published 02/19/2002

The age of molecular farming is upon us. It's been 16 years since researchers first coaxed green plants to make human proteins, and they've made tremendous strides since then. Not only have they proved that recombinant proteins made in plants are just as biologically active as their counterparts made in mammalian, yeast or bacterial systems, but also they've tested a few potential therapeutic drug candidates in early-stage clinical trials. Moreover, transgenic plants can be scaled up for manufacturing purposes just by planting more acreage. Molecular farming companies claim their systems can churn out more finished product than any commercial-scale cell culture facility -- at a fraction of the cost. But what will it take for biotech and pharmaceutical companies to adopt the technology?

There's a bright golden haze on the meadow
There's a bright golden haze on the meadow
The corn is as high as an elephant's eye
And it looks like it's climbing right up to the sky

From "Oh, What A Beautiful Morning"
Rogers & Hammerstein, Oklahoma! (1943)


Ah, yes -- Curly's opening lyrics from the ever-popular Broadway musical Oklahoma remain forever a glowing tribute to America's fertile heartland. But when Curly gazed out over acre upon acre of corn, that's exactly what he saw: Corn, pure and simple. Those days are long gone.

Thanks to stunning advances in agricultural biotechnology, a research scientist looking at those same fields 10 years ago would have seen not merely corn, but rather a staple crop that had been genetically engineered to resist insects and herbicides -- a great boon to farmers.

Today, the view's changed again: Curly's hypothetical elephant would be standing eye-to-eye with hundreds of little green factories, busily producing human pharmaceuticals. The age of molecular farming -- using plants as living bioreactors to produce recombinant proteins and monoclonal antibodies -- is upon us. If the technology proves out, it could be a godsend to those biotech and pharmaceutical companies that already find themselves in the midst of a manufacturing capacity crisis. Unlike even the largest cGMP commercial-scale manufacturing facility, fields of green plants can make tons of product every year. That's enough to supply commercial quantities of all the biopharmaceuticals in development or already on the market.

But we mustn't forget that others gaze at those cornfields, too -- and they see nothing but an environmental threat. If you thought Greenpeace and other activist groups were upset about genetically modified (GM) crops -- and their danger to animals, beneficial insects, soil microorganisms and the food chain -- just wait. From all reports, prior protests were but a prelude to what we'll witness when activists begin expressing their outrage about drug-producing plants, which, they say, could cause untold effects on human health if they happen to contaminate the food supply.


As unlikely as these concerns may be, they're not to be taken lightly. Just ask Julian Ma, a senior lecturer and honorary consultant in immunology and oral immunotherapy at Guy's Hospital in London. Ma's research involves the use of transgenic plants to make diagnostic and therapeutic antibodies as well as recombinant immune complexes. He depends on grants to support his work -- and funding is very tight. "Funding from the EU was good to begin with, but now it's dried up," he said. "The European view on GM crops is affecting everything."

That includes molecular farming. "A year ago there was a clear distinction between a plant engineered to make a pesticide and one engineered to make a drug," he continued. "Greenpeace was keeping quiet about pharmaceutical-producing plants and Golden Rice." (Golden Rice is nutritionally enhanced with beta-carotene, a precursor of vitamin A. For more details about agbio's efforts to engineer nutritional traits, see the Signals article "Agbio At Bat.")

But things have changed, Ma said. "Greenpeace no longer makes the distinction. It is anti-GM across the board."

And biotech companies have to take that to heart. The 20 or so molecular farming firms that are conducting, or planning, open-air field trials of transgenic, drug-producing plants are already proceeding with the utmost caution and attention to environmental issues. Some aren't even using edible crops. Others are confining their activities to greenhouses, which provide a completely controllable environment. (The tables in this article provide descriptions of many of the players in this arena.)


Though they differ in their approaches, all these firms have the same goal in mind: To harness the considerable abilities of transgenic plants to produce human biopharmaceuticals. Some are also using plants to make reagents for cell culture media, industrial enzymes and nutraceuticals. The technology's not especially new -- scientists achieved the basic feat of coaxing plants to make recombinant proteins about 16 years ago -- but it's been refined. Researchers have already demonstrated that recombinant proteins made in plants are just as biologically active as their counterparts produced in mammalian, yeast or bacterial cell culture. Moreover, a handful of these putative drugs have already made it into early-stage human clinical trials, where they've proven safe, perhaps even efficacious.

For instance, Meristem Therapeutics, based in Clermont-Ferrand, France, has engineered corn to produce recombinant mammalian gastric lipase for treating exocrine pancreatic insufficiency. Phase I clinical trials, conducted in the U.K., were completed in September 2000. Meristem's partner, Solvay Pharmaceuticals S.A., is currently shepherding the product through Phase IIa trials in patients with cystic fibrosis, whose mucus-laden pancreases are unable to secrete the digestive enzyme into the intestines.

As well, several years ago researchers at Cornell University's Boyce Thompson Institute for Plant Research conducted a small study in human volunteers of an edible vaccine for hepatitis B virus (HBV) infection. The technology, pioneered by Charles Arntzen (who is currently on the faculty at Arizona State University) involves genetically engineering potatoes to produce recombinant hepatitis B surface antigen (HBsAg), the same antigen (produced in yeast) that's used in Merck & Co. Inc.'s FDA-approved HBV vaccine. The putative vaccine, produced in the potato's flesh, is eaten raw.

Unfortunately, the British-based company involved in this collaboration -- Axis Genetics plc -- has since gone out of business. According to the Financial Times, the company may have been "the first corporate victim of public antipathy to genetic engineering." But edible vaccines haven't disappeared. Arntzen and his colleagues have conducted several other human studies of vaccine-containing potatoes: In each case, the vaccine elicited an immune response.


Plant-produced therapeutics and vaccines have been around longer than you may think: The first one entered the clinic more than four years ago. At the time, Seattle-based NeoRx Corp. was testing various monoclonals for use in its Avicidin cancer therapy. This treatment regimen consisted of first delivering a tumor-specific antibody that "painted" the tumor with a receptor that was subsequently targeted by a small molecule carrying a therapeutic agent -- initially radiation. One of those antibodies was produced in corn by Monsanto Co.'s Integrated Protein Technologies unit (formerly Agracetus Inc., which Monsanto acquired in 1996). The corn antibody entered human clinical trials in December 1997.

Why did NeoRx turn to plants? "We needed a cost-effective way to produce large quantities of monoclonal antibodies," explained Becky Bottino, the company's senior VP of technical operations. "In general, methods that use cultured cells growing in large tanks are pretty expensive. It also takes several months to produce one lot," she continued. So, the company initiated a multi-pronged program to investigate alternatives -- including transgenic animals and insect-cell systems, as well as transgenic plants. "We started programs in all three areas," Bottino said. "The corn program worked best."

Selected Molecular Farming Companies

Company	Technology	Plant(s)	Clinical trials	Collaborations
AltaGen Bioscience Inc. (Morgan Hill, CA and Richland, WA)	Stable transformation;  vegetative propagation; tubers act as master cell bank	Potato (leaves)	--------	U.S. Army; Three biotech companies (ND)
CropTech Corp. (Blacksburg, VA)	MeGA-PharM; Post-harvest manufacturing; synthesis of recombinant protein occurs in cGMP manufacturing facility, not in the field	Tobacco (leaves)	--------	ToBio LLC (2/00); Immunex (12/01)
Epicyte Pharmaceutical Inc. (San Diego)	Plantibodies: Identify antibody, sequence heavy and light chains, clone in bacterial plasmid, introduce into separate plants via biolistics or injury; mature plants sexually crossed; hybrids contain properly assembled antibody	Corn (seeds); Rice	First product, a topical anti-HSV Mab, should enter Phase I trials in early 2003	Dow Chemical Co. and Dow AgroSciences LLC (9/00); Medarex Inc. (6/01); Centocor Inc. (9/01)
Large Scale Biology Corp. (Vacaville, CA) (NASDAQ: LSBC)	Plants are infected with transgenic tobacco mosaic virus (TMV) or other mRNA viruses for transient gene expression	Tobacco (leaves)	Phase I clinical trial in B-cell non-Hodgkin's lymphoma (conducted by Ronald Levy, Stanford University) (11/00)	ProdiGene Inc. (2/01); Plant Bioscience Ltd. (6/01)

Suddenly, NeoRx was involved with two regulatory agencies: The FDA and the USDA. When Monsanto took its experimental plants out of the greenhouse and into the field for large-scale trials, "We had to get involved with the USDA," Bottino said. As well, NeoRx "worked with the FDA to come up with a plan to test the monoclonal antibody in humans." The company had to not only demonstrate that the product met traditional drug requirements (purity and lack of toxicity, for instance) but also that it didn't contain traces of pesticides or elements leached from the soil.

Once in patients, the corn-produced antibody "performed identically" to its non-corn (humanized murine antibody) counterpart, although it did exhibit biochemical differences, Bottino explained. But the trials on this particular antibody never progressed past Phase I. "We stopped the trials because the antibody didn't meet our criteria. It was an inherent problem with the protein we were trying to develop," she added, not the fact that the antibody was made in a plant. "No matter where the antibody was produced, it didn't have the desired cross-reactivity with the target organs."

In the end, NeoRx's pioneering foray provided a proof-of-concept for plant-made drugs. It also demonstrated that "the FDA will accept these materials for human use," Bottino explained. "It's a model for the industry."

Would she turn to plant-based systems again? "In the future, if we need a protein in large quantities, I wouldn't hesitate for a moment."


But using plants to make human therapeutics doesn't have to be restricted to thousand-kilogram endeavors. Just ask Large Scale Biology Corp., which is producing patient-specific cancer vaccines in transgenic tobacco plants. Since each patient gets a personalized treatment, the production "batches" are fairly small: The plants are grown in a greenhouse, not on a farm.

The Vacaville, CA company, best known for its proteomics expertise, is using Nicotiana benthamiana (a relative of the commercial tobacco plant) to produce single-chain antibody fragments for injecting into patients with B-cell non-Hodgkin's lymphoma. Each patient's cancer happens to be immunologically unique, sporting its own particular antigenic marker. This antigen, as it turns out, is actually an antibody, so Large Scale Biology's antibody-based vaccines are anti-antibodies (or anti-idiotype antibodies).

Company researchers developed a method to produce the idiotype regions of a patient's tumor-specific immunoglobulin as a single-chain variable region (scFv) protein by cloning the appropriate cDNA into a modified tobacco mosaic (TMV) vector for expression in tobacco plants. Once the constructs are optimized for expression, stability and secretion, it's only a matter of weeks before vaccine protein is available for use. "It takes 6-8 weeks from biopsy to a QC/QA-released pharmaceutical," explained Barry Holtz, the firm's senior VP of biopharmaceutical manufacturing. "We can respond quickly to patients."

Importantly, in November 2000, Large Scale Biology -- in collaboration with Stanford University's Ronald Levy -- started a 16-patient Phase I clinical trial of its personalized cancer vaccines. Phase II trials should begin later this year.

Large Scale Biology's plants, unlike most others under development by molecular farming firms, are not transgenic per se. Rather, it's the infecting virus that's transgenic. Thus, the tobacco plant serves as a temporary factory for making proteins of interest.

Selected Molecular Farming Companies

Company	Technology	Plant(s)	Clinical trials	Collaborations
Medicago Inc.	Expression cassette contains DNA of interest plus alfalfa regulatory sequences; transfection via electroporation	Alfalfa (leaves)	--------	Viridis S.A. (8/00); Wisconsin Alumni Research Foundation (10/00); Hemosol Inc. (2/01)
Meristem Therapeutics (Clermont-Ferrand, France)	Human gene or its cDNA linked to plant regulatory sequences, including promoter for tissue- and organelle-specific expression; integration into plant cells via Agrobacterium-mediated infiltration or injection of coated microparticles via gene gun	Corn (seeds) for quantities greater than several kilograms; Tobacco (leaves) for smaller quantities	Phase IIa clinical trials: recombinant gastric lipase for cystic fibrosis (trials conducted by Solvay Pharmaceuticals)	Adprotech Ltd. (10/00); Goodwin Biotechnology Inc. (3/01); Quintiles Transnational Corp. (3/01); Solvay Pharmaceuticals S.A.(6/01); Eli Lilly & Co. (10/01)
MPB Cologne GmbH (Cologne, Germany)	"Genetically optimized" potato tubers; gene switch technology based on ligand-inactivated recombinase that allows controlled regulation of gene expression and directed excision of DNA sequences (eliminates antibiotic marker genes from transgenic plants); post-harvest production in potato	Potato (tuber); Canola (seeds)	--------	Aventis CropScience (8/01); ATO B.V. (11/01)
Phytomedics Inc. (Dayton, NJ)	REPOST (recombinant protein secretion technology): rhizosecretion (from roots of hydroponically grown tobacco and tomato) and phyllosecretion (from exuded water on plant leaves)	Tobacco; Tomato	Yes, but the product is a botanical (extract of roots of perennial shrub); in Phase II/III trials for rheumatoid arthritis & other autoimmune diseases	Vilmorin Clause & Cie (6/01); Bristol-Myers Squibb Co. (9/01); DNAX Research Inc. [Schering-Plough Corp.] (1/02); Degussa AG (1/02)

Moreover, depending on how the vector is engineered, it's possible to target different areas of the plant (leaf vs. stem, for instance) as well as different subcellular components. That makes it possible to create secreted, correctly folded proteins; it also makes it easier to compartmentalize the foreign protein for manufacturing purposes. "We're borrowing all the machinery of the [plant] cell to make therapeutic proteins," Holtz explained. "Plant cells mimic human cells in many ways," added chairman and CEO Robert Erwin. "The proteins are folded correctly, cross-linked correctly and [biologically] active in almost all cases." However, he explained, plants do glycosylate differently. While the core glycans are identical, plant glycans can contain xylose and fructose, which are linked differently.

Does this affect the resultant protein's pharmacokinetics? Perhaps, Erwin said, so "each product has to be evaluated individually." However, even if the protein's slightly different, that might not be a bad thing. As an example, Erwin cited alpha galactosidase A (a potential therapy for Fabry's disease), which Large Scale Biology has produced in "very large quantities." Although this enzyme's glycosylation patterns are different than those on its human counterpart, it's actually demonstrated a higher activity in preclinical studies. As well, neither its immunogenicity nor its efficacy have been affected, Erwin said. And, if glycosylation does present a problem, added Holtz, then researchers can "humanize" the host plant through genetic modifications. In fact, the company's already done it, he added.


Indeed, an incorrectly glycosylated recombinant protein can be robbed of its activity, but glycosylation concerns pop up in mammalian cell-cultured products, too. The main issue concerning plant-made proteins is their safety and efficacy, explained Mich Hein, president of San Diego-based Epicyte Pharmaceutical Inc. And so far, so good.

It's too soon to know whether Meristem Therapeutics' recombinant gastric lipase or Large Scale Biology's cancer vaccines are efficacious, but they've already proven safe. However, Hein's got clinical data on a plant-produced monoclonal antibody that demonstrate efficacy, too.

Published in May 1998 (Ma J., et al., Nature Medicine 4: 601-606), the trial demonstrated that an anti-Streptococcus mutans secretory monoclonal antibody generated in transgenic tobacco plants, given topically, completely prevented re-colonization of human teeth with S. mutans (which causes dental cavities) for at least four months.

At the time, Hein was on the faculty of the Scripps Research Institute, where he and Andrew Hiatt (also an Epicyte founder and now its VP of R&D) invented the technology Epicyte now uses for making monoclonals in plants (Epicyte calls them Plantibodies). Monoclonals are a popular target for most every molecular farming firm, but they're the only target at Epicyte. Moreover, the company's focused on developing topically applied immunoprotective products that address infections at the mucosal epithelium -- infections caused by human immunodeficiency virus, herpes simplex virus, respiratory syncytial virus and Clostridium difficile (which causes diarrhea). All these programs are preclinical; "We're about one year away from entering our first clinical trial," Hein said.

To generate its transgenic plants, Epicyte identifies a disease-preventing antibody, determines its sequence, engineers the corresponding genes and introduces them into a plant germline (usually corn). For therapeutic purposes, the company concentrates on secretory IgA antibodies, which protect mucosal surfaces from infection; even though these antibodies are dimers, they can be properly assembled by the transgenic plant.

The genes for the antibody's heavy and light chains are cloned, put into bacterial plasmids that contain plant regulatory sequences and then transferred separately into plant cells via biolistics (shooting DNA-coated gold particles into the cells) or injury (a technology, called "whiskers," in which very sharp silica fibers puncture the cells, allowing uptake of the DNA through the tiny holes). These cells are then cultured and regenerate into fertile plants. Normally, researchers create two distinct lines, each containing one antibody chain, although in some instances, Hein said, both chains are introduced into the same plant. These sexually mature plants are then crossed, producing a properly assembled antibody in the hybrid progeny. And the seeds can be used to generate more plants -- which allows scalability to production level.

By engineering the appropriate plant regulatory sequences into the transgenic corn, it's possible to control when the transgene is turned on, and where. "We direct expression to the endosperm [of the kernel]," Hein explained. Once harvested, the kernels can be processed immediately to obtain the antibodies or stored for as long as two years, without any protein degradation.


While Epicyte's internal drug development programs center around secretory antibodies, the firm's technology can accommodate other antibodies, as well. "Plants can make any antibody you want," Hein said. And that's exactly what Epicyte's collaborators Medarex Inc. and Centocor Inc. (a Johnson & Johnson company) are hoping to find out. Epicyte and its partner Dow (The Dow Chemical Co. and Dow AgroSciences LLC) are gearing up to produce one of Centocor's therapeutic monoclonal antibodies (not identified) in corn; according to Hein, "the Centocor molecule will be into plants this year."

Epicyte's collaboration with Medarex is slightly different: The partners will jointly develop therapeutic antibodies, using Medarex's UltiMAb human antibody development system. They may use Epicyte's Plantibody technology for manufacturing antibodies emerging from the collaboration. "This agreement is more product-focused than technology-focused," explained Annarie Lyles, Medarex's director of business development. "It's likely we'll use Epicyte's manufacturing process, but we may use ours. It depends on the product and what makes the most sense."

Indeed, like many other biotechs vested in therapeutic monoclonals, Medarex is actively investigating alternate production technologies (not limited to transgenic plants). "We're enthusiastic about any technology coming along that would make antibodies work better or improve the cost of goods," Lyles said.

Selected Molecular Farming Companies

Company	Technology	Plant(s)	Clinical trials	Collaborations
PlantGenix Inc. (Philadelphia, PA)	Use of GS-X pumps (cell transporters) to facilitate accumulation of specific molecules in plant vacuoles	ND	--------	--------
ProdiGene Inc. (College Station, TX)	Agrobacterium-mediated transfection	Corn (seeds)	Plans to initial clinical trials for LtB (travelers' disease) vaccine and Aprotonin (protease inhibitor used in heart surgery) in 2002 or early 2003	Genencor International Inc. (10/97; expanded 3/99); Epicyte Pharmaceutical Inc. (6/98); Large Scale Biology Corp. (2/01); Avant Immunotherapeutics Inc. (5/01); Eli Lilly & Co. (5/01)
SemBioSys Genetics Inc. (Calgary, Canada)	Oleosin technology: genetically engineered oilseeds that produce value-added proteins attached to liposome-like oilbodies	Safflower (oilseeds)	--------	DowElanco Canada [now Dow AgroSciences Canada] (12/97); Novartis Agricultural Discovery Institute Inc. (1/00)

Having an alternate means to manufacture therapeutic proteins is appealing to Immunex Corp., too: When the company's arthritis drug Enbrel hit the market, demand for the product outstripped the supply in no time. As a result, Immunex is building more capacity. But it's also exploring alternative technologies. In December 2001, before Amgen Inc.'s stunning $16 billion acquisition bid, Immunex signed an agreement with CropTech Corp. to examine the feasibility of producing proteins in transgenic tobacco.

CropTech's transgenic technology, MeGA-PharM, incorporates a specific plant defense promoter that triggers synthesis of the recombinant protein after the plants have been harvested. "The promoter is turned on by a wound signal, for example, cutting a leaf," explained Karen Oishi, the company's VP of research. "The signal travels only about one millimeter from the wound site. It allows us to control when the protein is made." As well, the company's gene construct contains a plant signal peptide, which is fused to the protein of interest. "This allows the protein to be secreted out of the cell; as it's secreted, the signal is clipped off."

Since CropTech's transgenic plants are regenerated from single cells, 100 percent of the plant is transgenic, she added. "The entire biomass has potential." The company harvests its plants "way before they start to flower, when they're about two feet tall," Oishi said. "Then we let the plant regrow, so we can get multiple harvests from one stalk."

Once the plants are harvested, they're transported to a cGMP facility, where recombinant protein synthesis is triggered. The company recently opened a new production facility that is capable of manufacturing several kilograms of product. According to Rob Gustines, the company's VP of corporate development, "We're now designing a $40 million, large-scale manufacturing facility that can handle 600 kilograms -- equal to a mammalian cell culture facility that costs $250 to $400 million to build."


While the technologies and specific plants employed by these firms may differ, they all claim that transgenic plant-based manufacturing methods are far less costly than traditional biologics manufacturing.

For example, Epicyte's Hein said that his company's plant-based production technology can make the same annual quantity of drugs with 200 acres of corn that a $400 million factory would produce using a mammalian cell-based system. The costs required to genetically modify the plants and bring fields into production would amount to a few million dollars, he said, as opposed to hundreds of millions.

To meet current and future patient demands, many therapeutics -- especially those prescribed for treating chronic conditions -- will need to be made by the thousands, or hundreds of thousands, of kilos. Plant-based manufacturing can easily match this scale, and more, just by increasing the acreage. The transgenic plant companies are ready for it.

Take Large Scale Biology, for instance: While its personalized cancer vaccines are a relatively small-scale operation, there are plenty of other potential products -- such as alpha galactosidase A -- whose production will require acres and acres of tobacco plants. The company's been conducting UDSA-approved field trials at its Owensboro, KY site since 1991 to demonstrate that the technology is environmentally safe. "We've got a 10-year track record in the field [five of those involving very large scale trials], and there have been no adverse events," CEO Erwin said.

Large Scale Biology Corp.'s Owensboro, KY operations. Main processing bay,
upstream equipment; insert: field of inoculated tobacco.

To harvest the plants, Large Scale Biology's designed its own cutter and wagon, so as to not injure the leaves, Holtz explained. "We can harvest 6,000 pounds of plant leaves per hour." Thirty minutes later, the material's arrived at the processing facility. "We don't homogenize the leaves in the field. They act as a nice container for the therapeutic protein" until it gets to the factory -- which is a commercial-scale, high-capacity manufacturing facility capable of processing three tons of plant material per hour. There, the leaves are homogenized and processed rapidly; the protein fraction is isolated and stabilized within 18 minutes of homogenization. "Along with rapid isolation, we're not dealing with big volumes [of liquid]," Holtz said. "The spent biomass goes right back to the field, where it's used as compost." (By this time, the transgenic virus has been utterly destroyed, so it poses no environmental threat.)

According to Erwin, this entire process is very cost-effective. "We can replace all the upstream manufacturing requirements and infrastructure" of current biopharmaceutical manufacturing facilities with a field of plants. "After we get to the stage of having a concentrated extract of the protein, our downstream business looks like anybody else's."

Like Large Scale Biology, Epicyte also uses dedicated machinery and equipment to harvest and process its transgenic plants. Both firms -- and all others involved in this line of research -- pay close attention to environmental issues. They keep their transgenic crops as isolated as possible; both in the field and during processing, to eliminate contamination. "We want to know what our [corn] seeds are, where they are and where they're going," Epicyte's Hein said.

"Once we grind the corn, separating the endosperm from the other parts of the seed, we use standard methods to extract and purify the antibody," Hein explained.

It's actually Dow, Epicyte's partner, that does the heavy lifting here. "Dow's been in the manufacturing business for more than 20 years; it's got the expertise," Hein said. Moreover, "Dow will grow our first corn; we've handed the genes over to Dow and they're doing the initial transformation." Working with Dow, Epicyte is planning to build a cGMP facility to purify and manufacture clinical supplies of experimental therapies as well as commercial-scale quantities.


But, though these molecular farming firms seem to have all the pieces in place for large-scale operations, none has actually produced kilograms of recombinant proteins to date.

The first company to accomplish that feat will no doubt be ProdiGene Inc. In mid-February 2002, the College Station, TX company announced that it's begun commercial scale-up of trypsin, a protein that's widely used in cell culture and as an intermediary in the production of pharmaceuticals, including insulin. ProdiGene's goal is to market tens of kilograms of trypsin by the end of this year, and to meet full market demand, in the range of hundreds of kilos, by 2003.

The company's also using its transgenic corn system (at a much smaller scale) for vaccine development: It's made an edible vaccine for treating transmissible gastroenteritis virus infection in swine, which worked, and it's got a grant from the National Institutes of Health to develop an edible AIDS vaccine. As well, ProdiGene is gearing up to start human clinical trials of aprotonin, a protease inhibitor used in heart surgery.

But no matter the scale, ProdiGene's as mindful of environmental concerns as the other molecular farming companies. "We want to make sure that our crop [of transgenic corn] doesn't go anywhere else," explained John Howard, the company's CSO. "One of the main reasons for our announcement was to alert everyone to what we're doing. If any issues come up, we can deal with them. We're taking all kinds of extra precautionary measures and monitoring [our fields]."


But what if there were no fields to watch over? If all plants were grown in greenhouses, then environmental conditions could be controlled precisely. That's the approach taken by AltaGen Bioscience Inc., with an extra twist: The company grows its plants hydroponically, so it can even control the nutrients the plants receive. In fact, if conditions warrant, it can also grow the plants aseptically. AltaGen, which has just been formed through the merger of PhytaGenics Inc., based in Richland, WA, and Sierra BioSource Inc., of Morgan Hill, CA, intends to do large-scale manufacturing this way, too.

Armed with technology developed by Battelle Memorial Institute's Pacific Northwest National Laboratory and the U.S. Department of Energy, AltaGen's initially focusing on producing biopharmaceuticals having proven therapeutic value -- including hemoglobin, thrombin, Factor XIII, erythropoietin, interferon and human growth hormone -- as well as products for mammalian cell culture media -- bovine serum albumin and human transferrin. The firm's also got several partnerships, including one with the U.S. Army to produce elastin for arterial grafts.

Interestingly, AltaGen's making all these proteins in potatoes. It takes about 6-9 months for company researchers to go from inserting the gene of interest to producing a stable line, explained president and CEO Rick Srigley. "Potatoes grow quickly, and they propagate vegetatively." That means that the company avoids the time-consuming process of crossing and back-crossing plants to get stable lines.

"From the transformation event, we raise up several hundred clones, which we then screen for high levels of transgene product and desirable growth characteristics," explained Nick Lombardo, the firm's director of business development. "We maintain 4-10 prospective lines for further evaluation and scale-up." And scale-up is easy. "The plant line is immortalized as a potato tuber, which can be stored for a long time, like a master cell bank," Lombardo continued.

For most of the transgenic plant systems under development, greenhouses are just impractical for large-scale manufacturing. "With corn, you can get about six kilograms [of product] per acre per year. For 100 kilos, that's 16-18 acres of corn," Srigley said. "Tobacco is better, about 30-40 kilos per acre per year, but it's still in an open field." Alfalfa yields are similar to those from tobacco. However, AltaGen's production technology can yield 250 kilograms per acre per year, he said, all from the greenhouse. That's because it uses a semi-continuous "harvesting" process. "We start a batch of seeds every two weeks," he explained, "and do the initial product purification twice a week." After that, the product is sent to another company for final purification.


With transgenic plant technology's obvious benefits -- scalability, cost-effectiveness, human and/or animal-pathogen free -- why aren't the major biotech and pharma companies beating down molecular farmers' doors? According to Large Scale Biology's Erwin, "we need sufficient data to convince those companies. We have to remove their concerns about the risks of this process by moving products through the clinic and the FDA. We have to present compelling evidence that there's no risk associated with plant-based manufacturing."

One concern is how the FDA will regulate these products. There is precedent -- the agency's given the green light for several clinical trials. As well, most recombinant proteins are being grown in food crops, which are already recognized as safe. And the FDA's already issued guidelines for the use of transgenic animals, which should be a good a good starting point.

But perhaps potential customers, as well as the molecular farming companies themselves, will rest easier once the FDA and UDSA issue guidelines for transgenic plants -- a project that's been in the works for some time now. "We are anxiously awaiting FDA's guidance," said Epicyte's Hein. For instance, "what is the exact step in the manufacturing process where cGMP comes into play?"

Using transgenic plants to manufacture recombinant proteins and antibodies might not be the first choice for all the companies in this arena. But many are now investigating alternatives to mammalian, bacterial or yeast cell culture. After all, there's already a manufacturing crunch, and nearly 100 protein-based therapeutics are in late-stage clinical trials (not to mention hundreds more in earlier-stage trials). Even with very handsome budgets, many of these companies just won't be able to build manufacturing facilities fast enough to meet the demand.

According to AltaGen's Srigley, "Lots of biopharmaceutical companies are evaluating alternate technologies via feasibility studies. I suspect that once these feasibility studies bear fruit (in the next 6-12 months), there will be a very rapid acceptance of the technology."

"Interest in transgenic plant technology has escalated in the last nine months, for us and for the other companies as well," added CropTech's Gustine. "The industry is well aware of the manufacturing capacity crunch. Transgenics are getting a serious look."