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On-Demand Endotoxin-Free Production of Protein Cancer Therapeutics from Thermostable Freeze-Dried Cell-Free Protein Synthesis Reagents

Bundy, B. - Presenter, Brigham Young University
Wilding, K. M., Brigham Young University
Earl, C. C., Brigham Young University
Currently, shortages in certain FDA-approved protein therapeutics have put patients at risk of not receiving the drug they need. Cell-free systems has emerged as promising platform for on-demand and even on-site production of therapeutic proteins that could potential answer the challenge of protein therapeutic shortages and accessibility. The E. coli-based cell-free system is particularly attractive due to its low cost and robust nature. However, this system inherently contains endotoxins and here we report the use of an endotoxin-free E. coli strain to decrease the risk of endotoxin exposure from an on-demand system. The recent advent of lyophilized cell-free reagents also further simplifies on-demand applications, however its long-term stability at elevated temperatures is limited. Here we report the use of lyoprotectants to enhance the stability of lyophilized reagents and discuss the negative effects of lyoprotectants that have previously been reported for use with cell-free systems. Finally, we demonstrate the production of an FDA-approved protein therapeutic from a truly single-pot lyophilized, endotoxin-free, cell-free protein synthesis system after 45 days of storage at elevated temperatures.

Reference: Wilding KM, Zhao EL, Earl CC, Bundy BC. 2019. Thermostable Lyoprotectant-Enhanced Cell-free Protein Synthesis for On-demand Endotoxin-free Therapeutic Production. New Biotechnology. 53:73-80.