Volume 13, Issue No. 1
Planting
for the
Future
Plant-Made
Pharmaceuticals
Main Points
The Biotechnology Institute is pleased to present Your World’s fall 2003
issue exploring plant-made pharmaceuticals. Interest in this field is
blossoming as scientists recognize the tremendous potential of using
plants to manufacture useful proteins. It’s one of the many aspects of
biotechnology—the use of living organisms to benefit humanity.
We focus on these basics:
• What is involved in making plant-made pharmaceuticals (PMPs)?
• How are PMPs different from nutraceuticals and edible vaccines?
• Who can benefit from PMPs?
• Are plant-made pharmaceuticals safe and effective?
• How are policymakers dealing with PMPs?
Biotechnology is shaping the lives of people of all ages, and we hope this magazine cultivates an interest in a career that is part of this exciting technology.
Paul A. Hanle, President
Biotechnology Institute
Contents
Plants: Factories of the Future................................................................2
So You Want to Make a PMP . . . ............................................................4
Worth the Effort ......................................................................................6
Down on the ‘Pharm’ ..............................................................................8
The Chosen Ones..................................................................................10
Potent Plants.......................................................................................... 12
Career Profile Anne-Marie Stomp ........................................................14
Activity Microbial Bioassay....................................................................15
Glossary and Resources ........................................................................16
Volume 13, Issue No. 1
Fall 2003
Publisher
The Biotechnology Institute
Editor
Kathy Frame
Managing Editor
Lois M. Baron
Design
Dodds Design
Cover and Inside Illustration
©2003 John Michael Yanson
ALL RIGHTS RESERVED.
Troy Mashburn Photo
©2003 Keith Barraclough
ALL RIGHTS RESERVED.
Advisory Board
Don DeRosa, Ed.D., CityLab,
Director of Education,
Boston University Medical College
Lori Dodson, Ph.D.,
North Montco Technical Career Center
Anthony Guiseppi-Elie, Sc.D.,
Virginia Commonwealth University
Lynn Jablonski, Ph.D.,
GeneData (USA), Inc.
Mark Temons,
Muncy Junior/Senior High School
Sharon Terry, M.A., President,
Genetic Alliance
For more information
Biotechnology Institute
1840 Wilson Boulevard, Suite 202
Arlington, VA 22201
[email protected]
Phone: (703) 248-8681
Fax: (703) 248-8687
Biotechnology Institute
The Biotechnology Institute is an independent, national, nonprofit organization
dedicated to education and research about
the present and future impact of biotechnology. Our mission is to engage, excite,
and educate the public, particularly young
people, about biotechnology and its
immense potential for solving human
health, food, and environmental problems. Published biannually, Your World is
the premier biotechnology publication for
7th- to 12th-grade students. Each issue
provides an in-depth exploration of a particular biotechnology topic by looking at
the science of biotechnology and its practical applications in health care, agriculture, the environment, and industry.
Please contact the Biotechnology Institute
for information on subscriptions (individual, teacher, or library sets). Some back
issues are available.
Acknowledgments
The Biotechnology Institute would like to
thank the Pennsylvania Biotechnology
Association, which originally developed
Your World, and Jeff Alan Davidson,
founding editor.
The Biotechnology Institute acknowledges
with deep gratitude the financial support
of Centocor, Inc., and Ortho Biotech.
©2003 Biotechnology Institute. ALL RIGHTS RESERVED.
2
Planting for the Future: Plant-Made Pharmaceuticals
Plants: Factories
of the Future
A
cross the country, workers
have been reporting to manufacturing facilities that they
casually call plants, as in “I’ve got to
clock in at the plant at 5 a.m.” In the
not-too-distant future, we may see
plants—the kind with seeds, roots,
stems, and leaves—serving as factories
for plant-made pharmaceuticals.
Using biotechnology, scientists place into a
plant a gene that expresses a medically useful
protein, then the plant makes that protein
using its own biological machinery. These
protein-based drugs and vaccines (“biologics”) are called plant-made pharmaceuticals
(PMPs).
PMPs are true drugs, approved by the Food
and Drug Administration (FDA) in the United
States. They should not be confused with
nutraceuticals, which are compounds used as
part of a diet to improve health. And they are
not edible vaccines, that is, vaccines administered by eating a specific amount of a food.
Plant-made pharmaceuticals do not involve
eating the plant; the plant is merely used as a
factory for protein production. The proteins
for PMPs are always extracted from a plant.
Plants are naturally good protein makers,
and scientists know a lot about how plants go
about their work. Researchers also understand quite a bit about plant genetics, growth,
and development, which makes cultivating
genetically modified plants relatively simple.
Plants can be turned into economical and
renewable protein-manufacturing systems
with a few simple resources—water, air, sunlight, minerals, and the right set of genes. In
contrast, protein production in bacterial systems and mammalian cell cultures is very
costly and limited by the capacity of buildings
and equipment.
There are legitimate concerns about using
plants to produce therapeutic proteins,
including a low risk of food and feed contamination, exposure of farm workers to potentially harmful genetic material, and the
possibility that wildlife and insects will feed
on the altered crops. Scientists are working
hard to address these issues, and government
agencies have set up strict rules and routines
for confinement and cultivation methods that
minimize risks. The U.S. Department of
Agriculture (USDA) and the FDA oversee every
stage of PMP production.
Plant-made pharmaceuticals could dramatically benefit anyone who uses drugs or hopes
to find a drug to treat his or her illness. At the
very least, the science promises a new ability
to expand production, which might provide
more people with access to these drugs and
vaccines. Changing the balance of production
cost and profit also may make it possible to
offer drugs that are currently too expensive to
produce in mass quantities.
This technology holds tremendous potential to expand the range and availability of
pharmaceuticals to treat a wide array of
diseases. These diseases could go from
arthritis to asthma to cancer and beyond.
Some PMPs are already being tested in
humans. Clinical trials using proteins
encoded in corn to treat E. coli/travelers’ disease and cystic fibrosis, and a tobacco-based
PMP to treat non-Hodgkin’s lymphoma, are
under way. Scientists hope that several PMPs
will be commonly available in three to five
years. The following articles give you details
about this up-and-coming field.
Your World
3
How are plant-made pharmaceuticals produced?
So You
Want to
Make a
S
o how do you turn a plant into
a pharmaceutical factory? In
other words, how do you make
a plant start manufacturing a protein it has
never made before? And when it’s done,
how can you separate that protein from all
the other proteins the plant makes?
Surprisingly, this isn’t quite as hard as it
sounds. More than two decades of
research in genetic engineering—
including the knowledge of how
to move a gene from one living
organism to another—has
advanced these techniques
from obscure art to routine
science. While a great deal of
skill (and patience!) is still required, the
essential tools are well known and widely
available. And when it comes time to purify
the protein from the plant, the tools are even
more well known—people have been extracting useful substances from plants for centuries.
The first step in making any PMP protein is
to isolate the gene for the protein we want.
Once we have the gene, the rest of the process
looks like this:
•Add other bits of DNA to help control the
gene (to express the right protein) once
it’s inside the plant.
•Transfer the gene into plant cells grown in
a dish.
•Grow the plant cells up into whole plants.
•Harvest the plants, and isolate the protein.
Let’s look at each step in more detail.
Controlling a Gene
A gene is the segment of DNA involved in
making a protein. But a gene needs another
DNA sequence near its beginning to tell where and
when it should be “read”
(or in scientific terms,
where and when it should
be transcribed). Such a
sequence is called a promoter; it is the site on the
4
Planting for the Future: Plant-Made Pharmaceuticals
PMP...
DNA where RNA polymerase will bind and
begin transcription. If we want an antibody
gene to be expressed only in seeds, for
instance, we can add the same promoter that
plants use to turn on genes in their own
seeds. (See also “The Chosen Ones,” p. 10.)
We should also add another bit of DNA
called a signal sequence, which we’ll tag onto
the end of the gene. The signal sequence DNA
codes for a little tail of amino acids on our
protein that acts like an address label, sending
our protein to its destination within the
cell. Where shall we send it? One
option is the plant’s oil body. This
compartment stores oil, like
peanut oil or olive oil, and it
makes an ideal spot for proteins too, since the oil body
serves as a foundation that
protects the proteins.
Adding an oil body signal sequence to our
gene will direct our protein here after it is
produced. Other PMPs may be easier to isolate
if they are stored in the endoplasmic reticulum (ER), a membrane network within the
cell. We also add a termination sequence,
which is like a period at the end of a sentence; it tells the RNA to stop reading.
Transferring a Gene into the Plant
Two major methods are open to us. We
can stick the gene we’re making onto microscopic particles of gold, and use a gun to
shoot them into plant cells. Believe it or not,
this process, called biolistics, actually works
pretty well! Unfortunately the results are not
consistent (imagine the random results from
shotgun pellets), so biolistics is not widely
used.
A second method is to place a gene in a
small circle of DNA (plasmid) that is located
in the common soil bacterium, Agrobacterium
tumefaciens, and let the bacterium infect a
plant. When the bacterium
invades the plant, the plasmid
is transferred into the plant’s
own cells, carrying the gene
along with it.
Although it’s possible to
infect mature, fully developed
plants, it is much easier to infect
Green Machines
Chloroplasts are the green machines inside plant cells that convert sunlight, air, and
water into sugars. Chloroplasts have their own DNA and their own protein-making
machinery. Some researchers are investigating the use of chloroplasts for making
PMPs. This would isolate the introduced DNA within the chloroplast—providing a
measure of confinement for the PMP.
Therapeutic Proteins
PMP METHOD
Gene
Programming
Disposal of Biomass
Integration of Gene
Into Plant
Production of
Plant Material (Biomass)
Recovery and
Quality Control
Gene of
Interest
Energy
TRADITIONAL
MANUFACTURING
METHOD
Cell Culture
thousands of small pieces of plant tissue
growing in a lab dish. Such tissue cultures, as
they are called, provide a highly controlled
environment for manipulating plant cells.
Once the plants have taken up the gene, we
alter the mix of hormones in the growth
medium to encourage development. Within
several months, they are ready to be transplanted into soil and grown in a greenhouse
or a field.
Isolating the Protein
Once the plant has grown and made lots of
the protein we want, how do we get the protein out? First we harvest the plant part—
seeds, leaves, or roots—containing this
protein. If we’ve routed the protein to the oil
body in the seed, we might then grind the
extract from the oil body. If it’s in a leaf, we
will probably grind the leaf up to release its
contents.
From there, extraction will depend on the
particular chemical properties of the protein
we’re interested in and the tissue it’s in
(described in "The Chosen Ones,” p. 10).
Here are some possibilities:
Filtration: Removes plant fibers.
Ultracentrifugation: High-speed spinning
Bioreactor
Think About It
PMPs are expensive to
develop. How does a company keep someone else
from planting and profiting from their invention?
One strategy is to insert
a "signature sequence" of
DNA just after the signal
sequence. This puts the
company’s unique "fingerprint" on the gene. This
unique bit of DNA doesn’t
make any protein, but it
does indicate the origin of
the transgenic plant, discouraging theft. A signature sequence also makes
it traceable in cases of
contamination.
Fuel
Protein
separates the cell into different parts, such as
endoplasmic reticulum or chloroplasts.
Chemical extraction: Solvents attract the
proteins and exclude the cell’s other molecules to leave a crude protein mix.
Affinity chromatography: The protein mix
passes through a matrix—that is, a column
filled with an insoluble substance such as
beads. Molecules (ligands) attached to the
beads adsorb the desired protein. The ligand
holds the desired protein in place while all
the undesired materials in the protein mix are
washed off the column with a change in pH
or salt concentration (it is the chemical or
ionic change that causes materials to leave the
column). The final wash releases the desired
protein all nice and clean.
Extracting and purifying the protein are
often the most expensive steps in making a
PMP. Before they begin a project, companies
plan the entire manufacturing process from
start to finish, from gene construction
through final purification, to get the most for
the least cost.
Cultivating plants as protein factories is a
good example of how biotechnology uses living organisms to serve humanity.
—Richard Robinson
Your World
5
Why are people interested in PMPs?
Worth Effort
the
H
umans have used plants as medicines since the earliest
times. Centuries ago, if you were sick, a healer might have
used leaves, bark, or roots to treat your ailment. Until about
1900 or so, many doctors kept gardens of herb plants that they used
as medicines. Even today, about half of our medicines come from
chemicals extracted from plants or the plants themselves. Aspirin
production begins with a chemical found in willow bark, while morphine comes directly from a particular poppy.
Today, plant-based medicines make up a
much smaller portion of the modern medical
arsenal, but a modern twist may change that.
Scientists are using plants to produce a wide
range of high-tech medicinal proteins. Using
plants in this way offers several benefits.
© Kam Yu / Masterfile
Genes and Proteins
Every living cell contains genes.
Genes hold the information for cells
to make proteins, which the cells
use for a variety of purposes. Some
proteins help to make new cells,
some break down food for digestion, and others prevent disease. Many human diseases
arise when the body does not
The pancreas secretes a small protein called insulin. Insulin
lets glucose in the blood enter cells where it can be used for
energy. Researchers are studying how monoclonal antibodies can help diabetics.
6
Planting for the Future: Plant-Made Pharmaceuticals
Future Farmers of
America?
Plants: a solution to the shortfall in manufacturing capacity for new drugs
Molecule
Limitations
Plant Solutions
Hemoglobin
• Blood demand increases,
• Large-scale production
Donors decrease
• Serotypes incompatibility
• No concern about serotypes
• Security concerns (HIV; HVC; HBV) • No human pathogens
Antibody
• Expensive
• Production capacity limited
• Cost reduction
• Large-scale production
Enbrel
• Capacity shortage
• Large-scale production
Factor VIII
• Worldwide shortage
(40% of hemophilia patients
have access to the product)
• Large-scale production:
accessible to all patients
Insulin
• Too expensive for
non-industrialized countries
• Cost reduction: accessible to
all non-industrialized countries
if they need less! Using plants, pharmaceutical companies should be able to meet the
growing demand for biotech drugs.
Protein made from animal cell cultures for
use in medicine are often contaminated with
viruses. Most of these viruses are harmless,
but manufacturers must still
Did You Know...
spend a lot of time and money
Cultures of Chinese
to remove them. Contamination
hamster ovary cells
by viruses is not a problem
produce a wide variety
when plants are put to work
of human proteins.
making protein medicines.
Producing proteins in plants
is quite a new idea. Only about half a dozen
companies worldwide, and about 20 universities, are working on it. Like all new ideas,
this one will take time. However, the potential benefits—safer, more accessible medicines—are worth the effort.
—Angelo DePalma
Your World
7
Canadian Food Inspection Agency
make proteins correctly. For example, in one
form of diabetes, the body does not make
enough of a small protein called insulin.
People with Type 1 diabetes must take daily
insulin injections.
Most protein medicines today are made in
factories or laboratories, using cultures of
genetically engineered bacteria, yeast, or animal cells. Scientists give these cells new
genes—genes that are “foreign” to the cells.
These genes instruct the cell to make a
desired protein. Organisms containing foreign
genes are known as transgenic organisms.
Cell cultures are extremely expensive to
operate. To use them, biotechnology companies must buy sophisticated equipment or
construct special buildings, and hire highly
trained scientists and engineers to operate
everything properly. As a result, protein drugs
are not cheap. Some cost as much as $50,000
per gram! (A gram is the mass of a standard
paper clip.) One of the goals of biotechnology
is to make medically useful proteins (also
called therapeutic proteins) in plants at a
lesser cost than traditional mammalian cell
culture techniques.
Plants turn out to be ideal for making
biotech medicines. Most agriculturally useful
plants are inexpensive, easy to grow, and simple
to produce in large quantity. Thanks to greenhouses and advanced agriculture, a few seeds
from genetically engineered corn or wheat
plants may be grown into hundreds of thousands of identical plants in less than two years.
Producing therapeutic proteins in plants
could change the way certain drugs are made.
When traditional biotechnology companies
need to boost production, they must find a
way to make it in existing factories or build a
new facility. Sometimes biotech firms rent
manufacturing space from other companies or
hire another organization to make their product for them.
All these solutions take time and are very
expensive. By contrast, companies producing
medicines in green plants need only sow
more if they need more product, or plant less
Some of you who live on farms, or who know
farmers, may get excited about the prospect of
producing valuable proteins in your backyards.
Unfortunately, making medicines is not something
that just anybody can do in his or her spare time.
Only companies with experience making medicines
are permitted to manufacture drugs of any kind. Companies must prove their protein-making plants are free of disease, chemicals, and other substances that might
harm people who take the PMPs. Soil, water, fertilizer, growing, handling, and harvesting must conform to standards that are much stricter than for food plants. Yes,
it’s farming, but it’s much, much more. In addition, these plants won’t be sold on
the market the way current crops for food and animal feed are.
Is it safe to produce PMPs?
Down on th
Uncle Sam
Is Watching
W
ould you visit a reptile zoo
that didn’t have any
cages? The spitting cobra
exhibit alone should be enough to keep
you home, but you might not want a
harmless garden snake slithering
around your ankles either. At zoos—and
many other places—confinement is the
key to people feeling secure.
Currently, in order to first work
with a PMP company, growers
are required to complete a
training course given by the
Animal and Plant Health
Inspection Service (APHIS), the
arm of the U.S. Department of
Agriculture that is dedicated to
protecting crops from pests
and diseases.
It’s easy enough to keep a snake in
PMP compaThink About It
a cage, but other things are harder
nies are required How can the government to control—such as fields of
and companies control
plants. As you learn in other artito obtain a perthe potential for human
cles, scientists are now using
mit from APHIS
error in confinement
plants, such as alfalfa, corn, and
before planting
strategies?
soybeans, to produce proteins to
at the grower’s
be used for medicines, a breaksite. The applicathrough that could save lives. Unfortunately,
tion process is lengthy. The
all the potential benefits of PMPs will go to
waste if companies can’t assure the governcompany must describe the
ment and the public that the plants are safe.
new crop, list all new genes,
While many people see a big future for
and, most importantly, explain
pharmaceutical crops, the industry is still in
exactly how the crop will be
the experimental stage. Companies are concontained.
ducting research with different plants just to
As a final precaution, APHIS
see if they really can harvest the proteins they
want. In 2002, the government issued permits
inspectors also must visit each
for just 34 research plots that covered a total
PMP production site at least
of 130 acres.
five times throughout the
Every step of the PMP process—from hangrowing season and twice
dling seeds to planting and harvesting plants
more in the subsequent year.
to extracting proteins—is already carefully
regulated by some combination of the Food
and Drug Administration (FDA) and the U.S.
Department of Agriculture (USDA). In accordance with regulatory requirements, growers
or permit-holders must surround their crops
with a 50-foot buffer zone of fallow
(unplanted) land, use separate equipment,
like tractors, and separate storage facilities for
PMPs.
Some plants for PMPs are being grown
only in greenhouses, but if all plants grown
for PMPs are raised this way, the ability to
8
Planting for the Future: Plant-Made Pharmaceuticals
he ‘Pharm’
expand production easily—a main benefit of
PMPs—will be limited.
Other measures that growers take to ensure
the safety of the food supply include planting
pharmaceutical crops several weeks after other
nearby crops start growing. By the time these
plants start producing any pollen, it will be
too late to crossbreed with other crops.
Industry and the government are learning
from experience how to fine-tune their procedures. Two incidents highlighted how diligent
everyone involved needs to be. ProdiGene, a
Texas-based company, used fields in Nebraska
and Iowa to grow its plants. In Nebraska, soybeans were grown in the same field where
pharmaceutical corn had been grown the previous year. A few volunteer corn stalks (produced from corn seeds from the previous crop
that should have been weeded out by the
farmers) sprouted up with the soybeans. By
the time anyone suspected a problem, the
500,000 bushels of soybean crop were in the
warehouse. Under orders from the USDA, the
entire 500,000 bushels of soybean in that
warehouse were destroyed, and ProdiGene
had to pay the bill along with a hefty fine. A
similar problem cropped up at a test plot in
Iowa. In that case, ProdiGene had to buy up
and burn 155 acres of surrounding corn that
might have been pollinated by its plants.
Some people believe PMP companies
should plant only nonfood crops. However,
scientists know far more about the genes of
cultivated crops such as tomatoes and corn
than of, say, daisies. Other people say this
type of research should be kept out of agricultural hotbeds such as Iowa or Nebraska. But
these areas are popular with growers for a reason. A cornfield in Alaska would not be very
productive.
The technology is still in its infancy. All
the people involved—from government officials to leaders of biotechnology companies to
workers on the ground—are still trying to
find the best approach. Will America’s
Breadbasket ever become a medicine factory
for the world? The science is there.
—Chris Woolston
Three Models
For PMP
Production
Most companies that produce
PMPs set up their operation in
one of three ways:
• Hire a firm to develop and
grow genetically modified
plants that produce the protein
they’re interested in.
• Develop the modified plant
themselves and then hire a
farmer to sow it on his land;
the farmer oversees everything
from planting to delivering the
harvested crop.
• Buy land and set up an operation to do absolutely everything; everyone on the farm is
a company employee.
Your World
9
Which plants are used for PMPs?
the
Chosen Ones
I
n the world of
plant-made
pharmaceuticals, how do
researchers choose the plants
they’ll work with? For
example, why would a
scientist choose the alfalfa
plant instead of, say, a lilac
bush? Currently, manufacturing therapeutic proteins is
a challenging, time-consuming, and expensive process, so it’s
not surprising that the search is on for a
better alternative. But how do scientists
decide which plants to use and how and
where to grow them?
Plant-made pharmaceutical (PMP) research
is under way in the United States and Canada,
as well as in several European and Asian
countries. Corn, tobacco, rice, sugarcane, barley, tomatoes, soybeans, duckweed, alfalfa,
safflower, moss, lupine, carrots, lettuce, potatoes, spinach, and bananas are some of the
crops being studied.
There is no one perfect plant in which to
produce every therapeutic protein, and
researchers decide which plant to use based
more on economics than on matching plants
to specific diseases.
Some people worry that plants containing
therapeutic proteins might become mixed
with plants used for food and feed.
Researchers point out that we know a great
deal more about crop plants than about other
plants—including how to confine them.
Geography is also sometimes a factor—for
example, researchers might pinpoint a location where a disease is prevalent and then
find a crop that is common in that area.
Researchers also consider whether the final
processed protein will be as safe, as pure, and
work as well as one produced in culture.
Selected plants of course must be able to express
10
Planting for the Future: Plant-Made Pharmaceuticals
and store the desired protein until it is ready to
be processed without it breaking down or
being degraded (and thus becoming less
effective). Extracting the protein from the
plant must be doable—physically possible—in a cost-effective way. Finally, every
plant species has unique advantages and
disadvantages that researchers weigh in
deciding which to use.
Let’s consider an example.
Monoclonal antibodies (MABs), complex
proteins used to treat a variety of chronic conditions and life-threatening illnesses, are typically produced in cell culture, but they can
also be produced in corn. Corn can efficiently assemble, fold, and store large
proteins, including large quantities of MABs.
And the cost is favorable—$80 to $250
per gram of MAB produced in
corn, compared with $350
to $1,200 per gram of
MAB produced in cell
culture. About
two-thirds of
PMP research
involves
using corn.
Although corn
has a perceived disadvantage—pollen grains
containing genetic material
could become wind-borne to
neighboring fields—it has unique
confinement advantages. First, corn
has separate male and female flower structures; confinement can be accomplished by
removing the male structure before it begins
to shed pollen (detasseling). Second, corn can
also be bred to be sterile, which is a practice
currently used by PMP companies. Third, in
the event that corn is not sterile or detasseled,
the large size of corn pollen allows it to settle
quickly, which inhibits it from traveling great
distances to cross-pollinate. Finally, few wild
grass species in the United States can crosspollinate with corn.
Other crops have been studied for MAB
production, and research on other crops continues today, but so far none has been identified as a suitable host. Excessive water
content makes potatoes a poor candidate for
MAB production, for instance, and the addi-
tional steps required for protein extraction
and purification in soybeans makes them less
attractive than corn.
The Right Place
Remember, the gene for the desired protein, flanked by DNA sections that regulate
gene activity, can be inserted into the plant in
any random location. The DNA section that
precedes the gene is called the “promoter.” It
is like an “ON/OFF” switch because it determines when and where the protein is made in
the plant. Some promoters are “ON” all of the
time in all tissues, whereas other promoters
are “ON” only when they are in specific tissues. By choosing a promoter that is specific
to a particular location, scientists can target
where they want the protein to accumulate.
The DNA section that follows the gene, the
“termination sequence,” signals the end of the
gene sequence.
Seeds are most often targeted for PMP production because they are good vessels for protein
accumulation, storage, and transport. Leaves in
tobacco and alfalfa plants and tubers in potatoes
have also been sites of PMP production.
Picking the Process
The method of protein processing depends
on the plant being used, where the plant
stores the protein, the protein’s ability to withstand the processing procedure so that the
desired quantity can be harvested, the scale of
production, and whether drugs or plant-made
vaccines are being produced.
In PMP production, the protein is usually
mechanically extracted from the plant, and a
wide variety of equipment (such as grinders,
blenders, and large-scale separating systems)
is used. In plant-made vaccine production,
the protein is sometimes kept inside of the
plant cell and processed along with the plant
material into a powdered form. Later in
production, scientists may decide to place the
powder into gelatin capsules for oral
administration.
Matching the right protein to the right
plant is a crucial decision for the success of
plant-made pharmaceuticals.
—Kathleen Wildasin
From Field to Pharmaceutical
You’ve been asked to grow monoclonal antibodies (MABs) in corn and to estimate
how many seeds and how much land will be required to grow the seeds before
beginning the project. Your project director has given you a few clues about what is
already known about MAB production, but it’s your job to do the final math.
Let’s get started.
Pharmaceutical companies know that an average of 1 gram of monoclonal antibody
is produced annually per kilogram of corn seed:
1 g MAB
1 kg corn seed
They also know that an average of 175 bushels of corn can be produced per acre
and that there are 25.4 kg of seed per bushel. The following calculation shows how
much corn seed can be produced on each acre:
175 bushels of corn
acre
x
25.4 kg seed
bushel of corn
=
4,445 kg seed
acre
Knowing this information, it’s easy to figure out how many grams of MAB can be
produced per acre:
4,445 kg corn seed
acre
x
1 g MAB
1 kg corn seed
=
4,445 g MAB
acre
But only an average of 60 percent of the MAB can actually be recovered and purified:
4,445 g MAB x
acre
0.60
=
2,667 g MAB
acre
x
1 kg =
1,000 g
~2.7 kg MAB
acre
Now it’s your turn to do the math.
Your project director tells you that 3 g of purified MAB are needed to treat one
patient for one year. He estimates that 150,000 patients will need treatment each
year with your purified MAB.
(a) How many kg of purified MAB must be produced each year to meet the needs of
150,000 patients?
150,000 patients
x
3g
patient
x
1 kg
1,000 g
=
450 kg
(b) How many acres of land must be planted in order to achieve this level of production?
450 kg
x
1 acre
2.7 kg MAB
=
~167 acres
—KW
Your World
11
T
roy Mashburn, a sophomore in
Arlington, Virginia, has cystic
fibrosis, a genetic disease that
makes his body produce thick mucus
that clogs his lungs. That mucus also
obstructs his pancreas, keeping crucial
enzymes from reaching his intestines to
help digest his food.
To avoid malnutrition, Troy uses enzyme
replacement therapy. But that therapy isn’t
perfect. Because his medication is derived
from pig pancreases, it leaves Troy vulnerable to any pathogens—such as diseasecausing viruses or bacteria—the pig
carried. And the medicine’s effectiveness doesn’t last long,
because pancreatic proteins
take a beating in the highly
acidic human stomach.
Troy has a mild case of CF.
He leads a pretty normal life
and even plays on the
school’s JV ice hockey team.
But like other CF patients, he
has to take as many as 20 pills a
day. For some patients, the medication doesn’t work at all.
If a French company called Meristem
Therapeutics is successful, Troy and the other
70,000 children and young adults worldwide
who have cystic fibrosis may soon have a better option.
By using genetically engineered corn to
develop a protein called lipase, the company
bypasses the potential problems of animal tissue. And by making gastric lipase, the company produces a medication sturdy enough to
keep working even after a trip through the
stomach. Currently in European clinical trials,
the product could mean safer, more effective
treatment for cystic fibrosis patients.
Meristem is just one of the many companies
hoping to harness plant power in the fight
against disease. Like Meristem, some are creating PMPs to treat diseases. Others are using
plants to produce drugs or vaccines to prevent
diseases from happening in the first place.
You can’t buy PMPs at your local drugstore
yet. But the Texas firm ProdiGene has already
started marketing a corn-grown enzyme used
12
Planting for the Future: Plant-Made Pharmaceuticals
Why Do
N-Glycans
Matter?
to produce insulin for diabetics. Hundreds of
studies are under way to investigate PMPs’
ability to be used to tackle everything from
life-threatening diseases like cancer and HIV
to less serious problems like tooth decay and
the common cold.
Treating Disease
PMPs could eventually be used to treat
just about any disease currently treated with
drugs called “biologics,” a class of drugs that
In fact, California’s Large Scale Biology
Corporation is testing individualized vaccines
that train the immune system to fight cancer—
in this case, non-Hodgkin’s lymphoma—that is
already present. The company extracts proteins
from a patient’s own cancer cells, grows identical proteins in tobacco, and injects these back
into the patient to trigger an immune response
against malignant cells. Such tailor-made vaccines could one day offer an alternative to the
all-out attack of one-size-fits-all chemotherapy.
What diseases can PMPs combat?
Potent Plants
N-glycans are carbohydrate
chains found within the proteins
of both plants and animals. They
play an important role in protein
folding. Proteins that aren’t folded
correctly don’t work the way they
are expected to.
The core of the N-glycan
chain is identical in both animals
and plants. However, animal Nglycans have two additional substances (α 1,6 fucose and xylose
sugar residues) not found in plant
N-glycans. And plant N-glycans
carry two additional carbohydrates not found in animal N-glycans (α 1,3 fucose and xylose
includes such proteins as enzymes, horPreventing Disease
mones, and antibodies. Researchers are
Other researchers are using PMPs to prealready working on plant-made treatments
vent disease. Some are looking at vaccines
for a long list of conditions—Alzheimer’s disthat run interference with people’s own antiease, cancer, Crohn’s disease,
bodies rather than triggering a
Did You Know?
and many more.
their immune system to do the
About one in every 20
The fact that PMPs could be
protection. These aren’t edible
Americans is an unafquicker and cheaper to manuvaccines—fruit or other food
fected carrier of an
facture in large quantities makes
with vaccines built in. They’re
abnormal “CF gene.”
them an especially attractive
traditional vaccines whose proThese 12 million people
option where you need lots of
teins have been manufactured in
special protein to treat the disease. are usually unaware that a new way. Already in the
they are carriers. CF
That’s the case with chronic
pipeline are plant-made vacoccurs in approximately
diseases like rheumatoid arthricines for such conditions as
one of every 3,200 live
tis, where millions of people
diarrhea, hepatitis B, and
Caucasian births (in one
need long-term treatment. Or
cholera.
of every 3,900 live births
take obesity. A Canadian comResearchers are also looking
of all Americans).
at non-vaccine preventatives.
pany called SemBioSys Genetics
A California company called
is using genetically engineered
Planet Biotechnology, for example, is testing a
safflower to economically produce a peptide
plant-made product to battle tooth decay. The
that stimulates fat burning. The product
company uses tobacco to grow an antibody,
could one day become medicine for people
who are dangerously overweight.
purifies it, and creates a product that dental
Sometimes it’s individual patients who
hygienists and patients apply to teeth. The
need large quantities of protein—such as for a
product bonds to decay-causing bacteria and
topical gel—for treating a disease. California’s
prevents them from sticking to the teeth. In
Epicyte Pharmaceutical, for example, is trying
early trials, the product eliminated bacteria
to take advantage of plants’ superproductivity
for up to a year.
by creating a gel that people could spread on
Another of the company’s tobacco-grown
their skin to reduce the severity and duration
products—this one in the form of nose
of genital herpes symptoms. (A different form
drops—could prevent the common cold.
of this virus causes cold sores.)
In the fight against disease, plants could
Using plants could also make it financially
turn out to be people’s best friend.
feasible to produce just tiny amounts of very
—Rebecca A. Clay
specific proteins.
sugar residues).
In the creation of plant-made
pharmaceuticals, researchers are
focusing on the effect the two
additional plant N-glycan carbohydrates may have on medicines
used in people.
When plants are used as factories to create medicines, the
plant N-glycans can carry these
extra carbohydrates and pass
them along into the newly created
proteins contained in the medicines. The extra carbohydrates in
plant N-glycans may cause an
allergic reaction in people.
To create safe and effective
pharmaceuticals in plants,
researchers are learning how to
turn off, or deactivate, these carbohydrates.
They already know how to do
this for some plant-made pharmaceutical compounds planned
for human use. Laboratory testing
has indicated that these compounds should be safe in
humans, and some plant-made
pharmaceuticals are in the very
early stages of testing in people.
—Joene Hendry
Your World
13
Career Profile
A
ccording to
Anne-Marie
Stomp, being a
botanist is in her genes.
Raised in small-town
Connecticut by parents obsessed
with gardening, Stomp explains,
“As a kid, I would go on walks with
my mom and she would identify
plants by their Latin names. I knew
the Latin name of a flower but not
that it was a daisy.”
Today Stomp is not only a hard-core
gardener herself but also an associate
professor of forestry at North Carolina
State University in Raleigh. Just as she
did as a child, she spends her days learning about plants and sharing her discoveries.
Growing up, Stomp loved not only plants
but also animals, rocks, and every other
aspect of nature. Her training reflects those
diverse scientific interests: She earned an
undergraduate degree in food science from
the University of Connecticut in 1973, then a
master’s degree in biochemistry and biophysics from Connecticut in 1981, and finally
a doctorate in botany from North Carolina
State in 1985.
An entrepreneurial approach helped Stomp
narrow her focus once she hit graduate
school. Developing what she calls a “market
entry strategy” for herself, she decided that
tree-based biotechnology was an area with
lots of potential but not lots of competition.
Thanks to that business-like attitude, she convinced timber companies to pay for her graduate work.
Then she discovered why so few people
were in her field. “Doing biotechnology on
trees is like doing biomedical research with
whales,” confesses Stomp, explaining that
trees are too big and slow-growing to make
14
Planting for the Future: Plant-Made Pharmaceuticals
AnneMarie
Stomp,
Ph.D.
Associate Professor of
Forestry, North Carolina
State University
good research subjects.
The solution was duckweed, a
tiny aquatic plant that grows in
swamps. Soon Stomp had found
a way to genetically engineer the
fast-growing weed to produce
therapeutic proteins. The only
problem? There was no duckweed industry to support her
research. Undaunted, Stomp
simply created one.
In 1998, she put her entrepreneurial spirit to work
again by launching a
biotech company called
Biolex, Inc., to commercialize her discovery. The following year, she took a leave of absence and
spent the next three years doing everything
from designing the company’s labs to raising
money to acting as interim CEO.
Once she felt the company was on solid
ground, Stomp returned to academia. Since
the university holds the patent and investors
own the company, she won’t get rich. She
does have the satisfaction of having created
40 jobs, developed a process that could produce safer, cheaper forms of life-saving drugs,
and created a popular seminar called
“Anatomy of a Start-up Company” to share
with students what she learned along the way.
Now Stomp’s back to basic science, investigating duckweed’s interactions with other
swamp-dwellers. That research may lead
somewhere; it may not. In the meantime,
she’s doing what she loves.
And that’s just what students should do.
Do what you want, Stomp urges, not what
you think you should. “If you’re a cactus,
don’t try to be an orchid,” she says. “Find a
desert and be the best cactus you can be.”
She pauses. “See?” she asks. “I always
come back to plants!”
—Rebecca A. Clay
Activity
Microbial Bioassay
A
number of approaches have been historically used to find leads for new and potentially useful biologically
active natural products. Today, the drug-discovery process involves a sophisticated array of biological
assays, or “bioassays,” which range from live animal tests to cell culture methods to enzyme assays.
These assays are typically first used to identify a bioactive crude extract, then applied again as the mixture is purified, such as by chromatographic means, to correlate the activity with one particular substance. This “bioassayguided fractionation” yields a pure sample of a molecule that an organic chemist analyzes for structure and that
pharmacologists investigate for use in a medicine.
One of the simplest assays for antimicrobial activity is the spot disk assay. An aliquot of a test solution is applied
to a filter paper disk, then placed on an agar plate that has been preinoculated with a test microbe. A clear area of
no growth around the disk, or the zone of inhibition, indicates the presence of an antimicrobial substance in the
disk saturated by the test extract.
Objectives
(Record this in your research
notebook.)
Describe the steps involved
in the isolation of a natural product.
Describe and practice sterile
microbial techniques.
Be able to carry out a microbial bioassay.
Describe a positive and negative microbial bioassay
result.
Understand the use of positive and negative controls.
Carry out a research project
on unknown plant
specimens.
Procedure
Day 1 – Extraction and cell culture
Make a voucher sample of your specimen
(note location, date, taxonomic identification).
Make a 25 percent weight by volume
plant/solvent mixture.
Cover with aluminum foil, label, and place
overnight in a dark environment.
Inoculate a cell culture (bacteria or yeast) in a
sterile culture tube, and place in a 37°C
incubator.
Procedure
Day 2 – Extraction drying and cell inoculate
Decant extract (solvent) into clean beaker.
Introduce sodium sulfate (desiccant), allow to
sit for 10 minutes, decant sample into
labeled Epitube, cover with aluminum foil,
and store.
Use sterile forceps, dip an assay disk into
extract, remove, and place in aluminum
boat (label boat). Put boat into an incubator to dry.
Using sterile technique, introduce 50ml of a
24-hour-old cell culture onto agar plate,
spread to create an even distribution (lawn).
Allow culture to soak into agar surface for at
least 10 minutes.
Using sterile forceps, place positive, negative,
and extract disks onto the agar plate.
Spot disk array (area of halo)
Procedure
Day 3 – Read bioassay plate
Read results and record a drawing of what the
agar plate looks like in your research
notebook.
If your compound shows a zone of inhibition
(halo), measure the distance from the rim
of the extract disk to the outer margin of
the halo. Use the table below.
Answer these questions:
What is the study of a natural product?
What is a bioassay?
Why is it necessary to mass out the sample
and measure out the volume of alcohol?
What is an extraction?
What is a voucher? Why is it important to
document samples in this manner (i.e.,
voucher specimen)?
Why is it necessary to use a positive and a
negative control when performing a microbial
assay? What are the expected results of the
respective controls (i.e., in terms of zones of
inhibition, a.k.a. halos)?
What does it mean if a halo appears
around an experimental assay disk?
If a halo does not appear around the disk,
does this necessarily mean that the natural
product is not active? Explain.
If a halo is present, what is the next step in
the drug-discovery process? Explain.
Qualitative results
Quantitative results
Negative control
Positive control
Unknown
Activity © Mark Okuda, Silver Creek High School, with support for development and training from SCCBEP and BABEC. Photo from comstock.com.
Your World
15
You’ll find—
• Teacher’s guide
• Activity supplement: student and teacher procedures
• Links
• Information on subscriptions and previous issues
• Downloadable teacher’s guides for previous issues
These issues of Your World are available to download FREE—
• Exploring the Human Genome
• Gene Therapy
• Environmental Biotechnology
• Industrial Biotechnology
• Plant Biotechnology
• Health Care, Agriculture, and the Environment
Biologics: Drugs and vaccines developed with the use of living organisms; protein-based drugs and
vaccines.
Biomass: Plant materials and animal
waste used especially as a source
of fuel.
Edible vaccines: Vaccines produced
in food crops that can be eaten as
Resources
•• ••
•
Glossary
Harvest More Info
AgBiotech Buzz. Roundtable: Is There a Pharm in the Future?
<http://pewagbiotech.org/buzz/display.php3?storyID=3D68
(Have to subscribe, but subscription is free.)
Biology/Animations, Movies & Interactive Tutorial Links
<http://science.nhmccd.edu/biol/bio1int.htm#protein>
<http://www.lsic.ucla.edu/ls3/tutorials>
prescribed by a doctor for proper
doses.
Express: (v.) To translate a gene’s
Biotechnology Industry Organization. Fact Sheet.
<http://www.bio.org/pmp/>
message into a molecular product.
Maize: Indian corn. In the United
“Cures on the Cob,” by Margot Roosevelt, Time, May 26, 2003.
States, we say “corn” instead of
“maize.”
Nutraceutical: Any food or food ingre-
Cystic Fibrosis for Teens (The Nemours Foundation)
<http://kidshealth.org/teen/diseases_conditions/digestive/cystic_fibrosis.html>
dient believed to provide health
benefits.
Plant-made pharmaceutical (PMP): A
Monsanto Web Site
<http://www.Monsanto.com>
medically useful protein that is
extracted from a plant genetically
engineered to express that protein.
Pew Initiative on Food and Biotechnology
<http://pewagbiotech.org/>
Growing genetically engineered
plants for this purpose is sometimes called “molecular farming”
or “pharming.”
Therapeutic protein: A protein that is
medically useful.
“Pharm Farming: It’s Not Your Father’s Agriculture,” by Allan S. Felsot
<http://www.aenews.wsu.edu/July02AENews/July02AENews.htm#PharmFarming>
“Production of Antibodies, Biopharmaceuticals, and Edible Vaccines in Plants,”
by Henry Daniel
<http://www.wmrc.com/businessbriefing/pdf/lifescience2002/publication/daniell.pdf>
USDA and Biotechnology: Questions and Answers
<http://cofcs66.aphis.usda.gov:80/biotech/Bio_qa.html>
The Biotechnology Institute would also like
to thank its 2002 Donors and
Campaign Contributors.
Abgenix, Inc.
Alkermes, Inc.
Amersham Biosciences Corp
Amgen, Inc.
Aventis
American Society for Microbiologists
Biogen, Inc.
Biotechnology Industry Organization
Burrill & Company
Cell Genesys, Inc.
Centocor, Inc.
Ceres, Inc.
Connetics Corporation
CV Therapeutics, Inc.
Ernst & Young LLP
Genentech, Inc.
Genzyme Corporation
Government of Canada
Inspire Pharmaceuticals, Inc.
InterMune Pharmaceuticals, Inc.
Johnson & Johnson
Edward Lanphier
Ligand Pharmaceuticals, Inc.
Massachusetts Biotechnology Council
MdBio
Merck & Co., Inc.
Monsanto Fund
National Institutes of Health
National Science Foundation
Nektar Therapeutics
Northwestern University’s Kellogg Center
for Biotechnology
Oak Ridge National Laboratory
Onyx Pharmaceuticals, Inc.
Ortho Biotech, Inc.
Pfizer Foundation
Pfizer Inc
Physiome Sciences, Inc.
Sangamo BioSciences, Inc.
Schering-Plough Research Institute
State of Oklahoma
Syngenta Biotechnology, Inc.
University of Ulster
U.S. Department of Commerce
U.S. Department of Energy
Utah State University
Thomas G. Wiggans
Wyeth