BACKGROUND: The bacterium Escherichia coli is a commonly used host for the production of recombinant protein biopharmaceutical products. One class of such molecules is antibody fragments, typified by the Crohn's disease and rheumatoid arthritis therapy Certolizumab pegol (Cimzia(R)). Antibody fragments generated in E. coli are often directed to the periplasm, so that disulphide bonding can occur and release can be simplified. However, many recombinant protein products are prone to misfolding and mislocalization. Here, we optimized the production of a Fab fragment, D1.3, and its release from the periplasm of E. coli using osmotic shock. RESULTS: By minimizing stress imposed on the bacterial hosts and monitoring Fab, total protein and DNA concentrations of fractions isolated following osmotic release, we successfully targeted the majority of recombinant Fab to the periplasm and were able to rapidly define optimal harvest points. Coupled optimization of fermentation and release increased the Fab concentration of the periplasmic extract by more than 20-fold. CONCLUSION: Simultaneous optimization of fermentation and periplasmic release allowed rapid definition of operational space and generation of recombinant protein in a form compatible with downstream processing steps. This methodology could be used for optimization of the production of a range of periplasmically-targeted recombinant proteins. (C) 2015 Society of Chemical Industry