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要旨:
Influenza remains a major public health concern. Every year several million people are getting infected. Strategies to control influenza outbreaks are mainly focused on prophylactic vaccinations in conjunction with antiviral medications. Hence, every year large amounts of vaccines have to be produced. In the case of pandemic outbreaks these production processes have to be shifted to the appropriate pandemic influenza strain, reducing the production capacity of seasonal vaccines. Human influenza vaccines are traditionally produced in embryonated chicken eggs. This method has only a reduced possibility to be scaled up. Furthermore it potentially causes allergic reactions induced by egg proteins. Hence cell culture based vaccine production has been developed requiring new downstream processing strategies for virus purification. Studies have shown that Euonymus europaeus lectin (EEL) is a suitable ligand for an affinity capture step to purify different human influenza virus strains derived from Madin Darby canine kidney (MDCK) cell cultures [1] including two topical virus strains (A/Wisconsin/67/2005 (subtype H3N2), B/Malaysia/2506/2004: both from 33016 MDCK PF, Novartis Behring, Marburg, Germany; A/Puerto Rico/8/34 (subtype H1N1)). The lectin EEL was immobilized on polymer beads and binds to terminal alpha-galactose of glycosylated viral envelope proteins such as the hemagglutinin. Characterization of the capturing potential demonstrated very good host cell DNA and protein reduction, while the majority of the virus (around 90 %) could be recovered as product. The method indicated excellent reproducibility between various tested influenza virus strains as well as different cultivation batches. In addition, the impact of the host cells on purification by EEL-affinity chromatography has been evaluated. Therefore, Influenza A/Puerto Rico/8/34 virus was propagated in MDCK and Vero cells and screened for ligand binding. In a second part of the study different supports have been screened for EEL as ligand. This screening included stabilized reinforced cellulose membranes, polymer and porous glass particles, cellulose and agarose beads. Comparing virus binding abilities and product recoveries some of the tested materials indicated advantages for virus purification. The performance seems to be even superior to cellufine sulfate and heparin, two alternative affinity matrices currently used for influenza virus purification. Most virus binding was achieved by the cellulose membranes and the polymer based adsorbent. In addition, reinforced cellulose membranes have a far higher binding capacity than other tested adsorbents. With the tested virus strains our studies have shown that EEL-affinity chromatography is a valuable alternative to capture MDCK cell derived influenza viruses. Capturing studies will be completed using a reverse genetics derived reassortant H5N1 virus. In addition, kinetic measurements are being currently performed to characterize the virus ligand binding in more detail by surface plasmon resonance technology. [1] Opitz,L. et al. 2007.Vaccine 25:939-947.
