N-glycan analysis method for cell culture derived influenza virus hemagglutinin

The influenza A virus envelope protein HA is glycosylated by 3 to 9 N-linked glycans, depending on the virus strain. Thereby the glycosylation pattern 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-glycan fingerprints from influenza virus produced in different host cell systems and different influenza virus strains and subtypes are compared. Glycan structures were analysed by spiking glycans of known structures and sequential exoglycosidase digestion. 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