Abstract
Cell-free protein synthesis systems that can be lyophilized for long-term, non-refrigerated 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 and a low-cost energy substrate. As a model, we apply optimized formulations to produce conjugate vaccines for ∼$0.50 per dose after storage at room temperature (∼22 °C) or 37 °C for up to 4 weeks, and ∼$1.00 per dose after storage at 50 °C for up to 4 weeks, with costs based on raw materials purchased at the laboratory scale. We show that these conjugate vaccines generate bactericidal antibodies against enterotoxigenic Escherichia coli (ETEC) O78 O-polysaccharide, a pathogen responsible for diarrheal disease, in immunized mice. We anticipate that our low-cost, thermostable cell-free glycoprotein synthesis system will enable new models of medicine biosynthesis and distribution that bypass cold-chain requirements.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 95-107 |
| Number of pages | 13 |
| Journal | ACS synthetic biology |
| Volume | 12 |
| Issue number | 1 |
| DOIs | |
| State | Published - Jan 20 2023 |
Funding
The authors acknowledge Jessica Stark and Jasmine Hershewe for helpful discussions and contribution of cell-free reagents used in this work and Kosuke Seki for the development of the code used for maximum initial rate calculations. This work was supported by the Bill and Melinda Gates Foundation (OPP1217652 to M.P.D. and M.C.J.), Defense Threat Reduction Agency (HDTRA1-15-10052 and HDTRA1-20-10004 to M.P.D. and M.C.J.), Army Contracting Command (W52P1J-21-9-3023), and National Science Foundation (CBET-1936823 to M.P.D. and CBET-1936789 to M.P.D. and M.C.J.). K.F.W acknowledges the National Defense Science and Engineering (NDSEG) Fellowship Program (ND-CEN-013-096) sponsored by the Army Research Office. M.C.J. thanks the David and Lucile Packard Foundation. A.W. acknowledges support from the Cornell Postdoctoral Scholars Program. D.A.W. acknowledges support from the National Science Foundation Graduate Research Fellowship under grant no. DGE-1842165. S.E.S. acknowledges support from the Murphy Scholars Program of the Robert R. McCormick School of Engineering and Applied Science and the Office of Undergraduate Research at Northwestern University.
Keywords
- cell-free protein synthesis
- conjugate vaccine
- decentralized biomanufacturing
- glycosylation
- lyophilization
- lyoprotectant
ASJC Scopus subject areas
- Biomedical Engineering
- Biochemistry, Genetics and Molecular Biology (miscellaneous)
