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要旨:
Future requirements concerning process optimization and validation in mammalian cell culture will include an increased effort for detailed analysis of the production system. Not only metabolites in the cell culture supernatant but also cells are under closer investigation, ranging from single cell or proteome analysis to the investigation of the metabolome. Especially, if heading towards systems biology approaches, predominantly qualitative data sets are usually not sufficient.
In this work, we present a method to measure intracellular metabolite concentrations during cell growth and production. Several aspects to be considered are discussed, including the question of quenching metabolism, washing cells and the appropriate extraction procedure. Hereby, focus was put on reaching high metabolite concentrations in the extracts, but also on physiological reasonable metabolite ratios and recoveries. Furthermore, critical points like the cell type, varying cell size, medium, culture conditions or growth substrate are addressed. Main focus was put on an adherent MDCK cell line used for the production of an influenza vaccine. Intracellular concentrations of several metabolites from central carbon metabolism as well as a number of nucleotides were measured using an anion-exchange chromatography system coupled with conductivity and UV-detection. Results for the nucleotide ATP were compared with an independent enzymatic assay to assure reliable data. Additionally, the suitability of MS-detection coupled to chromatography has been tested to expand the spectrum of analyzed metabolites due to better sensitivity and selectivity.
Especially for adherent cells, metabolite pools are important numbers if dynamic modelling approaches are going to be followed. Changes are to be expected and should be incorporated into mathematical models.
Thus, we present data sets for the cultivation and virus production for MDCK cells showing a distinct dynamic behaviour within time. Additionally, some results for a suspension BHK cell line and adherent Vero cells will be shown.
