Monitoring and control strategies for inclusion body production in E. coli based on glycerol consumption

Abstract

The Gram-negative bacterium E. coli is the host of choice for the production of a multitude of recombinant proteins in industry. Generally, cultivation is easy, media are cheap and a high product titer can be obtained. However, harsh induction procedures using IPTG as inducer are often referred to cause stress reactions, leading to a phenomenon known as metabolic burden and expression of inclusion bodies. In this contribution, we present different strategies for determination of critical timepoints for product stability in an E. coli IB bioprocess. As non-controlled feeding during induction regularly led to undesired product loss, we applied physiological feeding control. We found that the feeding strategy has indeed high impact on IB productivity. However, high applied qs,C increased IB product titer, but subsequently stressed the cells and finally led to product degradation. Calculating the cumulated glycerol uptake of the cells during induction phase (dSn), we found an empirical value, which serves as a strong indicator for process performance and can be used as process analytical tool. We tested different approaches starting from offline control. Glycerol accumulation could be used as trigger to establish a model-based approach to predict titer and viable cell concentration for a model protein. This straight forward control and model-based approach is high beneficial for upstream development and for increasing stability.