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キーワード:
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要旨:
Optimization strategies for bioprocesses often need a thorough analysis of substrate, product and metabolite concentrations. In mammalian cell culture especially the amino acid concentrations are of interest, as essential amino acids should not be limiting. When switching to defined protein and serum free media these profiles become even more important for process optimization. We are interested in virus vaccine production processes where the infection of mammalian cells with virus represents a stress situation with metabolic changes. To better understand these changes and to improve virus yields, we started analyzing amino acids from typical mammalian cell culture broth samples using direct separation by anion-exchange chromatography with integrated pulsed amperometric detection. Existing gradient elution conditions were adapted, considering additions of 1 vol% peptone and 10 vol% foetal calf serum (FCS) to the medium as well as changing concentrations of glucose from 5.5 g/L up to complete consumption as were typical for an influenza vaccine production process using Madin Darby Canine Kidney (MDCK) cells. Samples had to be analyzed in two dilutions with water (1: 33.3 and 1: 200) due to the strongly varying amino acid concentrations in the samples as a result of the medium composition and cell metabolism. Validation results and problems with oxidation or fouling of the gold electrode will be discussed. The amino acid profiles of the cell culture medium during three parallel cultivations of MDCK cells in roller bottles demonstrated the reproducibility of the method for a specific application.
