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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 and controlling the glycosylation pattern during the virus production process can be crucial to obtain maximum production yields and to secure the immunogenicity of the antigens. In the present study we characterize the N-glycan structure of influenza A/NM/1/93 (H3N8) virus HA produced in Madin Darby canine kidney (MDCK) cells via the following method. In a first step viral proteins are separated by SDS-PAGE. HA N-glycans are than enzymatically cleaved from the protein in gel with PNGase F and labelled with 8-Aminopyrene-1,3,6-trisulfonic acid trisodium salt (APTS) by reductive amination. These fluorescently conjugated glycans are characterized by capillary gel electrophoresis (CGE), generating fingerprints of HA N-glycan mixtures with a detection limit in the low fmolar range. Further structural analysis of the HA N-glycans is obtained by sequential sequencing implementing a reagent array analysis method (RAAM). This involves the digestion of the oligosaccharides with specific exoglycosidases monitored by CGE. The HA N-glycanpool fingerprints of the cell culture derived virus and structural analysis results are compared to observations on egg derived H3N8 virus. Together, the methods represent a promising tool to monitor HA-glycosylation during the major steps of up- and downstream process during the influenza virus vaccine production.