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Abstract:
The envelope of influenza A is spiked with two glycoproteins: hemagglutinin (HA) and neuraminidase (NA). HA as the most abundant protein on the virus surface, triggers the strongest immunogenic response. Each HA monomer contains 3 to 9 N-linked glycans, depending on the virus strain. The functional role of these glycans is still not completely understood. However, previous glycosylation studies have shown that structural modifications of these glycans can influence virus attachment to the host cell, and therefore change viral replication dynamics and its immunogenicity. The glycosylation pattern of viral proteins is affected by the glycosylation machinery of the host cell and their cultivation conditions. Further modifications in the structure can occur during inactivation and downstream processing steps. Hence, monitoring the glycosylation pattern during the virus production process can be crucial to obtain maximum production yields and to guaranty the immunogenicity of the antigens.
In this study the HA N-glycosylation pattern of cell culture derived influenza A virus strains is analyzed via the following procedure. The virus is concentrated and purified by ?g-force-step-gradient-centrifugation? directly from cell culture supernatants. Afterwards viral proteins are separated by SDS-PAGE followed by enzymatical cleavage of HA N-glycans from the protein in gel with PNGase F. For monitoring by capillary gel electrophoresis with laser induced fluorescence (CGE-LIF), the N-glycanpool is labeled with 8-Aminopyrene-1,3,6-trisulfonic acid trisodium salt (APTS) by reductive amination. Fingerprints of HA N-glycan mixtures with a low detection limit allow a comparison of the HA N-glycosylation pattern of one influenza A virus strain produced under various cultivation conditions and between different influenza A virus strains produced under similar conditions. Spiking with oligosaccharides of known structures as well as enzymatical sequencing allows gaining structur al information on the N-glycanpool. Overall, the developed method presents a promising tool to characterize the N-glycosylation pattern of HA, in the low zeptomolar range, during the major steps of up- and downstream process while influenza virus vaccine production.
