Abstract

Cell-free protein synthesis systems that can be lyophilized for long-term, nonrefrigerated storage and transportation have the potential to enable decentralized biomanufacturing. However, increased thermostability and decreased reaction cost are necessary for further technology adoption. Here, we identify maltodextrin as an additive to cell-free reactions that can act as both a lyoprotectant to increase thermostability, as well as a lowcost energy substrate. As a model, we apply optimized formulations to produce conjugate vaccines for ~$0.50 per dose after storage at room temperature or 37 {degrees}C for up to 4 weeks and ~$1.00 per dose after storage at 50 {degrees}C for up to 4 weeks. We show that these conjugates generate bactericidal antibodies against enterotoxigenic E. coli (ETEC) O78 O-polysaccharide, a pathogen responsible for diarrheal disease, in immunized mice. We anticipate that our lowcost, thermostable cell-free glycoprotein synthesis system will enable new models of medicine biosynthesis and distribution that bypass cold-chain requirements.