Middle‐Down Nuclear Magnetic Resonance for Therapeutic Monoclonal Antibody Glycan Profiling

Abstract Recombinant monoclonal antibodies (mAbs) are widely used across therapeutic areas, and their glycosylation plays a critical role in product quality, manufacturing consistency, and biosimilarity assessment. A middle‐down nuclear magnetic resonance (NMR) method has been developed to profile mAb glycan distribution while preserving the covalent bond between glycan and mAb domains, e.g., the fragment crystallizable (Fc) domain. The workflow uses the immunoglobulin‐degrading enzyme from Streptococcus pyogenes (IdeS) to generate Fc fragments that are purified, denatured, and dissolved in urea prior to high‐resolution two‐dimensional 1 H– 13 C heteronuclear single quantum coherence (2D 1 H– 13 C HSQC) NMR spectrum collection. The resulting anomeric peak distribution reveals major and minor glycan species, including the trimannosyl core, high‐mannose variants, and branch‐specific galactosylation. Compared with traditional glycan mapping, which requires enzymatic cleavage on glycan and liquid chromatography separation, middle‐down NMR provides a non‐invasive analysis that preserves glycan integrity and enables comprehensive, semi‐quantitative monosaccharide profiling. The method requires 3 to 4 days with ∼4 to 5 hr of hands‐on time and can be readily implemented in regulated environments for development and quality control. Basic biochemistry and 2D NMR skills are enough to efficiently apply this protocol in a streamlined workflow. Published 2025. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1 : mAb‐Fc sample preparation Basic Protocol 2 : 2D NMR of HSQC peak profile