N-Glycan Fingerprinting of the NIST monoclonal antibody (NISTmAb)

Glycosylation on therapeutic antibodies is critically important for their drug efficacies. Here, using NISTmAb and Humira® as examples, we present methods of glycan fingerprinting for these antibodies. Glycans are first released with PNGase F or Endo S2, and then labeled by specific glycosyltransferases, including sialyltransferase ST6Gal1, fucosyltransferase FUT9, N-acetyl-glucosaminyltransferase MGAT3 and fucosyltransferase FUT8, with respective fluorophore-conjugated donor sugars. The labeled glycans are then separated by gel electrophoresis (SDS-PAGE). A fluorophore-labeled hyaluronan ladder is run along with the samples to reveal the relative mobility of each glycan band. Pretreatment of the samples with specific glycosidase or glycosyltransferase results in additional mobility shift of specific glycan bands, which allows identification of some of these bands. Particularly, we report the identification of α-Gal epitopes, core-6 fucosylated glycans, paucimannose glycans and likely some bisecting/tri-antennary glycans on NISTmAb. Overall, our methods could serve as quick assessments of glycosylation on therapeutic antibodies.