Accelerating characterization of conformational stability for biologics in Discovery space by combining SEC‑HDX‑MS with molecular modeling
4 affiliations
- 1Discovery Analytical Research, MRL, Merck & Co., Inc.
- 2Digital Insights
- 3Merck & Co., Inc.
- 4Discovery Pharmaceutical Sciences, MRL, Merck & Co., Inc.
Abstract
Conformational stability is a critical attribute for biologics discovery and manufacturability assessment, yet conventional approaches often require substantial material, extensive preparation, or lack sufficient structural resolution for early decision-making. Here, we present a native size-exclusion chromatography hydrogen–deuterium exchange mass spectrometry (SEC-HDX-MS) workflow integrated with molecular modeling to rapidly characterize conformational stability of therapeutic proteins in the discovery space. The workflow was evaluated across monoclonal antibody and bispecific antibody modalities. From a sequence variant comparison, SEC-HDX-MS showed that elevated aggregation did not uniformly reflect global conformational instability; instead, only one variant displayed increased deuterium incorporation, which modeling linked to an elongated, solvent-exposed CDR-H3 loop with increased hydrophobicity. In a representative proprietary mAb (“mAb X”), SEC-HDX-MS revealed that thermally-induced isoaspartate accumulation reduced conformational stability, whereas high-pH stress showed behavior comparable to controls despite higher overall deamidation. Molecular dynamics simulations explained this destabilization through disruption of a backbone Asn43–Ala40 hydrogen bond and altered local coordination after isoAsp formation. Finally, application to a complex bispecific antibody confirmed the workflow’s sensitivity for detecting conformational differences in more complex formats. Collectively, this integrated approach connects global HDX measurements with molecular-level interpretation, requires minimal preparation and limited material, and is compatible with partially purified samples. The workflow enables rapid conformational stability assessment to support candidate ranking, protein engineering, formulation development, and CMC risk assessment during biologics discovery.
Related
- Metaheuristic Optimization of Temporal Surrogates for Early Prediction of Protein Conformational Stability from Short Molecular-Dynamics Trajectories
- Accelerating protein design by scaling experimental characterization
- Characterization of the N7 RNA cap methyltransferase from Trichomonas vaginalis and inhibitor discovery
- Discovery of Novel HDAC8 Inhibitors Through Shape-Based Virtual Screening: Experimental Validation and Characterization of a Promising Quinoline-Based Hit Scaffold
- Computational Discovery and Characterization of Cancer-associated Variants in DNA Transaction Enzymes: Impact of a Glioma–associated mutation on Thymine-DNA Glycosylase
BibTeX
@misc{gao2026accelerating,
title = {{Accelerating characterization of conformational stability for biologics in Discovery space by combining SEC‑HDX‑MS with molecular modeling}},
author = {Zhuo Gao and Elizabeth F. Bayne and Michael Hartmann and Alexander M. Curreri and Gregory F. Pirrone and Alexey A. Makarov},
howpublished = {ChemRxiv},
year = {2026},
month = oct,
doi = {10.26434/chemrxiv.15010843/v1},
url = {https://chemrxiv.org/doi/full/10.26434/chemrxiv.15010843/v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Accelerating characterization of conformational stability for biologics in Discovery space by combining SEC‑HDX‑MS with molecular modeling
AU - Zhuo Gao
AU - Elizabeth F. Bayne
AU - Michael Hartmann
AU - Alexander M. Curreri
AU - Gregory F. Pirrone
AU - Alexey A. Makarov
PY - 2026
DA - 2026/10/22
PB - ChemRxiv
DO - 10.26434/chemrxiv.15010843/v1
UR - https://chemrxiv.org/doi/full/10.26434/chemrxiv.15010843/v1
AB - Conformational stability is a critical attribute for biologics discovery and manufacturability assessment, yet conventional approaches often require substantial material, extensive preparation, or lack sufficient structural resolution for early decision-making. Here, we present a native size-exclusion chromatography hydrogen–deuterium exchange mass spectrometry (SEC-HDX-MS) workflow integrated with molecular modeling to rapidly characterize conformational stability of therapeutic proteins in the discovery space. The workflow was evaluated across monoclonal antibody and bispecific antibody modalities. From a sequence variant comparison, SEC-HDX-MS showed that elevated aggregation did not uniformly reflect global conformational instability; instead, only one variant displayed increased deuterium incorporation, which modeling linked to an elongated, solvent-exposed CDR-H3 loop with increased hydrophobicity. In a representative proprietary mAb (“mAb X”), SEC-HDX-MS revealed that thermally-induced isoaspartate accumulation reduced conformational stability, whereas high-pH stress showed behavior comparable to controls despite higher overall deamidation. Molecular dynamics simulations explained this destabilization through disruption of a backbone Asn43–Ala40 hydrogen bond and altered local coordination after isoAsp formation. Finally, application to a complex bispecific antibody confirmed the workflow’s sensitivity for detecting conformational differences in more complex formats. Collectively, this integrated approach connects global HDX measurements with molecular-level interpretation, requires minimal preparation and limited material, and is compatible with partially purified samples. The workflow enables rapid conformational stability assessment to support candidate ranking, protein engineering, formulation development, and CMC risk assessment during biologics discovery.
ER -