Intensified upstream processing by a phosphate-regulated, auto-inducible expression system in E. coli W3110 for recombinant Fab production

Escherichia coli is a microbial expression system widely spread in industrial manufacturing systems for the production of biotherapeutics. Most of these recombinant protein production processes are established in fed-batch operation mode. While many sectors of the chemical industry have implemented intensification strategies, the bio-pharmaceutical sector lacks a profound change towards intensified operations due to a decrease of cellular productivity during extended cultivation periods. In this study, intensified bioreactor cultivations of an auto-inducible E. coli strain W3110 as a promising candidate for the production of a recombinant fragment antigen binding (Fab) are demonstrated. The main goal was to investigate the impact of the natural phosphate limitation on the activity of the alkaline phosphatase promoter (phoA) in terms of cell-specific productivity in different processing modes. Following a cell-physiological characterization in fed-batch operation, the transition to intensified upstream processing modes, such as repetitive fed-batch and chemostat was established successfully. A comprehensive understanding of phosphate limitation and precise control of critical parameters, such as growth rate and phosphate-to-substrate uptake enabled the intensified process to match specific product titers and surpass process efficiency (in terms of space-time yield) compared to fed-batch mode. The auto-inducible phoA promoter in E. coli W3110 enabled stable Fab production under intensified bioprocess operations. These findings highlight the suitability of this expression system for repetitive fed-batch and chemostat operations to support broader adoption of intensified strategies in the bio-pharmaceutical industry.