Analytical Glycomics

Andras Guttman, Horvath Csaba Memorial Institute of Bioanalytical Research, University of Debrecen, Debrecen, Hungary

 Analytical glycomics represents a toolset for comprehensive characterization of glycans including profiling, micro- and macroheterogeneity analysis with special respect to linkage and positional isomers, as well as oligosaccharide sequencing. Such detailed analysis of the carbohydrate moieties of glycoproteins is of high importance as glycosylation changes can impact the effect of biopharmaceuticals and, on the other hand, may serve as novel biomarkers for the biomedical field. Therefore, high performance glycoanalytical techniques are of great demand for the analysis of therapeutic antibodies, especially during clone selection, process development and lot release. Analysis of complex carbohydrates is a very challenging task due to the lack of their chromophore / fluorophore activity and, in many instances, easily ionizable groups, necessitating derivatization before analysis. Full glycosylation characterization may also require sequencing with consecutive exoglycosidase digestion steps, followed by analysis of the resulting products.

This lecture covers the state-of-the-art of liquid phase separation methods for full structural elucidation of protein N-glycosylation, mostly focusing on capillary electrophoresis and its combination with mass spectrometry. Assisted by the emerging field of glycoinformatics, the identity of the glycan structures can be assigned relative to their electrophoretic migration time based glucose unit (GU) values. With the aid of such database, reliable, precise and traceable glycan structural assignment of biotherapeutics and other glycoproteins of biomedical importance can be obtained. Glycosylation characterization of the recently emerging class of biopharmaceuticals is discussed with the main goal to demonstrate structural and functional equivalence of the products focusing on the analysis of effector function determining and immunogenic epitopes.