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Industry adoption of plant-made biologics remains slow, but plant-based technology gains an advantage by mainstream exposure.
While Chinese hamster ovary (CHO) cells are the workhorse of biologics production, other cell expression systems are starting to gain ground, such as plant-based expression systems. Though an older technology in and of itself, plant-based manufacturing has recently been validated in the mainstream biopharmaceutical industry, but there is still a lack of biomanufacturers using such platforms for good manufacturing practice (GMP) production.
In particular, a newer emerging platform, the plant-made pharmaceutical platform (PMP), has come into the mainstream spotlight with the February 2022 approval of Canada-based Medicago’s COVID-19 vaccine in Canada, COVIFENZ (plant-based virus-like particles, recombinant, adjuvanted) (1).
An important criterion for any expression system used to manufacture biologics products is the comparability of the end product with a validated expression system that is well known and widely used in the industry, such as mammalian CHO cells.
Plant cells are highly developed eukaryotic cells; each cell has the same machinery as a mammalian cell to express heterologous protein sequences accurately. Plants do, however, have slightly different post-translational modifications such as minor glycoform differences.
According to one industry source, if the glycoform on a particular therapeutic is a key to functionality, the glycoform can now be modified in the plant cell expression system to be as fully functional as the mammalian counterpart. This has been demonstrated in the literature for over a decade. One advantage in the plant system seems to be the consistency of the glycoform pattern expressed.
Plants also tend to produce a more homogenous product with post-translational modifications. Mammalian cells produce multiple glycoform patterns, which can be affected by culture conditions, including scale-up to larger reactor systems. Many biologics do not have any post-translational modifications.
Since the plant is a singular bioreactor that is grown the same way every time, consistency is achieved. To scale-up, simply grow more plants, this industry source says.
For most recombinant proteins, a plant-based expression system yields products that are highly comparable to mammalian/CHO systems, says Tom Isett, CEO of iBio. Isett also points out that, particularly in regards to monoclonal antibody (mAb) development, plants can deliver a more homogeneous N-linked glycosylation pattern. This characteristic provides greater assurance that the glycoform of choice is appropriately represented in the final product, likely improving efficacy, Isett notes.
“More simply put, it turns out that adding or removing sugars, or carbohydrates, to proteins in the development of antibodies can in some instances make them more stable and perform better. This process, called glycosylation, seems to be more easily controlled in plants than in CHO,” Isett states. “We typically see more homogeneous glycan types with plant-expressed antibodies. This is particularly important in the production of next-generation mAbs, biobetters, and fast-follower products.”
In addition, Isett points out that, for the development of mAbs for immuno-oncology, an afucosylated molecule (a mAb lacking the sugar, fucose) can yield enhanced potency via greater antibody-dependent cellular cytotoxicity (ADCC). Isett explains that his company uses a version of its plant-based bioreactor to deliver afucosylated molecules, “which have proven to be highly comparable, if not superior, to CHO expression systems.”
Because there is no limit to the size of a gene insert, plants can express both large and complex biomolecules that do not express well or cannot be expressed at all in conventional mammalian cell or bacterial expression systems, including immunoglobulin M molecules, says Leanne Williams, PhD, business development manager, Leaf Expression Systems.
Another benefit for plant-based expression of antibodies is that plants produce a vast range of stable and consistent post-translational modifications, including glycolysis. Although plants produce a slightly different glycosylation to mammalian systems, data have shown that these changes do not usually affect antibody functionality. “In the event that human glycosylation patterns are important or necessary for functionality, there are accessible plant lines that have been modified to produce human glycolysis patterns,” Williams states.
The approval of COVIFENZ makes a case for the use of plant-based manufacturing beyond just mAbs; rather, vaccines can now also be manufactured using a plant bioreactor system.
Isett points out that his company has used its plant-based technology (the FastPharming System) to rapidly produce subunit vaccine candidates. “In fact, at the outbreak of COVID-19, we were able to produce two spike-based vaccine candidates in just five weeks, a speed that rivals that of the mRNA [messenger RNA] platforms. This is consistent with the original DARPA [Defense Advanced Research Projects Agency] ‘Blue Angels’ project—upon which our facility was built—with the aim to be able to rapidly produce medical countermeasures in the event of a pandemic,” Isett remarks.
Isett says that iBio has continued to utilize plant-based expression technologies to produce a next-generation COVID-19 vaccine candidate. “Specifically, we believe that a vaccine that uses the more stable nucleocapsid protein of the virus can provide more durable protection against the frequently changing spike protein that commercially available vaccines attempt to provide immunity against,” he states.
In terms of production parameters, the upstream part of the company’s process (i.e., growing and transfecting the plants with the protein of interest) is much simpler and more robust than cell culture-based systems because fewer components are required, there is no need to maintain sterile boundaries, and there is no risk of exogenous viral contamination. Meanwhile, the downstream purification process is essentially the same in both mammalian and plant-based platforms. “That said, it’s the design of the antigen that will ultimately deliver us a more durable, effective, vaccine, than the first-generation versions that don’t seem to give us more than six months of immunity,” Isett says.
Vaccines are considered ideal targets for plant-made systems since the development time, time to clinic, and subsequent time to production can be much less than traditional mammalian systems. Although mRNA vaccine production has not necessarily been a large focus for plant systems, it has been demonstrated that plants are a strong platform for mRNA production, says an industry source. This achievement should portend well for plant-made production of mRNA vaccine candidates, the source adds.
Given the necessity of protein sub-unit vaccines for many applications, plant-based production is here to stay and will be part of the vaccine landscape for a long time even with the welcome success of mRNA vaccines. To achieve wide-spread immunity and to mitigate risk of new variants, it is important to develop these vaccine production systems globally, including parts of the developing world that cannot afford or support such efforts, the industry source notes.
The plant-based system works well, particularly for production of virus-like particle (VLP) vaccines, which includes COVIFENZ, adds Williams. “Production parameters between the systems suggest that plant-based expression processes offer a commercially viable option to conventional bioreactor-based processes, particularly when considering the ease of scale-up, which is achieved by simply increasing the number of plants, negating the need for complex scale-up processes required by traditional bioreactor systems,” Williams states.
She further explains that post-translational modifications (e.g., glycosylation patterns) are also more consistent in the plant systems because there is not the variation in culture parameters that occur in bioreactor scale-up processes. Williams also points to lower production costs in plant-based systems, saying that production time is predicted to be approximately 25% faster than a conventional bioreactor system. “In addition to the above points, a VLP vaccine is stable at 4 °C, which enables the vaccine to be transported more easily than the mRNA-based vaccines that are stored at either -20 °C or -70 °C,” she states.
Investment (e.g., equipment, technologies, facility, personnel) would be far less capital intensive for the upstream processing portion of plant-based biologics production, compared to investment needed to set up comparable upstream processing for mammalian cell-based systems (both single-use and traditional stainless-steel reactor facilities).
The ability to grow plants as reactors with high precision in automated and vertical hydroponic and aeroponic systems have been well developed, according to the industry source. This source also emphasizes that these plant manufacturing systems have no animal/human inputs or intervention, and, thus there are no concerns for adventitious virus contamination in these systems.
The upstream capital expenditure for a plant system is less than 35% of a traditional staged bioreactor train, whether single-use or stainless-steel, the industry source states. Furthermore, scale-up is not incremental in plant-based systems as additional capacity does not require a linear increase in robotics and personnel. Meanwhile, as explained earlier, downstream processes are comparable between plants and traditional bioreactors. Other advantages of the plant-made system include much lower energy consumption, lower water usage through recycling, and extremely low waste treatment costs, according to that industry source.
Furthermore, when comparing manufacturing with single-use systems, one must be mindful of the environmental impacts, points out Isett. Specifically, while single-use systems are considered by some to be more environmentally friendly than the cleaning associated with stainless-steek, at the end of the day, single-use systems use a significant amount of plastic disposables, Isett explains. He also notes that such significant plastic use likely contributes to biopharmaceutical industry being 55% more emissions intensive than the automotive industry.
Conversely, the key raw materials with a plant-based manufacturing system are considered “all-natural”: purified water, stone wool for the plants’ root substrate, and seeds, Isett lists. Isett says his company is still looking at ways to bring modern continuous purification bioprocessing techniques to the fore in downstream processing as well.
While afucosylated mAbs for immuno-oncology are undoubtedly well suited to having a plant-based production system, it has been demonstrated that plant-based technologies also have the ability to produce other recombinant proteins, says Isett. Isett uses the example of Safi Biosolutions, for which iBio was able to produce important cytokines and growth factors for that company’s bioproduction red blood cells and neutrophils in cell culture, with the aim to replace donated blood transfusions. Additionally, Isett points out that there are certain molecules that simply cannot be produced effectively in CHO cells, either because of their construct, or because aggregation and other issues occur with other expression platforms.
One significant technological advantage is the fact that plant cells, in most cases, are not affected by highly active human proteins, while mammalian cell-based systems can be affected by a build-up of a potent, heterologous protein, says the industry source. Plant systems are therefore ideal for producing highly active antibodies and antibody conjugates. Proteins can be compartmentalized in plant cells in the endoplasmic reticulum or trans-golgi spaces and can be secreted into the lamellar, cellulosic cell wall. Innovative downstream extraction and separation systems have been developed to take advantage of these properties, the source explains.
Widespread adoption of plant-based manufacturing technologies in the biopharmaceutical industry remains elusive, however. The biggest hurdle has been attracting capital investment to these platforms. The biopharmaceutical industry is slow to change, especially in production systems. Many biologics have been developed and demonstrated to be active by a relatively small number of academic laboratories and companies, and only a few have gone through the entire regulatory approval cycle, the industry source points out.
This source says that successes by companies such as Medicago and Protalix are welcome and showcase the platform; however, the plant-made pharmaceutical community, as a whole, has not put forth a unified effort for marketing the platform to the mainstream.The potential of the platform is still not widely understood, the industry source states.
On the regulatory front, both FDA and the European Medicines Agency are well informed about plant-made products and have been supportive and helpful in evaluating clinical trials and manufacturing systems, the source adds. Moreover, few, if any, clinical trials involving plant-made products have been put on clinical hold for toxicity; however, at this point in time, there are few, if any, contract development and manufacturing organizations that currently manufacture biologics for human clinical trials under current GMP compliance.
The COVID pandemic has played a role in bringing new technological platforms to the forefront, including a plant-made vaccine, baculovirus vaccines, and mRNA vaccines. In this relatively short period, these three platforms have gained a more mainstream prominence, the industry source adds. The hope is that this attention will result in investment and subsequent development of these systems for routine development and production of biologics.
The biopharmaceutical industry has traditionally been slow to innovate for a number of reasons, primarily patient safety/risk and quality/regulatory risk, according to Isett. The standard mammalian cell culture-based expression systems have been around for more than 30 years and continue to be perceived to be a relatively low-risk way to produce protein therapeutics. Furthermore, since upstream expression systems have achieved significantly high yields, the onus has been put on downstream processing to solve debottlenecking issues.
“That said, the question is whether we have ‘walked past’ quality and sustainability as well as speed as major unmet needs with traditional bioproduction. Let’s face it, in the midst of a terrible time for the biotech markets, now—more than ever—we need to find ways for developers of new therapeutics and vaccines to cross the funding ‘valley of death’ between preclinical and Phase II to find faster, less expensive ways to bring better new molecules to the clinic. The way to do that is with expression systems that expedite time-to-clinic and allow poor projects to fail faster, and good projects to proceed to IND [investigational new drug] application more cost effectively and with a greater probability of success,” Isett remarks.
Producing therapeutic and diagnostic products in plants is not new, says Williams. This technology has been around for the past 30 years, but it is not well established and is met with skepticism by many in what is a very highly regulated industry, she emphasizes. “This [skepticism] is by far the biggest hurdle plant-based expression systems have had to overcome. That said, the recent development of a successful COVID-19 vaccine has proved that the industry is open to change, and recognizes the need for a variety of production platforms in order to adapt to our growing and changing world,” she adds.
1. Medicago, “Medicago and GSK Announce the Approval by Health Canada of COVIFENZ, an Adjuvanted Plant-Based COVID-19 Vaccine,” Press Release, Feb. 24, 2022.
Feliza Mirasol is the science editor for BioPharm International.
Vol. 35, No. 7
When referring to this article, please cite it as F. Mirasol, “Plant-based Expression Systems are Gaining Mainstream Advantage,” BioPharm International 35 (7) 21–24 (2022).