The Production System That Does Not Need a Cell
Cell-free protein synthesis uses the transcription and translation machinery extracted from cells, primarily ribosomes, transfer RNAs, aminoacyl-tRNA synthetases, transcription factors, and energy regeneration systems, to produce proteins in vitro from a DNA or RNA template without the need for living cells whose growth, maintenance, and genetic regulation impose constraints on the protein synthesis conditions and product range that conventional cell-based expression systems can achieve. The cell-free system runs the translation machinery directly, adding the DNA template, amino acids, and energy substrates to a cell extract that contains all the molecular machinery required for protein production, and accumulating the protein product in the reaction mixture over hours rather than the days to weeks that cell culture expression systems require from initial culture inoculation through fermentation, harvest, and purification. The elimination of the cellular container whose integrity requirements constrain the process conditions of conventional protein expression creates the freedom to adjust reaction composition, temperature, redox environment, and supplementary factors in ways that optimise production of the specific protein being synthesised rather than compromising between the cell's survival requirements and the protein production conditions that the target protein's folding and modification chemistry requires.
The commercial applications of cell-free protein synthesis have diversified from the early academic use as a protein research tool into the pharmaceutical development, diagnostics, and synthetic biology applications whose commercial value justifies the investment in cell-free system development and optimisation that academic laboratories and commercial companies have been making. The pharmaceutical development application, in which cell-free synthesis enables the rapid production of small quantities of candidate proteins for initial characterisation, activity screening, and structural analysis without the cell line development and expression optimisation that conventional cell-based production requires, is the commercial context that is growing most rapidly and whose time and cost savings relative to conventional protein production create the clearest quantifiable commercial benefit.
Sutro Biopharma and the Clinical Production Platform
Sutro Biopharma is the commercial company that has advanced cell-free protein synthesis furthest toward clinical and commercial pharmaceutical production, using its XpressCF and XpressCF+ cell-free systems to produce antibody-drug conjugates and other complex biotherapeutics whose site-specific chemical modification is enabled by the cell-free system's compatibility with unnatural amino acid incorporation at defined positions in the protein sequence. The site-specific conjugation capability that unnatural amino acid incorporation enables creates antibody-drug conjugates whose linker and payload attachment at a defined single site creates a more homogeneous product than the conventional lysine or cysteine conjugation methods that attach the drug payload at multiple variable positions on the antibody surface. Sutro's clinical pipeline of ADCs produced using its cell-free platform and its partnerships with Bristol-Myers Squibb, Merck, and other pharmaceutical companies for ADC development create the clinical validation of cell-free protein synthesis as a pharmaceutical manufacturing platform rather than a research tool. Its regulatory engagement with the FDA for the clinical and eventual commercial use of cell-free produced biopharmaceuticals is establishing the quality and regulatory framework that cell-free pharmaceutical production requires.
Tierra Biosciences, a California company applying cell-free protein synthesis to the agricultural biologics and pharmaceutical research markets, uses its cell-free platform to produce proteins for pesticide discovery, crop protection research, and pharmaceutical target validation at the speed that iterative protein engineering and screening requires. Its cell-free synthesis turnaround of days rather than the weeks that cell-based production requires creates the protein engineering cycle speed that directed evolution and combinatorial protein library screening benefit from, allowing more design-test-learn cycles within a given research timeline than conventional production enables. Synvitrobio, a cell-free synthetic biology company, applies cell-free systems to the production of complex biomolecular machines including ribosomally synthesised and post-translationally modified peptides whose production in cell-based systems is complicated by the cellular toxicity of many RiPP natural products.
The Diagnostics and Point-of-Care Application
The diagnostics application of cell-free protein synthesis, in which freeze-dried cell-free reactions can be embedded in paper-based or lateral flow diagnostic formats and rehydrated in the presence of a nucleic acid target to produce a detectable reporter protein, creates the cell-free biosensor whose on-demand protein production in a field-deployable format enables the detection of pathogen nucleic acids, environmental contaminants, or other molecular targets without the laboratory infrastructure that conventional nucleic acid amplification and detection requires. The SHERLOCK and DETECTR CRISPR-based diagnostic platforms that were adapted for COVID-19 detection use cell-free transcription and translation in some configurations to amplify and report the CRISPR cleavage signal, demonstrating the diagnostic cell-free application whose point-of-care deployment potential has attracted substantial research and commercial development investment since the pandemic demonstrated the need for rapid, deployable molecular diagnostics.
Top 10 Companies in Cell-Free Protein Synthesis Globally
- Sutro Biopharma: US biopharmaceutical company with XpressCF and XpressCF+ cell-free platforms for antibody-drug conjugate production; its clinical ADC pipeline produced by cell-free synthesis and its partnerships with Bristol-Myers Squibb and Merck create the most commercially advanced cell-free biopharmaceutical production programme whose FDA regulatory engagement is establishing the framework for cell-free pharmaceutical manufacturing approval.
- Tierra Biosciences: US cell-free protein synthesis company with agricultural biologics and pharmaceutical research applications; its day-scale protein production turnaround and its application in crop protection discovery and pharmaceutical target validation create the commercial cell-free synthesis service for iterative protein engineering workflows.
- Synvitrobio: US cell-free synthetic biology company with applications in RiPP natural product synthesis and complex biomolecular machine production; its cell-free approach to toxicity-limited natural product synthesis and its synthetic biology toolkit create the commercial cell-free platform for synthetic biology applications that cell-based production cannot practically achieve.
- Agilent Technologies: US life science tools company with cell-free expression system products for protein research; its in vitro transcription and translation kits and its automated liquid handling integration create the commercial cell-free protein expression toolkit for high-throughput research applications.
- Thermo Fisher Scientific: US life science company with PURExpress and other cell-free expression systems for research; its cell-free protein synthesis kits and its protein characterisation instruments create the complete in vitro protein research workflow that cell-free synthesis enables for academic and pharmaceutical research.
- New England Biolabs: US enzyme and reagent company with PURE system cell-free protein synthesis reagents; its reconstituted PURE cell-free system whose defined component composition creates reproducibility advantages over crude extract systems and its enzyme toolkit for cell-free synthetic biology applications create the reagent supply for the most rigorous cell-free research applications.
- Green Biologics (RIBO): UK biotechnology company with cell-free systems for biosensor and diagnostic applications; its freeze-dried cell-free reaction diagnostic format and its point-of-care biosensor application create the diagnostics cell-free synthesis commercial position whose field-deployable format addresses the point-of-need detection applications that laboratory-based nucleic acid diagnostics cannot serve.
- Creative BioMart: US protein production company with cell-free protein synthesis services for research customers; its cell-free expression service and its rapid turnaround capability create the contract service for researchers whose protein needs are too small for cell-based production optimisation but whose protein characteristics are too demanding for commercial cell-free kit systems.
- Profacgen: US contract protein production company with cell-free synthesis services alongside conventional cell-based expression; its cell-free service offering for difficult-to-express proteins including membrane proteins, toxic proteins, and selenoprotein-containing sequences creates the contract research service position for the protein classes whose cell-free production advantage over cell-based production is greatest.
- Cell Free Sciences (Ehime University): Japanese cell-free synthesis technology company with bilayer systems for membrane protein production; its lipid bilayer-integrated cell-free synthesis whose production of membrane proteins in their functional lipid environment creates the specialised cell-free product for the pharmaceutical and structural biology applications where membrane protein production in functional form is the primary technical challenge.