Production of bioactive recombinant monoclonal antibody fragment in periplasm of Escherichia coli expression system

Abstract

The microbial expression system (Escherichia coli) is the most widely studied host for the production of biotherapeutic products, such as antibody fragments, single chain variable fragments and nanobodies. However, recombinant biotherapeutic proteins are often expressed as insoluble proteins, thereby limiting the utility of E. coli as expression system. To overcome this limitation, various strategies have been developed, such as changes at DNA level (codon optimization), fusion with soluble tags and variations in process parameters (temperature), and inducer concentration. However, there is no “one size fits all” strategy. The most commonly used approach involves induction at low temperature, as reducing the temperature during cultivation has been reported to increase bioactive protein production in E. coli. In this study, we examine the impact of various process parameters, such as temperature and inducer concentration, as well as, high plasmid copy number vector for achieving enhanced soluble expression of TNFα inhibitor Fab. An interaction amongst these parameters has been observed and their optimization has been demonstrated to result in expression of 30 ± 3 mg/L antibody fragment using E. coli. This case study illustrates how process optimization can contribute toward making biotherapeutics affordable.