Fast and Cell-Free-ious! Cell-Free Protein Synthesis in Biologics Manufacturing and Research

Imagine a world where life-saving drugs and vaccines could be produced in mere hours, bypassing the complexities that come along with cell culture. It may seem like fiction, but it is one of the most life-changing realities of today.

Welcome to the revolutionary realm of Cell-Free Protein Synthesis (CFPS), where scientists break the boundaries of biologics manufacturing and research. About 60 years ago, this revolutionary technique aided scientists, in reading the genetic code and finding the connection between mRNA and protein synthesis. (Read the original paper here!)

In recent years, CFPS has emerged as a breakthrough technique with important implications for biologics manufacturing and research and development (R&D). Unlike traditional cell-based systems, it operates in vitro, utilizing cell lysates or purified components to manufacture proteins without the restrictions of living cells.?This methodology presents several benefits, such as expedited protein synthesis, effortless genetic manipulation, compatibility with synthetic amino acids, and intricate protein architectures. As a result, CFPS has gained popularity as a diverse platform for manufacturing biologics, ranging from therapeutic proteins and vaccines to enzymes and diagnostics.

What is Cell-Free Protein Synthesis?

Cell-free protein synthesis (CFPS) is a method of expressing proteins that allows for the production of a specific protein without the need for living cells. It utilizes a solution that includes the necessary cellular components for protein synthesis, such as ribosomes, tRNAs, enzymes, cofactors, and amino acids along with crude cellular lysates from microorganisms, plants, or animals. This solution is employed to transcribe and translate a given nucleic acid template, such as plasmid DNA, linear DNA, or mRNA. CFPS, in a basic biochemical reaction, produces desired proteins in a matter of hours, whereas typical in vivo protein expression methods can take several days or even longer.

Cell-free protein synthesis harnesses the transcription and translation machinery of living cells to enable protein synthesis in vitro through the expression of natural or synthetic DNA. In the 1960s, the process of translation was discovered?in living cells and was extracted to?establish the genetic code embedded?into living cells. This also indicated that the sophisticated pieces of the machinery of life could be rebuilt in test tubes, without the necessities of a cell membrane or cell reproduction [2].

Cell extracts can be generated from any type of cell. Nevertheless, the most commonly employed cell-free systems are derived from the bacterium Escherichia coli, wheat germ, rabbit reticulocytes, or insect cells. By including amino acids, an energy source, salts, NTPs,?and other cofactors, it is possible to synthesize practically any protein from a DNA or RNA template.

Case Studies:

CFPS has many potential applications, including high throughput monoclonal antibody screening, portable system for vaccine production, therapeutic proteins, difficult-to-express proteins (eg. membrane proteins, toxins), genetic circuits, and incorporating non-natural amino acids in proteins.[3][4][5].

In a work by Jaroentomeechai et al., they successfully created a technology for one-pot biosynthesis of N-linked glycoproteins in the absence of living cells. This was accomplished by uniting cell-free transcription and translation with the necessary reaction components for N-linked protein glycosylation through a process of crude extract enrichment. The ability to modularly reconfigure and quickly interrogate glycosylation systems in vitro should make the CFPS technology a useful new addition to the glycoengineering toolkit for increasing our understanding of glycosylation and, in the future, advancing applications of on-demand biomolecular manufacturing.

Individual expression and assessment of antigen-specific hits is one of the major bottlenecks in the antibody discovery process. Hunt AC et al. devised a unified process for screening antibody fragments by merging techniques for cell-free DNA assembly and amplification, cell-free protein synthesis, and binding characterization.

This workflow possesses two prominent characteristics. Firstly, it is rapid. The full workflow can be accomplished within a few hours. Furthermore, each workflow phase was meticulously designed with a focus on automation and maximizing throughput. This procedure possesses several constraints and holds promising possibilities for further expansion. One drawback of the current workflow is that the sdFab (synthetically dimerized Fab) antibody fragment format is not the final therapeutic format of the antibody. Therefore, the sequences need to be reformatted to full-length IgGs before they can be expressed on a larger scale, either using cells or cell-free methods, for further development. This workflow's enhanced speed and throughput will allow researchers to efficiently and quickly screen a huge quantity of antibodies. This will facilitate the selection of extremely potent candidates that can be reformatted as IgGs, produced at higher scales, and further developed. This approach has the potential to assist in identifying effective medical countermeasures in future pandemics, as well as in the creation of binding proteins for therapeutic, diagnostic, and research purposes [1].

Advantages of CFPS in Biologics Manufacturing

1. Speed: One of the most significant advantages of cell-free protein synthesis (CFPS) is its ability to accelerate the protein production process. Traditional methods of protein expression rely on culturing living cells, which can be time-consuming due to the need for cell growth and maintenance. In contrast, CFPS bypasses these steps, allowing for rapid synthesis of proteins directly from genetic templates. This speed is particularly beneficial in research and development settings, where quick iterations and rapid prototyping are essential for innovation and efficiency.
2. Flexibility: CFPS offers unparalleled flexibility in protein production, enabling the synthesis of proteins that are challenging or impossible to express in living cells. This includes proteins with toxic properties or those that require the incorporation of non-natural amino acids. By removing the constraints of cellular machinery, CFPS allows researchers to experiment with a wider range of protein structures and functions, facilitating advancements in protein engineering and the development of novel therapeutics.
3. Scalability: Another noteworthy benefit of CFPS systems is their capacity to be scaled from small-scale research applications to industrial-scale production. From large-scale bioreactors to microplate-based high-throughput screens, CFPS systems are easily adaptable to a variety of sizes and forms. Since CFPS is scalable, it can accommodate the needs of both exploratory research and the commercial biologics manufacturing industry. This makes it an adaptable platform for protein production at various stages of development.
4. Safety: CFPS improves the safety and purity of the produced proteins by lowering the possibility of contamination from cell-derived products. Traditional cell-based protein manufacturing can introduce contaminants such as host cell proteins, DNA, and endotoxins, which require substantial downstream processing to eliminate. CFPS lowers these risks by eliminating the requirement for living cells, resulting in cleaner production processes and greater purity products. This safety component is critical for the manufacture of therapeutic proteins, where stringent purity standards are mandatory to ensure patient safety.[12]

Applications of CFPS in Research and Development

1. Protein Engineering: Cell-free protein synthesis (CFPS) is a valuable technique in protein engineering that allows researchers to efficiently generate and evaluate a wide range of protein variations without the constraints of living cells. CFPS circumvents the requirement for cellular machinery, enabling the inclusion of non-natural amino acids and the production of proteins with unique functions, hence augmenting the variety and possibilities of designed proteins. This approach optimizes the repetitive cycle of designing, testing and improving proteins, which speeds up the synthesis of proteins with enhanced stability, activity, and therapeutic potential. As a result, it pushes the limits of what can be achieved in protein engineering.[9]
2. High-throughput screening: Cell-free protein synthesis (CFPS) is an effective method for conducting high-throughput screening, allowing for the quick and simultaneous generation of many protein variations. This feature greatly speeds up the process of identifying and describing possible drug candidates, enzymes, and functional proteins.[11].
3. Synthetic Biology: A flexible platform is provided by CFPS for the quick design, testing, and optimization of genetic circuits. CFPS creates a controlled environment without the complications of living cells, which allows for the exact manipulation of experimental factors. This enables effective troubleshooting and adjustment of synthetic pathways and regulatory networks. This is particularly beneficial for developing complex metabolic pathways and novel biomolecules, as researchers can swiftly examine the functionality and interactions of numerous genetic components. CFPS expedites the progress of pioneering biotechnological applications and enhances our comprehension of biological systems. [10].

Challenges and Limitations:

Despite the significant advancements in cell-free protein synthesis (CFPS) technology, several challenges and limitations persist in its application for producing biologics. One of the primary concerns is the high cost of the process, which is largely attributed to the expensive cell lysates and the need for large-scale production facilities. Additionally, ensuring the quality of the synthesized proteins remains a significant challenge, particularly in terms of post-translational modifications (PTMs) such as glycosylation. Optimizing energy sources and reaction conditions to enhance protein folding and stability is another area of ongoing research. Furthermore, the production of glycosylated proteins, which are crucial for many biologics, remains a significant challenge due to the complexity of glycosylation pathways and the need for precise control over the process.

To overcome these limitations, researchers are exploring strategies such as the use of more cost-effective cell lysates, the development of novel energy sources, and the implementation of advanced reaction optimization techniques. Additionally, the integration of machine learning algorithms and computational design tools is helping to improve the efficiency and productivity of CFPS systems, ultimately enabling the production of high-quality biologics at a lower cost and with greater ease. [6][7][8].

Conclusion

In summary, cell-based systems excel in yield and quality but are slower and more complex. CFPS offers speed, simplicity, and potential scalability, but researchers must address quality limitations. The choice depends on the specific goals and constraints of biologics production.

References:

1.?????? Hunt AC, V?geli B, Hassan AO, Guerrero L, Kightlinger W, Yoesep DJ, Krüger A, DeWinter M, Diamond MS, Karim AS, Jewett MC. A rapid cell-free expression and screening platform for antibody discovery. Nat Commun. 2023 Jul 3;14(1):3897. doi: 10.1038/s41467-023-38965-w. PMID: 37400446; PMCID: PMC10318062.

2.?????? Garenne, D., Haines, M.C., Romantseva, E.F. et al. Cell-free gene expression. Nat Rev Methods Primers 1, 49 (2021). https://doi.org/10.1038/s43586-021-00046-x

3.?????? Zawada JF, Burgenson D, Yin G, Hallam TJ, Swartz JR, Kiss RD. Cell-free technologies for biopharmaceutical research and production. Curr Opin Biotechnol. 2022 Aug;76:102719. doi: 10.1016/j.copbio.2022.102719. Epub 2022 May 12. PMID: 35569340.

4.?????? Chiba CH, Knirsch MC, Azzoni AR, Moreira AR, Stephano MA. Cell-free protein synthesis: advances on production process for biopharmaceuticals and immunobiological products. Biotechniques. 2021 Feb;70(2):126-133. doi: 10.2144/btn-2020-0155. Epub 2021 Jan 20. PMID: 33467890.

5.?????? Jaroentomeechai, T., Stark, J.C., Natarajan, A. et al. Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery. Nat Commun 9, 2686 (2018). https://doi.org/10.1038/s41467-018-05110-x

6.?????? Dondapati SK, Stech M, Zemella A, Kubick S. Cell-Free Protein Synthesis: A Promising Option for Future Drug Development. BioDrugs. 2020 Jun;34(3):327-348. doi: 10.1007/s40259-020-00417-y. PMID: 32198631; PMCID: PMC7211207.

7.?????? Batista AC, Soudier P, Kushwaha M, Faulon JL. Optimising protein synthesis in cell-free systems, a review. Eng Biol. 2021 Feb 21;5(1):10-19. doi: 10.1049/enb2.12004. PMID: 36968650; PMCID: PMC9996726.

8.?????? Dondapati SK, Stech M, Zemella A, Kubick S. Cell-Free Protein Synthesis: A Promising Option for Future Drug Development. BioDrugs. 2020 Jun;34(3):327-348. doi: 10.1007/s40259-020-00417-y. PMID: 32198631; PMCID: PMC7211207.

9.?????? Kanter G, Yang J, Voloshin A, Levy S, Swartz JR, Levy R. Cell-free production of scFv fusion proteins: an efficient approach for personalized lymphoma vaccines. Blood. 2007 Apr 15;109(8):3393-9. doi: 10.1182/blood-2006-07-030593. Epub 2006 Dec 12. PMID: 17164345; PMCID: PMC1852255.

10.??? Hodgman CE, Jewett MC. Cell-free synthetic biology: thinking outside the cell. Metab Eng. 2012 May;14(3):261-9. doi: 10.1016/j.ymben.2011.09.002. Epub 2011 Sep 18. PMID: 21946161; PMCID: PMC3322310.

11.??? Kelwick R, Webb AJ, MacDonald JT, Freemont PS. Development of a Bacillus subtilis cell-free transcription-translation system for prototyping regulatory elements. Metab Eng. 2016 Nov;38:370-381. doi: 10.1016/j.ymben.2016.09.008. Epub 2016 Sep 30. PMID: 27697563.

12.??? Zemella A, Thoring L, Hoffmeister C, Kubick S. Cell-Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems. Chembiochem. 2015 Nov;16(17):2420-31. doi: 10.1002/cbic.201500340. Epub 2015 Oct 19. PMID: 26478227; PMCID: PMC4676933.

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