(667d) Removing Endotoxins from E. coli-Based Cell-Free Systems: Towards Enabling on-Demand Distributed Production of Therapeutics

Very recently researchers demonstrated the ability to lyophilized the reagents for E. coli-based cell-free protein synthesis. The resulting long-term stability of these reagents at room temperature has greatly simplified the use of this system for the on-demand on-site production of protein therapeutics. Indeed, this technology has been combined with DNA synthesis technology such that small robotic systems have been developed where an amino acid sequence is digitally inputted and a product protein is produced in a matter of hours using E. coli-based cell-free protein synthesis. This rapid turnaround time would be especially important for personalized cancer therapeutic and pandemic threat response applications. The low cost, robust, and rapid nature of E. coli-based cell-free systems makes it one of the most promising systems for on-demand therapeutic production. However, the presence of endotoxins at the extremely high concentrations of millions of endotoxin units per milliliter is a challenge for on-site distributed production. Here we report the utilization of liquid-liquid phase separation, affinity chromatography, and genetically engineered E. coli to remove endotoxins from cell-free reagents prior to protein production to reduce the risk of endotoxin contamination. This technology would eliminate additional endotoxin purification steps following protein production as well as additional quality control steps which would be otherwise necessary for on-demand on-site protein therapeutic production applications.