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PPL Therapeutics’ (PPL; Edinburgh,Scotland, UK) announcement on June 18that it was postponing trials of its lead ther-apeutic protein, α1-antitrypsin, signals thelatest setback for the production of anapprovable drug in a transgenic animal sys-tem. The company’s shift to focus its clinicalefforts instead on fibrinogen, a proteinmerely derived from human plasma, illus-trates the daunting economic and reg-ulatory uncertainties of manufacturingproteins in the milk, blood or eggs of genet-ically modified (GM) animals or in the tis-sues of GM plants.

Human proteins produced by recombi-nant means in bacterial or mammalian cellculture bioreactor facilities have proven to bevery effective therapeutic agents, but they arealso among the most expensive drugs.Amgen’s (Thousand Oaks, CA, USA)Neupogen, for example, which is used tostimulate white blood cell production in can-cer patients, tops the prescription drug list at$2,800 per vial.

The market for human protein therapeu-tics is expanding, but increasing productioncapacity of conventional facilities takes aboutfive years and $300–500 million. Setting up aGM (transgenic) herd for production, on theother hand, costs tens of millions and takes acouple years. New production methods areneeded both for scalability and because manyhuman proteins can be made correctly onlyin higher organisms.

“It’s clear this situation will continue forsome time and pharmaceutical companieswill need to look at alternative forms of man-ufacture,” says Lyle Hohnke, partner at Tullis-Dickerson (Greenwich, CT, USA), a venturecapital firm that invests in three biomanufac-turing companies, Epicyte (San Diego, CA,USA), Prodigene (College Station, TX, USA)and Avigenics (Athens, GA, USA). “What’snot quite as clear is which of these platformsis going to win.”

Yet, many company officials who spoke toNature Biotechnology say that it’s not so mucha competition as a market opening up withroom for unique platforms to fill the needs of

pharmaceutical firms. In fact, two leaders inthe field, GTC Biotherapeutics (GTC;Framingham, MA, USA) and Pharming(Leiden, The Netherlands) signed a cross-licensing deal in June 2002 that gives each cer-tain rights to the other’s intellectual property.

Burden of proofBecause no protein produced in a transgenicanimal has been taken all the way throughregulatory approval and commercialization,many unknowns face the industry. For exam-ple, differences in how humans and otherorganisms process proteins (e.g., via glycosy-lation or farnesylation) could lead to allergicor other unwanted immune reactions. Of thefew proteins produced in animals that havemade it into trials (see Table 1), none hasshowed any such problems. However, it isclear that regulators will be closely scrutiniz-ing these products in the postmarketingphase, as the formulation has been docu-mented to affect toxicity of erythropoietin(see p. 956).

GTC’s vice president for corporate com-munications, Tom Newberry, expects regula-tors to inspect the first filings very closely andcarefully owing to the lack of previousknowledge from earlier filings. There mayalso be regulatory or public perception sur-

prises as products move into the market-place, according to Hohnke.

The lack of an approved product todemonstrate the feasibility of protein pro-duction in GM animals or plants makesinvestors and commercial partners wary.“The scalability is there and the economicsare there, but until you’ve done it, you haven’tdone it,” says Hohnke. Drug companies alsowant to know that a platform can both meettheir production demands and navigateuncharted regulatory territory. “Potentialpartners are taking a cautious approach andwant to see how the regulatory agenciesreview and deal with some of the lead prod-ucts coming up for approval,” says SamirSingh, vice president at Pharming, whichexpects to file for European approval of theirlead product, recombinant human C1inhibitor, by the end of 2004.

The burden of proof lies squarely on theshoulders of the pioneering transgenic com-panies with products in trials like GTC,Pharming and PPL. But critics say that mis-takes made early on have slowed progress.Some leading companies like Pharming andPPL miscalculated by trying to develop toomany products at once or by focusing onproducts with messy clinical endpoints, con-founding clinical trials.

NATURE BIOTECHNOLOGY VOLUME 21 NUMBER 9 SEPTEMBER 2003 965

Barnyard biotech—lame duck orgolden goose?Recent setbacks in product development at PPL Therapeuticsillustrate the economic challenges and regulatory uncertainties thatface protein production in GM animals.

Table 1 Transgenic products currently in clinical development

Company/ System Product and indication Stagesponsor

GTC Goats Antithrombin III for antithrombin III deficiency Phase 3

MM-093 mAb for myasthenia gravis, rheumatoid Phase 1

arthritis and multiple sclerosis

PPL Sheep α1-antitrypsin against cystic fibrosis and edema Phase 2c

on hold

Meristem Plants Gastric lipase against steatorrhea Phase 2a

Therapeutics

Prodigene/NIH Plant Vaccine derived from Escherichia coli LtB toxin subunit Phase 1

Pharming Rabbits Human C1 inhibitor against hereditary angioedema Phase 1

Human lactoferrin against bacterial or hepatitis C infections Phase 1

Epicyte Plant mAb product against respiratory syncytial virus Phase 1

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Hard lessonsPPL may have learned that lesson the hardway. Their emphysema indication for α1-antitrypsin requires measurement of theslowing of lung disease progression instead ofan obviously cured condition as an end point,says William Velander, biomedical engineer atVirginia Tech (Blacksburg, VA, USA). Thestumble may have cost PPL its biomanufac-turing program for the foreseeable future, asinvestors and partners retreat. Without fund-ing, PPL backed down from building a $67million production facility to supply α1-antitrypsin for the phase 3 trials. BayerBiological Products (Research Triangle Park,NC, USA), which was partnering with PPL,pulled out of funding the trials, anticipatingsupply problems.

Velander also says that, in their effort toraise venture capital, companies rushed to usehigh-volume milk animals without determin-ing which animals would give the best proteinexpression. For example, the coagulation fac-tors that make up some of the industry’s lead-ers are some of the most complex proteins inthe human body. Making a simpler proteinthat would express well in the mammarygland and sail through clinical trials would“convince people of the advantages of thetechnology,” he says.

Several companies have restructured tofocus on bringing at least one promisingproduct to market. Singh says Pharming willfocus limited resources on the C1 inhibitorbecause it has a clear indication for a geneticdisorder, hereditary angioedema, thatresults from a lack of the natural protein.Pharming also has brought a human lacto-ferrin protein through phase 1 trials, but,Singh says, that product has many possible,more complex applications that Pharmingwill explore in partnership. GTC has also putits efforts into a protein used as a replace-ment for patients with a hereditary defi-ciency. The anticoagulant antithrombin III

is in the lead to be the first animal-deriveddrug approved in Europe.

Industrial plantsMany of the pioneering transgenic companiesexploited GM animals as expression systems,but an increasing number of companies is nowdeveloping plants as factories for biophar-maceuticals. Three companies, Prodigene,Meristem Therapeutics (Clermont-Ferrand,France) and Epicyte, already have products intrials (Table 1). Prodigene and Epicyte haveeach secured four biotech or pharmaceuticalpartners in the last three years while Meristem

has seven. Among the plant expression sys-tems, grains seem very promising options,although issues remain in containment andtoxicology (see Box 1). A recent industryreport entitled A Strategic Evaluation ofTransgenic Plant and Animal BiomanufacturingSystems by Revelogic (Ft. Collins, CO, USA)suggests that rice may have strong advantagesbecause of its high expression level and veryminimal risk of outcrossing.

Epicyte is producing human monoclonalantibodies (mAbs) in corn and has chosen topursue targets that have already been vali-dated as good therapies by other drugs. “Wemade a strategic change over the last year inresponse to the investment community andantibody markets,” says Debra Robertson,executive director of intellectual property.The company decided to switch from a mAbtopical gel product for herpes simplex virus,to mAb products against validated antigensfor respiratory syncytial virus (RSV) infec-tion, rheumatoid arthritis and lymphoma.Robertson says the RSV product is on track tofile for approval at the end of 2004.

Prodigene and Meristem also have prod-ucts in clinical trials, including Prodigene’soral vaccine for a bacterial toxin that causestraveler’s diarrhea and Meristem’s gastriclipase against steatorrhea (excessive fat in the

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Table 2 Selected list of companies working on transgenic protein production

Transgenic system Company

Cattle Gala Biotech (Sauk City, WI, USA)GTC (Framingham, MA, USA)Hematech (Westport, CT, USA)Pharming (Leiden, The Netherlands)

Chickens Avigenics (Athens, GA, USA)Vivalis (Nantes, France)TranXenoGen (Shrewsburg, MA, USA)

Goats GTCNexia Biotechnologies (Montreal, QC, Canada)

Rabbits PharmingBioProtein Technology (Paris, France)

Sheep GTCPPL Therapeutics

Plants AltaGen Bioscience (Morgan Hill, CA, USA)Crop Design (Zwijnaarde, Belgium)Crop Tech (Blacksburg, VA, USA)Dow Agrosciences (Indianapolis, IN, USA)EpiCyte (San Diego, CA, USA)Medicago AB (Uppsala, Sweden)Meristem Therapeutics (Clermont-Ferrand, France)Monsanto (St. Louis, MO, USA)MPB Cologne (Cologne, Germany)ProdiGene (College Station, TX, USA)Ventria Biosciences (Sacramento, CA, USA)SemBioSys Genetics (Calgary, CA, USA)

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Figure 1 Comparisonof various productionsystems. Source:Revelogic, Inc.(www.revelogic.com).

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stool). Prodigene’s trials for the vaccine, spon-sored by the National Institute of Allergy andInfectious Disease (Bethesda, MD, USA), rep-resent the first safety test of an oral vaccinederived from plants. Such vaccines, a goal ofseveral plant-based companies, may be easierto use in developing countries.

Economics of scaleWhatever the transgenic system employed(see Table 2), the next step is to show an eco-nomic advantage. Experts think establishingthe cost-effectiveness of the technology willbe easy, given the heavy capital investmentsrequired for bioreactor facilities and lowerproduction costs of most GM-derived prod-ucts compared with cell culture. Additionally,the field has many advantages over traditionalmanufacturing methods that seem to ensureeventual success for many different platforms.

First and foremost, volume is a key advan-tage. Hohnke points out that recent successesof antibody products that require largeamounts to be therapeutic will necessitate theuse of GM organisms. Copious amounts ofmilk, eggs or seeds can be produced from rel-atively small herds, flocks or crops with rela-

tively small initial and maintenance costs. TheRevelogic report notes that production of 100kg of a generic therapeutic protein wouldrequire 450 acres (182 hectares) of corn, 8,125chickens or 60 cows—tiny numbers com-pared with commercial agriculture.

While milk-secreted products are in thelead now, eggs potentially offer higher levelsof expression at the lowest production cost.The egg system has lagged behind because ofthe difficulties in creating transgenic chickensthat expressed therapeutic proteins.Yahshwant Deo, CEO and president ofAvigenics (Athens, GA, USA) says now thatthe hurdle has been cleared, the system ispoised to catch up to other platforms.

Downstream purification of proteinsextracted from the egg white and milk is madesimple because the protein contaminants arefew and known. On the other hand, plants arefree of prion contaminants and store easilywithout refrigeration. All platforms offerunique production characteristics, greatersafety and more consistent supply thanblood-derived products (see Box 1). Becauseof these differences, each platform may findproducts aptly suited to its parameters.

“We are not necessarily even competingwith other transgenic platforms becausethere are subtle differences in the productioncharacteristics that set each platform apart,”says Singh. He says in some ways transgeniccompanies will have to work together to offeran array of platforms to drug companies “sothey can benefit from whichever platformmakes sense for their particular product” (seeFig. 1).

The need to expand manufacturing capac-ity for human protein therapeutics dictatesthat GM organism ‘factories’ are coming—ones that can meet supply needs by simplyand cheaply planting more fields or breedingmore animals. With improvements in trans-genic technology, developing herds and cropsand boosting protein expression levels isbringing new players like rice and chickensinto the game.

An approval will be “a pivotal moment forthe industry,” says GTC’s Newberry. “Wherefirst the doctors are rewarded because a) itworks and then b) it gets accepted, and thequestions surrounding [the technology] willbe resolved—at least to some degree.”

Kendall Powell, Denver

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Box 1 Regulatory considerationsTransgenic platforms must comply with regulatory guidelines forprotecting the food supply chain from drug contamination andthe environment from outcrossing of drug-producing GMorganisms with non-GM populations. Last year, the industryfaced its first regulatory scare when the US Department ofAgriculture (USDA; Washington, DC, USA) destroyed 500,000bushels of soybeans that had been contaminated by a few GMcorn plants carrying a pig vaccine grown by Prodigene (CollegeStation, TX, USA) in Iowa and Nebraska (see Nat. Biotechnol.21, 3–4, 2003). The violation cost the company a $250,000fine from the USDA and $3 million in compensation to cover thedestroyed soybeans.

Although some company officials point to it as an example ofthe system working to catch infractions before reaching the foodsupply, others say they welcome the tighter restrictions ongrowing GM ‘pharma’ crops the USDA issued after the incident.The rules—like growing crops a half a mile (805 meters) fromother crops—are designed to keep GM crops from cross-pollinating food crops or getting mixed into seed meant forhuman or animal consumption.

Planting outside of the US Corn Belt has been a strategy usedby Epicyte and others to avoid regulatory slips. Company officialspoint out that the downstream regulations for the purity andsafety of the protein product are no different than those requiredfor traditional bioreactor products. The industry is also aware ofthe need to keep the public’s perceptions of GM-derived productspositive and has worked together for the most part to shape policy.

Animal companies must also worry about the health of theiranimals and exposure to infectious disease that could potentiallybe transmitted through the protein product.

“There used to be a common misconception that transgenicproduction animals …are walking around mingling with othercows and people just like a Far Side cartoon,” says Pharming’sSingh. He says that drug companies now have a betterunderstanding of the regulatory and safety guidelines in place fortheir production animals, which are housed in isolated shower-infacilities and monitored around the clock. Keeping animalfacilities in prion disease–free countries is another waycompanies have combated fears of contamination. KP

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Transgenic maize used to produce therapeutic proteins.

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