Methods to Determine the Level of Afucosylation in Recombinant Monoclonal Antibodies

摘要

Glycosylation is a common posttranslational modificationnand a key quality attribute of recombinant monoclonalnantibodies. In particular, the level of core-fucosylation isncritical to several Fc mediated biological functions. However,nit is challenging to accurately determine the level ofnafucosylation due to its low level and possible distributionnin multiple oligosaccharide species. Methods that cannaccurately determine the level of afucosylation werendeveloped in the current study. Digestion of monoclonalnantibodies with Endoglycosidases F2 and H, which cleaventhe glycosidic bond between the two primary GlcNAcnresidues, reduced complicated oligosaccharide populationninto two groups with either only GlcNAc or GlcNAcnand the core-fucose residue. Analysis of the digestednantibodies by LC-MS provided a quick estimate of the levelnof afucosylation. Alternatively, PNGaseF released oligosaccharidesnwere labeled with 2-aminobenzamide, digestednwith Endoglycosidases F2 and H followed bynnormal phase HPLC with fluorescence detection. Thenresults demonstrated that the latter method can providenan accurate quantitation of the level of afucosylation.