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A process scheme for the harvesting and concentration of cell culture-derived human influenza A virus is presented. The scheme comprises of two static filtration steps, chemical inactivation by beta-propiolactone and cross-flow ultrafiltration. Human influenza A virus A/PR/8/34 (H1N1) was produced in roller bottles with serum-free medium using MDCK cells as a host. Cultivations resulted in specific hemagglutination (HA) activities of 393 HAU (100 El)-1 and turbidities of 0.479 O.D. measured as the extinction of light at 700 nm (mean values are presented). The concentrations of soluble protein and DNA in the harvests were 72 Eg ml-1 and 5.73 Eg ml-1, respectively. An average product yield of 79% based on HA activity was achieved after clarification by depth (85%) and microfiltration (93%). The turbidities of cell culture supernatants were reduced to 2% of their initial value. Concentration with 750 kDa hollow-fibre modules by a factor of 20 resulted in 97% recovery of the product when operated at a constant flux of 28 l m-2 h-1 and a wall shear rate of 9500 s-1. The amount of protein and DNA could be reduced to 16% and 33% of their initial amount, respectively. An overall product yield of 77% was achieved. Clarified supernatants and concentrates were further analyzed by non-reducing SDS-PAGE and agarose gel electrophoresis. Particle volume distributions of concentrates were obtained by dynamic light scattering analysis. From the results it can be concluded, that the suggested process scheme is well suited for the harvesting and concentration of cell culture-derived influenza A virus.
Copyright © 2006 Wiley Periodicals, Inc. [accessed 2013 November 14th]
