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Abstract

Despite the huge potential that Cell-Free Protein Synthesis (CFPS) holds as a manufacturing technology, especially for difficult-to-produce proteins, there are several technical hurdles that must be overcome. These are: high cost of reaction mixture, lack of studies on scalable cell-extract production and variability in protein yields. A minimal CFPS system was developed using low-cost chemical feedstock and cell-extracts generated from growth on defined media to address the cost issues by eliminating expensive biomolecules from the reaction mixture. A scalable protocol for consistent CFPS performance was developed at the 1L bioreactor scale by developing a dimensionless factor based on harvest of E. coli 1.8-2 doublings after induction with a fixed ratio of protein expression inducer Isopropyl β- d-1-thiogalactopyranoside to cell optical density. Furthermore, rapid high-quality cell-extract generation was demonstrated using a microfluidic device. The utility of the minimal CFPS system was demonstrated with the expression of the pertussis vaccine components Fim2 and Fim3 which are near impossible to produce in-vivo with E. coli. A rapid screening method was also developed to identify suitable chaperone composition and mixtures for the efficient production of Short-chain Fragment Variables (SCFVs). In conclusion, this work demonstrates that scalable, low-cost CFPS of difficult to produce therapeutics is possible and provides a framework and scalable factors for further adoption of this manufacturing method.