Toward site-specific characterization of structural perturbations on glycosylated Fc using NMR at natural abundance.

Monoclonal antibodies (mAbs) are leading therapeutic agents due to their high specificity and limited side effects. Ensuring their structural integrity under stress and maintaining batch consistency require robust quality control. Methyl 2D NMR has emerged as a powerful tool to probe mAb structure at natural isotopic abundance, enabling spectral fingerprint comparisons across production batches to detect subtle structural changes. However, extracting atomic-level structural information requires assignment of methyl resonances to their amino acids. While such assignments are available for several antigen-binding fragments (Fabs), no comprehensive assignment has been reported for the crystallisable fragment (Fc). In this study, we present the methyl group assignment of the 50-kDa Fc fragment of an immunoglobulin G1 (IgG1) antibody. Using cell-free expression, strategic isotopic labelling, and high-quality 2D and 3D NMR experiments, we successfully assigned 94% of methyl resonances of a non-glycosylated Fc. Given that therapeutic mAbs are typically produced in Chinese Hamster Ovary (CHO) cells, we transferred this assignment to the methyl spectrum of a glycosylated Fc fragment obtained by the enzymatic cleavage of a CHO-produced mAb at natural abundance, achieving 83% assignment coverage. This assignment was then used to investigate the impact of methionine oxidation on Fc structure at atomic resolution using NMR. The methyl group assignment transforms 2D methyl NMR fingerprinting into a powerful tool for quality control. It enables the direct comparison of spectra acquired on mAbs produced at natural abundance, allowing the detection and localisation of chemical modifications and structural changes without the need for isotopic labelling. This approach offers a robust solution for monitoring the structural integrity of therapeutic antibodies throughout development and manufacturing.