Cell-free protein production on a larger scale

The use of recombinant proteins for therapeutic, diagnostic, technical, agricultural, and cosmetic applications is constantly increasing. Most recombinant proteins are produced in mammalian cells or microbes (bacteria and yeast). However, numerous promising protein candidates that are difficult to produce (or even toxic) in living cells are lost during screening and evaluation procedures before suitable industrial production processes can be established. Therefore, some industrial companies are considering the use of cell-free expression systems to identify protein candidates that cannot be produced using current prokaryotic and eukaryotic cell-based expression platforms. Although such cell-free in vitro transcription and translation (IVTT) systems are well suited for rapid and reproducible high-throughput screening and can provide microgram quantities of protein for initial analytical testing, the production of milligram or even gram quantities using cell-free expression systems is currently nearly impossible. However, for the next step of product development, larger quantities of protein must be made available to further characterize the protein candidates before the final protein production process is defined.

The planned ThinkBig project aims to close this gap in the development process by establishing methods that enable scaling cell-free protein expression from the current milliliter range to 1–10 liters. Since many pharmaceutical and technical proteins are of eukaryotic origin, the focus will be on a newly developed cell-free lysate from tobacco BY-2 cells, which can be produced faster and more cost-effectively while yielding significantly higher protein amounts than other cell-free systems, such as the commonly used wheat germ extract.

Within the ThinkBig project, new methods will be developed for the large-scale production of cell-free lysates and the implementation of innovative bioreactor strategies for in vitro transcription and translation reactions in the 1–10 L range. This will enable the rapid provision of proteins in gram-scale quantities within 1–2 days. The project will also address industrial and regulatory compliance requirements, for example in the production of therapeutic proteins for clinical testing.

The practical application of the approach will be demonstrated through the production of an antimalarial immunotoxin, human epidermal growth factor (hEGF) for cosmetic use, and the technical enzyme enterokinase. Although there is high demand for these proteins, none can be produced in high yields using cell-based expression systems.

The project partners will also develop commercialization strategies with the industrial partner LenioBio GmbH to market the cell-free process for large-scale protein production as well as the proteins produced during the project.

Thus, the project covers the entire value chain, providing methods for large-scale cell-free protein biosynthesis that can also be applied to the production of other proteins whose further development is hindered by low yields in conventional cell-based systems.