pharmaceuticals traditionally used for the treatment of disease have been made
through the expression of protein in bacterial, fungal, and mammalian cell
cultures. Recently, the possibility to produce more diverse and complex
pharmaceutical proteins in plants has reached the laboratory benches of
scientists and companies worldwide. These pharmaceuticals are known as
plant-made or plant-based pharmaceuticals. In this article, we will discuss
pharmaceutical proteins, how genetic engineering makes it possible to produce
proteins in plants, why plants are desirable for the production of
pharmaceutical proteins, and which plant systems are being considered for their
production. Additionally, we will discuss risk and regulatory issues
associated with this new technology.
essential to all living organisms for function, structure, and regulation of
the body. Protein development in a cell begins with DNA transcribing into RNA,
and RNA translating into proteins. Proteins are made up of amino acids that are
arranged in different combinations and lengths. The differences in
arrangements and lengths of amino acids determine the function of the protein.
Some examples of proteins include hormones, enzymes, and antibodies.
Many people suffer
from infectious, inflammatory, and cardiovascular diseases — and these numbers
are growing. Protein-based drugs are the fastest growing class of drugs for
the treatment of these diseases in humans and other diseases in animals. The
reasons for this are because the numbers of people with diseases such as
diabetes are growing and new technologies are making proteins easier to produce.
The current methods of production of proteins for pharmaceutical application
(mammalian, bacterial, and fungal cell cultures) are predicted to fall short of
demand in the near future (Rogers 2003).
Insulin was the
first pharmaceutical protein produced using genetically engineered bacteria
(Thomas et al. 2002). Insulin originally was isolated from cows and pigs that
were slaughtered for food. This method was inefficient and caused some
patients to develop allergies from the animal-derived insulin. Today it is
made from the human gene that codes for the insulin protein and is expressed
and cloned in the bacterium, Escherichia coli. Large quantities of E.
coli are now grown in fermentation vats to make tons of human insulin
available to the growing number of diabetic patients.
engineering also has made it possible to use plants as factories for
pharmaceutical protein production. Plant-made pharmaceuticals are made by
inserting a segment of DNA that encodes the protein of choice into plant
cells. The plants or plant cells are essentially factories used to produce the
desired proteins and are only grown for the purpose of pharmaceutical
There are two
common methods of transformation (the process by which DNA from one organism is
incorporated into the DNA of another organism) that have been established
through biotechnology to produce transgenic plants, which, in turn, could be
used to create the plants used to make pharmaceutical proteins. The
transformation techniques include the Agrobacterium tumefaciens-mediated
transformation system and biolistics, also called particle bombardment.
tumefaciens is a bacterium that naturally infects plants and causes crown
gall disease. It is very useful for the production of transgenic plants
because it has the amazing ability to transfer a segment of its DNA, called
T-DNA, into the nucleus of the plant cells. The T-DNA from A. tumefaciens
is then integrated into the plant and transcribed, causing crown gall disease.
Scientists have used A. tumefaciens to their advantage by inserting
their DNA of interest between the T-DNA to create a plant with qualities such
as herbicide resistance, or pesticide resistance, as well as many others (Nester
et al. 1984, Binns and Thomashow 1988). Crown gall disease does not occur in
the plant after being transformed by A. tumefaciens because the plasmid
has been constructed to disarm the tumor-inducing properties that cause the
disease, and instead it expresses the DNA of interest.
Biolistics, or the
Particle Bombardment Transformation System, employs the use of metal particles
either of gold or tungsten. The metal particles are coated with the DNA that
is to be transferred into the plant cells. Using a pressurized system, a
plastic bullet coated with metal and DNA particles is released from the
biolistics machine when the pressure is released. The bullet is shot, and
within seconds, stopped by a shield causing the metal coated DNA particles to
be knocked off the bullet. The particles are then ultimately forced to be
inserted into the plant cells below.
Once the genes
have been transferred into the cells, the cells expressing the gene of interest
must be selected. In most cases, a selectable marker is included with the DNA
construct, which will confer resistance to an antibiotic or herbicide. The
plant cells that survive the antibiotic or herbicide application are the ones that
have successfully received the gene of interest (Purdue Agricultural
Plant Systems vs. Mammalian and
Bacterial Cell Cultures
Plants and plant
cells offer several advantages in the production of proteins for pharmaceutical
purposes. Unlike mammalian cells and bacterial cells, plants are capable of
post-translational modification and other assembly steps that are needed for
biological activity in complex multi-component proteins such as antibodies.
This in turn allows for more diversity and possibilities of drugs that could be
conceived from the proteins produced in plants (Rogers 2003, Goldstein and
Thomas 2004). When a protein is glycosylated, this means a certain sugar
molecule is added to its structure, causing it to fold in a specific and unique
orientation. The way the protein is folded determines its function and
activity. Plants are capable of producing more complex proteins with no
assistance because they are capable of glycosylation on their own, whereas
mammalian cells need assistance to glycosylate complex proteins. The system
needed to maintain and create complex proteins in mammalian cells is expensive
and difficult to maintain, which makes plant systems a more attractive source
for protein production. (Fischer et al. 2003).
are difficult and expensive to grow; therefore, bacterial cells are used to
produce proteins when glycosylation is not required. Bacterial cells still
have the potential of being contaminated, and require facilities to be built to
keep up with the growing demand of proteins needed for drug production. This
is why plants are an excellent alternative to mammalian and bacterial cell
cultures; they have to ability to glycosylate complex proteins and are
inexpensive to grow in large quantities.
pharmaceutical production has the potential to provide large amounts of protein
for the pharmaceutical industry. The types of plants used for pharmaceutical
protein production depend largely on their final application. To date, the
plants that have successfully been transformed include tobacco, potato, tomato,
corn, soybeans, alfalfa, rice, and wheat. However, it is important to
emphasize that no commercially available pharmaceutical products are currently
produced in plants (Ma et al. 2003).
Tobacco was the
first plant to be genetically engineered and has the advantage of being used as
a plant biopharmaceutical for exactly that reason; the methods for gene
transfer and expression are well established for this plant. Tobacco can also
be cropped multiple times per year, giving it an edge over other plants in
producing a large biomass (Fischer et al. 2003). The protein of interest can
also be targeted to the seed. In tobacco, up to one million seeds can be made
on a single plant (Daniell et al. 2001). One concern associated with using
tobacco as a protein factory is that it has toxic alkaloids associated with it,
which could be present in the final drug that is administered to the patient.
Another concern is that if the protein is targeted for the leaf tissue, it must
be frozen or dried and then processed immediately, making it difficult to
harvest and store for preservation of the protein (Ma et al. 2003).
Legumes, such as
alfalfa and soybean, and cereal crops, such as corn and rice, have been
considered as ideal candidates for plant biopharmaceuticals because the protein
can be targeted to accumulate in the seed and the seed can be harvested and
stored for an extended amount of time. Legumes are good candidates because
they naturally produce large amounts of protein in their seeds, making the
final protein recovery much larger. When considering a cereal crop as a
protein factory, yield is the main quality of importance, as well as ease of
transformation and speed of production scale-up (Ma et al. 2003).
The application of
the proteins made in plants include, antibodies, vaccines, hormones, enzymes,
interleukins, interferons, and human serum albumins. Monoclonal antibodies
have the potential to be among the first protein pharmaceuticals commercially
produced in plants (Rogers 2003). Antibodies are produced in vertebrate immune
systems to recognize and bind to antigens with amazing specificity. Because
antibodies possess this specificity they can be used as diagnostic tools as
well as prevention and treatment of diseases (Fischer et al. 2003). There are
at least six types of plant-derived recombinant antibodies that have moved to
the preclinical stage of testing, and one more advanced antibody in phase II
clinical trial (Ma et al. 2003). A possible use for monoclonal antibodies is
to one day attach a chemotherapeutic agent onto the antibody and allow it to
specifically bind to a tumor cell and eventually the chemotherapeutic agent will
destroy the tumor cell (Rogers 2003).
application for plant-made pharmaceuticals is the production of vaccines.
Subunit vaccines are made up of specific macromolecules that induce a
protective immune response against a pathogen. Subunit vaccine technology has
increased the safety of administering vaccines because it does not involve the
use of live or weakened viruses. Currently, it is very expensive to produce
subunit vaccines as well as to store them because they are not heat-stable.
Because the vaccines are not heat-stable, this limits where they can be sent
for use and unfortunately makes them unavailable in the developing countries
where vaccines are needed the most. Producing the vaccines in plants
eliminates the heat-stability issue because the vaccinogenic plant tissue can
be administered raw, dried, or in an encapsulated form; all of these forms can
be stored and shipped at room temperature (Sala et al. 2003). The risk of
contamination with animal pathogens during production is also eliminated
(Walmsley et al. 2000). Oral delivery of the vaccine is another reason that
vaccines produced in plants are an attractive possibility because they can
eliminate injection-related risks. Overall, the fact that the vaccines can be
stored as seeds are advantageous because large amounts of vaccines can be
produced in limited time and storage is less of an issue because the seed is a
stable form that will not degrade the protein over time.
The choice of the
crop will determine the way the vaccine is finally administered because only
some plants can be consumed raw whereas others must be processed. With
processing there is the potential that heat or pressure treatments could
destroy the protein. Cereal crops are an attractive species for expressing subunit
vaccines because they can produce proteins in their seeds, which are stable for
long storage periods. For animal vaccines the plant choice could be chosen
based on what is eaten as a major part of its diet, therefore eliminating the
need to process the protein and the risk of destroying it as well (Daniell et
In the U.S., a
three-agency coordinated framework including the Food and Drug Administration
(FDA), the United States Department of Agriculture (USDA), and the Environmental
Protection Agency (EPA) is responsible for regulating all products made from
biotechnology. Currently, the FDA regulates pharmaceuticals in the U.S., but
with the introduction of plant-made pharmaceuticals the USDA plays a key role.
Each agency has its own regulatory scope. For example, the FDA is responsible
for overseeing the production of pharmaceuticals for their safety to humans and
animals. The USDA evaluates genetically engineered plants and animals to
assess whether they will pose unacceptable risks to U.S. agriculture and the
environment. For plant-based pharmaceuticals, EPA will play a lesser role, but
will help ensure safety to the environment. The activities of the FDA and USDA
include not only products that are registered, but also experimental products
grown in laboratory, field, and greenhouse environments. Specific regulations
are currently evolving, but at this time the specific responsibilities and
activities of the two agencies remain unclear (Peterson and Arntzen 2004).
The production of
proteins in plants to be used in the pharmaceutical industry will largely
follow the same regulatory requirements that have already been established for
non-plant produced pharmaceuticals. However, there are key challenges for the regulation
of plant-based pharmaceuticals. These challenges primarily are associated with
the fact that some production systems will include manufacturing pharmaceutical
proteins in the open environment. Further, many production systems will involve
food. These are unique aspects of pharmaceutical manufacturing –– aspects that
will present novel risks that must be considered. As Peterson and Arntzen
(2004) stated, “Because of this, questions of intra- and inter-species gene
flow, allergen exposure to the public and non-target organism exposure come
risks associated with plant-based pharmaceuticals include: pollen transfer to
related species, contamination of non-transgenic crops intended for the
consumption by humans, allergic reactions to the drugs produced from the
genetically engineered plant, and persistence of genetically engineered
material to persist in the environment and accumulate in non-target organisms
(Daniell et al. 2001). Risk assessment of plant-made pharmaceuticals should be
reviewed on a case-by-case basis because the plants used to produce proteins
each have different risks associated with them (Peterson and Arntzen 2004).
To address the
out-crossing potential of transgenic crops to a receptive crop species or weedy
relative, physical isolation distances have been considered. This can mean
containment in a greenhouse setting or buffer zones between fields of the same
species. Some species have enclosed flower structures, reducing the distance
needed in between fields and therefore are being evaluated as good candidates
for producing plant-made pharmaceuticals. In the case of corn –– a wind
pollinated crop –– because it has many benefits for being a plant
biopharmaceutical, the tassels can be manually removed and male-sterile
varieties could be used to contain the pollen. The transgenic plants can also
be planted at different times from food crops to make sure they flower at
different times, which decreases the potential for pollen transfer (Thomas et
Goldstein and Thomas (2004), “virtually every pharmaceutical product currently
on the market can cause allergic reactions in some people.” The FDA approved
the drugs because their benefits outweighed their allergenic potential. This
will be the same approach taken on plant-made pharmaceuticals; the FDA will be
concerned if, and only if, the plant-made pharmaceuticals are more allergenic
than pharmaceuticals not derived from plants (Rogers 2003). However, there is
a question of the allergic risk for people not taking the drug but happen to
inhale or topically receive pollen from the genetically engineered plant. Will
the contact from the genetically engineered plant cause a reaction or
immunity? Because these proteins are in the environment does not necessarily
make them environmental contaminants or even human health hazards. The
specificity of the proteins produced in plants potentially makes the risk
organisms are organisms not intended to be affected by the plant-produced
protein. These organisms include insects, mammals, birds, fish, plants, and
other wildlife in and around the area where the genetically engineered plant is
growing. Animals potentially could be affected by genetically engineered
plants, by eating or landing on the plant or plants nearby that have the pollen
from the genetically engineered plants on them. There are ways to prevent or
reduce the risk of these non-target organisms from being affected by the
genetically engineered plant. Planting border crops that are not genetically
engineered near the engineered crops, or containing the plants in greenhouse
facilities are a couple preventative ways to keep non-target organisms
unharmed. Fortunately, many of the plant-made pharmaceutical proteins
currently produced or being considered are species-specific (such as
antibodies) which reduces their effects (Goldstein and Thomas 2004).
the evolving regulatory environment and the need to evaluate the human-health
and environmental risks of plant-based pharmaceuticals on a case-by-case basis
necessitate using a robust, transparent science-based methodology. Therefore,
Peterson and Arntzen (2004) recommended using the established paradigm of
environmental risk assessment. Additionally, Wolt and Peterson (2000) argued
that the risk assessment framework was sufficiently robust to evaluate risks
from most applications of biotechnology.
assessment is a formalized basis for the objective evaluation of risk in a
manner in which assumptions and uncertainties are clearly considered and
presented. Risk assessment proceeds in a systematic, stepwise fashion that
includes the following five steps: (1) problem formulation; (2) hazard
identification; (3) dose-response relationships; (4) exposure assessment; and
(5) risk characterization. Hazard and dose are considered in relation to
exposure to determine risk or what additional information is needed to
calculate or refine risk estimates (NRC 1983).
Plants used as
protein factories to assist the pharmaceutical industry in drug manufacturing
have the advantage of producing proteins which could not be made using other
systems, reducing the cost of production, and increasing the amount of proteins
available to be used to make drugs. With this increased availability and
potentially lower cost, more patients will be able to receive the drugs they
need. Plant systems are capable of producing large quantities of protein and
have an advantage over mammalian and bacterial cell cultures because of the
ease of scale-up that is possible with the equipment and land already in
existence for these plants. These systems should be assessed for risk using the
risk assessment paradigm. Risk analysis (which includes risk assessment, risk
management, and risk communication) is a flexible and robust framework, making
it ideal for addressing the myriad issues associated with this new technology.
A.N., Thomashow, M.F. 1998. Cell biology of Agrobacterium infection
and transformation of plants. Annual Review of Microbiology 42:575-606.
H., Streatfield, S. J., Wycoff, K. 2001. Medical molecular farming: production
of antibodies, biopharmaceuticals and edible vaccines in plants. Trends in
Plant Science 6:219-226
R., Twyman, R. M., Schillberg, S. 2003. Production of antibodies in plants and
their use for global health. Vaccine 21:820-825.
D.A., Thomas, J.A. 2004. Biopharmaceuticals derived from genetically modified
plants. QJM: An International Journal of Medicine 97:705-716.
J.K-C., Drake, P.M.W., Christou, P. 2003. The production of recombinant
pharmaceutical proteins in plants. Nature Reviews Genetics 4: 794-805.
E.W., Gordon, M.D., Amasino, R.M., Yanofsky, M.F. 1984. Crown gall: a
molecular and physiological analysis. Annual Review of Plant Pathology
National Research Council. 1983. Risk Assessment in the Federal Government:
Managing the Process. Washington, DC: National Academy Press.
R.K.D., Arntzen, C.J. 2004. On risk and plant-based biopharmaceuticals.
Trends in Biotechnology 22:64-66.
Agricultural Biotechnology. 2004. Moving Genes into Plants. http://www.agriculture.purdue.edu/agbiotech/genetransfer.html
K.K. 2003. The Potential of Plant-Made Pharmaceuticals. http://www.plantpharma.org/ials/index.php?id=1
F., Rigano, M. M., Barbante, A., Basso, B., Walmsley, A.M., Castiglione, S.
2003. Vaccine antigen production in transgenic plants: strategies, gene constructs
and perspectives. Vaccine 21:803-808.
S.J., Lane, J.R., Brooks, C.A., Barker, D.K., Poage, M.L., Mayor, J.M.,
Lanphear, B.J., Drees, C.F., Jilka, J.M., Hood, E.E., Howard, J.A. 2003. Corn
as a production system for human and animal vaccines. Vaccine 21:812-815.
B.R. Van Deynze, A., Bradford, K.J. 2002. Production of Therapeutic Proteins
in Plants. Agricultural Biotechnology in California Series 8078. University of
A.M., Arntzen, C.J. 2000. Plants for delivery of edible vaccines. Current
Opinion in Biotechnology 11:126-129.
J.D., Peterson, R.K.D. 2000. Agricultural biotechnology and societal decision-making:
the role of risk analysis. AgBioForm 3:39-46.