iCFree project: improved cell-free systems to accelerate therapeutic proteins production at minimal cost

Funded by one of the 5 grand challenges launched by the PIA program of the French government to date: Biomedicines: improving yields and controlling production costs.

Healthcare systems are increasingly dependent on access to critical, high-value proteins, which are often environmentally damaging and complex to obtain domestically. The emergence of functionalized and customized proteins is enabling a radical transformation of therapies, which are becoming more individualized and complex. 

Nevertheless, the production costs of new biological drugs are skyrocketing compared to chemical drugs. These costs are also very susceptible to the technology used and the production batch size. As far as cell and gene therapies are concerned, these techniques are not yet fully industrialized and optimized. To meet these needs, progress is necessary.

Cell-free systems have lately revolutionized the field of synthetic biology as they provide a fast and robust platform for protein production. Here we aim to develop and optimize cell-free protein synthesis systems (CFPSs) to produce therapeutic proteins which are difficult to synthesize in vivo.

Project description

iCFree uses machine learning tools to develop and optimize CFPSs. To reach the commercial market for protein production, CFPS systems must evolve from a laboratory level to a robust production platform. To that end, several factors need to be optimized, like the synthesis of high-quality functional proteins with proper folding and post-transcriptional modifications, the cost of production, and the scalability. Addressing these factors requires more detailed understanding of the CFPSs components to optimize lysate quality, the reaction conditions, and the buffer elements.

CFPS will be considered to express therapeutic proteins in three different systems:

  • proCFPS: E. coli extract.
  • pureCFPS: Pre-defined purified bacterial components. This enables the integration of non-canonical amino acids (ncAAs).
  • euCFPS: hybrid mammalian cell or tissue lysate.

The expected outcome of the iCFree project is an optimized CFPS system for the production of any user given protein and biomanufacturing objective based on productivity, cost and functionality.

Machine-learning-driven optimization

Recently, we proposed a strategy based on machine learning to explore large combinatorial spaces (~4,000,000 cell-free buffer compositions) varying several components at one time to efficiently find an optimum for cell-free productivity. That latter work demonstrated that when compared to a standard cell-free preparation protocol (Sun, Zachary Z et al), protein productivity was increased by 34 folds, and cost was reduced by lowering buffer components concentrations.

Here we will expand upon that strategy optimizing for the multi-objective of maximizing productivity and minimizing cost while maintaining functionality. For the three proposed CFPS systems, an optimization machine-learning-based workflow (image below) will help determine the stoichiometry of specific supplementary components such as chemicals, enzymes, DNA, tRNAs, or riboproteome.

Provided a gene sequence, within less than 10 days the systems will enable on-the-fly synthesis, transcription-translation of the gene, quantification-purification of the corresponding protein for further scale up or small-scale on-site and on-demand production at point-of-care facilities.



The project is driven by a consortium that is composed of four partners with complementary expertise in cell-free systems and protein production.

The MICALIS institute (INRAE, AgroParisTech, University of Paris Saclay) has several years of experience and published records in optimizing prokaryotic cell-free systems with machine learning for biomanufacturing and developing biosensors.

The I2BC institute (CEA, CNRS, University of Paris Saclay) brings the know-how in the recombinant production of peptides and proteins and in the biological incorporation of ncAAs into peptides.

The CarMeN institute (INSERM, UCBL1, Lyon) has a strong background in in vitro eukaryotic transcription and translation mechanisms.

Synthelis is a biotech that offers more than 10 years of expertise in protein production using cell-free systems. To date, the team has successfully produced over 230 difficult-to-express proteins using its cell-free technology.