Boosting the Activity of CHO-Based Cell-Free Protein Synthesis Factories for High-Throughput in Vitro Production of Functional Antibodies

Cell-free protein synthesis (CFPS) is an emerging research field with high potential for rapid and flexible production of therapeutic proteins. Among CFPS systems, mammalian based systems have been receiving an increasing attention due to their ability to produce post-translationally modified therapeutics. Although CHO cells are the market-dominating cell line in cell-based production systems, only little is yet known about their performance in CFPS systems.

In this study, we have achieved the successful design and implementation of an in-house mammalian CFPS factory for more robust and easy to prepare CFPS using CHO cell extracts. A standardized workflow for the preparation of highly-active CHO lysates has been established and different IRES-mediated expression constructs (circular and linear) have been generated. Protein expression is optimized via EMCV IRES-mediated translation initiation and the addition of a poly-A tail for further mRNA stabilization. Using the transcriptional and translational machinery present in the CHO lysates, our cell-free factory achieves protein yields of up to 40 µg/ml in single 25 µl coupled batch reactions with continued protein production for up to 8 hours.

To address the yield limitations of CFPS, we have explored the supplementation of accessory proteins (APs) to our in-house system to evaluate the boosting effect on lysate activity for improved production. Different conditions and strategies for implementing the accessory proteins (purified protein addition, transient expression) have been compared for their effect on yield, reproducibility, and cost efficiency. A strong synergetic effect on protein synthesis of both APs seems to be present with up to a 50-fold increase in expression levels of tGFP observed. Subsequently, we explored the CFPS of antibodies including Herceptin (Trastuzumab). In order to demonstrate the importance of this novel platform for commercial applications, we aim to further extend our product portfolio to difficult-to-express proteins highlighting the advantages of CFPS over cell-based production.