Methyl-specific NMR of therapeutic antibodies: cost-effective isotopic labeling strategies in CHO cells for high-resolution structural characterization.

Monoclonal antibodies (mAbs) are essential therapeutic agents whose efficacy depends critically on the integrity of their higher-order structure (HOS). Subtle perturbations in HOS, arising from post-translational modifications, chemical degradation or formulation conditions, can impair antigen binding or induce immunogenicity. Nuclear Magnetic Resonance (NMR) spectroscopy, and particularly methyl NMR, offers a powerful means to probe the structure and dynamics of large proteins, such as antibodies, at atomic scale resolution. While 2D 1H-13C methyl correlation spectra at natural abundance provide valuable structural fingerprints, their limited resolution and sensitivity hamper sophisticated NMR studies. Isotopic labeling of methyl groups with 13CH₃, combined with tailored deuteration, greatly enhances spectral quality. However, such labeling strategies remain underdeveloped in mammalian systems like CHO cells, which are the standard hosts for therapeutic antibody production. Here, we report an optimized labeling strategy enabling selective incorporation of the ¹³CH₃-labeled methyl groups from the six methyl-bearing amino acids into antibodies expressed in CHO cells. This includes enzymatic synthesis of regio- and stereoselectively labeled isoleucine and valine with partial deuteration and an optimized protocol for their incorporation into CHO-produced proteins. When applied to an anti-LAMP1 therapeutic antibody, this strategy yields highly resolved methyl NMR spectra, enabling comprehensive HOS assessment. It allows the transfer of previously assigned Fab and Fc methyl resonances, resulting in approximately 84% of methyl signals being assigned in the intact mAb. This work establishes a robust framework for NMR-based structural analysis of glycosylated antibodies directly from mammalian expression systems.