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キーワード:
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要旨:
For production of the biologicals using microorganisms or cell cultures, downstream processing next to the actual biosynthesis is often the most expensive and time consuming step. Based on the physico and bio/chemical properties of the desired product and the impurities from the cultivation broth a broad range of separation methods can be applied. Here we report the development and optimisation of downstream processing methods for the purification of equine influenza virus from microcarrier cultures to produce inactivated vaccines. The process involves depth filtration, chemical inactivation, ultrafiltration (UF) and gelfiltration (GF) operated in a sequence. Hemagglutinin (HA) and neuraminidase (NA) are the two surface membrane proteins of the virus with HA as the major antigenic component. Analysis of overall recovery shows that this method suffers from a comparatively low purification factor for HA with an overall yield of about 34% while NA activity increases slightly during purification. Reported recoveries based on an ELISA assay used in quality control of a commercial process are in the range of 60-70% 1. Elution profiles show a broad HA peak and sharp NA as well as protein peaks. This indicates the destruction of the intact virus, which is possibly due to the following reason(s): inactivation process, release of HA molecules due to high shear stress involved in the UF, adsorption of HA to cellular membranes during virus replication in the bioreactor etc. The level of contaminating proteins is reduced by 96%. Experimental work is on progress to further characterize individual processing steps and to develop methods to characterize virus size distributions. While a combination of ultrafiltration and gel filtration steps seems to be robust and reproducible enough for industrial applications, recovery and purity of the final product as well as overall process economy could possibly be improved by the integration of an Expanded Bed Affinity Chromatography method. At present we investigate the use of several base matrices2 and ligands required for the specific binding of whole virions or viral membrane proteins. Eventually, this will allow us to compare different downstream processing and to evaluate recovery, purity and process efficiency of various purification schemes.
