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Abstract

The envelope of influenza A virus 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 HA glycosylation pattern in virus production processes can be used e.g. as a tool to ensure the immunogenicity of the antigens. In this study, a sensitive and reproducible N-glycan profiling method for cell culture derived influenza virus HA is presented. We are able to monitoring potential variations of the glycosylation pattern, concerning N-glycan type and amount, due to the production process. The method includes virus purification directly from cell culture supernatant, protein separation by SDS-PAGE, endo- and exoglycosidase-cleavage of N-glycans, desalting and Capillary gel electrophoresis (CGE-LIF). HA N-glycosylation is analyzed on two levels: first generating fingerprints and second performing structural analysis by spiking N-glycans with know structures as well as enzymatic sequencing. The impact of host cells used for virus production on HA N-glycosylation is presented. In addition, data from a comparative analysis of HA N-glycan fingerprints of different influenza A virus strains and subtypes are shown together with results obtained from structural N-glycan analysis. The developed method presents a promising tool to characterize and compare N-glycosylation patterns of HA during the major steps of up- and downstream processing in influenza virus vaccine production.