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Lyophilization: The Key to Portable Biomolecular Manufacturing

Synthetic biology applies rational design principles of engineering to molecular biology to build genetic devices, which have begun to impact the diagnostic and therapeutic space. This approach has helped to create whole-cell biosensors, genetically modified probiotics, and a growing capability for cell-based biomolecular manufacturing.

Vaccines are one of the most powerful tools in disease prevention, with childhood diseases, such as polio, smallpox, and diphtheria, largely contained owing to systematic immunizations. However, distribution to the developing world is challenging, primarily due to costs and cold-chain requirements. While some vaccines can be lyophilized, temperature-controlled distribution cannot be avoided for others. Therefore, vaccines and other protein-based biomolecules must be globally distributed from centralized foundries and, most often, require a cold chain for stability. These limitations impact distribution costs and highlight the challenge of delivering the benefits of these technologies to developing regions.

A new method is proposed for the safe deployment of genetically encoded tools using Freeze Dried-Cell Free (FD-CF) expression machinery on paper, generating a platform that retains the fundamental protein synthesis capability of live cells while remaining abiotic, sterile, and portable. In combination with toehold switch RNA sensors, this platform might be used a new class of low-cost diagnostic tools.

Freeze Dried-Cell Free (FD-CF) reactions offer additional venues for the distributed use of synthetic biology apart from diagnostics, such as the exciting prospect of the portable manufacture of pharmaceuticals, therapeutic proteins, and other biomolecules. In recent years, in vitro biosynthesis from fresh or frozen lysates has developed remarkably, including the biomanufacture of difficult molecules that cause cell toxicity and the incorporation of non-canonical elements. These advances have thus far been tied to laboratory settings where the necessary skills and equipment are found. Building off of this foundation, the proposed use of Freeze Dried-Cell Free systems, with their long-term activity at room temperature (>1 year) and ease of operation, could alleviate both the restrictions of livecell biosynthesis and cold-chain distribution requirements. Recent reports draw emphasis to a pressing need for the decentralization of therapeutic biomanufacturing, offering novel alternatives that, nonetheless, require expensive, large equipment and highly skilled operators or yet rely on production from living cells.

Previous work has demonstrated protein production from lyophilized reactions, which strongly supports the notion of advancing this concept further toward on-demand, local biomanufacturing. In addition to portability, the Freeze Dried-Cell Free format has all of the advantages that are innate to in vitro biosynthesis. Moreover, with buffers, cellular machinery, and molecular instructions all compressed into a single Freeze Dried reaction pellet, on-demand, on-site activation would only require the addition of water and yields product within 1–2 hr, without the need for specialized equipment and skill. This system could be applied for global health and personalized medicine, making scalable molecular synthesis available to anyone with Freeze Dried reagents and DNA-encoding biosynthesis instructions.

Looking forward, we envision a system that merges Freeze Dried-Cell Free diagnostics with biomanufacturing, such as their combination into wound dressings, allowing Freeze Dried-Cell Free systems to autonomously monitor patients for infection and respond with therapeutic biosynthesis. It is also intriguing to consider a more distant future in which new technology would allow for on-site DNA synthesis. Coupled with Freeze Dried-Cell Free reaction pellets, this capability would provide users with both the physical genetic program and the manufacturing tools in a form of ‘‘molecular 3D printing.’’ Scenarios of extreme conditions, including war zones or space travel, may leave individuals with unanticipated diagnostic or therapeutic needs, whereupon electronic transmission of biosynthesis instructions could be implemented on site. In summary, the Freeze Dried-Cell Free format provides a new venue for biomanufacturing that promises to stimulate further innovation in synthetic biology and, perhaps ultimately, to have a transformative effect on the distribution of global health.

Original article: Portable, On-Demand Biomolecular Manufacturing


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