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Abstract

The presented approach, allows the characterization of N-glycosylation patterns, shown exemplarily by hemagglutinin of influenza A/PR/8/34 (H1N1) virus, produced in Madin Darby Canine Kidney (MDCK) cells. The envelope of influenza A contains two glycoproteins: hemagglutinin (HA) and neuraminidase (NA). The functional role of their glycans is still not completely understood, but it is known, that structural modifications of these glycans can influence viral replication dynamics and immune response after vaccination. The glycosylation pattern of viral proteins can be affected by the virus strain, by the glycosylation machinery of the host cell, by cultivation conditions and via incipient degradation of the glycoproteins during virus inactivation and downstream processing. Hence, the ability of monitoring and thereby controlling the glycosylation pattern during the virus production process, is a prerequisite for yield enhancement in vaccine production and to ensure sufficient immune-response after vaccination. Within this work, the N-glycans are analyzed in two stages: first - via glycome fingerprints and second - via structural sequenzing of the glycans. For the generation of fingerprints we are utilizing a capillary gel electrophoresis DNA-sequenzer with laser induced fluorescence detection (CGE-LIF) and a matrix-assisted-laser-induced-ionization time-of-flight mass spectrometer (MALDI-TOF MS). Besides glycome fingerprints, additional structural information is obtained, spiking the samples with a series of N-glycans with known structures. Further structural analysis of the HA N-glycans is obtained by consecutive sequencing the glycan ladder utilizing reagent array analysis method (RAAM) in combination with CGE-LIF, achieving a limit of detection down to the lower zeptomolar range. The developed procedure allows monitoring of N-glycosylation patterns of relevant glycoproteins during the major steps of up- and downstream processing in influenza virus vaccine production.