Expression of biopharmaceuticals through the culturing of transgenic organisms is the basis of therapeutic biotechnology. Production platforms based on mammalian cells, yeast, and bacteria lead the way, with plants barely above noise levels in terms of commercial relevance.
Most efforts to date have used transgenic plants, which pass their acquired characteristics on to their progeny. A second approach, based on transient expression, may provide greater flexibility in terms of facility utilization and multi-product production, and significant advantages in time-to-market as well.
iBio is one firm espousing transient expression. In March, 2018, the company expanded its capabilities to include the cGMP contract manufacturing of therapeutic Fc fusion proteins, an emerging class of therapeutic protein with eleven FDA approvals and many more molecules in development pipelines.
The technique
iBio scientists engineer vectors for the desired protein sequence and transfer them to agrobacteria, which infiltrate mature plants and transfer the gene to the plant cell nuclei, which over the course of about five days churn out the therapeutic protein. Downstream steps are similar to purification of proteins expressed in mammalian cells.
iBio employs transient transfection, meaning that the agrobacterium hijacks the plant’s protein without conferring permanent changes to the plant’s genetics—the plant cannot pass protein production capability on to offspring.
Thus, issues related to human consumption of genetically modified plants disappears, as does the potential for Frankenplant-type scenarios in which traits are transmitted willy-nilly to plants outside the production area. “FDA views the plants as a raw material,” says iBio CEO Robert Erwin.
iBio conducts its cGMP processes indoors for biopharmaceuticals, but applications are opening up for producing food or industrial proteins outdoors.
Like all genetic engineering, transient transfection requires getting genes into cells in some manner. Spraying the transfected agrobacterium onto plants works, but iBio has devised a much more efficient, and elegant technique. It submerges plants into a suspension of the bacterium, and applies a vacuum to remove gases lurking within the plant structure. Releasing the vacuum creates a positive relative pressure that forces agrobacterium into cells via interstitial openings.
Yield and productivity for fed-batch CHO cultures are usually expressed volumetrically, i.e. grams-per-liter. Bioprocessors sometimes break down this value as the product of cellular productivity times cell density. For plant-derived proteins, yield is sometimes expressed as grams per unit of biomass, and somehow time must be factored in.
But where it ultimately counts—in cost of goods—plant-derived biopharmaceuticals compare very nicely with fed-batch cell culture. In the quantities needed for clinical trials and for initial commercial product launches, plant-made antibodies can be produced as bulk pharmaceutical (active pharmaceutical ingredient) for less than half the cost of fed-batch culture processes.
Flexible facilities and capital costs are huge concerns for therapeutic bioprocessing companies, and this is where expression in green plants has a sizable advantage over fixed-tank or even single-use bioprocessing.
“Scaleup is extremely predictable, unlike CHO, and the transition from lab to pilot to commercial production is much shorter,” Erwin explains. “Issues related to geometry or footprint of scaling do not exist.”
Capital costs are much lower as well, at least upstream, as controlled growing areas replace highly classified manufacturing space, green plants fill in for expensive stainless steel or plastic bioreactors, cleaning and disposal costs are practically eliminated, fluid volumes at harvest are significantly lower compared with cell culture, and manufacturers retain the option of “growing” new products or expanding existing production in almost no time.
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These advantages reduce significantly downstream, however.
“Achieving equivalent output from CHO is much more expensive,” Erwin notes, “but downstream, with the exception of viral safety steps, everything is pretty much the same as for cell culture processes, and cost or operational advantages disappear for fill/finish.”
Costs, flexibility
A group at the University of California, Davis, headed by Somen Nandi and Karen McDonald, also uses Nicotiana species and transient transfection. Using the engineering modeling package Superpro Designer, Nandi has performed extensive cost analysis of protein production in plants, and arrived at some interesting numbers.
Nandi’s “base case” cGMP facility, producing 300 kg of monoclonal antibody per year at a yield of 1g of protein per kg of biomass, with 65% downstream recovery of target antibody, entails a capital investment of $122 million, compared with more than $200 million for a facility built around single-use bioprocessing, and double that figure for a stainless steel-only facility. COG comes in at $121 per gram in this model, a 50% reduction.
According to Nandi, safety concerns regarding post-translational modifications, particularly glycosylation, were once an issue with plant-made biopharmaceuticals, but that is no longer the case. One solution is to knock out the plant genes coding for undesirable glycans, for example fucose and xylose, and proceed as normal with transient, expression, harvest, and purification.
“The other approach is to go with glycans that plants produce naturally,” Nandi says. He cites Elelyso®, a replacement enzyme developed by Protalix for treating Gaucher disease. Elelyso, the first FDA-approved plant-derived biopharmaceutical, is expressed in carrot cell culture and administered by injection. Although the product is produced in single-use bioreactors and not cultivated in soil or hydroponically, Protalix estimates facility capital outlays of just $20 million.
“There’s actually no evidence that fucose and xylose are harmful when taken orally or IV, but we can eliminate those glycans if we need to,” Nandi tells Biocompare. “If plant glycans were really a problem we’d all be dead anyway.”
Why the wait
Trade publications have been publicizing drug-making plants for three decades. The science is sound and the economics compelling, which begs the question of why this idea has not been widely commercialized. Conspiracy theories sound more reasonable year by year, but our experts have more reasonable explanations.
“First of all FDA and overseas regulators are not the problem, nor is anything wrong with the science or product safety,” says Erwin. “Unfortunately, many early developers lacked the resources to take products through clinical testing. At the first sign of trouble their larger commercial partners, when they existed, walked away.”
“I believe that commercial success will eventually occur, and we already have the necessary products in the pipeline,” Erwin says, “but it will depend on markets outside the United States and Europe. Developing countries are not wedded to mammalian cell culture, and are driven by costs to a much greater degree than U.S. and European markets, where biomanufacturers have existing infrastructure that’s fully capitalized and must be put to use. That is not the case in the rest of the world.”
Commercializing plants as production systems will depend on achieving a critical mass of effort in this area.
“Very few people are seriously working on plant-derived biopharmaceuticals relative to traditional cell culture-based production,” Nandi explains. “And yes, developers have for too long ignored or not prepared for clinical development. But at its core biopharmaceutical is ultra-conservative. Once a process, or a manufacturing platform is established, they’re loath to modify it.”