The Promise And Challenges Of Cell-Free Protein Systems

By Matthew Coleman, Amy Rasley, Abisola Abisoye-Ogunniyan, and Nicholas Fischer — Lawrence Livermore National Laboratory

The production of subunit vaccines traditionally depended on live cell-based systems that involve cultivating cells to produce antigens or other key vaccine components. While these methods have proven effective, they often face challenges such as high costs, lengthy production timelines, and susceptibility to contamination.

As global health needs evolve, particularly in response to emerging infectious diseases, there is an increasing demand for more efficient and scalable production methods for rapidly producing subunit vaccines. Cell-free protein systems (CFPS), which leverage cell lysates or purified enzymes to synthesize proteins without the need for living cells, have emerged as a promising solution.¹

This technology offers significant advantages over traditional methods, enabling faster production, increased flexibility, and improved scalability.^2,3 By eliminating the need for living cells, CFPS can transform the vaccine manufacturing landscape allowing for more rapid and targeted responses to global health challenges. Our work at Lawrence Livermore National Laboratory (LLNL), has been instrumental in advancing vaccine technology through the development of nanolipoprotein particles (NLPs, also referred to as nanodiscs).^4,5 Our research over the last decade and a half has focused on utilizing synthetic biology to create nanoparticles that can effectively deliver membrane bound proteins as effective vaccine antigens.

LLNL’s collaborative work exemplifies the innovative application of NLP technology in vaccine development, offering promising avenues for creating effective and stable vaccines against various pathogens. LLNL researchers have also shown that NLP subunit vaccines can be freeze-dried, stored at room temperature for extended periods, and rehydrated without losing their efficacy. This property enhances the practicality and accessibility of vaccines developed using NLP technology.

Cell-Free Protein Synthesis And Vaccine Development

CFPS technology can represent a significant shift in the way vaccines are produced, enabling the synthesis of complex biomolecules more rapidly and efficiently. By utilizing cell lysates or purified enzymes, CFPS bypasses the biological constraints of living systems. This approach holds particular promise for producing membrane proteins, which are often key targets in vaccine design. However, membrane proteins pose significant formulation challenges due to their complex structures and poor solubility.

Cell-free systems overcome these barriers by enabling the production of these proteins in a more stable and functional state, paving the way for the development of more effective vaccines.

Cell-free protein biomanufacturing systems represent a significant advancement for developing vaccine technology by enabling rapid and efficient synthesis of complex biomolecules. CFPS offers freedom to tune and tailor the individual vaccine constituents, including antigens, adjuvants, and other components of vaccine formulations. In particular, chemical modifications can be incorporated using CFPS that would not be feasible in live cells, providing new tools for biomolecule conjugation, visualization, and stabilization.

The precision and modularity of CFPS make it possible to produce vaccines that are tailored to specific pathogens or personalized for individual patients. Furthermore, its ability to scale production rapidly is invaluable during outbreaks or pandemics, where speed is critical to saving lives.

One area where CFPS technology has shown significant potential is in the production of NLPs. NLPs are synthetic nanoparticles that mimic the structure of natural lipoproteins, providing a stable platform for delivering antigens and adjuvants in a biocompatible format. These particles are particularly useful for stabilizing and providing a supporting scaffold for membrane proteins,^6-8 which are critical in the development of vaccines targeting viruses and bacteria. By incorporating membrane proteins into NLPs, researchers can ensure that the antigens maintain their native conformation, which is crucial for eliciting a strong and specific immune response.

Figure 1: Cell-free expression can be used to generate membrane protein (purple) supported within an NLP nanodisc (green), which is formed by the apolipoprotein corralling the lipids (yellow) to form a supporting scaffold. Source: Matthew Coleman

 

Traditional subunit vaccine development

Traditional subunit vaccine production relies on live cell-based systems, such as bacterial, yeast, or mammalian cell cultures, to express and purify antigenic proteins for immunization. These methods have been widely used due to their ability to produce well-characterized vaccine components with proven efficacy. However, they come with several challenges that can hinder large-scale vaccine development.

The production process is often time-consuming and resource-intensive, requiring optimized growth conditions, extensive fermentation or cell culture techniques, and sophisticated downstream purification to remove contaminants such as endotoxins, host cell proteins, or misfolded proteins. Ensuring the proper folding and post-translational modifications of complex antigens, particularly in non-mammalian expression systems, adds another layer of difficulty that can possibly be addressed by cell-free protein expression.⁹

Additionally, scalability remains a significant issue, as expanding production to meet global demand—especially during pandemics or outbreaks — can be constrained by bioreactor capacity, supply chain limitations, and regulatory hurdles.¹⁰

Despite these challenges, traditional subunit vaccine platforms have been instrumental in the development of vaccines for diseases such as hepatitis B and human papillomavirus (HPV), demonstrating their effectiveness and reliability in immunization strategies.

Ongoing advancements in bioprocessing, recombinant protein expression, and adjuvant formulation continue to improve the efficiency and immunogenicity of subunit vaccines, but the need for more flexible and rapid production methods remains an ongoing area of exploration in the field of vaccinology.

Nanolipoprotein Particles In Vaccine Development

Nanolipoprotein particles have become a central tool in vaccine development due to their versatility and ability to stabilize challenging antigens. NLPs consist of a lipid bilayer surrounded by scaffold proteins, which create a nanoscale platform that mimics the native cellular environment.¹¹ This allows for the effective incorporation of membrane proteins and enhances their stability, solubility, and functionality.

The concept of using nanodiscs, self-assembled systems composed of phospholipids and scaffold proteins,⁶ was first introduced in the early 2000s and was first expanded for use in CFPS systems in 2008.^12,13 This innovation has been critical for studying membrane proteins in their native-like state and has had a profound impact on vaccine research, especially for proteins that are difficult to solubilize in traditional systems.

 

CFPS can be used to simultaneously express both the membrane protein and NLP, allowing the membrane protein to be captured within the NLP bilayer during translation. By providing a native lipid environment, the NLP supports proper folding and reduces aggregation of the membrane protein.

NLPs also offer several advantages as delivery platforms for adjuvants, which are substances that enhance the body's immune response to an antigen.^14,15 Their unique nanostructure allows for precise control over their size, surface charge, and composition, enabling optimization for efficient uptake by antigen-presenting cells, such as dendritic cells.

Decorating NLPs with adjuvants and immunostimulatory molecules, such as Toll-like receptor agonists, can boost the immune response in response to the vaccine antigen. This multifunctionality makes NLPs an ideal platform for developing vaccines that are not only effective but also simpler to formulate, potentially reducing the number of adjuvants needed and minimizing side effects.

Application Of CFPS In Nanolipoprotein Particle Production

The modularity and scalability of NLP production make them an ideal choice for use in large-scale vaccine manufacturing. NLPs can be synthesized using well-established biochemical techniques, ensuring that they can be produced consistently and on a large scale. This scalability is particularly important in the context of pandemic preparedness, where the need for rapid production of vaccines is critical.

CFPS technology, by enabling the production of NLPs in a cell-free environment, allows for the rapid synthesis of these particles in a controlled and efficient manner. This is achieved by combining DNA encoding for apolipoproteins and membrane proteins with lipids in a cell-free reaction chamber, eliminating the need for purified proteins or detergents, which simplifies the production process.

Recent studies have demonstrated the potential of CFPS-produced NLPs incorporating a promising membrane protein vaccine candidate.¹⁶ MOMP, the major outer membrane protein of Chlamydia trachomatis, is a key target for vaccine development. The research shows that NLPs incorporating MOMP can elicit strong immune responses in preclinical models, including the production of MOMP-specific antibodies and protective immunity against chlamydia infection.^17-19 These examples highlight the potential of CFPS and NLPs for developing vaccines against difficult-to-target pathogens. Other efforts are ongoing to extend CFPS for vaccines against other pathogens.^20,21

Translation Of CFPS To Large Scale

The successful translation of CFPS technology from laboratory research to large-scale vaccine production hinges on overcoming several regulatory and manufacturing challenges.

One of the primary concerns is ensuring batch-to-batch consistency, as variations in lysate preparation, reagent stability, and reaction conditions can impact the reproducibility of vaccine production. Regulatory agencies such as the U.S. FDA and the EMA require rigorous validation of manufacturing processes to guarantee product safety, potency, and purity. Additionally, GMP must be established for CFPS-based vaccines, necessitating the development of standardized protocols for lysate production, protein expression, and purification.

Another key consideration is the immunogenicity and stability of CFPS-produced antigens, which must be extensively evaluated in preclinical and clinical trials to meet regulatory approval standards. Unlike traditional cell-based systems with well-documented regulatory pathways, CFPS represents a novel approach, requiring ongoing collaboration between researchers, industry stakeholders, and policymakers to establish clear regulatory guidelines.

Furthermore, ensuring the scalability and cost-effectiveness of CFPS technology is crucial for global vaccine distribution, particularly in resource-limited settings where affordability and storage stability are critical factors. As regulatory frameworks evolve to accommodate emerging biomanufacturing technologies, proactive engagement with regulatory bodies will be essential to streamline approval processes and facilitate the rapid deployment of CFPS-based vaccines in response to global health threats.

Challenges And Future Directions

While the potential of CFPS and NLPs in vaccine production is clear, there are several challenges that must be addressed to optimize their use. One key focus area is improving the scalability and cost-effectiveness of production. Although CFPS offers significant advantages in terms of speed and flexibility, the current processes still require optimization to achieve the high yields necessary for large-scale vaccine production. Advances in reactor design, energy regeneration systems, and gene codon optimization could help increase the efficiency of cell-free reactions, reducing costs and improving scalability.

Another area for improvement is the stability of NLPs under various storage conditions. For vaccines to be effective in global health campaigns, they must be stable and easy to transport, even in regions with limited access to refrigeration. Research into improving the physicochemical properties of NLPs, such as size, surface charge, and lipid composition, could help increase their stability and improve their biodistribution in the body. Furthermore, enhancing the loading capacity of NLPs to deliver more antigens and adjuvants without compromising their stability would increase their effectiveness as vaccines.

Finally, for clinical translation, it is essential to establish robust safety profiles and standardized manufacturing protocols for CFPS-produced vaccines. Regulatory approval processes must be streamlined to enable the rapid deployment of cell-free vaccines, particularly in response to emerging infectious diseases.

Conclusion

Cell-free protein synthesis technology represents a groundbreaking advancement in vaccine production, offering a potentially more efficient, flexible, and scalable approach compared to traditional cell-based systems. The use of nanolipoprotein particles as a vaccine delivery platform further enhances the potential of this technology by stabilizing membrane proteins and improving immune responses. We have successfully applied this to the development of a protective chlamydial vaccine. While challenges remain in optimizing scalability, stability, and cost-effectiveness of CFPS combined with NLPs, ongoing research is paving the way for their widespread adoption in vaccines as well as biotechnology. As global health challenges continue to evolve, CFPS combined with NLPs could play a key role in enabling faster, more targeted, and cost-effective vaccine production, ultimately transforming the field of vaccinology.

References:

1. Katzen F, Chang G, Kudlicki W. The past, present and future of cell-free protein synthesis. Trends Biotechnol. 2005 Mar;23(3):150-6. doi: 10.1016/j.tibtech.2005.01.003. PMID: 15734558.
2. Sullivan CJ, Pendleton ED, Sasmor HH, Hicks WL, Farnum JB, Muto M, Amendt EM, Schoborg JA, Martin RW, Clark LG, Anderson MJ, Choudhury A, Fior R, Lo YH, Griffey RH, Chappell SA, Jewett MC, Mauro VP, Dresios J. A cell-free expression and purification process for rapid production of protein biologics. Biotechnol J. 2016 Feb;11(2):238-48. doi: 10.1002/biot.201500214. Epub 2015 Dec 7. PMID: 26427345.
3. Yang WC, Patel KG, Wong HE, Swartz JR. Simplifying and streamlining Escherichia coli-based cell-free protein synthesis. Biotechnol Prog. 2012 Mar-Apr;28(2):413-20. doi: 10.1002/btpr.1509. Epub 2012 Feb 1. PMID: 22275217.
4. Cappuccio, J.A., Blanchette, C.D., Sulchek, T., Arroyo, E. S. Hinz, A.K. Chromy, B.A. Fletcher, J., Katzen, F., Kudlicki, W., Bench, G., Hoeprich, P.D. and Coleman, M.A. (2008) Self-assembly of solubilized functional integral-membrane proteins in nanolipoprotein complexes using cell-free co-expression. Mol. Cell. Proteomics. 7, 2246-2253.
5. 37. Katzen F. Fletcher, J., Kang, D., Peterson, T., Cappuccio, J.A., Blanchette, C.D., Shulchek, T., Chromy, B., Hoeprich, P., Coleman, M.A. and Kudlicki, W. (2008) Cell-free expression of integral membrane proteins into discoidal membranes. J. Proteome Res. 7:3535-3542.
6. Bayburt, T. H., Grinkova, Y. V., & Sligar, S. G. (2002). Self-Assembly of Discoidal Phospholipid Bilayer Nanoparticles with Membrane Scaffold Proteins. NANO LETTERS, 2(8), 853–856. DOI:10.1021/nl025623k.
7. Nath, A., Atkins, W. M., & Sligar, S. G. (2007). Applications of Phospholipid Bilayer Nanodiscs in the Study of Membranes and Membrane Proteins. BIOCHEMISTRY, 46(8), 2059–2069. DOI:10.1021/bi602371c.
8. Denisov, I. G., & Sligar, S. G. (2017). Nanodiscs in Membrane Biochemistry and Biophysics. CHEMICAL REVIEWS, 117(6), 4669–4713. DOI:10.1021/acs.chemrev.6b00690.
9. 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.
10. Dudley QM, Karim AS, Jewett MC. Cell-free metabolic engineering: biomanufacturing beyond the cell. Biotechnol J. 2015 Jan;10(1):69-82. doi: 10.1002/biot.201400330. Epub 2014 Oct 15. PMID: 25319678; PMCID: PMC4314355.
11. Chromy BA, Arroyo E, Blanchette CD, Bench G, Benner H, Cappuccio JA, Coleman MA, Henderson PT, Hinz AK, Kuhn EA, Pesavento JB, Segelke BW, Sulchek TA, Tarasow T, Walsworth VL, Hoeprich PD. Different apolipoproteins impact nanolipoprotein particle formation. J Am Chem Soc. 2007 Nov 21;129(46):14348-54. doi: 10.1021/ja074753y. Epub 2007 Oct 27. PMID: 17963384.
12. Cappuccio, J.A., Blanchette, C.D., Sulchek, T., Arroyo, E. S. Hinz, A.K. Chromy, B.A. Fletcher, J., Katzen, F., Kudlicki, W., Bench, G., Hoeprich, P.D. and Coleman, M.A. (2008) Self-assembly of solubilized functional integral-membrane proteins in nanolipoprotein complexes using cell-free co-expression. Mol. Cell. Proteomics. 7, 2246-2253.
13. Katzen F. Fletcher, J., Kang, D., Peterson, T., Cappuccio, J.A., Blanchette, C.D., Shulchek, T., Chromy, B., Hoeprich, P., Coleman, M.A. and Kudlicki, W. (2008) Cell-free expression of integral membrane proteins into discoidal membranes. J. Proteome Res. 7:3535-3542.
14. Fischer NO, Rasley A, Corzett M, Hwang MH, Hoeprich PD, Blanchette CD. Colocalized delivery of adjuvant and antigen using nanolipoprotein particles enhances the immune response to recombinant antigens. J Am Chem Soc. 2013 Feb 13;135(6):2044-7. doi: 10.1021/ja3063293. Epub 2013 Jan 31. PMID: 23331082.
15. Weilhammer DR, Blanchette CD, Fischer NO, Alam S, Loots GG, Corzett M, Thomas C, Lychak C, Dunkle AD, Ruitenberg JJ, Ghanekar SA, Sant AJ, Rasley A. The use of nanolipoprotein particles to enhance the immunostimulatory properties of innate immune agonists against lethal influenza challenge. Biomaterials. 2013 Dec;34(38):10305-18. doi: 10.1016/j.biomaterials.2013.09.038. Epub 2013 Sep 27. PMID: 24075406; PMCID: PMC7172747.
16. He, H., Felderman, M., Evans, A.C., Geng, J., Homan, D., Bourguet, F., Fischer, N.O., Li, Y., Lam, K.S., Noy, A., Xing, L., Cheng, R.H., Rasley, A., Blanchette, C.D., Kamrud, K., Wang, N., Gouvis, H., Peterson, T.C., Hubby, B. and Coleman, M.A. (2017) Cell-free production of a functional oligomeric form of a chlamydia major outer membrane protein (MOMP) for vaccine development. J. Biol. Chem. jbc.M117.784561. doi:10.1074/jbc.M117.784561.
17. Tifrea, D.F., Wei, H., Pal, S., Evans, A.C. Gilmore, S.F., Fischer, N.O., Rasley, A., Coleman, M.A., and de la Maza, L.M. (2021) Induction of protection in mice against a Chlamydia muridarum respiratory challenge by a vaccine formulated with the major outer membrane protein in nanolipoprotein particles. Vaccines. 9(7): 755. doi: 10.3390/vaccines9070755.
18. Mohagheghi, M., Abisoye-Ogunniyan, A., Evans, A. C., Peterson, A. E., Bude, G. A., Hoang-Phou, S., Vannest, B. D., Hall, D., Rasley, A., Weilhammer, D. R., Fischer, N. O., He, W., Robinson, B. V., Pal, S., Slepenkin, A., de la Maza, L., & Coleman, M. A. (2024). Cell-Free Screening, Production and Animal Testing of a STI-Related Chlamydial Major Outer Membrane Protein Supported in Nanolipoproteins. Vaccines, 12(11), 1246. https://doi.org/10.3390/vaccines12111246
19. Weyant, K.B., Jaroentomeechai, T., Moeller, T.D., Hoang-Phou, S., Rivera-De Jesus, M., Singh, R., Liao, J., Locher, C., Putnam, D., Gilmore, S., de la Maza, L.M., Coleman, M.A. and DeLisa, M.P. (2023) A modular vaccine platform enabled by decoration of bacterial outer membrane vesicles with biotinylated antigens. Nat Commun 14, 464. https://doi.org/10.1038/s41467-023-36101-2
20. Stark JC, Jaroentomeechai T, Moeller TD, Hershewe JM, Warfel KF, Moricz BS, Martini AM, Dubner RS, Hsu KJ, Stevenson TC, Jones BD, DeLisa MP, Jewett MC. On-demand biomanufacturing of protective conjugate vaccines. Sci Adv. 2021 Feb 3;7(6):eabe9444. doi: 10.1126/sciadv.abe9444. PMID: 33536221; PMCID: PMC7857678.
21. Hu VT, Kamat NP. Cell-free protein synthesis systems for vaccine design and production. Curr Opin Biotechnol. 2023 Feb;79:102888. doi: 10.1016/j.copbio.2022.102888. Epub 2023 Jan 13. PMID: 36641905.
22. M.A. (2024) Cell-free screening, production and animal testing of a STI-related chlamydial major outer membrane protein supported in nanolipoproteins. bioRxiv, 2024.08. 20.608363.

About The Authors:

Matthew A. Coleman, Ph.D., is a senior staff biomedical scientist at Lawrence Livermore National Laboratory and an adjunct professor in the Department of Radiation Oncology at the University of California, Davis. His research focuses on radiobiology, biodosimetry, and nanobiotechnology, particularly the development of nanolipoprotein particles for membrane protein studies. Dr. Coleman has authored more than 120 publications and holds multiple patents in biomarker discovery and biotechnology. He earned his B.S. in biology from the University of Massachusetts and a Ph.D. in biophysics from Boston University.

Amy Rasley, Ph.D., is a senior staff scientist in the Physical and Life Sciences Directorate at Lawrence Livermore National Laboratory. Her research encompasses host-pathogen interactions, immunology, and infectious diseases, with a focus on the development of cell-based and cell-free platforms for diagnostics and countermeasure discovery as well as vaccine development. She is chair of the Institutional Animal Care and Use Committee (IACUC) and is also a member of the editorial advisory board for Current Immunology Reviews and serves as associate editor for Frontiers in Immunology. She earned her B.S. in biology from Weber State University, Utah, and her Ph.D. in immunology from the University of North Carolina Charlotte.

Abisola Abisoye-Ogunniyan, Ph.D., is a staff research scientist at Lawrence Livermore National Laboratory, specialized in understanding immune responses to nanolipoprotein particle-based vaccines and therapeutics in development and preclinical studies. She earned her B.S. in microbiology from the University of Jos, Nigeria, and later pursued her M.S. in biology and Ph.D. in integrative biosciences at Tuskegee University, where she gained interdisciplinary expertise in molecular biology, immunology, and biotechnology. Through her research, Dr. Abisoye-Ogunniyan contributes to improving global health outcomes and advancing scientific innovation.

Nicholas Fischer, Ph.D., is a senior staff research scientist at Lawrence Livermore National Laboratory. His research focuses on the development of nanoparticle-based therapeutics and vaccines, including nanoparticle formulation, functionalization, characterization, and evaluation. He earned his Ph.D. from the University of Massachusetts – Amherst in cellular and molecular biology.