A strategy for scalable antibody production: the combination of <i>Escherichia coli</i> expression, translation-enhancing peptide and automated refolding system

Monoclonal antibodies are essential tools in biological research, diagnostics, and therapeutics. While mammalian expression systems are widely used, microbial hosts such as Escherichia coli offer cost-effective and scalable production of antibody fragments but often yield inclusion bodies requiring refolding. In this study, we report a combined strategy using a translation-enhancing SKIK peptide tag and a leucine zipper-fused Fab format to improve expression in Escherichia coli, together with a continuous, dialysis-based automated refolding system. Compared with the conventional stepwise method, the automated process recovered model antibodies with equivalent antigen-binding activity and refolding yield (approximately 98%), while reducing processing time to 40%, waste volume to 69%, and chemical consumption to 47% of the modified stepwise process. The apparatus is simple to set up and operate, making it applicable from laboratory to industrial scales. This resource-efficient and scalable approach provides a practical alternative for scalable antibody fragment production in microbial systems.