Fast Intrinsic NMR Reporters of Molecular Changes Correlating with Biological Activity in Stressed Therapeutic Monoclonal Antibodies

Abstract

The biological activity and stability of monoclonal antibodies (mAbs) can be potentially compromised by chemical and conformational changes and assessing the structural integrity of mAbs is therefore a critical task in biopharmaceutical development. Monitoring higher-order structure (HOS) in formulated mAbs remains a critical analytical challenge. Here, we present an integrated nuclear magnetic resonance (NMR)−based framework for characterizing stress-induced modifications in therapeutic antibodies and correlating them with functional activity. In this study, adalimumab, trastuzumab and bevacizumab in their respective formulations were subjected to controlled light and thermal stress alongside matched unstressed controls, and analyzed using 1D and 2D NMR spectroscopy, liquid chromatography/mass spectrometry (LC/MS), mass photometry, and cell-based bioassays. MS peptide mapping revealed stress-dependent oxidation, deamidation and isomerization with mAb-specific susceptibilities, while NMR experiments such as 1D 1H diffusion-filtered (1D DF), 1D 1H excitation sculpting with perfect echo (1D ESPE) and 2D 1H−13C Heteronuclear Multiple−Quantum Correlation (2D XL-AFHMQC), clearly distinguished samples subjected to different types of stress. Specifically, multivariate statistical analysis performed on 1D DF NMR spectra correlated quantitatively with biological activity and 2D XL-AFHMQC NMR spectra revealed some specific signatures of thermal and light stress. Notably, rapid 1D ESPE experiments, requiring only minutes of acquisition time, selectively detected NMR signals from mobile side-chain resonances from tyrosine, histidine, and asparagine/glutamine residues, together with formulation-derived histidine signals, that acted as intrinsic reporters of stress-induced molecular perturbations. Together, these results establish that naturally mobile molecular reporters captured by NMR provide a rapid, non-destructive readout of antibody integrity, linking structural perturbations, chemical modifications, and biological activity.