Chinese hamster ovary (CHO) cell lines are one of the most-used cell-based protein expression systems for therapeutics, thanks to their ability to support post-translational modifications such as glycosylation. That makes them valuable for producing antibody-drug conjugates (ADCs), monoclonal antibodies (mAbs), and therapeutic vaccines.
Despite CHO cells’ expanding applications, other protein expression systems may be at least as valuable in terms of rate of replication and ease of manufacturing. Cell-free expression systems are gaining traction, and use of the widely used Escherichia coli (E. coli) remains strong. Manufacturers also are assessing possibilities in avian-, plant-, fungal-, and insect-based cell cultures.
Cell-free systems
“The idea of expressing protein in bacterial extract goes back to the founding of Genentech in 1975, where the first products were produced in bacteria,” Hans-Peter Gerber, PhD, CSO of Sutro Biopharma, says. “Simple peptide growth factors were small and easy to manufacture, but when they moved to antibody therapeutics, which are larger molecules, they realized a quick bacterial approach wouldn’t work.” The solution was to engineer cell expression systems and to look at alternative systems.
Sutro Biopharma is among those companies that engineered bacterial extracts into a cell-free expression system.
“We use the cell-free system to make oncology medicines, specifically oncology ADCs, for targeted chemotherapy,” Jane Chung, CEO of Sutro Biopharma, tells GEN.
“The design of an ADC is very complex because it requires engineering not only an antibody, but a linker and a payload,” Chung points out. These components must be considered both individually and as a part of the whole. “A cell-free system gives far greater flexibility and control of the design, including where the linker payloads are attached to the antibodies.”
Such precise configuration enhances consistency of the medicine that’s delivered to patients, Chung says. Specifically, “When we configure a drug to antibody ratio (DAR) as a DAR 8, it’s a DAR 8—not an average,” thus ensuring patients receive precise, consistent dosages.
Additionally, Gerber says, “When we produce proteins with cell-free extracts, we can make more proteins faster, test them quicker and learn faster—especially with AI and machine learning—and, recently, make them as cheap or cheaper even than [with] CHO cells.”
Producing ADCs in a cell-free system is relatively easy, he continues. “We can generate 10,000 unique ADC molecules because all we have to do is make different DNAs and code for the different ADCs. We put unique DNA sequences into 10,000 tubes of extract, and they’re conjugated overnight and tested for activity within two weeks. The best you can do with CHO cells—and I’ve done that for 25 years—is to generate 10 to 100 ADC molecules within three to six months, and then select the lead candidate from among them.”
“High throughput screening is a key differentiator for us on the cell-free platform,” Chung observes, helping Sutro identify the optimal lead candidate from a wider field and thus de-risk development.
Reaching this point involved certain challenges, of course, that can be addressed with engineering. For example, “Bacteria don’t naturally form cysteine bridges between two protein chains, so we had to engineer them,” Gerber elaborates. The resulting proteins also needed to meet the same or higher quality than those of CHO cells. The U.S. Food & Drug Administration (FDA), he says, reached out to Sutro to collaborate on generating standard ADCs with precise DARs that serve as reference compounds for ADCs derived from CHO cells.
Post-translational modifications—like glycosylation—of proteins and antibodies also can be challenges in cell-free systems. But, Gerber points out, preventing glycosylation can be a “huge advantage” because glycan-induced toxicities are avoided.
Industry-wide, Gerber predicts more companies will move into cell-free mammalian systems.
- coli systems
- coli is the go-to organism for large-scale production of enzymes at New
England Biolabs, according to Brad Landgraf, PhD, process development group leader, formulation and purification discovery. While CHO cells are often the system of choice for complex mammalian proteins, E. coli is a go-to system for a plethora of other proteins.
“E. coli is the first organism you would try and has been our workhorse organism for making recombinant proteins,” Landgraf says. “It’s simple, fast, and easy. The genetic system is very well understood, and we know how to manipulate it very well. It’s relatively cost-effective and it grows quickly, doubling every 15 minutes at 37°C.” The platform produces enzymes required for vaccine manufacturing, rather than the vaccines themselves.
The main limitation of E. coli is its inability to install complex post-translational modifications, such as glycosylation. Kluyveromyces lactis (K. lactis) or Pichia pastoris (P. pastoris) are alternative systems in those instances, he says, “even though they’re very simple yeasts.”
Avian systems
GeoVax is transitioning from primarily avian cells to a continuous avian suspension system, AGE.1. “That places us squarely within the broader industry shift toward more scalable and resilient non-CHO manufacturing platforms,” David Dodd, CEO, tells GEN.
By transitioning modified vaccinia Ankara (MVA) vaccine production from primary chicken embryo fibroblasts (CEF) cells to the AGE.1 cell system, GeoVax expects to “improve consistency, scalability, yield, production speed, and long-term supply reliability,” Dodd says.
Before making the transition, the company evaluated four avian cell lines that had already been reviewed by regulators. AGE.1, which the company ultimately selected, showed “a tenfold increase in productivity and held that advantage across [about] 15 passages, and it was more than five times better than the other avian cell lines.”
Developing MVA vaccines in the AGE.1 cell expression system provides the surge capacity needed for both pandemic and niche applications, while enabling the vaccines to engage multiple targets and elicit both antibody and T cell responses, he says. GeoVax is using this approach with its SARS-CoV-2 vaccine, targeting the original strain through Omicron strains in a single vaccine. “We’re getting eight to 12 months of protective immunity, versus three to six for mRNA,” Dodd says.
Plant-based systems
Protalix is, to date, the only company to have received approval from the FDA and other regulators to manufacture and market plant cell-derived protein therapeutics. Two drugs have been developed in-house, approved, and out-licensed, and a third is in Phase II trials, says Dror Bashan, CEO.
The company is developing and manufacturing protein therapeutics using ProCellEx®, a proprietary plant cell-based expression platform. In it, plant cells are cultivated in suspension using disposable bioreactors that enable efficient, scalable, and cost-effective production of recombinant therapeutic proteins.
Plant-based systems “are an alternative way to produce complex proteins in a way that addresses some of the limitations inherent to mammalian-based expression systems,” Ori Kalid, PhD, vice president of R&D, says.
Advantages include competitive costs, robust production that survives fluctuations in growth conditions, a high safety factor due to the lack of mammalian pathogens in plants, and the ability to produce difficult-to-express proteins.
“Producing proteins in plant cells is very simple compared to CHO because of simpler culture conditions and media,” Uri Hanania, PhD, senior director of plant cells and genomic development, says.
Initially, Protalix used only very simple, well-known vectors to express proteins, but found that they yielded very low levels of recombinant proteins. Since those early days, the company’s scientific team has developed advanced systems that integrate highly effective viral vectors into proprietary, genetically engineered platforms compatible with Nicotiana benthamiana cell lines. “Expression levels are about 100 times greater than the initial system,” Hanania says, “reaching grams per kilogram cell scale, depending on the protein to be produced.”
ProCellEx “provides mammalian-like post-translational modification patterns,” Kalid adds, “and enables controlled production of key glycosylation patterns that are critical for protein activity and biodistribution.” Additionally, it provides “a robustness that enables the expression of proteins that may be toxic to mammalian systems, such as specific enzymes, cytokines, hormones, and other proteins.” Notably, “The glycan profile produced by the plant system… [enabled by genetically-modified host cell lines] is less complex than that produced in mammalian cells, thus providing a regulatory advantage,” Kalid adds.
“The expression level for this plant-based system varies among protein targets, and the ability to produce antibodies appears to be limited. However, because Protalix manufactures highly potent enzyme therapies for rare diseases, which require modest production volumes, this constraint has limited practical impact,” Kalid says. It is readily scalable and is being continuously improved, he adds.
Fungal systems
Dyadic’s fungal cell expression platform, C1, is a proprietary Thermothelomyces heterothallica-based system for producing mammalian-like glycoproteins used in mAb development. It enables faster, lower-cost production of complex proteins for mAbs, vaccines, and reagents than CHO cells, while operating across a broader range of temperatures and pH levels, says Mark Emalfarb, founder and CEO.
Using its C1 expression system, the company has completed Phase I trials in Africa for a COVID vaccine. That trial shows “we’re hitting the proof points in terms of yields and analytics,” Emalfarb says.
The C1 system is attracting interest from international organizations as a potential way to quickly scale up production for pandemic responses and for regional outbreaks. It has a doubling time of about two hours, compared with more than 20 hours for either CHO cells or baculovirus. Emalfarb adds, “Our manufacturing process is seven days in the fermenter, compared to 12 to 14 days for other processes.” In a pandemic scenario—for avian flu, for example—he says the C1 expression system could produce 300 million vaccine doses within two weeks using one 15,000-liter bioreactor.
In terms of mAbs, the C1 platform can move from DNA to purified material in about eight weeks, compared with more than 20 weeks for CHO production systems, Emalfarb says. The difference is that C1 cells can enter production and purification immediately after the product strain is selected, whereas CHO systems require clone isolation and screening, followed by creation of a working cell bank and seed train before production can begin.
Dyadic is partnering with the Gates Foundation, CEPI, and the Fondazione Biotecnopolo di Siena to increase the speed of vaccine development, and with other organizations, including the National Institutes of Health, on other vaccine or mAb projects.
Future
Novel organisms will continue to emerge as cell expression systems, but the main workhorses—including E. coli, CHO cells, P. pastoris, and K. lactis—are likely to remain so for some time.
Taken together, these approaches suggest that while CHO cells will remain foundational, alternative expression systems are increasingly reshaping how biologics are developed and manufactured. Landgraf says, “I really think the future of protein production is in cell-free [production]. The reason is that it’s very simple. You basically add DNA to your reaction and it makes the protein within hours, and you often can go directly from the protein synthesis reaction straight into an activity or functional assay to characterize that protein without extensive purification.”
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