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Abstract:
The ability of monitoring and thereby controlling the glycosylation pattern of viral membrane-proteins during the vaccine production process, is a prerequisite for yield enhancement in vaccine production and to ensure sufficient immune-response after vaccination. The glycosylation pattern of viral glycoproteins can be affected by virus strain, host cell glycosylation machinery, cultivation conditions and via incipient degradation of the glycosylation during virus inactivation and downstream processing.
The presented approach allows the characterization of N-glycosylation patterns, shown exemplarily on hemagglutinin of influenza A/PR/8/34(H1N1) virus, produced in MDCK-cells. The procedure includes: virus purification and concentration directly from cell-culture supernatant, followed by preparation, separation and identification of viral (glyco-)proteins and their N-glycan pools, utilizing: g-force-step-gradient-centrifugation, SDS-PAGE, enzymatical cleavage, fluorescent labeling, CGE-LIF (employing a DNA-sequenzer) and MALDI-TOF MS. The N-glycans are analyzed in two stages: glycome fingerprints and structural identification. Generation of fingerprints with CGE-LIF, enables LODs down to the lower zeptomolar range. Additional structural information are obtained, spiking the samples with a series of N-glycan standards, measured with CGE-LIF and MALDI-TOF MS. This proceeding enables monitoring of N-glycosylation patterns of relevant glycoproteins during major steps of up- and downstream processing in influenza virus vaccine production.
