Increased purity and refolding yield of bacterial inclusion bodies by recursive high pressure homogenization

Therapeutic proteins are often produced intracellularly in the bacterium Escherichia coli in aggregated form, known as inclusion bodies, which must be solubilized and refolded to recover the biologically active product. However, the industrially established refolding method of batch dilution is still carried out at low protein concentrations, leading to high water consumption, large equipment footprints, and poor sustainability. An underexplored way to increase refolding yields and thereby enable higher product concentrations after refolding is the optimization of cell lysis and inclusion body washing. In this study, we developed and evaluated a "recursive high pressure homogenization" approach, wherein wash steps and additional homogenization cycles were combined to enhance the final purity of inclusion bodies before solubilization. The degree of cell lysis, nucleic acid release and final inclusion body purity were compared to conventional "linear washing", where all homogenization cycles are completed prior to the washing of inclusion bodies. In total, seven process variations were compared and the resulting batches of inclusion bodies were solubilized and refolded to investigate the effect of early downstream processing on the product concentration in the final refolds. For the most efficient process variant, the recursive high pressure homogenization protocol led to the highest measured product concentration of 855 mg/L after refolding. Thus, the proposed recursive high pressure homogenization approach led to an estimated 18 % reduction of CO₂ footprint caused by urea, and increased the product yield per biomass from 5.17 g/kg to 7.84 g/kg compared to its linear wash counterpart - without introducing non-standard equipment or chemicals.