High-Throughput N-Glycan Analysis with Rapid Magnetic Bead-Based Sample Preparation.

Abstract

N-glycan profiling of therapeutic glycoproteins is essential to ensure the activity and efficacy of these promising new-generation drugs. The N-linked glycan moieties of these entities highly affect circulation half-life, immunogenicity and receptor-binding activity as well as physicochemical and thermal stability properties. In addition, more than half of the biopharmaceuticals are glycoproteins representing multibillion dollar worldwide business, further emphasizing the importance of their analysis. In the biomedical field, on the other hand, revealing disease-related glycan structure alterations holds the promise of the discovery of new biomarkers for early diagnostics. Therefore, there is a great demand for widely applicable, high-throughput sample preparation and analysis methods for N-glycan profiling of glycoproteins. One of the newest exciting developments of the field is the magnetic bead based glycoprotein sample preparation technique. A detailed protocol of this method is given in this chapter in conjunction with rapid capillary electrophoresis analysis of the prepared samples by laser induced fluorescence detection (CE-LIF). N-glycans are digested by the endoglycosidase PNGase F and the released carbohydrates are labeled with the charged fluorophore dye of aminopyrenetrisulfonate (APTS). Effective glycan capture by magnetic microparticles enabled fast, easily automated sample preparation both in individual (single vial) and 96-well plate formats, including excess dye removal. Rapid separation of APTS labeled IgG glycans is also shown utilizing an optimized CE-LIF protocol.