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要旨:
In most biotechnological processes, metabolism of production strains is still far from being completely understood concerning molecular and biochemical reactions, leading in the worst case to suboptimal product yields. One approach to gain further insights into these complex cellular systems is the analysis of metabolite pools, in particular from energy metabolism. Most of the analytical techniques used for quantitative determination of intracellular metabolic intermediates require quenching of metabolism followed by an extraction. However, to assure reliability of the measured metabolite concentrations, this procedure needs to be optimized with respect to media, organism and metabolites of interest. In this work, the biological source is an adherent MDCK cell line, used for the production of influenza vaccine. A typical cultivation comprises a batch period of about four days of growth followed by medium exchange and virus infection. Depending on the status of the culture, significant changes in metabolite pools are expected due to e.g. substrate depletion, waste product accumulation or viral infection. Here we focus on the quantitative determination of nucleotides and metabolites of the central carbon metabolism. However, in advance to these measurements, a suitable procedure for quenching and extraction has to be identified to assure an acceptable accuracy of the method. This is discussed in the following. For the quantitative measurements we used an anion exchange chromatography system (DX-320, Dionex, Idstein, Germany), designed for the separation of inorganic ions, organic acids, and energy phosphates using conductivity and UV detection. In standard runs, a wide range of anionic intracellular metabolites could be separated and quantified even in concentrations below the micromolar range. Various sample preparation procedures mentioned in literature were tested in a systematic approach for their extraction capability, but especially the adherent cell line and the diverse properties of the metabolites posed some problems. In addition, serum and high ionic load of the medium complicated the finding of a suitable method in combination with the analytical instrumentation used. Some methods could be excluded from recovery studies by spiking standards in both PBS and medium. A final decision for one quenching and extraction method will be taken depending on maximum measured intracellular metabolite concentrations and in agreement with results for similar organisms in literature.
