A middle-down NMR protocol for therapeutic mAb glycan profiling

Recombinant monoclonal antibody (mAb) use is growing across various therapeutic areas. During production, host cell enzymes glycosylate target mAb proteins. Monitoring glycan distribution is essential for ensuring mAb product quality, manufacturing consistency, and assessing biosimilarity between manufacturers. A middle-down NMR method has been developed to profile mAb glycan distributions, where glycans remain covalently attached to the mAb’s fragment crystallizable (Fc) domain. This technique uses the immunoglobulin-degrading enzyme IdeS from Streptococcus pyogenes to cleave the Fc domain. The purified Fc domain is then denatured and dissolved in urea, and a high-resolution 2D ¹H-¹³C HSQC NMR spectrum is collected. The resulting anomeric peak distribution reveals major and minor glycan species, including the trimannosyl core, high-mannose structures, and branch-specific galactosylation patterns. Compared to the traditional glycan mapping method, which involves enzymatic cleavage and liquid chromatography (LC) separation, the middle-down NMR approach offers a non-invasive, comprehensive analysis that preserves glycan integrity and ensures full monosaccharide coverage. The method takes 3-4 days to complete, with approximately 4-5 hours of hands-on time. It can be easily implemented in regulated environments for both development and quality control workflows, especially where high-resolution glycan profiling is crucial. Basic biochemistry and 2D NMR analysis expertise are required to perform this method.