Roche’s biggest biotechnology plant is in Penzberg
Bacteria produce anticoagulants to order
The new Biologics Building at the Biotechnology Research
and Production Centre of Roche Diagnostics Boehringer Mannheim
GmbH in Penzberg, which was inaugurated in July 1998, came
on stream in early 1999. The facility manufactures erythropoietin
and recombinant plasminogen activator (rPA) as well as monoclonal
antibodies. The hormone erythropoietin is the active principle
of the drug Recormon®, which stimulates the production of
red blood cells and is therefore used in the treatment of
anemia. The rPA in the thrombolytic drug Rapilysin® dissolves
blood clots and optimises the treatment of acute myocardial
infarction. It is the first recombinant drug to be researched,
developed, and manufactured from scratch in Germany. The new
production complex features two fermenters, each with a capacity
of ten cubic meters, for extracting protein from mammalian
cells. The fermenters used for manufacturing bacteria-derived
drugs have capacities of up to five cubic meters each.
1. Treatment of anemia associated with cancer and premature
birth
The human growth hormone erythropoietin (EPO) is
secreted primarily by the kidneys. It binds to receptors located
on the surface of precursor cells in bone marrow that develop
into red blood cells. In doing so, it protects the precursor
cells from premature death or failure to mature. Patients
with chronic kidney disease often develop a deficiency of
red blood cells, or anemia, the symptoms of which include
general weakness, fatigue, and in some cases serious heart
problems.
Many cancer patients also suffer from anemia following chemotherapy
or bone-marrow transplantation. Many premature infants with
a birth weight of 750 g to about 1500 g also tend to be anemic.
Neonates weighing less than 1000 g need blood transfusions
during the first week of life, in many cases every two or
three days. In fact, the only available method of treating
anemia has been the transfusion of blood from other individuals.
But this exposes the patient, who is often immunologically
compromised, to the risk of infection and iron overload. In
such cases and where a patient donates his/her own blood before
planned surgery, erythropoietin (EPO) can be beneficial.
Since the early 1990s EPO has been available for the treatment
of anemia in the form of Recormon® with epoetin beta,
a substance that is identical to a natural hormone in the
body. It prevents further progression of anemia and promotes
the maturation and differentiation of red blood cells by selectively
stimulating erythroid precursor cells.
When researchers at Boehringer Mannheim succeeded in elucidating
the genetic information for the structure of the hormone,
they laid a cornerstone for the biotechnological production
of recombinant human (rh) EPO for use as a pharmaceutical.
The substance is synthesised from genetically modified mammalian
cells measuring around 10 to 20 micrometers in diameter. The
cells concerned are Chinese hamster ovary cells derived
from a selected laboratory cell line with a doubling time
of about 18 to 24 hours. The cells secrete the EPO protein
into the culture fluid of large fermenters — complete with
all the structures needed for correct function, including
sulfur bonds and sugar side chains. From there it is separated
from the other constituents by centrifugation and purification
steps.
2. Third-generation thrombolytic drug
It should be possible to achieve better product yields with
the help of genetically improved strains of microorganisms
that grow much more rapidly. The microorganisms generally
used are selected strains of Escherichia coli, a naturally
occurring denizen of the intestines of humans and animals.
These organisms are used, for example, to produce reteplase
(rPA), the active ingredient of the thrombolytic agent
Rapilysin® 10 U, which is used in the treatment of
patients with acute myocardial infarction. Unlike natural
human tissue-type plasminogen activator (tPA, alteplase),
it contains no sugar side chains and has a more potent thrombolytic
action. This structural modification also results in a longer
half-life, greater plasminogen specificity, and lower fibrin
affinity, which allows the substance to penetrate much more
readily into blood clots (thrombi).
Normally, small clots are dissolved at a healthy vessel
wall because a balance exists between clotting factors and
factors that oppose them. This balance is meant to ensure
that the blood remains fluid as long as the vascular system
is intact but that, if an injury does occur, affected vessels
are quickly sealed by the formation of blood clots. If necessary,
the initially inactive proenzyme plasminogen as well as plasminogen
activators such as tPA bind to thrombi with high affinity
and gather at the site of the clotting process, where they
convert plasminogen to active plasmin.
The danger posed by a clot in a blood vessel is much greater
in the presence of atherosclerosis. If the clot is swept away
from its site of origin by the bloodstream, it may become
lodged in the smaller vessels of the lungs or brain, triggering
pulmonary embolism or a stroke, respectively. If the clot
blocks one of the coronary vessels supplying the heart, the
result is a myocardial infarction, in which heart muscle
cells starved of an adequate blood supply die. This is especially
liable to happen if the coronary vessels are narrowed by high
blood pressure. Myocardial infarction is one of the commonest
causes of death in Europe and the USA.
3. Custom-made clones for biotechnological production
When microbial or animal cells with selected characteristics
are cultivated, it is essential that they be present in a
pure culture that is free of contamination by foreign organisms.
Single cells are placed in a liquid culture solution or on
a solid agar substrate in Petri dishes and allowed to proliferate.
Grown at a suitable temperature and given an optimal supply
of nutrients, bacteria, which are usually just a few micrometers
(thousandths of a millimeter) in diameter, have a doubling
time of around 20 minutes. On a Petri dish a single cell gradually
forms a colony of several million cells that is visible to
the naked eye. As the cell density increases, the initially
clear culture fluid turns cloudy.
Microorganisms that concentrate the desired product within
their cells are separated from the culture solution and then
digested by mechanical or chemical means. Destroying the cell
wall releases the genetic material consisting of DNA (deoxyribonucleic
acid), which can then be precipitated out of the cell extract
by the addition of alcohol.
The DNA is then cut into segments at specific sites with
the help of restriction enzymes. In this way the DNA molecule
is divided into a selected number of fragments of a given
size. Also, a specifically defined segment, for example one
containing a selected gene, can be snipped out of the DNA
molecule. The DNA segment containing the desired gene is separated
and with the help of the enzyme DNA ligase is then joined
to another DNA segment that serves to introduce the gene into
the cells of the production strain. The recombinant DNA segment
thus obtained is incorporated into the DNA of the bacterial
strain, which then synthesises the drug.
As the process does not take place in every cell, those cells
that carry the error-free recombinant segment have to be selected.
Only these possess the genetic information needed to synthesise
the desired protein. When the bacterium translates the imported
genetic information for the drug using its own protein-synthesising
machinery, the gene is said to be expressed. On dividing,
the protein-synthesising bacterium passes on the inserted
instructions to its daughter cells. The result is a clone.
The custom-made bacterial strain is transferred stepwise
to larger and larger culture vessels containing liquid nutrient
solution and, after optimisation of the culture conditions,
it is cultivated on the desired production scale in fermenters.
To obtain the product, the bacterial cells are centrifuged
out of the culture medium. The cells are then digested and
the product is purified.
4. DM 177 million invested
In some cases the bacteria produce the desired protein in
such large quantities that it conglomerates into insoluble
structures known as inclusion bodies, which are visible
under a microscope. To isolate the product, the separated
cells are digested under high pressure and the compressed
proteins in the inclusions are dissolved and separated out.
The proteins are made to unfold by addition of salts to the
solution. Stepwise reduction of the salt concentration then
restores the functional three-dimensional shape of the proteins.
The new production facilities in which these steps
are carried out comprise five modular building units.
One module is used for the fermentation and purification of
products from Chinese hamster ovary cells. Two others are
used for the fermentation and purification of products from
bacteria such as Escherichia coli. A central module
supplies the entire complex with starting materials and resources.
During the entire production process all the steps run fully
automatically in closed systems. The production steps can
be monitored continuously on a computer screen. The
production modules, representing an investment of DM 177 million,
came on stream in early 1999.
5. Immunoreagents, enzymes, and biochemicals from Penzberg
A wide range of immunoreagents, enzymes, and synthetic biochemicals
are produced in Penzberg. These products are used in research,
in diagnostic tests, in the pharmaceutical and chemical industries,
and in food analysis. Examples of enzyme products are
lipases, which react with fats, and hexokinases and glucose
oxidases, which react with carbohydrates. They also include
Taq DNA polymerase for the polymerase chain reaction (PCR),
a technique for amplifying and identifying selected genes.
The company’s first genetically engineered enzyme for diagnostic
purposes was also produced at this site.
Immunoreagents include monoclonal and polyclonal
antibodies, immunogenes, hormones, and growth factors. Synthetic
biochemicals include sugars with phosphate components,
peptides, coenzymes, and pigment-containing enzyme substrates.
Nucleotides and even biocides are also produced. Many of the
reagents are used in diagnostic tests for identifying pathogens,
for the early detection of diseases, or for assaying metabolic
products such as blood sugar and cholesterol.
Arterosclerotic plugged blood vessel
Bright-field micrograph of a transverse section of a blood
vessel at approximately 180x magnification.
Source: Heinz Günter Beer, Oberasbach
Production of therapeutic proteins with microorganisms
Fed-batch fermentation and harvest of inclusion bodies
for example: plasminogen-activator from the genetically
modified bacterium E. coli
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