Cost-effective production process of scFv antibody fragments against Shiga toxin 2 via recombinant E. coli

• Antibody fragment for preventive measures and monitoring programs to reduce risks to the health of consumers. • Anti-Stx2 scFv production using recombinant E. coli pLys. • Proposed process flow design. • Projected low cost production (<US$ 300/g of active) at high-cell density culture. Shiga toxin (Stx)-producing Escherichia coli (STEC) and its subgroup enterohemorrhagic E. coli are significant pathogens responsible for diarrhea, which can progress to hemorrhagic colitis and hemolytic uremic syndrome (HUS), the leading cause of acute renal failure in children. Early diagnosis is crucial for effective clinical management, as antibiotic treatment is not recommended for STEC infections. The present study aimed to establish a cost-effective biotechnological platform for cultivating recombinant E. coli to produce scFv antibody fragments against Stx2 for diagnostic applications. The method was first evaluated through shake flask experiments and subsequently scaled up to bench-scale bioreactors operated in both batch and fed-batch modes using defined culture media. Optimal production conditions were achieved by inducing recombinant E. coli pLys at 18 °C for 18 h with 0.1 mM IPTG, resulting in a yield of 3.0 to 4.0 mg scFv/g cell biomass. A fed-batch, high-cell-density procedure with E. coli pLysS achieved a maximum production up to 150 mg scFv/L. A preliminary economic assessment demonstrated the production potential at a value of around $250/g scFv. Economic analysis also highlights that the relative cost of capital investment becomes important as production processes intensify. Therefore, technical parameters such as productivity (scFv mass/bioreactor volume * time) and scFv concentration (mass scFv mass/bioreactor volume) should be prioritized to maximize their values. Similarly, optimization of the recombinant E. coli microbial platform should be pursued to increase the Yp/x level.