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OCR for page 62
Biotecir~oZo~ for Healed Care
J. PAUL BURNETT
Biotechnology has been an applied science in the pharmaceutical in-
dustry for a long time. The antibiotics that are used so extensively today
in clinical medicine are products of fermentation or biotechnology. These
substances have been produced on a very large scale for the last 30 or
40 years. Until a few years ago, however, the organisms used in bio-
technology within the pharmaceutical industry were all isolated from
nature. Existing organisms were selected using screening procedures
designed to detect organisms producing useful substances.
Today the tools of biotechnology have changed. Molecular biologists
have provided ways of designing and manipulating organisms to produce
substances in which there is specific interest, rather than simply accepting
what nature has already provided. This advance primarily accounts for
the recent excitement within the pharmaceutical industry insofar as bio-
technology is concerned.
In the pharmaceutical industry biotechnology generally encompasses
two primary areas. The first is immunology, in which the discovery of
hybridomas and cell-fusion technology have allowed the production of
monoclonal antibodies. This has already led to some very important
new diagnostic techniques, and it offers the promise of therapeutic ap-
plications in the future, but so far none of the latter has really been
reduced to practice. This discussion focuses on the second area of bio-
technology or biology that forms an important segment of the new
technology in the pharmaceutical industry, namely, the area of recom-
binant DNA (deoxyribonucleic acid), where many discoveries have al-
62
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BIOTECHNOLOGY FOR HEALTH CARE
63
ready been reduced to practice and where at least one product is already
on the market.
The concept of recombinant DNA is based on the relatively well-
understood function of DNA within all cells. A brief review of some
basic concepts of molecular biology can help clarify the ways in which
molecular biology and biotechnology can be used. Figure 1 shows the
management information system of all cells. All cells contain genetic
information stored in DNA. This information is transcribed from the
DNA into a working pattern called messenger RNA (ribonucleic acid).
This pattern is used in the cell to produce proteins. Proteins are the
molecules in the cell that give rise to all of the characteristics that are
recognized for particular cells.
Some of these protein molecules are enzymes, or biocatalysts, that
catalyze the formation of all of the other molecules in the cell. Some
DNA
(Genetic Information)
Messenger RNA
Proteins
—-—Transcription
—-—Translation
Enzymes Structural Proteins Biological Messengers
1. Low-molecular-weight
metabol ites
a. Sugars, fatty acids,
amino acids, alcohols,
etc.
b. Antibiotics, antifungals
2. Macromolecules
1. Physical structure 1. Hormones
2. Carrier molecules 2. Mediators
FIGURE 1 Management information system of all cells. DNA controls the synthesis of
cellular proteins, which subsequently determine the phenotypic characteristics of the cell.
OCR for page 64
64
NEW FRONTIERS IN BIOTECHNOLOGY
-
/ \ Plasmid DNA
/ Protein \
~ ~Ribosomes i0 / - \
DNA In\ ib / / 00 Protein l
(~:~ ;y / / ~ /
\ `, imRNA / / mRNA~/ /
\ / ~ ''<~ iRibosomes
~ ~ it' /
~ I) ~Chromosomal DNA
Animal Cell
Bacterial Cell
FIGURE 2 Pictorial presentation of management information of cells. In addition to
chromosomal DNA, bacterial cells often contain small, autonomously replicating DNA
molecules called plasmids.
of the proteins serve a structural function, giving rise to the physical
appearance in the structure of the cell. Others serve messenger func-
tions, carrying messages back and forth between the cells. The sum and
substance of a cell is the complement of proteins that it contains.
Figure 2 presents much the same information as that in Figure 1, but
in pictorial form, and it introduces an additional concept. On the left-
hand side of the figure is a typical animal cell where DNA is in the form
of chromosomes in the nucleus. The messenger RNA is made in the
nucleus and goes out to the cytoplasm where it serves as a pattern for
protein synthesis. The same process occurs in bacterial cells (right-hand
side of Figure 2) except that bacterial cells have small DNA molecules
(known as plasmids) that have particular advantages for use in biotech-
nology. As opposed to the chromosomal DNA, which is a very large
and very fragile molecule, the plasmid DNAs are very small molecules.
They can be isolated in a test tube. They can be cut apart and put back
together. DNA can be added to them or subtracted from them. And,
finally, plasmids can be put back into a cell in a functional form. Thus,
plasmids form the cornerstone, if you will, of the application of molec-
ular biology to biotechnology and recombinant DNA.
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BIOTECHNOLOGY FOR HEALTH CARE
BIOTECHNOLOGY IN THE PHARMACEUTICAL INDUSTRY
65
Figure 3 indicates how, in a general way, one might use recombinant
DNA to produce a substance via biotechnology in the pharmaceutical
industry. In the upper left of the figure is a recombinant DNA organism
being made by combining an animal gene with a plasmid DNA, followed
by introduction into a microorganism. This first step, introducing the
recombinant DNA into the microorganism, is a laboratory process. In
the laboratory one would generate test-tube-scale cultures that contain
the transformed cells that will now produce the protein coded by this
animal gene. The next stages are development and production processes.
This culture must be scaled up from the test-tube stage to the bioreactor,
or fermenter, stage. The product must then be purified and packaged
in suitable clinical form. Finally, before the product is ever subjected
to clinical use, it is extensively tested in animal systems.
Is is important to point out that the bulk of this overall process begins
CUT
PLASMID
Q ANIMAL
<=3 ~ GENE
PURIFICATION
J
RECOMBINANT
DNA
PASSAGE _
THROUGH
ADSORBING
COLUMNS _
PACKAGING
GROWTH IN
LARGE TANKS
.
0 0 0 0
INSERTION LABORATORY
INTO BACTERIA TESTS
me) ' 1(~)
/ ~ :~;
5~
3 1
THERAPEUTIC VALUE
TESTED IN ANIMALS
~ ~-
CLINICAL
USE
FIGURE 3 Production of pharmaceuticals by recombinant DNA. Recombinant DNA
can be used to add new genes to microorganisms, and these can be grown in fermentation
tanks to produce proteins on a large scale. Purification and extensive testing in animals
precede clinical application in human beings.
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66
NEW FRONTIERS IN BIOTECHNOLOGY
TABLE 1 Amino Acid Residues and Molecular
Weight of Human Polypeptides Potentially Attractive
for Biosynthesis
Polypeptide
Amino Acid Molecular
Residues Weight
Insulin 51 5,734
Proinsulin 82
Growth hormone 191 22,005
Calcitonin 32 3,421
Glucagon 29 3,483
Corticotropin (ACTH) 39 4,567
Prolactin 198
Placental lactogen 192
Parathyroid hormone 84 9,562
Nerve growth factor 118 13,000
Epidermal growth factor 6,100
Insulinlike growth factors
(IGF-1 and IGF-2) 70, 67 7,649, 7,471
Thymopoietin 49
after the genetic engineer has completed his or her work. In a sense,
the contribution of the molecular biologist, although crucial, is a small
portion of the total process.
Using the general process just described, a number of different types
of molecules that have potential therapeutic use can now be made. The
general categories of these substances include hormones and growth
factors, pain-relieving proteins, plasma proteins, enzymes, proteins in
the immunology area, and possibly even new types of antibiotics.
Table 1 lists some of the growth factors and hormones that one might
consider producing by this technology. The genes for almost all of these
proteins have now been cloned, and it is possible today to use those
genes to produce these proteins in microorganisms. One at least, human
insulin, has now been produced on a large scale and is a marketed
product. It will be useful here to illustrate how genetic engineering is
actually used to produce human insulin.
Production of Human Insulin
Theoretically there are two ways in which one could go about pro-
ducing the insulin molecule (see Figure 4~. Insulin consists of two dif-
ferent protein chains, the so-called A chain and the so-called B chain.
One could produce the normal precursor of insulin, proinsulin, that is
OCR for page 67
67
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OCR for page 68
68
NEW FRONTIERS IN BIOTECHNOLOGY
FIGURE 5 Schematic presentation of process for producing biosynthetic human insulin.
Strains of E. cold have been engineered to produce insulin A and B chains. The initial
cellular protein product is a chimeric protein in which the insulin polypeptide chain is
attached at the carboxyl terminus of a protein coded by the tryptophan operon. Cleavage
by cyanogen bromide releases the insulin chain from the chimera.
found in the pancreas. Proinsulin is a molecule that contains insulin but
also contains an extra connecting peptide linking the two chains together.
In the pancreas gland, this so-called connecting peptide is clipped out,
leading to the production of insulin that is then released into the blood
circulation system. One can mimic this process today in the laboratory
and actually even in production, but up to the present it is not being
used to produce human insulin on a large scale.
~ .. . . _
. .
Presently the A chain and the B chain are made individually, and
then these are coupled in the plant to produce the bioactive insulin.
Figure 5 shows the process schematically.
In separate plasmids the genes have been introduced individually for
the A chain and B chain of insulin, and then these plasmids have been
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BIOTECHNOLOGY FOR HEALTH CARE
69
transformed into bacterial cells. The Escherichia cold are then grown in
large fermentation facilities. The product that is initially made is a large
chimeric protein consisting of the A chain or B chain attached to the
end of a naturally occurring E. cold protein.
This protein is subjected to a cleavage reaction in which the A chain
and the B chain are chemically cleaved away from the rest of the chimeric
molecule. Then, following several further purification steps, these two
chains are combined, and the biosynthetic human insulin is recovered
and purified.
Large amounts of the gene product of this plasmid accumulate in the
E. coli. A thin-section electron micrograph of E. cold producing human
insulin polypeptide shows dense areas, which are deposits of that protein
within the cell. The protein is produced in very substantial amounts and
can occupy a major portion of the cell.
This is one of the advantages of biotechnology today by using ap-
propriate control systems and regulatory systems on the plasmid being
dealt with, one can make the protein of interest a major portion of the
total protein of the microorganism. It can become a very efficient proc-
ess.
Figure 6 shows crystals of the final product. It is a crystalline protein,
F~GuRE 6 Crystals of biosynthetic human insulin produced by the process described in
Figure 5.
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70
NEW FRONTIERS IN BIOTECHNOLOGY
and it has all of the characteristics of the insulin that is circulating in all
of our bodies.
There are at least two advantages to being able to produce human
insulin as opposed to continuing to use the pork and beef insulin that
is currently used in many diabetic patients. First, the chemical structure
of pork and beef insulin differs slightly from that of human insulin.
Thus, there is the possibility of an improved therapy by using a molecule
identical to the insulin that is already circulating in human bodies. The
second advantage relates to the fact that currently produced pork and
beef insulins are really by-products of the meat industry. Their produc-
tion is subject to all of the economic pressures of the meat industry in
terms of supply of pancreas glands. By production in microorganisms
an essentially limitless supply of human insulin is available; the supply
is no longer subject to the particular economic pressures of the beef and
pork markets.
The following are some of the plasma proteins that one might consider
producing by this technology:
· Albumin
· Globulins a,,B,~y
· Lipoproteins a, ,B
· Plasminogen
· Fibnnogen
· Prothrombin
· Transfernn
Albumin, for instance, is a protein that can now be manufactured using
recombinant-DNA technology. At least one company is working to scale
this process up to commercial levels. Many of the genes for other pro-
teins in the plasma protein series have also been cloned.
Other Uses of Recombinant DNA
Following is a brief discussion of examples of other ways in which
recombinant DNA could be used to make products useful in the phar-
maceutical industry.
Table 2 lists enzymes that are now used clinically. Probably the most
important group today is that of the enzymes and cofactors involved in
hemophilia proteins like Factor VIIIc, which is used in hemophilia A.
A large number of research groups are trying very hard to clone the
gene for this protein; although the goal has not yet been reached, it can
be expected that this will be accomplished in the near future.
One area of particular excitement is the fibrinolytic area. Blood-
clotting problems, thrombosis, are an important aspect of clinical med-
OCR for page 71
BIOTECHNOLOGY FOR HEALTH CARE
TABLE 2 Ethical (Prescription) Enzymatic Products Currently
Employed in the United States
71
Enzymes
Therapeutic Category Involved Indications
Approximate
Mfrs. Sales
(millions of
dollars)
Blood clotting factors
Antihemophilic Factor Factor VIII Hemophilia A
(AHF)
Plasma thromboplastin Factor IX Hemophilia B
component
Gastrointestinal digestive Pepsin, pancreatin "Nervous" or other
indigestion
Pancreatin: lipase, Inadequate fat digestion;
trypsin, amylase cystic fibrosis
Wound debriding agents Fibrinolysin and
deoxyr~bo-
nuclease
Trypsin
Subtilains
Collagenase ~ <1
Streptokinase and Clot lysis, reduction of 1
streptodornase edema and inflammation
Chymotrypsin "Possibly effective" for
Bromela~ns . .
Papain ep~s~otomy
Streptokinase
Urokinase
Fib ri no lysin
Hyaluronidase
Chymotrypsin
Asparaginase
Penicillinase
Oral proteolytic
preparations
Thrombolytic
Absorption promoter
Ophthalmic surgery
Cancer chemotherapy
Allergic drug reaction
40
4
11
4
( Removal of purulent
exudates and eschar 2
2
3
| Lysis of intravascular <2
blood clots
Rarely for IM or SC in
Cataract removal
Leukemia
Destroy penicillin
1j. <1
<1
<1
<1
icine. A number of enzymes, such as streptokinase or urokinase, will
degrade blood clots. The problem with both of these enzymes is that
although they hydrolyze fibrin and destroy clots, they also lead to gen-
eralized bleeding in the patient. A new protein has recently been dis-
covered, called the tissue activator of plasminogen (TPA), which is very
specific and will hydrolyze fibrin only when it is in the form of a clot.
This enzyme does not cause the side effect of generalized bleeding. The
gene for TPA has been cloned, and one can now produce this protein
using recombinant-DNA technology.
One of the most exciting areas for the application of biotechnology
within the pharmaceutical industry is in immunology, where it will be
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72
NEW FRONTIERS IN BIOTECHNOLOGY
_
Factors Affecting Inflammation Helper Factors May Be Suppressor Factors May
May Be Useful in Useful in Be Useful in
Patients with overwhelming Tumor patients Allergy
infection
Postsurgical immune Aging Autoimmune disease,
suppression SLE, arthritis
Burn patients Diabetics M.S., thyroiditis,
myasthenia gravis, etc.
Postsurgical peritonitis Dialysis patients Transplantation
Tumor patients Immunodeficiency
disease
Aging Al lergy
Immunodeficiency disease NK cell activity
Dialysis patients Burn patients
Thyroiditis Trauma
M.S.-EAE Postsurgical
immune suppression
Allergy Chronic diseases—
hepatitis, parasitic
Hodgkin's disease
FIGURE 7 Clinical applications of cytokines.
possible to produce some interesting vaccines. Instead of discussing these
possibilities, however, let us turn to a group of proteins called cytokines.
A cytokine is a molecule made in a cell as a result of some sort of
stimulus. It can result from various types of stimuli. A cytokine is elab-
orated by the cell producing it. It leaves that cell and acts as a messenger,
one of the functions of proteins mentioned earlier. It then stimulates a
second type of cell to cause some sort of biological effect. It may affect
the growth of the cell, it may affect the movement of the cell, or it may
activate the cell to perform a specific function. For instance, in the
phagocytic series it may activate a cell to become more active phago-
cytically. Figure 7 illustrates some of the areas where cytokines might
be used clinically. Many cytokines are involved in the inflammatory
response. Others promote the immune response, and still others are
known to suppress the immune response. It is thought that the clinical
states shown in Figure 7 represent those in which many of these cytokines
might be used therapeutically. The important point with regard to this
figure is that it represents a very large number of clinical diseases.
Figure 8 shows some of the cytokines that are produced by one par-
ticular type of human-cell, the lymphocyte. It can be seen, for instance,
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BIOTECHNOLOGY FOR HEALTH CARE
· Mediators Affecti ng Macrophages
— Migration Inhibitory Factor (M I F)
— Macrophage Activating Factor (MAP}
— Chemotactic Factor {CF)
· Mediators Affecting Lymphocytes
— Allogenic Effect Factor {AEF)—Katz
Mitogenic Factor {MF)
_ _ . . . .. .
Factors Enhancing Antibody Formation
Factors Suppressi ng Anti body Formation
— T-Cell Replacing Factor (TRF}
· Chemotactic Factors for Basoph i Is ~ BC F
and Eosinophils (ECF)
· Mediators Affecting Other Cells
— Cytotoxic Factor (Lymphotoxin)
— Cal lagen-Produci ng Factor
— Osteoclast—Activating Factor
· I nterferon ~ I F ~
FIGURE 8 Cytokines produced by lymphocytes.
73
that there are mediators made that affect macrophages, the cells that
destroy invading organisms in the body. There are mediators that affect
other lymphocytes and cause them to do a variety of things. Some may
enhance, for instance, antibody formation. There are Chemotactic fac-
tors elaborated by the lymphocyte that affect cell movement. One of
the cytokines that has been most publicized in the recent past is inter-
feron, which is made by the lymphocyte, among other types of cells.
All of the examples just examined illustrate the type of products that
can reasonably be expected to be produced by biotechnology in the
pharmaceutical industry. However, exciting as these present applications
for recombinant DNA may be, it appears that the total number of drugs
that will be produced by this technology as proteins will be limited. In
the future the greatest value of the application of biotechnology and
recombinant DNA within the pharmaceutical industry will probably
come about as we begin to understand life at the molecular level.
In addition to offering a way of producing molecules, recombinant
DNA offers the molecular biologist a way of cloning and isolating genes,
characterizing these genes, and understanding their function at a genetic
level. In fact, today it is sometimes much easier to isolate the gene for
a particular protein and to learn the structure of the protein from the
gene than it is to isolate the protein itself.
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NEW FRONTIERS IN BIOTECHNOLOGY
All of the processes of life, and especially those orderly processes
such as differentiation and development seen in the nodal growth of
plants and animals, are ultimately controlled by DNA. This is also true
of the disorderly processes that we recognize, for instance, malignant
cell growth. As we define these life processes at a genetic level, it is
expected that it will be possible to design new small molecules that may
be produced by traditional chemical means which will be the drugs of
the future; but the discovery of these drugs will hinge on the application
of molecular biology.
Following is one example of how that might happen and of one area
where this might be applied. Genes known as oncogenes have been
discovered; they have been found in a variety of tumor viruses from
various types of animals. It is also known that there are corresponding
genes in normal cells that seem to be very similar in structure to the
oncogene of the tumor virus. When a tumor virus invades a cell, the
function of the oncogene is to produce a protein that leads then to the
transformed or malignant phenotype. The function of the corresponding
gene in the normal cell is to regulate the growth and function of the
cell.
We believe that it will be possible in the future, by understanding
how these genes are genetically regulated and how these proteins func-
tion, to develop small molecular inhibitors that may be intriguing new
types of chemotherapy for treating cancer. It is certainly too early to
say what form these will take or exactly what structure they might be,
but it is an interesting example at least of a way in which recombinant
DNA might lead indirectly to new drugs.
CHALLENGES TO BE MET
Today we can use DNA technology to produce drugs that have im-
portant clinical uses, and in the future we can expect products, as yet
unimagined, that will flow from our increased understanding of the
biological processes brought about by the use of recombinant-DNA
technology as a research tool. However, further problems must be solved
in order for there to be broad application of biotechnology to the pro-
duction of proteins in the pharmaceutical industry. These challenging
problems involve the following:
· Fermentation
Regulation of protein production
New host organisms, including mammalian cells
Fermentation technology development
· Protein recovery and purification
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BIOTECHNOLOGY FOR HEALTH CARE
75
Many of the problems faced today in the application of recombinant
DNA within the industry relate not to molecular biology and to how
one genetically engineers a cell, but to how the production of protein
in the fermentation vessel itself is regulated once that cell has been
genetically engineered.
We know today that there are proteins that probably will not be able
to be produced economically in bacterial cells. We believe that there
will be occasions in the future where mammalian cells will have to be
used to produce those proteins. So the biotechnologists or bioengineers
in the fermentation industry will have to learn to deal with mammalian
cells on a large scale or in mass culture.
It is my belief that the companies that develop their fermentation
technology in general to the greatest degree will be the most successful
in applying recombinant DNA and biotechnology.
There will be need for great improvement in protein recovery and
purification. Many of the techniques used today on production scale,
for instance, to produce human insulin, really mimic the techniques of
the laboratory. There is great opportunity for new innovation particu-
larly in the area of protein recovery and purification.
And finally, it appears that the organizations that will most success-
fully apply biotechnology in the future will be the companies that can
bring about the closest collaboration between the genetic engineer and
the biochemical, electrical, and other engineering disciplines.
OCR for page 76
Representative terms from entire chapter:
human insulin
