FACTORS AFFECTING THE FERMENTATIVE PRODUCTION OF A LYSOZYME-BINDING ANTIBODY FRAGMENT IN ESCHERICHIA-COLI

Fed-batch fermentation for production of a single-chain Fv antibody fr agment (scFv) expressed as a recombinant periplastic protein from Esch erichia coli was investigated. A high cell density of 50 g dry cell we ight per liter was routinely achieved in a 14-L vessel by controlled e xponential feeding of glucose to impose a constant specific growth rat e. Following biomass accumulation, induction of the tac promoter by ad dition of IPTG was accompaied by a linear feed of yeast extract. The c oncentration of yeast extract feed was found to be highly influential upon both concentration and location of active product. Although scFv fragments were specifically targeted to the periplasmic space, at yeas t extract feed rates of 0.72 g/h the final location was largely extrac ellular (68% to 79%). Total concentrations (extracellular + periplasmi c) were of the order of 5 to 8 mg/L. A ten-fold increase in yeast extr act supply increased total scFv concentration to almost 200 mg/L and 7 8% of this yield was retained in the periplasm. Control of such leakag e of the recombinant product is fundamental to process design of downs tream operations for product recovery. (C) 1997 John Wiley & Sons, Inc .