A middle-down NMR protocol for therapeutic mAb glycan profiling

Recombinantly produced monoclonal antibody (mAb) drugs (including biosimilar versions) use continues to expand for multiple therapeutic areas. During the biologic manufacturing process, target proteins can be glycosylated by cellular enzymes in host cells. Assessment of mAb product quality includes its glycan distribution which is a key attribute to monitor drug manufacturing consistency and assess biosimilarity between different manufacturers. A “middle-down NMR” analytical procedure has been developed for profiling mAb drug glycan distributions where the glycans remain covalently linked to the mAb fragment crystallizable (Fc) domain instead of the more traditional approach of cleaving the glycans from the mAb for analysis. The method uses a specific immunoglobulin-degrading enzyme from streptococcus pyogenes (IdeS) that cleaves the Fc domain from the mAb. The purified Fc domain protein is then dissolved in deuterated urea for collection of a high resolution 2D 1H-13C HSQC NMR spectrum. The resulting anomeric peak distribution signals reveals major and minor glycan species including the trimannosyl core, high-mannose, and branch specific galactosylation patterns. In comparison to the classical glycan mapping method which involves enzymatic cleavage of glycan from mAb and liquid chromatography (LC) separation, the middle-down NMR method provides a non-invasive analytical characterization approach to ensure integrity and complete coverage of monosaccharide distribution. The middle-down NMR method takes 3-4 days, but the hands-on time is approximately 4-5 h. The protocol could be implemented in mAb drug development and quality assurance as an orthogonal method to profile glycan distribution. The expertise required would be basic biochemistry and two-dimensional NMR analysis experience.