Plant-Made Pharmaceuticals
Biopharming might be as easy as literally growing drugs on trees, if it wasn’t for the international regulations,
technical challenges, and safety issues.
Mariam Andrawiss, PhD
Andrawiss is a freelance writer based in London
During the September 2005 United Nations World Summit, Secretary-General Kofi Annan gave a ringing
endorsement for advancing the Millennium Development Goals (MDGs). These goals, seeking to cure a host of
global socioeconomic ills by 2015, form the basis of a mutual pact between developing and developed
countries.
Among the eight MDGs, two are directly related to vaccination and infectious diseases. A third seeks to provide
access to affordable essential drugs in developing
countries and to make the benefits of new technologies available in
cooperation with the private sector. Millions of people die or suffer
unnecessarily each year from diseases or conditions for which effective
medicines or vaccines exist. Essential drugs can save lives and improve
health, but only if they are accessible, affordable, and of good quality.
Genetically modified alfalfa plants grow
in a greenhouse. (All photos courtesy
of A. Wigdorovitz, Instituto Nacional de
Tecnologia Agropecuaria, Buenos Aires,
Argentina)
Recent studies suggest the promise of molecular farming, the
production of pharmaceuticals in plants, as a successful way of making
a range of proteins used in research and diagnostics.
Producing pharmaceutical proteins in plants presents several
advantages: the costs can be lower than traditional methods based on
microbial fermentation, insect, and mammalian cell cultures. Some
studies have suggested that recombinant proteins can be obtained at less than 10% of the cost of microbial
fermentation systems and at 0.1% of the cost of mammalian cell cultures. When high-yield proteins are
produced, the economic advantages of plants seem difficult to match.
Another major advantage of drug production in plants is its potential for fast scalability. Biolex Therapeutics,
Pittsboro, N.C., produces recombinant human therapeutic proteins in a small green aquatic plant called Lemna.
"From the transformation event to the production of the transgenic plant, we can come out with a production
line within three to four months," says Lynn Dickey, PhD, the company's research vice president.
Plants as biofactories
Using plants, it is technically possible to grow, harvest, and process pharmaceutical proteins on a large scale.
The genetically modified crop containing edible vaccine, for instance, can be consumed raw or partially
processed. By targeting protein expression into specific organs of the plant such as grains, one creates a stable
storage system. By targeting the recombinant protein to subcellular regions of the cells, such as the endoplasmic
reticulum, a favorable environment is created for its appropriate folding and
Unripe Law & Order
As early as in 1993, the US Patent and
Trademark Office (USPTO) filed a patent for
the invention of vaccines in edible transgenic
plants that was finally issued in 1996 to
Edible Vaccines Inc. (today Prodigene). Since
then, many other applications have been
registered (see www.uspto.gov). The
granting of a patent does not automatically
allow the patent holder to exploit the
invention commercially. For that, specific
national regulations need to be fulfilled.
Although recombinant proteins obtained from
plants are subject to the same set of
marketing authorizations than any other
drug of other origins, this technology
requires the approval of farming of
genetically modified (GM) crops, which varies
from one country to another. The United
States, Argentina, Australia, Canada, China,
and South Africa are countries with the
biggest transgenic crops areas.
Intellectual property laws, health and safety
regulations, and their enforcement differ
greatly from nation to nation. The pace at
which these norms are carried out does not
satisfy molecular plant researchers. "If
Pharma-Planta does not reach its milestones
within these five years, the problem would
have been our failure to reach an agreement
with regulators, because the regulators so
far have been really slow in putting down the
rules," says Julian Ma, PhD, St. George’s
Medical School, London, and scientific
coordinator of the Pharma-Planta consortium
(see "The Pharma-Planta Project" sidebar).
Appropriate international legal agreements
are required to control "biopiracy."
Developing countries with less regulatory
structures need help in the risk assessment
of environmental impact, biosafety, and
socioeconomic effects of GM crops. However,
according to the Nuffield Council on Bioethics
(www. nuffieldbioethics.org), the United
States and other countries blocked the
implementation of a Biosafety Protocol that
had been examined by more than 100
parties to the Convention on Biological
Diversity. The countries that rejected it
already have extensive commercial GM
crops.
assembly, thus increasing the amount of recombinant proteins
produced. Additionally, targeting the recombinant protein to the
membrane concentrates the product. As a consequence, the cost of
downstream purification is minimized. Finally, using plants for
the production of drugs reduces the risk of contamination with
human or animal pathogens, unlike production via animal or
human sources.
So far, pharmaceutical proteins produced in plants can be
classified into three groups: human biopharmaceutical proteins,
including growth hormone, human serum albumin, β-interferon,
and erythropoietin; recombinant antibodies such as IgG1 and IgM;
and recombinant subunit vaccines, from the hepatitis B envelope
proteins and the rabies virus glycoproteins to the cholera toxin B
subunit.
High-expression levels of recombinant proteins determine the
commercial viability of molecular farming. Yields are therefore
crucial, as witnessed by the collapse of the US company
CropTech in 2003, due to its inability to achieve the expression
levels required for commercial feasibility. "Yield is a critical
point," says Hilary Koprowski, MD, Thomas Jefferson University,
Philadelphia, who pioneered the expression of animal proteins in
plants. "You can overcome it by using larger amounts of plants,
which are much easier to use than mammalian cells, but we still
would like to increase the yields."
In determining yield, the choice of plant for production is critical.
Four groups can be distinguished: leafy crops such as tobacco,
alfalfa, lettuce, and soybean; cereal and legume seeds; fruit and
vegetables; and finally fiber and oil crops (see table below). Each
of them has pros and cons and is generally chosen as a function of
the recombinant proteins to be produced.
Tobacco for proteins
Tobacco is the most widely used species for the production of
recombinant pharmaceutical proteins at the research laboratory
level. Plant biotechnology company Chlorogen, St. Louis, chose
this plant to produce its recombinant proteins. "Tobacco has been
around for a long time; it is sort of a laboratory rat," said David
Duncan, PhD, president and CEO. "It is easy to transform, and
next to sugar cane, tobacco is one of the largest biomass producers
on earth. Tobacco is also a plant that we can harvest up to five
times per growing season and it grows back from the same plant.
Another advantage of tobacco over other plants is that it is a self-pollinator, so even if an internal mechanism
such as the maternal inheritance breaks down, the possibility of cross-contamination is limited."
To introduce the gene encoding for the protein of interest, a process named transformation, two general methods
are used. One is a vector, in which the plant parasite Agrobacterium tumefaciens operates to introduce the
foreign gene into the host genome. The other is the gene gun, in which DNA-coated projectiles are accelerated
into the plant tissues.
After the transformation process, the efficiency of all stages of gene expression and protein stability needs to be
maximized. Both the promoter sequence and the polyadenylation signal sequence are key elements for a high
level of transcription. The strong and constitutive cauliflower mosaic virus promoter 35S (CaMV35S) is often
chosen. Specific promoters, that is promoters that are only active in
certain organs of the plant, have also been used and have the advantage
of avoiding protein accumulation in vegetative organs, preventing both
toxicity in the host plant and contact with nontarget organisms. Finally,
inducible promoters allow the production of the desired protein only for
a certain period of time.
Subcellular targeting is another important factor influencing protein
yields. Compartments within the cell can influence the processes of
folding, assembly, and post-translational modification. These all affect
proteins' stability and thus determine the final yield. Comparative
targeting experiments have shown that targeting to the secretory
pathway using N-terminal signal peptides derived from plant or animals
is a suitable zone for folding and assembling for full-size
immunoglobulins and single-chain Fv fragments. Several important
factors for protein folding and assembly are indeed present in the
endoplasmic reticulum: an oxidizing environment, a lack of proteases,
and plenty of molecular chaperones. In the absence of a targeting
signal, proteins accumulate in the extracellular space.
Different stages of the alfalfa
transformation. The recombinant
binary vector was introduced in alfafa
plants using the Agrobacterium
tumefaciens system.
Targeting chloroplasts
Chloroplasts are alternative organs targeted in plants. Several examples of chloroplast-based molecular farming
have been reported. The ability to genetically transform chloroplasts is significant for two important reasons.
First, chloroplasts are inherited maternally and therefore are not transferred via pollen to other sexually
compatible plants.
This is vitally important given environmental concerns about gene transfer. Second, protein production using
chloroplast transformation is several hundred times more effective when compared with the currently used
nuclear transformation of plants. "Back in the 1980s, it was discovered that there are about 100 chloroplasts per
cell and that there are about a hundred copies of DNA within each chloroplast," says Duncan. "In other words, it
was discovered that you could have up to 10,000 copies on the foreign gene. This is what we call
hyperexpression."
However, chloroplasts are not able to perform many of the posttranslational modification tasks such as
glycosylation. Plant-derived recombinant proteins tend to lack the terminal galactose and sialic acid residues
normally found in mammals, but have the carbohydrate group α-(1,3) fucose, which has a (1,6) linkage in
animal cells and β-(1,2)
xylose, which is absent in mammals although present in invertebrates.
This "foreign" glycan structure has affected the use and acceptance of
plant-made proteins. To improve this, attempts have been made to
humanize the glycosylation process by using purified human galactosyl
and sialyl transferases to modify plant-derived protein in vitro.
Finally, production costs of recombinant proteins depend on the
required purity because more than 85% of expenditure reflects
downstream processing rather than production per se. Plants are
advantageous because several recombinant proteins can be used in raw
or partially processed material, which decreases the costs significantly.
click the image to enlarge
Plants used as biofactories for the
production of pharmaceutical proteins
Several strategies at the molecular level have been developed to reduce
downstream processing costs, such as concentrating the proteins to the
membrane of the cell, which are then purified by membrane
fractionation. Secretion systems are also advantageous because the
recovery of proteins does not necessitate the disruption of plant cells.
Market opportunities
The fast scalability and the lower production costs of plant-made pharmaceuticals have scientists and the
industry excited about the future of biopharming. "Once proper expression is achieved, this is just a win-win
situation" says Kisung Ko, PhD, a collaborator of Koprowski working on the generation of clinical-grade
monoclonals in tobacco. "I am very optimistic about this," adds Duncan. "I think we are turning the corner, even
in Europe." Their enthusiasm is certainly shared by business analysts. For them, plant biopharming is emerging
as a genuine competitive force in the large-scale production of recombinant proteins.
Last December, consultants Frost & Sullivan published an analysis of the biopharming market, mostly focused
on North America and Europe. Impelled by the growing demand for biopharmaceuticals and the general public
acceptance of transgenic crops, North America is anticipated to become the largest market for plant molecular
farming. Frost & Sullivan's report predicts that the US market alone might be worth $2.20 billion in less than a
decade. "The gradual relaxation of the moratorium on transgenic crops in Europe is a good indication that the
public and regulatory perception toward transgenic crops is improving, and this trend is expected to drive the
market," the report states.
Among pharmaceutical proteins, the remarkable success of therapeutic monoclonal antibodies (mAbs), which
account for more than half of the mAbs approved by the FDA since 1997, has certainly prompted a frantic quest
for new medical applications. In oncology alone, the NIH is currently recruiting participants to complete more
than 250 clinical trials. Diverse needs, such as for lymphoma and melanoma anti-cancer treatments and
Alzheimer's disease therapeutics, are increasing demand for these molecules. Routine immunizations need to be
implemented in many regions such as Oceania, sub-Saharan Africa, and South Asia.
However, as of June 2005, neither mAbs nor vaccines produced in plants are manufactured yet for any market.
The Tufts Center for the Study of Drug Development in Boston has only one record of a plant-made therapeutic
monoclonal antibody in clinical studies. This antibody is CaroRxe from Planet Biotechnology Inc., Hayward,
Calif., which is used for prevention of dental caries. According to Tufts, only two companies are producing
vaccines in plants that are in clinical studies. Large Scale Biology Corp., Vaccaville, Calif., has already
achieved preclinical validation for its human papillomavirus vaccine program, and ProdiGene Inc., College
Station, Texas, developed a vaccine against a virulent strain of E. coli.
Tough competition
Even though the academic and biotech biopharming community is using the most innovative and ingenious
biotechnical means to turn plants into biofactories, competing effectively in the pharmaceutical market is still
difficult. Most biopharming companies are not necessarily interested in developing their own products, says
Janice Reichert, PhD, senior fellow
The Pharma-Planta Project
The Pharma-Planta project (www.pharmaplanta.org) is a consortium of 39 research
groups from academic and industrial
institutions in Europe and South Africa
founded in 2003. Their goal is to enter plantmade pharmaceuticals into clinical trials. The
Pharma-Planta scientists have signed a
"Statement of Intent on Humanitarian Use"
in which they agree that to the best of their
ability, they will make knowledge that may
be created by their program freely available
for the achievement of humanitarian
purposes. "I think this [statement] is a very
important thing," says Julian Ma, PhD, St.
George’s Medical School, London, and
scientific coordinator of the Pharma-Planta
consortium. "We have an enormous range of
vaccines available in the United Kingdom
that are not made available for the
developing countries. When we think about
the benefits of molecular farming, benefits in
terms of the scale and the low cost of
at the Tufts center. At the moment, they are just trying to come up
with proof-of-concept for their diverse methods of plant
expression. Reichert believes what they are currently lacking is
another firm that has gone through the process of finding a right
mAb candidate. "Most of these companies do not have their own
mAb. What most of them need is a partner that wants to explore
plant production."
Sue Mayer, DVM, PhD, executive director of GeneWatch UK, a
non-profit group that monitors developments in genetic
technologies, says people often forget that the product has to work
clinically. "People think all they need to do is to make a vaccine in
a plant and they will be all right, but of course that vaccine has to
work and has to stimulate the right immune response. Our feeling
is that it is the biggest challenge."
production, clearly the main beneficiaries will
be those in developing countries."
"Most of the targets that we are looking at
for Pharma-Planta are mostly relevant for the
developing world," says Ma. "In the UK, one
person died of rabies last year. But if you go
to Southeast Asia or Africa, rabies is a
serious danger, killing thousands of people
every year."
The program still has three years to
accomplish its objectives. Other therapeutic
molecules have been freely licensed before in
developing countries. Merck, for instance,
has been providing its antiretroviral
treatments in Africa for free. However,
lengthy research projects, strict biosafety
regulation check ups, expensive clinical
trials, and costly processing of patents and
licensing are undoubtedly going to make
Pharma-Planta’s goals difficult to attain.
Koprowski thinks the biopharming community needs to gain the
confidence of the pharmaceutical industry and investors. "When
the pharmaceutical industry realizes the possible future of
growing vaccines in plants, then the community will gradually gain confidence and be able to produce them,"
he says, adding that profits will make them confident. "The first year, profit of a vaccine is very low. You have
to gain market share. You have to educate doctors and veterinarians to use it. But in my opinion, these are the
vaccines of the future."
But Reichert says more needs to be done: "I was in Montreal early last year for the Plant-Made Pharmaceuticals
Conference, and even among themselves they were not convinced they can actually do plant production yet.
There is potential, but there are serious issues that need to be addressed."
Indeed, one important constraint for cost-effective biopharming is good manufacturing of the final product. The
quality of the final product is measured by its structural authenticity and to its homogeneity across bushels,
given that conditions of soil and weather will change from crop to crop.
Another major constraint are multiple biosafety concerns that need to be addressed, as with other genetically
modified crops. Although pharmaceutical proteins produced in plants are generally considered safer than those
of microbial and animal origin because they lack endotoxins and pathogens, biosafety concerns include the
potential co-purification of pesticides and the unpredictable consequences of using viral DNA sequences
employed in the transformation of the plant.
Environmental threats
The possibility of huge amounts of recombinant protein being infiltrated into soil and groundwater is a major
concern among environmentalists. Cross contamination could also occur between the genetically modified
plants and their wild-type counterparts, leaving microorganisms in the soil, insects, and animals. "I would not
advocate growing plants that produce vaccines in open fields. These plants should be grown in greenhouses.
Then you can hold all your safety problems there," says Koprowski.
"If they use the economical argument, the advantage starts to go away if you have to build a contained
greenhouse," says Reichert.
Mayer agrees: "It is very cheap to grow something in the fields, but production in high-containment facilities is
far from sustainable. I think people have exaggerated the potential cost savings if you are going to do it in a way
that has a good safety control. I think it is extremely unlikely that licensing will be given to grow genetically
modified crops outdoors."
Even though regulatory and safety issues need to be addressed, molecular farming has much to offer for
developed and developing countries. Two years ago, the European Community funded the Pharma-Planta
project (www.pharma-planta.org). The Pharma-Planta scientists have signed a "Statement of Intent on
Humanitarian Use" in which they express their intention to develop products that specifically solve health needs
of the poor in developing countries. This year, the Bill and Melinda Gates Foundation set up a The Grand
Challenges in Global Health intiative and a committee of medical specialists. The experts indentified the urgent
need for needle-free delivery systems and heat-stable preparations as particularly pressing. Molecular farming
could be the way to overcome obstacles like storage, transportation, and delivery to developing countries. The
latter could produce their own plant-based vaccines and distribute them. "Vaccines seem like a better fit for the
plant-made pharmaceuticals," says Reichert. Vaccines that could be produced and administered through edible
plants seem like a science-fiction dream to prevent disease in rural African areas. But in Western countries,
where the market for vaccines is roughly a hundredth of the market for therapeutics, it seems unlikely that
biotech companies would look into tackling these issues without the support of either public or nonprofit
funding.
"I think there are indeed niche opportunities. They complement and maybe fill a few unmet needs, like perhaps
production in developing countries," says Reichert. "I do not think they will ever displace normal grounds of
production, like E. coli and CHO cells. Not unless there are major-major advances in the efficiency of the
production of the material" Mayer says GeneWatch thinks that people should not use food crops. "It is just
asking for troubles. We do think that containment is important, and if trials are going to go head outdoors,
serious attention needs to be made on the potentially adverse environmental effect."