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

•Lyoprotectant combinations enhance lyophilized extract viability under 50 °C storage.•Lyoprotected single-pot cell-free protein synthesis (CFPS) retains >45% activity after 90-day ambient storage.•Lyoprotected single-pot CFPS systems maintain viability for 46 days at 50 °C.•Active, endotoxin-free crisantaspase is produced from stored CFRxlyo systems.