Journal of Applied Bacteriology 1986,61,99-116 2287103186

A Review
The application of genetically engineered micro-organisms in the
production of drugs

R os E * Seurle Research and Development, PO Box 53, Lune End Road, High
S .B . P R I M
Wycombe, Buckinyhamshire H P l 2 4 H L , U K

Received 3 March 1986

1. Introduction, 99
2. The role of the micro-organisms in the production of drugs, 100
3. Proteins as novel therapeutic agents, 100
4. Technical problems associated with the production of
recombination-derived protein drugs, 101
4.1 Post-translational and other modifications, 102
4.2 Inclusions, 102
4.3 Choice of purification method, 104
4.4 The impact of fermentation, 106
5. The good news: purification fusions, 107
6. The influence of development costs, 108
6.1 Human insulin, 109
6.2 Human growth hormone (somatotropin), 1 I 1
6.3 Interferon-alpha, , 111
6.4 Some proteins which are commercially attractive, 111
6.5 Modified proteins as therapeutic agents, 111
7. Regulatory aspects of drug development, 112
8. The future: recombinant DNA technology and the production
of small molecules, 113
8.1 Cloning all or part of a biosynthetic pathway, 113
8.2 New synthetic routes, 113
8.3 Novel antibiotics, 114
9. References, 115

1. Introduction
Since the advent of recombinant DNA technology it has become possible to produce for the first time
substantial quantities of human proteins normally present in the body in trace amounts. Despite the
fact that over 200 biotechnology companies are developing human proteins as therapeutic agents, in
10 years only three proteins have reached the market place: human insulin, human somatotrophin
and interferon-alpha,. The delay in developing other proteins as human medicines is due to a
mixture of technical and regulatory problems, cost considerations and the need to administer most
proteins parenterally. The future role for recombinant DNA technology in the pharmaceutical
industry is the improvement of existing processes for the production of low molecular weight com-
pounds.
* Present address: Amersham International, Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA,
UK.
Lecture to the Society’s Winter Meeting on 8 January 1986.
100 S . B. Primrose
2. The role of the micro-organismsin the production of drugs
An association between micro-organisms and the pharmaceutical industry can be traced back to the
introduction of vaccination as a means of combatting infectious diseases. Vaccines are drugs and
consist of either live, attenuated or killed micro-organisms. More recently, micro-organisms have
been used in the manufacture of therapeutically useful compounds. Table 1 gives some representative
examples of the kinds of compounds produced and the range of micro-organisms involved.

Table 1. Representative examples of the use of micro-organisms in

the production of pharmaceuticals
Product or process Organism
Production of antibiotics
penicillin Penicilliurn chrysoyenum
colistin Bacillus colistinus
tetracycline Streptomyces nureofaciens
fusidic acid Fusidium coelcineum
Steroid transformations
11 alpha-hydroxylation Rhizopus nigricuns
I I beta-hydroxylation Curuularia lunata
1-dehydrogenation Arthrobacler simplex
side-chain cleavage Mycobacterium sp.
Production of vitamins
vitamin B,, Propionibacterium shermanii
riboflavin Eremothecium ashbyii
Pharmacologically active agents
ergot alkaloids Clauiceps purpurea
cyclosporin Tolypocladiuni inflatum
mevinolin Aspergillus terrens
Production of vaccines (bacterial) Appropriate bacterial species

In many instances the micro-organisms carrying out the kinds of reactions listed in Table 1 are
subjected to extensive genetic modifications to increase the efficiency of the process. Usually this
involves repeated rounds of mutagenesis and selection but it can also include such techniques as
limited genetic exchange between related species or even cell fusion. The last decade has seen the
development of the very powerful recombinant DNA technology, often loosely referred to as ‘genetic
engineering,’ and it would be expected that this would have had a significant impact on the pro-
duction of pharmaceuticals by micro-organisms. Certainly in the last 10 years over 200 new biotech-
nology companies based around recombinant DNA technology have been formed and many of these
are specializing in pharmaceuticals. Despite this there is only a handful of drugs on the market which
are produced by genetically engineered micro-organisms. Why should this be? There are two basic
reasons. First, the primary product of a cloned gene is a protein and very few of the existing drugs on
the market are proteins. Rather, most are low molecular weight organic compounds which in this
review are referred to as ‘small molecules’. If proteins are to be developed as novel drugs then a whole
series of technical, regulatory and commercial hurdles have to be overcome. Second, the techniques of
genetic engineering have largely been developed in organisms which traditionally are not used to
produce drugs, e.g. Escherichia coli, Bacillus subdilis and Saccharornyces cereuisiae. Only in the last
few years have these techniques been adopted for use in the kinds of micro-organism listed in Table 1.

3. Proteins as novel therapeutic agents

Before the advent of recombinant DNA technology the use of proteins as drugs was restricted to the
animal insuiins, human somatotropin, urokinase and streptokinase, and blood fractions such as
human serum albumin, factor VIII, etc. Many other proteins have properties which suggest that they
 Genetically engineered micro-organisms 101
Table 2. Human proteins with therapeutic potential
Protein Application
Interferon (alpha, beta and gamma) Treatment of virus infections and cancer
Human growth hormone Pituitary dwarfism
Insulin Diabetes
Tissue plasminogen activator Thrombosis
Urokinase Thrombosis
Beta-endorphin Analgesic
Interleukin-2 Cancer therapy
Alpha, -antitrypsin Emphysema
Relaxin Facilitate childbirth

have potential as therapeutic agents and some of these are listed in Table 2. The reason why they
have not been developed in the past has been their limited or non-availability: since many are
hormones they are present in tissues only in trace amounts. Since the introduction of the relevant
cloned genes into bacteria such as E . coli enables production of human proteins at the gram per litre
level, commercial manufacture is now technically feasible. An example of the manufacturing scale
required is shown in Table 3. The fact that there has not yet been an increase in the number of
protein drugs on the market is a reflection on the technical problems and the regulatory hurdles
which have to be overcome and the return on investment which can be expected.

Table 3. Manufacturing scale required to produce proteins for therapeutic purposes

Assume patient dose of protein is 100 pg/d ( - 1 pg/kg)
Assume protein yield is 1 g/l culture and recovery of pure protein is 10%
Then 1 1 culture yields 1000 doses
100 I culture yields 1oOOOO doses
loo0 1 culture yields 1 00OooO doses

4. Technical problems associated with the production of recombinant-derived

protein drugs
Ail drugs have to be manufactured to extremely tight specifications and proteins are no exception.
For most countries, existing regulations governing the manufacture and quality of human and
veterinary drugs are not applicable to proteins, particularly those of recombinant origin, and new
regulations are being prepared. In the interim each new drug application for a recombinant-derived
protein is being considered on a case-by-case basis. The kind of specification which will be set is
shown in Table 4 but it must be emphasized that the absolute quality required should depend on the
route of administration, the intended use and the proposed duration of therapy.

Table 4. Draft specification for recombinant proteins which are

to be used as pharmaceuticals
~~

1. Greater than 95% pure

2. Absence of microheterogeneity
3. Absence of chemicals used in purification
4. Absence of IgG if monoclonal antibodies used in purification
5. Absence of endotoxin
6. Absence of bacterial and plasmid DNA
7. Consistent three-dimensional structure
In practice, ‘absence’means ‘below a specified limit’.
102 S. B. Primrose
4. I P O S T - 'r R A N S L A T I 0 N A L A N D 0 T H E R M 0 D I F I c' A T O N S

After their synthesis many proteins undergo enzymatic modification, often referred to as post-
translational modification. For example, many proteins are glycosylated and the pattern of glycosyla-
tion may differ depending on the tissue of origin. It is difficult to get micro-organisms to mimic this
glycosylation; although S . cereoisiae can glycosylate proteins the pattern of glycosylation is diflerent
rrom that occurring in human tissues. A different kind of modification is thc protease cleavage of the
singlc human proinsulin polypeptide into the A and B chains of the active insulin molecule. Again
this modification cannot bc achieved in micro-organisms. Yet another modification is exemplified by
calcitonin in which the carboxy-terminal amino acid is amidated. In this particular instance an
enzymatic technique (Bradbury rt al. 1982) can be used to generate authentic calcitonin (Fig. I).
f:in:illy, some proteins contain unusual amino acids, e.g. nisin (Gross & Morel1 1967) and phenylala-
nine ammonia lyase (Hansen & tlavir 1970), but it is not clear whether these are generated sponta-
neously o r by post-translational modification.

H2N - 7
glycine residue

r----~--l
C H - C O ~ N H - C H ~-COOH
-.-.----.I
I

enzyme

I
HZN - R
I
CH-CO-N = CH-COOH

' 2 0 '1;-n+oneous

R
I CHO
HzN
CH - CO-NH, + I
COOH
Fig. I . EnLymatic amidation of proteins. Proteins are synthesized with an additional glycine residue at the
C-terminus. Following enzymic dehydrogenation the resultant imino bond undergoes spontaneous hydrolysis.

By contrast there are protein modifications that occur with recombinant organisms but which do
not occur in human tissues. Thus protein synthesis in bacteria is initiated with N-formylmethionine,
whereas this does not occur in eukaryotes. With some proteins this N-formylmethionine is removed
post-translationally, with some it is not, and with others there is variable removal. For example,
human somatotropin synthesized in E . coli contains N-formylmethionine at the N-terminus, unlike
thc corresponding protein derived from the human pituitary gland. Since this methionine residue
subsequently cannot be removed, the recombinant product has to be registered as N-formyhethionyl
human somatotropin. In addition, during the fermentation or extraction process many recombinant
proteins are partially digested at the C- or N-terminus and such modified proteins need to be
removed during the purification process.

4.2 INCLUSIONS

Biochemists are taught that when handling proteins they should keep them solublc and native: this
will facilitate their purification and the retention of full specific activity. However, when they are
over-expressed in E. coli many foreign proteins form insoluble aggregates (Williams et a/. 1982)
known as inclusions (Fig. 2) which consist of a mixture of denatured protein and nucleic acid. I f a
 Genetically engineered micro-organisms 103

Fig. 2. Inclusions formed in Eschrrichin c d i following over-expression of a foreign protein (urogastrone). a, Phase
conlrast micrograph of whole cells. Note that the cells resemble spore-forming bacilli; b. electron micrograph of
thin section of cells in which the urogastrone inclusions have been identified with a n immuno-gold stain.
I04 S . B. Primrose
production process is to be developed it is essential that this protein be solubilized and renatured-a
task akin to recreating egg white from a meringue! Not all foreign proteins form inclusions in E . coli,
e.g. human growth hormone, and in other instances it is possible to prevent inclusion formation by
changing the fermentation conditions. By contrast, most E. coli proteins d o not form inclusions when
over-expressed in E . coli but why this should be is not known. O n the other hand, there are no
reports of inclusion formation in S . cereuiside but as yet the levels of expression of foreign proteins
are not as good as those obtained in E . coli.
What can be done to overcome the inclusion problem? There are three solutions. Firstly, try
changing the fermentation conditions, but as yet there are no ground rules about what changes to
make. Secondly, secrete the protein from the producer cell so that it is present in the culture medium
in a much lower concentration. Thirdly, extract the protein from the cell and renature it.
Secreting proteins from cells is relatively easy to achieve. Proteins which are secreted by micro-
organisms are synthesized with an extra 20-25 amino acids at their N-terminus (Talmadge er al.
1980). These extra amino acids are known as a ‘signal sequence’ and the signal sequences from
dif‘erent organisms have a striking similarity to one another. These signal sequences are involved in
the mechanics of secretion and during passage of the protein through the membrane the signal
sequence is removed. However, the majority of proteins secreted from E. coli are transported to the
periplasmic space rather than the culture medium (Villa-Komoroff et a[. 1978). In this location they
are still concentrated, thus defeating the original intention, and additionally are subject to protease
degradation (Talmadge & Gilbert 1982). In order to get secretion into the growth medium it is
necessary to use cloning hosts such as Gram-positive bacteria, yeasts and filamentous fungi. Unfor-
tunately the devclopment of expression systems for these organisms is not as far advanced as for E .
cali (see, for example, Old & Primrose 1985). It is worth noting that the use of filamentous bacteria
(e.g. actinomycetes) and fungi may be particularly advantageous when protein secretion is desired
because filamentous organisms have a large surface area to volume ratio. It should be borne in mind
that secretion might not always be the ideal solution, for proteins in the growth medium will be very
dilute and subject to shear.
Thc protein contained in inclusions can be solubilized by treatment with sodium dodecyl sulphate
(SDS), guanidinium HCI or urea but these treatments are not without their attendant problems.
Traces of SDS can remain bound to proteins and this may not be acceptable if the protein is to be
administered parenterally. Guanidinium HCI is a very powerful solubilizing agent but is highly
corrosive, particularly if it has to be used at elevated temperatures. If urea is used it often is as a 5
mol/l alkaline solution and prolonged contact can result in chemical modification to the protein. In
addition, special procedures may be needed to handle SDS and urea, both of which form noxious
dusts.
Once extracted, thc desired protein needs to be renatured. Although there is a wealth of classical
literature on the renaturation of proteins this relates to dilute, purified ‘model’ proteins. Industrially,
one is required to renature concentrated, unpurified proteins which are anything but ‘model’. Where
success has been achieved it often is a testament to the skill of the protein chemists involved.

4.3 CHOICE OF P U R I F I C A T I O N METHOD

There are four properties of a protein which are exploited for purification: charge, size, hydropho-
bicity and affinity for ligands. Of these, size is the least useful property to be utilized for protein
purification. Size separation involves gel filtration and this has two basic problems when used on a
large scale. Firstly, the volume of the protein-containing eluate is larger than the volume of the
protein load, i.e. dilution and not concentration occurs. Secondly, very large columns are required
unless the protein load is highly concentrated. By contrast, ion exchange is a technique admirably
suited to scale-up; a large variety of different resins is available, a large concentration factor can be
achieved and there is a lot of industrial experience in their use.
Many different systems for affinity purification have been described and Table 5 gives some
representative examples. Perhaps the best known system of affinity purification is the use of matrix
bound monoclonal antibodies. Figure 3 shows the kind of purification which can be achieved with
 Genetically engineered micro-organisms 105
Table 5. Ligands for affinity purification of proteins
Ligand Specificity
5’ adenylic acid Enzymes using NAD’ or ATP as cofactor, e.g.
2’, 5’ adenosine dehydrogenases, kinases
diphosphate
Protein A F, region of immunoglobulins
Cibacron blue Any protein with affinity for nucleotides
Heparin Specificity not known but in complex mixture
of proteins only a few will bind
Metal ions Specificity not known. Especially effective
for gamma globulins, beta-lipoproteins, inferons

such a system. However, monoclonal antibodies are not without their disadvantages. If they are to be
used in large scale purification substantial quantities of the antibody will be required. Preparation of
such antibody can occasionally be a formidable task, particularly if it has only a limited capacity for
re-use! In addition, if the protein being purified is to be used parenterally then the antibody will need
to be free of adventitious agents, e.g. viruses, mycoplasmas and must not leach off the matrix to
which it is bound.

Fig. 3. Polyacrylamide gel electrophoresis of an Escherichio coli extract containing interferon before and after
immunoaflinity chromatography. The E . coli extract which was loaded onto the immunoaflinity column is shown
in track 2 and the unbound protein is shown in track 3. The interferon which was eluted from the column is
shown in track 4; note the greatly reduced level of contaminating protein although the amount of protein loaded
in track 4 was three-fold higher than in tracks 2 and 3. Molecular weight markers are shown in tracks I and 5.
106 S. B. Primrose

- \
-0 95 O/o
)I
m
L

0
21
e, m
3
2
a
90% f
m
0
e
a,

85%

I 80%

-1
I
'
2
uL
3 4 5 6 7
A
i - i
8 9 10
Number of manipulative steps

Fig. 4. Influence of the numher of manipulative steps and the step efliciency on the overall recovery of purified
protein. Note that if the average step efficiency is 80'X, and 10 manipulative steps are required the final product
recovery will he approximately I O ' X ~ .

The sequence in which the different purification steps are arranged is also important for the
number of unit operations/manipulative steps needs to be kept to a minimum if high recoveries are to
be achieved (Fig. 4). Thus an ion exchange step followed by hydrophobic interaction chromatography
is a better arrangement than the converse. Proteins are eluted from ion exchange resin in solutions of
high ionic strength whereas during hydrophobic interaction chromatography the salt concentration is
reduced. With the converse arrangement additional steps such as dialysis would be required. In
designing the purification sequence attention should also be paid to the way in which the protein will
be formulated. Usually the protein will be supplied as a freeze-dried powder mixed with excipients
such as physiologically acceptable buffers or human serum albumin. However, salts or other chemi-
cals used in purification first may have to be removed and again this can involve additional manipu-
lative steps.

4.4 T H E I M P A C T OF F E R M E N T A T I O N

One impact of fermentation, as indicated above, is that by changing the fermentation conditions it is
sometimes possible to prevent the formation of inclusions. Another impact is on the extent of
proteolytic degradation. Figure 5 shows HPLC analyses of whole cell extracts from two different
fermentations or a recombinant E . c d i strain producing urogastrone. In one, cell extracts show no
intact urogastrone molecules despite the fact that the radioimmune assays indicated that the urv-
gastrone titre was the same in both fermentations. This result also underscores the importance of
choosing the correct analytical procedures for evaluating fermentations. It should be noted that in
some instances turnover of recombinant proteins is so rapid that special techniques have to be
adopted for their detection, far less their production. For example, somatostatin has to be 'protected'
by producing it a s a beta-galactosidase fusion product (Fig. 6, Itakura el ul. 1977).
 Genetically engineered micro-organisms 107

Good fermentation

Poor fermentation

Fig. 5. HPLC analysis of urogastrone in two different crude Escherichia coli extracts. The titre of urogastrone as
determined by radio immunoassay was the same in both fermentations but in only one was a significant amount
of undegraded urogastrone (shaded area) present.

5. The good news: purification fusions

Recombinant DNA technology can be used to minimize or eliminate some of the problems described
above by providing methods to facilitate protein purification. The beta-galactosidase-somatostatin
fusion protein just described can be used as an example. One method of purifying somatostatin
would be to use an affinity matrix for beta-galactosidase as a first step (Ullman 1984) followed by
‘release’ of the somatostatin from the fusion protein by treatment with cyanogen bromide (Fig. 6). A
second purification step, say ion exchange chromatography, would permit further purification of the
somatostatin, hopefully free of the beta-galactosidase fragments. In principle, this method can be
applied to any beta-galactosidase fusion protein. Of course, the method is not restricted to beta-
galactosidase-containing fusion proteins: other kinds of fusion proteins, for example those containing
staphylococcal protein A can be purified with an appropriate matrix (Nilsson et u1. 1985).
 -
108 S. B. Primrose

NH2
p- qolactosidase
Met - Alo - Gly - Cys - Lys - Asn - Phe
I
- Phe
S \
I Tr P
S I
LY s
I /
HOOC - C y s - Ser -Thr - Phe --Thr

NH2-
1 Cyonoqen
bromide

Ah-Gly-Cys-Lys-Asn-Phe
I
- Tr P
S \
I LY s
S /
I Thr
HOOC - C y s - Ser -Thr - Phe ’
Active somatostatin
Fig. 6. Production of somatostatin as a beta-galactosidase fusion product and its release by cyanogen bromide
treatment. A synthetic somatostatin gene with an additional codon for methionine at the 5’ end was fused to the
Escherichia rnli beta-galactosidase gene. Upon expression in E. coli a beta-galactosidase-somatostatin Fusion
product is produced.

There are two disadvantages with the above method. Firstly, a method is required which will
precisely excise the desired protein from the fusion protein. This is not always easy: the use of
cyanogen bromide, as shown in Fig. 6, is restricted to those proteins which do not contain methi-
mine. Other methods are available but there is n o generalized method. Secondly, the desired protein
has to be purified free of the beta-galactosidase moiety or fragments derived from it. Thus the second
purification step has to be tailored to each new protein. Both these problems are eliminated by the
use of poly-arginine tailing (Sassenfeld & Brewer 1984). In this method the gene sequence encoding
the desired protein is modified by the inclusion at the 3’ end of a number of codons for arginine
(Smith et ul. 1984). This is a routine procedure given the simplicity of chemical synthesis of genes.
When such genes are expressed the resultant proteins have a polyarginine ‘tail’ which makes them
more basic. On ion exchange chromatography such proteins are separated from the bulk of the host
cell proteins which are more acidic (Fig. 7a). The polyarginine tail is then removed with the enzyme
carboxypeptidase B which, for convenience, can be immobilized. The de-tailed protein is re-
chromatographed on an identical ion exchange resin to separate it from any remaining contami-
nating proteins which will be more basic (Fig. 7b). The power of this technique is shown in Fig. 8.

6. The influence of development costs

The cost of developing a new chemical entity as a drug for human therapy is enormous. Figure 9
shows how this cost increased in the period 1962 to 1979. In part this escalating cost is due to greatly
increased requirements for safety testing largely as a result of the thalidomide tragedy, and in part to
inflation. 1t should be noted that the costs of drug discovery are only a small part of the total
development costs. Thus there is no reason to suppose that the development costs for a new bio-
logical entity, such as a protein produced as a result of recombinant DNA technology, will be
significantly different from those for a new chemical entity. Therefore, before choosing to develop a
recombinant-derived protein due attention must be paid to its marketability and its potential to
produce sales far in excess of the development costs. A few selected examples are discussed in more
detail below; these include the three recombinant-derived products currently on the market-human
insulin, human growth hormone and interferon-alpha, . It should be noted that an adequate return
on investment is unlikely if the drug is not approved for use in Western Europe and/or the United
States.
 Genetically engineered micro-organisms 109

Fig. 7. Schematic representation of the chromatographic behaviour of a polyarginine-tailed protein before and
after treatment with carboxy-peptidase B. a, Before tail removal the protein (shaded) elutes at high salt molarity
free of the bulk of the host cell proteins (solid line); b, after tail removal the desired protein elutes at much lower
salt molarity.

6.1 H U M A N INSULIN

Insulin plays a key role in the body’s regulation of carbohydrate, fat and protein metabolism.
Diabetes mellitus is due to a deficiency in insulin synthesis and secretion and the severity of the
metabolic disturbance is directly related to the extent of the insulin deficiency. Patients with extreme
deficiency require long-term replacement therapy with insulin which can be derived from bovine or
porcine pancreas. Most preparations contain a mixture of beef and pork insulins in variable propor-
tions; although both are immunogenic in man, immunological resistance to insulin action is uncom-
mon. When it does occur, antibodies are directed against the beef component and may be overcome
by changing to a pure pork preparation.
Human insulin differs from animal insulins by a single amino acid and may be produced by
chemical modification of bovine or porcine insulin or by recombinant DNA technology (Goeddel et
110 S . B. Primrose
1 2 3 4 5 6

Fig. 8. Purification by ion exchange chromatography

of a protein by polyarginine tailing. The figure shows
polyacrylamide gel electrophoresis of: I , crude lysate
prior to application to the ion exchange resin; 2,
unbound protein; 3, protein eluted with 0.75 mol/l
NaCl ; 4, fraction from track 3 after carboxypeptidase
digestion; 5, protein which fails to bind to ion
exchange resin after digestion; 6, material which elutes
from second ion exchange column with 0.75 mol/l
NaC1. The major band in track 5 is the desired
protein. The bands in track 6 are the proteins which
contaminated the polyarginine-tailed protein after the
lirst ion exchange step.

72

60

48
E
-
0
-
0
V
% 3€
u)
C
0
-
._
s 24

1.2

I I
1962 1979
Years
Fig. 9. Increase in development costs required to bring a new drug to the market place during the period
1962 1979.
 Genetically engineered micro-organisms 111
al. 1979b; Wetzel et al. 1981). From the foregoing, however, it should be clear that there is little to be
gained by the use of human insulin however produced. Although diabetics are prone to such dis-
orders as retinopathy, ischaemic heart disease and gangrene of the lower limbs there is no evidence to
suggest that these will not occur at the same frequency regardless of the type (animal or human) of
insulin used.

6.2 H U M A N GROWTH HORMONE (SOMATOTROPIN)

Pituitary dwarfism is a direct result of a deficiency in human growth hormone production. Only
growth hormone from humans is active in man; animal somatotropins are without effect. Until the
advent of recombinant DNA technology, somatotropin was extracted from human pituitaries
removed at autopsy and this restricted supply. More recently there has been concern about the
transmission of Creutzfeldt-Jakob syndrome to treated individuals and pituitary-derived human
growth hormone has been withdrawn from the market. Fortunately adequate supplies can be produc-
ed from recombinant E . cnli (Goeddel er al. 1979a) but since pituitary dwarfism is a relatively rare
disorder, commercial viability will depend on other applications. A potentially large market for
somatotropin is the treatment of osteoporosis which afilicts over 20% of post-menopausal women.

6.3 INTERFERON-ALPHA

The commercial problems associated with this product are similar to those associated with human
growth hormone. Although interferon-alpha, has been extensively tested against a wide range of
human cancers, only in the treatment of hairy cell leukaemia has there been a consistently high
success rate (Bonnem & Spiegel 1984). Clearly other indications are required. This interferon is
effective in the short-term prophylaxis of rhinovirus colds but whether large scale prophylaxis of this
kind is desirable or practicable remains to be seen.

6.4 S O M E P R O T E I N S W H I C H A R C~O M M E R C I A L L Y A T T R A C T I V E

Relaxin is a 53 amino acid insulin-like molecule which prepares the endometrium for parturition. It
also inhibits uterine contraction permitting foetal development. Porcine relaxin has been used clini-
cally to decrease the time of labour and the incidence of Caesarian delivery due to foetal distress. A
dose of 1-2 mg is required per patient and this can be obtained from a single pig oirary. However, a
recombinant source would be more practicable (Stewart et a/. 1983). The real attraction of course is
that pregnancy is a very common disease!
Thrombosis, the blockage of blood vessels by clots, is the commonest cause of death in the
developed world; more than half of all deaths are due to coronary thrombosis, stroke or pulmonary
embolism. The basis of a blood clot is a network of fibres of the protein fibrin. This network is
destroyed by the fibrinolytic enzyme plasmin which is formed in the blood from an active precursor,
plasminogen. Urokinase is a plasminogen activator which is synthesized by cells which line the
urinary tract and which can be isolated from human urine. Streptokinase is a bacterial product which
is similar to urokinase. However, urokinase and streptokinase are relatively non-specific: they cleave
all plasminogen molecules and this can lead to haemorrhages. Tissue-specific plasminogen activators
are much more selective (Jaffe & Sobel 1986) since they have little afinity for circulating plasminogen.
Because tissue plasminogen activators and plasminogen bind avidly to fibrin in close proximity to
each other, plasminogen is activated on the fibrin surface and clot lysis ensues. Given the high
incidence of thrombosis, the market for tissue plasminogen activator is potentially huge.

6.5 MODIFIED PROTEINS AS THERAPEUTIC AGENTS

One of the advantages of producing human proteins in micro-organisms, as opposed to isolating

them from human tissues, is that it is possible to generate modified proteins with beneficial proper-
ties. For example, cumulative damage to lung tissue by leucocyte elastase is throught to be
I12 S. B. Primrose
responsible for the development of emphysema, an irreversible lung disease characterized by loss of
lung elasticity. The primary defence against elastase damage is alpha,-antitrypsin, a glycosylated
serum protein of 394 amino acids. Oxidation of methionine residue 358 located at the active site
efYectively inactivates the protective function and this is probably the major cause of emphysema. By
replacing methionine 358 with valine, an active, oxidation-resistant alpha,-antitrypsin has been gen-
erated which could be beneficial as it might reduce the amounts of enzyme which would need t o be
administered (Rosenberg et a/. 1984).

7. Regulatory aspects of drug development

Not only is the cost of drug development considerable, so too is the time required for development
(Fig. 10). Again, the increase in the time taken to get approval of a drug is directly related to demands
for increasing numbers of safety tests. For proteins produced by recombinant DNA technology two
basic safety problems can be envisaged. Firstly, immunological complications produced in treated
individuals as a result of administration of impure proteins or proteins which have not been subjected
to post-translational modification such as glycosylation. The immunological complications which
might arise from administration of novel proteins such as the modified alpha,-antitrypsin described
above remain to be determined. Secondly, toxicological complications arising from administration of
natural proteins far in excess of those found in the normal individual. The time taken to produce a
protein to the standard outlined in Table 4 and to accomplish the necessary immunological and
toxicological tests will not be much less than that for safety evaluation of a conventional chemical
entity.

I 1 I
1960 1965 I970 1975
Years

Fig. 10. Increase in time required to develop a drug during the period 1955-1980.
 Geneticully engineered micro-orgunisms 113
8. The future: recombinant DNA technology and the production of small molecules
Many genetic engineering companies, often working in association with major pharmaceutical firms,
were not slow to realize the potential of recombinant DNA technology. Thus it is now possible to
produce many human proteins in commercially realistic amounts. There is a down side to this,
however. As indicated above, the market size for most human proteins is too small to justify their
development unless they achieve orphan drug status. More importantly, most proteins will have to be
administered parenterally and this will greatly reduce their marketability. Human insulin and tissue
plasminogen activator notwithstanding, the pharmaceutical industry prefers drugs which are orally
active and can be self-administered because the sales potential is much greater. Such orally active
drugs are generally low molecular weight organic chemicals. Since many of these molecules are
already produced with the aid of micro-organisms (Table 1) there is plenty of scope for process
improvement by means of recombinant DNA technology.
The ways in which recombinant DNA technology can be used to facilitate the production of small
molecules with pharmacological activity are shown in Table 6. Since these techniques are still in their
infancy the examples quoted are only intended to be illustrative: the commercial viability of each
remains to be ascertained.

Table 6. Recombinant DNA technology and the production of low molec-

ular weight compounds
1. Improve efficiency of existing synthetic route by increasing levels of
enzymes catalyzing rate-limiting steps, e.g. antibiotics, amino acids
2. Introduction of synthetic systems for known molecules enabling pro-
duction in micro-organisms for the first time, e.g. indigo, vitamin C
3. Novel combinations of biosynthetic pathways enabling production of
novel antibiotics

8.1 CLONING ALL OR PART OF A BIOSYNTHETIC PATHWAY

Many pharmaceutically useful molecules such as antibiotics are synthesized by micro-organisms and
are the end-product of long biosynthetic pathways. By definition there will be at least one enzymic
step in the pathway which is rate-limiting. One way to increase the productivity of the biosynthetic
pathway is to increase the level of the rate-limiting enzyme and this is most easily achieved by cloning
the relevant gene. Of course, some other step in the pathway will then become rate-limiting! If the
overall flux through the biosynthetic pathway is low then it is possible to clone all the genes
controlling biosynthesis.

8.2 NEW SYNTHETIC ROUTES

The best example is provided by the manufacture of vitamin C. The conventional process starts with
glucose and comprises one microbiological and four chemical steps. By cloning a single gene from
Corynebacterium in Erwinia, the process can be simplified to a single microbiological and a single
chemical step (Fig. 11 : Anderson et al. 1985). Another variant of this concept of new synthetic routes
is provided by the microbial synthesis of another small molecule, indigo (Ensley et al. 1983). Cloning
of a single Pseudomonas gene, that encoding naphthalene dioxygenase, resulted in the generation of
an E. coli strain able to synthesize indigo from tryptophan (Fig. 12).
114 S. B. Primrose
(a )

D-Glucose
chemical
- D-Sorbitol
Acelobacter
submydons
Z- L-Sorbose
I

Ascorbic
acid
chemical 2-keto -L-
gulonic acid
chemicaI
chemical

Di
-2-
I
aceketo-
tone L -

gulonic acid

CHO COOH COOH

HOHO! Er winio
Endoqenous
~ : /H
.O- 2,5 DKG
reductase ~ ~ o { ~ ~
OH enzymes OH (Coryneboctefium1

OH HO

CH,OH CHzOH CHZOH

0-Glucose 2,5 - d iketo- 2-keto- L-
D- gluconic acid gulonic acid

Fig. 11. New, recombinant route for vitamin C production. a, The conventional Reichstein-Grussner synthesis; b,
new synthesis following introduction into Erwinia of the gene from Corynebucteriurn encoding 2, 5-diketo-u-
gluconic acid reductase.

8.3 NOVEL ANTIBIOTICS

Pharmaceutical companies have used a whole battery of techniques to induce microbial cultures t o
synthesize novel antibiotics, e.g. mutational biosynthesis, inter-generic and inter-specific mating and
protoplast fusion between different antibiotic-producing species. Recombinant DNA technology has
added a new dimension to this search. The Streptomyces coelicolor gene cluster encoding the biosyn-
thesis of the isochromanequinone antibiotic actinorhodin has been cloned and transferred t o a
variety of Streptomyces sp. producing different isochromdnequinones. This resulted in the synthesis of
at least three new antibiotics (Hopwood et al. 1985).
 Genetically engineered micro-organisms 115

~74 Indigo

’0
N Dd

\
oxzbrtion
rpntaneous
elimination

of water
O
~H
Q
-

N
-

H
c

OH

Fig. 12. Chemical transformations leading to production of indigo by Escherichia coli after introduction of cloned
Pseudomonas gene encoding naphthalene dioxygenase. Tryptophan present in the growth medium is converted to
indole by endogenous tryptophanase.

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