(307g) Boosting the Activity of CHO-Based Cell-Free Protein Synthesis Factories for High-Throughput in Vitro Production of Functional Antibodies

Chiara Heide^1,2,3, Gizem Buldum^1,5, Oscar Ces^2,4, Cleo Kontoravdi¹, Karen M. Polizzi^{1, 3}

¹Department of Chemical Engineering, ²Department of Chemistry, ³Imperial College Centre for Synthetic Biology, ⁴Institute of Chemical Biology, Imperial College London, SW7 2AZ, UK, ⁵Department of Bioengineering, Marmara University, Göztepe Campus, Istanbul, Turkey

Keywords: cell-free protein synthesis • CHO cells • antibodies

The synthesis of therapeutic proteins has traditionally been dominated by cell-based expression systems. Among these, mammalian cell lines have become the favoured choice for the synthesis of biologically active proteins due to the necessity for appropriate post-translational modifications (PTMs) (Brödel et al., 2014). Nearly 70% of all recombinant protein therapeutics are industrially produced in Chinese hamster ovary cells, which make them the most well-established mammalian host cell line today (Jayapal et al., 2007).

However, working with these living cells has a considerable downside, as protein synthesis is always strongly dependent on host cell metabolism in determining the product yield and quality. Process conditions are a compromise between the conditions that are essential for cell growth and viability, and the preferred conditions for the synthesis of a functional target protein (Stech et al., 2013). Attention has hence been drawn to cell-free protein synthesis (CFPS) systems, which do not require intact host cells (Carlson et al., 2012). Cell-free systems have evolved from an analytical tool into a powerful complementary approach to cell-based production systems. Particularly, in the context of high-throughput production of protein libraries (Griffiths and Tawfik, 2003), and synthesis of difficult-to-express proteins, such as membrane (Katzen et al., 2009) or even toxic proteins (Bechlars et al., 2013), cell-free synthesis has shown to offer the potential for rapid, flexible, and reliable protein production. Due to the ability to perform human-like PTMs for protein therapeutics, mammalian-based cell-free platforms have been receiving increasing attention (Brödel and Kubick, 2014). Despite optimization efforts, low production yields and poor cost efficiency remain the biggest challenges of CFPS platforms.

We have achieved the successful design and implementation of an ‘in-house’ mammalian CFPS factory for more robust, easy to prepare and highly-active 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.

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