Among the challenges in upstream bioprocessing is the cellular waste generated as a by-product of cell culture. One approach that potentially offers the benefit of a “cleaner” biologic product in upstream processing is the use of cell-free expression systems. By producing less waste upstream, there is the potential benefit of having less burden downstream in the separation and purification steps. However, the practicality of using cell-free expression systems in the commercial manufacture of biologics requires proper assessment.
Traditional cell culture struggles
Traditional cell culture systems continue to deal with challenges, a major one being waste disposal, confirms Tim Ding, senior scientist in the Antibody and Protein Production Division at Creative Biolabs, a US-based custom service provider of antibody production and engineering. In the prolonged process of biologic production, the dying cells will inevitably release cytosolic content to the culture medium, essentially “contaminating” the batch and introducing heterogeneity. The lysing event also risks ruining the production because of the protease that are released, which will also increase the workload for downstream separation and purification, Ding says.
Traditional cell-culture based biomanufacturing is a mature technology; however, it is tied to a living cell, which necessitates a great deal of complexity. That complexity ranges from the creation of a master cell bank all the way to manufacturing at scale, product recovery and purification, and, finally, product formulation, according to a group commentary provided by James Swartz, James H. Clark professor in the School of Engineering and professor of Chemical Engineering and Bioengineering, Stanford University; Michael Jewett, Walter P. Murphy professor of Chemical and Biological Engineering and director of the Center of Synthetic Biology, Northwestern University; Matthew DeLisa, the William L. Lewis professor of Engineering and director of the Cornell Institute of Biotechnology, Cornell University; and Govind Rao, professor of Chemical, Biochemical, and Environmental Engineering and director of the Center for Advanced Sensor Technology, University of Maryland Baltimore County.
The professors explain that protein therapeutics (biologics) produced by cell culture face limitations, such as high product variability created by glycoform (micro- and macro-) heterogeneity; difficulty in engineering glycosylation pathways because of cell viability constraints; the inability to completely decouple cell growth from protein synthesis; and inconsistency in batch-to-batch runs based on the inherent variability in living cellular factories.
Limitations also include expensive centralized facilities, which are needed for good manufacturing practice (GMP); limited volumetric productivities due to the need to propagate cells in batch-mode bioreactors; and a timeframe of at least 18 months to go from finalized DNA-customized cells to commercial-scale volumes of product, according to the professors.
Cell-free in cell culture
“Cell-free systems offer an alternative approach that can address these limitations in traditional cell-based cell culture,” says Jewett. “They shorten design–build–test–learn cycles and enable portable, on-demand biomanufacturing at the point-of-use.”
“Recent technical advances in cell-free protein synthesis technologies have been shown to promote increased yields of therapeutically relevant proteins/glycoproteins,” says Swartz. The professors point to studies of these technical advances that have shown the following results:
Protein yields exceed grams of protein per liter of reaction volume; batch reactions last multiple hours; and reaction scale has reached 100 L (1)
• Cost-effective energy metabolism has been realized (2)
• Industrial applications are being advanced (3)
• Protein screening workflows have been accelerated (4)
• Controllable glycosylation reactions have been integrated with cell-free expression (5)
• More than 50 therapeutic glycans have been synthesized (6)
• Engineered strains that produce homogenous human N- and O-glycans have been developed and used to source lysates for cell-free expression (7)
• Medicines have been manufactured using on-demand, portable platforms comprised of detoxified lysates that contain endotoxin levels well below those in FDA-approved products (8,9).
“While transformative breakthroughs have set the stage to create a disruptive protein biosynthesis technology, gaps still exist,” Rao cautions.
The primary advantage of a cell-free system is speed (hours versus days or weeks to express product), simplicity (non-living system, hence easier to deal with), single-product expression (typically, only one gene product is being expressed, unlike the entire genome in a living cell), and, finally, potential for distributed and just-in-time biomanufacturing by virtue of using lyophilized cell extracts, the professors state. The professors collectively believe that cell-free protein expression offers increased control and flexibility compared to traditional cell-based bioprocessing. DeLisa also emphasizes that “cell-free biomanufacturing technology could enhance disaster responsiveness, enable timely response to emergent/pandemic threats, facilitate diagnostic development, and enable distributed biomanufacturing globally.”
Ding adds that the fact that no extra energy is “wasted” to maintain cell proliferation and survival in cell-free synthesis is another advantage. “The energy and nutrients in the cell-free systems are used exclusively to make desired biologic products. Thus, this high-efficiency expression could be theoretically achieved at very high yield per volume. On the other hand, minimal waste or contaminants are produced during the expression, which allows for a much easier purification process,” he states.
Cell-free expression comprises three major components, explains Ding:
• An energy source (i.e., an energy regenerating apparatus)
• Building blocks, such as nucleotides, amino acids etc.
• Transcription/translation machinery.
Scale-up of cell-free expression technologies is already seeing implementation. Companies, such as Sutro Biopharma, a US-based clinical-stage drug discovery, development, and manufacturing company, for instance, are already making microbial cell-free extracts and products at scale as well as having several products in clinical trials for which GMP compliance has been demonstrated, the professors state. Meanwhile, LenioBio, a Germany-based biotechnology company specializing in protein expression solutions, is making plant cell extracts. Swiftscale Biologics, another US-based company, is building the infrastructure to help creative drug developers advance previously impractical drug programs using cell-free biomanufacturing, the professors explain.
While new technologies are being developed, Jewett adds that he “anticipates that cell-free systems will promote better access to medicines through decentralized production. The future is bright.”
1. A.D. Silverman, A.S. Karim, and M.C. Jewett, Nat Rev Genet 21, 151–170 (2020).
2. M.C. Jewett, et al., Molecular Systems Biology 4, 220 (2008).
3. B.C. Bundy, et al., Current Opinion in Chemical Engineering 22, 177–183 (2018).
4. T. Hadi, et al., Sci Rep 10, 10279 (2020).
5. T. Jaroentomeechai, et al., Nat. Commun. 9, 2686 (2018).
6. J.M. Hershewe, W. Kightlinger, and M.C. Jewett, Journal of Industrial Microbiology and Biotechnology 47 (11) 977–991 (2020).
7. J.M. Hershewe, et al., Nat. Commun. 12, 2363 (2021).
8. J.C. Stark, et al., Science 7 (6) eabe94442021 (2021).
9. R. Adiga, et al., Nat Biomed Eng 2, 675–686 (2018).
10. J.F. Zawada, et al., Biotechnology and Bioengineering 108 (7) 1570–1578 (2011).
11. J.C. Stark, et al., Science Advances 7 (6) eabe9444 (2021).
About the author
Feliza Mirasol is the science editor for BioPharm International.