Carbohydrate analysis of a chimeric recombinant monoclonal antibody by capillary electrophoresis with laser-induced fluorescence detection.

A general method for the analysis of asparaginyl-linked (N-linked) carbohydrate moieties of an IgG1 monoclonal antibody is described here. The antibody, rituximab, is a mouse/human chimeric antibody to human CD20 antigen. The glycans present on rituximab are neutral complex biantennary oligosaccharides with zero, one, and two terminal galactose residues (G0, G1, and G2, respectively). To monitor the variation of the glycosylation during manufacture, the glycans were first enzymatically released from the antibody via digestion with peptide-N-glycosidase F, then derivatized with a charged fluorophore, 8-aminopyrene-1,3,6-trisulfonic acid and further separated by capillary electrophoresis with laser-induced fluorescence detection. All observed glycans were fully resolved, including the positional isomers of G1. The exact nature of the isomers in terms of the location of the terminal galactose was further characterized via multiple enzymatic digestion steps including mannosidase with activity toward specific Man(alpha 1,3) linkage. The optimization and several key parameters, i.e., enzymatic digestion and derivatization, in the assay development will be discussed. Moreover, to ensure that the assay can be used in routine lot release testing, the assay was validated and found to be accurate and precise. The analytical approach described is suitable for characterization as well as routine testing of the N-linked glycan content in any IgG1 monoclonal antibody and glycoproteins in general.