Thermostable lyoprotectant-enhanced cell-free protein synthesis for on-demand endotoxin-free therapeutic production

Cell-free protein synthesis has emerged as a promising platform for the production of therapeutic proteins due to its inherently open reaction environment, flexible reaction conditions and rapid protein synthesis capabilities. In recent years, lyophilized cell-free systems have widened the application space of cell-free technology by improving reagent stability outside of cold-chain storage. Current embodiments of the system, however, demonstrate poor stability at elevated temperatures. Lyoprotectants have long been recognized for the ability to preserve the activity of biological molecules during drying processes, but the application of this technology to lyophilized cell-free systems has been limited and has failed to address the negative effects that such lyoprotectants may have on cell-free systems. Here, several lyoprotected, lyophilized cell-free protein synthesis systems are demonstrated using antiplasticized sugar glasses as lyoprotectants, showing significant improvement over standard lyophilized systems or trehalose-preserved systems. Furthermore, we demonstrate for the first time, preservation and therapeutic expression, specifically of FDA-approved crisantaspase, from a truly single-pot lyophilized, endotoxin-free, cell-free protein synthesis system, exemplifying the potential for on-site therapeutic synthesis.