Spatiotemporal Control of Protein Refolding Through Flash-change Reaction Conditions

Protein refolding is vital for protein production; howev-er, a scalable method has not yet been developed. We introduce an innovative refolding approach using a flow microreactor (FMR) that allows precise control of buffer pH and solvent content. Using interleukin-6 as a model, the system yielded an impressive 96% pure refolded protein and allowed gram-scale production. This FMR system allows flash changes in reaction conditions, ef-fectively circumventing protein aggregation during re-folding. To the best of our knowledge, this is the first study to use an FMR for protein refolding, which offers a more efficient and scalable method for protein produc-tion . The study results highlight the utility of the FMR as a high-throughput screening tool for streamlined scale-up and emphasize the importance of understanding and controlling intermediates in the refolding process. The novelty of this approach is derived from the unique ability of the FMR to control both spatial and temporal aspects of protein refolding.