Optimization of a cascaded continuous cultivation mode using a Design of Experiment approach

Abstract

In many branches of diverse industrial sectors continuous processing is already the golden standard. In biotechnology continuous processing is becoming more and more realistic and first products are already implemented in the market. However, most industrial processes for recombinant protein expression in Escherichia Coli are still performed in fed batch mode. The aim of time-independent processing and thereby increasing space-time yields supports a change of cultivation mode towards continuous processes. However, implementation of biological systems is not as easy as in other branches, where no living cells are used for production. Shifts in the transcriptome and the proteome, in combination with mutation rates are issues, which need to be considered and cause decreasing productivity during the process. Having two cascaded continuous bioreactors to separate cell growth and recombinant protein expression turned out to be a solution to increase not only the space-time yield, but also to avoid a fluctuating productivity. Even though the cascaded cultivation system is already published in literature, little knowledge about this process strategy is given yet. As it is crucial to find out the link between optimal cultivation conditions for a stable overall specific productivity (at high product titers), the aim of this study was to optimize a cascaded continuous cultivation mode for a recombinant protein, expressed in E. coli, using a Design of Experiment (DoE) approach. Thereby the process parameters dilution rate and the ratios of carbon source (glycerol) to inducer (lactose) were altered. In this study we were able to achieve a long-term stable production and determined the dilution rate as significant impact factor on the process performance. The highest productivity after 120 h of 4.68 ± 1.61 mg g-1 h-1 was achieved by applying a dilution rate of 0.2 h-1 and a ratio of qs,glycerol = 0.4 g g-1 h-1 to qs,lactose = 0.1 g g-1 h-1.