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Background Bioprocesses like the cell-based production of biologicals, i.e. mainly recombinant proteins and monoclonal antibodies, require optimal culture conditions to obtain a high yield of quality products. The performance of a bioreactor highly depends on the cell characteristics as well as on the composition of the cell culture medium and the process conditions. As the metabolic activity of the cells is very high during fermentation, the external and internal metabolite compositions vary tremendously throughout the process. The quantification of a wide range of metabolic substrates and products is a prerequisite to understand and optimize the underlying cell-based activities. Furthermore, metabolite quantification reveals the composition of biologically derived cell culture supplements, thus serving as a tool to monitor supplement quality or providing the base for the formulation of a chemically defined medium supplement.