A novel high-throughput methodology for the simultaneous optimization of many cell culture media components is presented. The method is based on the media blending approach which has several advantages as it works with ready-to-use media. In particular it allows precise pH and osmolarity adjustments and eliminates the need of concentrated stock solutions, a frequent source of serious solubility issues. In addition, media blending easily generates a large number of new compositions providing a remarkable screening tool. However, media blending designs usually do not provide information on distinct factors or components that are causing the desired improvements. This paper addresses this last point by considering the concentration of individual medium components to fix the experimental design and for the interpretation of the results. The extended blending strategy was used to reshuffle the 20 amino acids in one round of experiments. A small set of 10 media was specifically designed to generate a large number of mixtures. 192 mixtures were then prepared by media blending and tested on a recombinant CHO cell line expressing a monoclonal antibody. A wide range of performances (titers and viable cell density) was achieved from the different mixtures with top titers significantly above our previous results seen with this cell line. In addition, information about major effects of key amino acids on cell densities and titers could be extracted from the experimental results. This demonstrates that the extended blending approach is a powerful experimental tool which allows systematic and simultaneous reshuffling of multiple medium components.

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