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Updated: Nov 13, 2025
Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics
Published on: April 17, 2017
Stability of antibody drug conjugate formulations evaluated using solid-state hydrogen-deuterium exchange mass
Eunbi Cho1, Brendan M Mayhugh2, Jayasree M Srinivasan2
1Department of Industrial and Physical Pharmacy, Purdue University, Robert E. Heine Pharmacy Building, 575 W Stadium Ave, West Lafayette, IN 47907, United States.
Abstract:
Antibody drug conjugates (ADCs) have been at the forefront in cancer therapy due to their target specificity. All the FDA approved ADCs are developed in lyophilized form to minimize instability associated with the linker that connects the cytotoxic drug and the antibody during shipping and storage. We present here solid-state hydrogen-deuterium exchange with mass spectrometric analysis (ssHDX-MS) as a tool to analyze protein structure and matrix interactions for formulations of an ADC with and without commonly used excipients. We compared results of the ssHDX-MS with accelerated stability results using size-exclusion chromatography and determined that the former technique was able to successfully identify the destabilizing effects of mannitol and polysorbate 80. In comparison, Fourier-transform infrared spectroscopy results were inconclusive. The agreement between ssHDX-MS and stressed stability studies supports the potential of ssHDX-MS as a method of predicting relative stability of different formulations.
Insights
Solid-state hydrogen-deuterium exchange with mass spectrometry (ssHDX-MS) can predict antibody drug conjugate (ADC) formulation stability. This method identified destabilizing excipients, aligning with stressed stability studies.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Pharmaceutical Sciences
Background:
- Antibody drug conjugates (ADCs) are crucial in targeted cancer therapy.
- Current FDA-approved ADCs utilize lyophilized formulations to ensure stability during storage and transport.
- Linker stability is critical for ADC efficacy and safety.
Purpose of the Study:
- To evaluate solid-state hydrogen-deuterium exchange with mass spectrometry (ssHDX-MS) as a tool for analyzing ADC protein structure and matrix interactions.
- To assess the impact of common excipients on ADC formulation stability.
- To compare ssHDX-MS results with accelerated stability data.
Main Methods:
- Solid-state hydrogen-deuterium exchange with mass spectrometry (ssHDX-MS) was employed.
- Formulations with and without common excipients (mannitol, polysorbate 80) were analyzed.
- ssHDX-MS data was compared with accelerated stability results from size-exclusion chromatography (SEC).
- Fourier-transform infrared spectroscopy (FTIR) was used for comparative analysis.
Main Results:
- ssHDX-MS successfully identified the destabilizing effects of mannitol and polysorbate 80 on the ADC formulation.
- The results from ssHDX-MS showed strong agreement with accelerated stability studies.
- Fourier-transform infrared spectroscopy (FTIR) provided inconclusive results regarding formulation stability.
Conclusions:
- ssHDX-MS is a valuable tool for analyzing protein structure and matrix interactions in ADC formulations.
- ssHDX-MS demonstrates potential for predicting the relative stability of different ADC formulations.
- This technique can aid in the development of more stable and effective ADC drug products.
