Analysis of Influenza A Virus Hemagglutinin (HA) Glycosylation by Capillary Gel electrophoresis – Laser induced Fluorescence

Glycosylation is the most common posttranslational modification of proteins in eukaryotes. The virus envelope protein HA is glycosylated by 3 to 9 N-linked glycans, which are important for virus replication and its immunogenicity. During vaccine production process the glycosylation pattern can be affected by the cell cultivation conditions as well as downstream processing. For obtaining maximum production yields and securing the immunogenicity of the antigens, monitoring the glycosylation pattern during the production process can be crucial. In this study HA N-glycosylation of cell culture (MDCK-cells) derived influenza A/PR/8/34 (H1N1) virus is analyzed. The method includes virus purification directly from cell-culture-supernatant, protein separation by SDS-PAGE, endo- and exoglycosidase-cleavage, SEC, CGE-LIF and MALDI-TOF-MS. N-glycans are analyzed on two levels: first generating fingerprints and second performing structural analysis by spiking N-glycans with know structures as well as enzymatical sequencing. 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