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Zusammenfassung:
The presented study aims on the development of a capture step for the purification of human Influenza viruses using lectin-affinity chromatography. This enables a high product concentration factor and a separation of the target biomolecule from the major bulk of contaminants with a single step. In the following studies a Madin Darby canine kidney (MDCK) cell produced human Influenza A/PR/8/34 virus has been chosen as a model strain. The Influenza A virus surface possesses two viral spike glycoproteins: the hemagglutinin (HA) and the neuraminidase (NA). The HA is the most abundant and immunogenic surface glycoprotein. Based on HA glycoanalysis some adequate lectins have been chosen for lectin binding screening via lectin blots. Therefore, the proteins of the concentrated cultivation broth were separated by SDS-PAGE analysis and transferred onto PVDF-membranes. Treatment of the membrane with biotinylated lectins and detection by chemiluminescence indicated the lectin affinity to viral and host cell glycoproteins. The most specific binding to the viral glycoprotein HA was achieved via the galactose specific lectins Erythrina cristagalli lectin (ECL, gal(betta1,4)glcNAc) and Euonymous Europaeus Lectin (EEL, gal(alpha1,3)gal). These lectins have been selected for further investigations of chromatographic separations. The virus and the viral membrane glycoproteins from concentrated cell culture broth adsorbed to the lectin-polymer-matrix specifically. The majority of host cell proteins did not bind to the lectins. These proteins were washed out from the column. Viral proteins were desorbed from the column by competitive elution with the appropriate carbohydrate. HA recoveries of up to 80% based on activity measurements have been achieved with this procedure with a high degree of removal of host cell proteins and nucleic acids. On that account, lectin affinity chromatography represents a promissing tool for an effective capture step for the downstream processing of MDCK-cell derived Influenza vaccines.
