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.

Protein L (PpL) is a universal binding ligand that can be used for the detection and purification of antibodies and antibody fragments. Due to the unique interaction with immunoglobulin light chains, it differs from other affinity ligands, like protein A or G. However, due to its current higher market price, PpL is still scarce in applications. In this study, we investigated the recombinant production and purification of PpL and characterized the product in detail. We present a comprehensive roadmap for the production of the versatile protein PpL in E. coli.