Item

Abstract

Within this work, the utilization of a capillary DNA-sequencer for N-glycan analysis [1] is presented. With this highly sensitive nano-separator, based on capillary gel electrophoresis with laser induced fluorescence detection (CGE-LIF), the N-glycans can be analyzed in two stages: first - comparing “glycan-pool fingerprints” and second - structural elucidation via database matching. The developed procedure allows monitoring and elucidation of N-glycosylation patterns of relevant glycoproteins. Besides workflow and proof of principle, suitability and potential of the method are depicted. The methods performance is exemplarily shown via elucidation of glycosylation patterns of pharmaceutical relevant glycoproteins (e.g. interferons, immunoglobulins, …). In more detail, feasibility and performance of this method is demonstrated exemplarily for hemagglutinin (HA), an important glycoprotein of the influenza virus membrane. The HA glycosylation patterns of different influenza virus strains, replicated in different mammalian cell lines for vaccine production were elucidated. Results concerning the influence of the host cell line on complexity and composition of the HA N-glycosylation pattern, are presented. Besides a strong host cell dependence of HA N-glycosylation that could be shown, a significant change in N-glycan type attached to HA was observed, comparing different virus types and subtypes [2]. [1] Schwarzer, J.; Rapp*, E.; Reichl, U. Electrophoresis, 2008, 29, 4203-4214. [2] Schwarzer, J.; Rapp*, E.; Hennig, R.; Genzel, Y.; Jordan, I.; Sandig, V.; Reichl U. Vaccine, 2009, in press.