Hydrogen-Deuterium Exchange Dynamics of NISTmAb Measured by Small Angle Neutron Scattering

Róisín B. Donnelly; Sai Venkatesh Pingali; Luke Heroux; Robert G. Brinson; Norman J. Wagner; Yun Liu

doi:10.1021/acs.molpharmaceut.3c00751

Hydrogen-Deuterium Exchange Dynamics of NISTmAb Measured by Small Angle Neutron Scattering

Róisín B. Donnelly, Sai Venkatesh Pingali, Luke Heroux, Robert G. Brinson, Norman J. Wagner, Yun Liu

Research output: Contribution to journal › Article › peer-review

Abstract

Understanding protein dynamics and conformational stability holds great significance in biopharmaceutical research. Hydrogen-deuterium exchange (HDX) is a quantitative methodology used to examine these fundamental properties of proteins. HDX involves measuring the exchange of solvent-accessible hydrogens with deuterium, which yields valuable insights into conformational fluctuations and conformational stability. While mass spectrometry is commonly used to measure HDX on the peptide level, we explore a different approach using small-angle neutron scattering (SANS). In this work, SANS is demonstrated as a complementary and noninvasive HDX method (HDX-SANS). By assessing subtle changes in the tertiary and quaternary structure during the exchange process in deuterated buffer, along with the influence of added electrolytes on protein stability, SANS is validated as a complementary HDX technique. The HDX of a model therapeutic antibody, NISTmAb, an IgG1κ, is monitored by HDX-SANS over many hours using several different formulations, including salts from the Hofmeister series of anions, such as sodium perchlorate, sodium thiocyanate, and sodium sulfate. The impact of these formulation conditions on the thermal stability of NISTmAb is probed by differential scanning calorimetry. The more destabilizing salts led to heightened conformational dynamics in mAb solutions even at temperatures significantly below the denaturation point. HDX-SANS is demonstrated as a sensitive and noninvasive technique for quantifying HDX kinetics directly in mAb solution, providing novel information about mAb conformational fluctuations. Therefore, HDX-SANS holds promise as a potential tool for assessing protein stability in formulation.

Original language	English
Pages (from-to)	6358-6367
Number of pages	10
Journal	Molecular Pharmaceutics
Volume	20
Issue number	12
DOIs	https://doi.org/10.1021/acs.molpharmaceut.3c00751
State	Published - Dec 4 2023

Funding

This study was funded by the National Science Foundation (DMR-1935956). SANS experiments were performed under beamline proposals #275821.1 and #28784.1 at the Bio-SANS instrument, which is supported by the U.S. Department of Energy, Office of Science, through the Office of Biological and Environmental Research under contract FWP ERKP291, using the High Flux Isotope Reactor facility, a DOE Office of Science User Facility operated by the Oak Ridge National Laboratory. The laboratory resources at the Institute for Bioscience and Biotechnology Research in Rockville, Maryland, are gratefully acknowledged. Certain commercial equipment, instruments, or materials (or suppliers, or software, ...) are identified in this paper to foster understanding. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose. Y.L. acknowledges the support provided by the Center for High Resolution Neutron Scattering, a partnership between the National Institute of Standards and Technology and the National Science Foundation under agreement no. DMR-2010792. This article was authored by UT-Battelle, LLC, under contract DE-AC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan). This study was funded by the National Science Foundation (DMR-1935956). SANS experiments were performed under beamline proposals #275821.1 and #28784.1 at the Bio-SANS instrument, which is supported by the U.S. Department of Energy, Office of Science, through the Office of Biological and Environmental Research under contract FWP ERKP291, using the High Flux Isotope Reactor facility, a DOE Office of Science User Facility operated by the Oak Ridge National Laboratory. The laboratory resources at the Institute for Bioscience and Biotechnology Research in Rockville, Maryland, are gratefully acknowledged. Certain commercial equipment, instruments, or materials (or suppliers, or software, ...) are identified in this paper to foster understanding. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose. Y.L. acknowledges the support provided by the Center for High Resolution Neutron Scattering, a partnership between the National Institute of Standards and Technology and the National Science Foundation under agreement no. DMR-2010792. This article was authored by UT-Battelle, LLC, under contract DE-AC05–00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan ( http://energy.gov/downloads/doe-public-access-plan ).

Funders	Funder number
DOE Public Access Plan
United States Government
National Science Foundation	DMR-1935956, DE-AC05–00OR22725, DMR-2010792
U.S. Department of Energy
National Institute of Standards and Technology
Office of Science
Biological and Environmental Research	FWP ERKP291
Oak Ridge National Laboratory

Keywords

HDX
NISTmAb
SANS
monoclonal antibody
protein dynamics
stability

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10.1021/acs.molpharmaceut.3c00751

Cite this

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abstract = "Understanding protein dynamics and conformational stability holds great significance in biopharmaceutical research. Hydrogen-deuterium exchange (HDX) is a quantitative methodology used to examine these fundamental properties of proteins. HDX involves measuring the exchange of solvent-accessible hydrogens with deuterium, which yields valuable insights into conformational fluctuations and conformational stability. While mass spectrometry is commonly used to measure HDX on the peptide level, we explore a different approach using small-angle neutron scattering (SANS). In this work, SANS is demonstrated as a complementary and noninvasive HDX method (HDX-SANS). By assessing subtle changes in the tertiary and quaternary structure during the exchange process in deuterated buffer, along with the influence of added electrolytes on protein stability, SANS is validated as a complementary HDX technique. The HDX of a model therapeutic antibody, NISTmAb, an IgG1κ, is monitored by HDX-SANS over many hours using several different formulations, including salts from the Hofmeister series of anions, such as sodium perchlorate, sodium thiocyanate, and sodium sulfate. The impact of these formulation conditions on the thermal stability of NISTmAb is probed by differential scanning calorimetry. The more destabilizing salts led to heightened conformational dynamics in mAb solutions even at temperatures significantly below the denaturation point. HDX-SANS is demonstrated as a sensitive and noninvasive technique for quantifying HDX kinetics directly in mAb solution, providing novel information about mAb conformational fluctuations. Therefore, HDX-SANS holds promise as a potential tool for assessing protein stability in formulation.",

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Hydrogen-Deuterium Exchange Dynamics of NISTmAb Measured by Small Angle Neutron Scattering

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