Stefan Kittler

Various fermentation strategies in industrial biotechnology are applied to produce recombinant target proteins using Escherichia coli. These proteins are often expressed as inclusion bodies (IBs), resulting in a high purity, high stability, and high product titers. In state-of-the-art fed-batch processes, product formation takes place in a short period of time. Sterilization, cleaning, and biomass growth are time consuming steps and reduce the space-time yield. Thus, the interest in establishing continuous cultivations, facilitating higher space-time yields, has been increased in recent years. In this protocol, we provide information and a guide to set-up the production of recombinant proteins in fed-batch, as well as in chemostat continuous cultivations using E. coli. Key words E. coli Inclusion body Fed-batch Chemostat Recombinant protein production

Auto-inducible promoter systems have been reported to increase soluble product formation in the periplasm of E. coli compared to inducer-dependent systems. In this study, we investigated the phosphate (PO₄)-sensitive phoA expression system (pAT) for the production of a recombinant model antigen-binding fragment (Fab) in the periplasm of E. coli in detail. We explored the impact of non-limiting and limiting PO₄ conditions on strain physiology as well as Fab productivity. We compared different methods for extracellular PO₄ detection, identifying automated colorimetric measurement to be most suitable for at-line PO₄ monitoring. We showed that PO₄ limitation boosts phoA-based gene expression, however, the product was already formed at non-limiting PO₄ conditions, indicating leaky expression. Furthermore, cultivation under PO₄ limitation caused physiological changes ultimately resulting in a metabolic breakdown at PO₄ starvation. Finally, we give recommendations for process optimization with the phoA expression system. In summary, our study provides very detailed information on the E. coli phoA expression system, thus extending the existing knowledge of this system, and underlines its high potential for the successful production of periplasmic products in E. coli.

In the past 30 years, highly specific drugs, known as antibodies, have conquered the biopharmaceutical market. In addition to monoclonal antibodies (mAbs), antibody fragments are successfully applied. However, recombinant production faces challenges. Process analytical tools for monitoring and controlling production processes are scarce and time-intensive. In the downstream process (DSP), affinity ligands are established as the primary and most important step, while the application of other methods is challenging. The use of these affinity ligands as monitoring tools would enable a platform technology to monitor process steps in the USP and DSP. In this review, we highlight the current applications of affinity ligands (proteins A, G, and L) and discuss further applications as process analytical tools.