note, “Pressurized Online Pepsin Digestion of mAb IgG2 for Hydrogen Deuterium Exchange Mass Spectrometry”. Features the Enzymate™ Digestion Column. |
Waters has turned what was a highly manual analytical process, where success was dependent on technical skill, into an integrated and automated system solution. This system leverages high-pressure nano- to microscale UPLC separations and high-resolution MSE mass spectrometry to answer important questions about biotherapeutic protein dynamics, conformation, and interactions, including:
Waters offers the only commercially-available complete system for HXMS studies, pairing our ACQUITY UPLC M-Class System with HDX Technology with a Waters mass spectrometer such as the Xevo G2-S QTof, and HDX application-specific software, DynamX.
THE SYSTEM
The unprecedented system-level design integrates all the steps necessary to accurately, reproducibly – and most of all, easily – perform protein conformation studies. The Waters UPLC HDX MS system removes the barriers to adopting high resolution LC and MS for routine studies of protein conformational changes, making HDX a core competency for your laboratory.
ACQUITY UPLC® M-Class with HDX Technology
A core element of the system is the HDX Manager, which is purpose-built to manage samples separated under higher operating pressures, at 0° C and with no compromise in deuterium recovery. Its improved chromamatographic resolution is paired with a state-of-the-art mass spectrometer such as the Xevo® G2-S QTof or SYNAPT® G2-Si HDMS systems. The system also integrates on-line proteolysis with trapping capability and MSE technology for maximum sequence coverage of complex mixtures of peptic digest.
Enzymate™ Online Digestion Column
An immobilized pepsin column packed with mechanically strong BEH particles, the Enzymate BEH Pepsin Column (2.1 x 30 mm, 5 µm) is capable of operating at 15,000 psi, whose digestion efficiency is enhanced at both low and high digestion pressures.
HDX Phosphorylase B Check Standard
Used for digestion and separation experiments.
Mass Spectrometry
Ion mobility separation (IMS) is used in the UPLC/MS HDX workflow, providing additional, orthogonal separations to chromatography and mass dimensions. Overlapping interfering ions are successfully resolved by IMS and displayed in DynamX HDX Software.
DynamX Data Analysis Software v 3.0
In HDX studies, data are produced across multiple time points, multiple species, and with replicates. Curating this data manually is not time-efficient and requires expert interpretation. Interpretation of this data is a repetitive process that requires counting and measuring spectra.
DynamX Software is designed to systematically select spectra with predetermined criteria and measure the mass change of the deuterated form. The software automation was greatly simplified by utilizing the sharper peaks and better separation available with UPLC, and the comprehensive nature of the MSE detection. This automation, along with the capability to sort and display data, has been an important advance.
DynamX tracks all peptides that are reproducibly found in replicates, ensuring consistency in monitoring the deuterium exchange. The software also calculates the amount of deuteration and displays the results in convenient comparative views: uptake curves, a butterfly chart, and a difference chart. Data processing time is significantly reduced from months for manual processing to hours for automated processing.
DynamX software helps researchers assess possible conformational changes in their proteins quickly. It simplifies hydrogen-deuterium exchange interpretation by performing these tasks:
PRINCIPLES OF HDX
Hydrogen deuterium exchange (HDX) MS works in many applications, including understanding how small-molecule therapeutics bind to protein targets, and performing epitope mapping. With this technology, deuterium in a solution replaces the amide hydrogen in a biomolecule’s backbone, and MS measures these changes. The results reveal information about protein structure.
Basic principles of hydrogen deuterium exchange
Amide hydrogen on the backbone of a biomolecule exchanges with deuterium in solution at different rates partly dependent on conformation. These changes can be measured by MS and therefore the degree of activity at various sites can be inferred. These locations can be identified and mapped to the primary sequence, which also allows easier visualization. Relative folding and dynamics can be determined from the different rates of uptake at different locations.
Learn more: "Performing Hydrogen/Deuterium Exchange with Mass Spectrometry" in BioPharm International.
Using UPLC/MSE provides better coverage
Using MSE means that a comprehensive dataset can be generated. All of the peptides are measured without bias by peptide signal intensity. This is an important feature for HDX studies where key information might be found in low-intensity peptides.
HDX by mass spectrometry opens new windows into the dynamics of biomolecules by providing information on the relative deuterium uptake of different conformations of a protein, or locations within a protein. Recent developments have made HDX with mass spectrometry a more accessible tool to study the dynamics of higher order structure (HOS) of biotherapeutics.
Industry and regulatory trends have very clearly moved towards an emphasis on conformation and the relationships between biomolecules. Therefore HDX is a key tool that is being rapidly adopted in more areas of biotherapeutic analysis.
MSE is the ultimate technology for comprehensive, reproducible profiling and characterization.
Learn more.
"Analytical tools for characterizing biopharmaceuticals and the implications for biosimilars" by HDX thought leaders Steven Berkowitz and John Engen.
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Professor John Engen at Northeastern University is a pioneer in the use of hydrogen deuterium exchange and ion mobility mass spectrometry, HDX MS.
Learn about Dr. Engen's lab