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Hybridoma Technology and Monoclonal Antibodies
165
QUESTIONS
1. Write a note on production and application of mAbs.
2. Describe hybridoma technology.
3. What are mAbs? Write their applications.
4. Define the terms mAbs, hybridoma technique and hybridization.
5. Write a note on formation and selection of hybrid cells.
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HISTORICAL DEVELOPMENT OF ANTIBIOTICS
The term antibiotic was coined by Selman Waksman in 1942 to describe any substance produced
by a microorganism that is antagonistic to the growth of other microorganisms in high dilution.
This definition excluded substances that kill bacteria, but are not produced by microorganisms
(such as gastric juices and hydrogen peroxide). It also excluded synthetic antibacterial compounds,
such as the sulphonamides.
The mixtures with antimicrobial properties that were used in treatments of infections were
described over 2000 years ago. Many ancient civilizations, including Indian, Egyptian and Greek
used specially selected moulds and plant materials and extracts to treat infections. In ancient days,
the treatments for infections were primarily based on medicinal folklore.
The earliest evidence of humans using plants or other natural substances for therapeutic purposes
comes from the Neanderthals, who lived over 50,000 years ago. In northern Iraq, archaeologists
uncovered evidence of human remains that had been buried with a range of herbs, some of which
are now known to be antibacterial. Many of these herbs are still used by the inhabitants of this
region.
The first prescription for treating infections has come from the Egyptians around 1550 BC.
Written as ‘mrht’, ‘byt’ and ‘ftt’, it was a mixture of lard, honey and lint, and was used as an
ointment for dressing wounds. Honey is antibacterial, as it kills bacterial cells by drawing water
out of them. In addition, the enzyme inhibin, which is found in honey, converts glucose and oxygen
into hydrogen peroxide, a well-known disinfectant.
Antibiotics
12
CHAPTER
Historical Development of Antibiotics
Antimicrobial Spectrum
Standardization of Antibiotics
Screening of Soil for Organisms
Producing Antibiotics
Fermenter or Bioreactor
Design of Industrial Fermentation Process
Fermenter Design and Control
Isolation of Mutants
Factors Infl uencing Rate of Mutation
Isolation of Fermentation Products
Questions
Textbook of Pharmaceutical Biotechnology
168
The use of moulds dates back to the ancient Egyptians, and perhaps even earlier. An Egyptian
physician, quoted in the Ebers Papyrus around 1550 BC, stated that if a wound rots, then bind on it
spoiled barley bread. The Egyptians used all kinds of moulds to treat surface infections. The ancient
Chinese also used moulds to treat boils, carbuncles and other skin infections.
Inorganic substances have been used to treat infections since ancient times. Copper was widely
used by the Greeks, Egyptians and Romans, often in combination with honey. Modern scientific
tests have proved that copper is, indeed, antibacterial. A skin infection known as impetigo, which
is caused by the Staphylococcus aureus bacteria, is being treated in France with Eau Dalibour, a
combination of zinc and copper. This prescription dates from the time of Jacques Dalibour, surgeon
general of the army of Louis XIV.
Wine and vinegar have been popular treatments for infected wounds since the time of
Hippocrates. Vinegar is an acid and a powerful antiseptic. The antibacterial properties of wine
cannot be fully attributed to its alcohol content, as this is very low. Recent chemical analysis of
wine has shown the presence of an antibacterial substance called malvoside.
Antagonistic activities by fungi against bacteria were first described in England by John
Tyndall in 1875. Ehrlich from Germany (1880) noted that certain dyes coloured human, animal or
bacterial cells, whereas others did not. He then proposed the idea that it might be possible to create
chemicals that would bind to and kill bacteria without harming the human host. After screening
hundreds of dyes against various organisms, he discovered a medicinally useful drug, the synthetic
antibacterial, salvarsan.
In 1928, Alexander Fleming observed antibiosis against bacteria by a fungus of the genus
Penicillium. Fleming postulated that the effect was mediated by an antibacterial compound named
penicillin, and that its antibacterial properties could be exploited for chemotherapy. Prontosil,
the first commercially available antibacterial antibiotic, was developed by Gerhard Domagk and
colleagues in 1932 (he received Nobel Prize for medicine for his discovery in 1939). The work was
carried out at the Bayer Laboratories of the IG Farben conglomerate in Germany. Prontosil exhibited
relatively broad effect against gram-positive cocci but not against enterobacteria. The discovery
and development of this first sulphonamide drug opened the era of antibacterial antibiotics.
In 1939, Rene Dubos reported the discovery of gramicidin from Bacillus brevis. It was one of
the first commercially manufactured antibiotics in use during World War II treating wounds and
ulcers.
The scientists in the 19th century began to closely examine various curative substances and
discovered that there were good or beneficial bacteria. This could be isolated and encouraged for
their growth in the laboratory. The bacterial agents from such bacteria were then tested for their
ability to treat disease. This pattern of research and clinical trials continued into the 20th century
(and, in fact, continues even today), leading to the production of the antibiotic drugs.
Late 1800s: The growing acceptance of the germ theory of disease, a theory which linked bacteria
and other microbes to the causation of a variety of ailments. As a result, scientists began to search
for drugs that would kill these disease-causing bacteria.
Antibiotics
169
1871 Joseph Lister (a surgeon) began researching the phenomenon that urine contaminated
with mould would not allow the successful growth of bacteria. In 1890, German doctors,
Rudolf Emmerich and Oscar Low, were the first to make an effective medication named
pyocyanase from microbes. It was the first antibiotic to be used in hospitals.
1928 Sir Alexander Fleming observed that colonies of the bacterium S. aureus could be
destroyed by the mould Penicillium notatum, demonstrating antibacterial properties.
1935 Prontosil, the first sulpha drug, was discovered by German chemist Gerhard Domagk.
1942 The manufacturing process for penicillin G procaine was invented by Howard Florey
and Ernst Chain. Penicillin could now be sold as a drug. Fleming, Florey and Chain
shared the 1945 Nobel Prize for medicine for their work on penicillin.
1943 American microbiologist Selman Waksman isolated streptomycin from soil bacteria, the
first of a new class of drugs called aminoglycosides. Streptomycin could treat diseases
like tuberculosis (TB). However, the side effects were often too severe.
1955 Tetracycline was patented by Lloyd Conover, which became the most prescribed broad-
spectrum antibiotic in the United States.
1957 Nystatin was patented and used to cure many disfiguring and disabling fungal
infections.
1981 SmithKline Beecham patented amoxicillin or amoxicillin/clavulanate potassium tablets.
The antibiotic was first sold in 1998 under the trade names of Amoxicillin, Amoxil and
Trimox. Amoxicillin is a semisynthetic antibiotic.
Discovery of Penicillin and Other Antibiotics
In 1928, while attempting to grow the bacteria Staphylococcus spp. on an agar plate, Fleming noticed
that the growth of this bacterium was inhibited by a mould that had accidentally contaminated the
plate. He decided to identify the mould that was eventually called P. notatum. Fleming was excited
by this discovery. He cultured the mould in a special broth and injected the broth into some of
his patients, who had various infectious diseases. The results were encouraging, and the broth
proved to be nontoxic. Unfortunately, Fleming had not made enough of this broth, making his
experiment rather limited. When he presented a paper on his findings in 1929, his colleagues in the
medical profession were not particularly impressed or interested. It took two other gifted researcher
doctors—Florey and Chain, working at Oxford University in the late 1930s and early 1940s—
to realize the importance of Dr. Fleming’s findings. It was their pioneering work that brought
penicillin into clinical use. Florey, an Australian doctor, had gone to Oxford on a scholarship to
study pathology. Chain was a German chemist who had fled from the Nazis in the 1930s and had
come to rest in England. In 1945, Fleming, Florey and Chain shared Nobel Prize for their work on
penicillin.
Penicillin was later produced in oral form and was added to many products, including salves,
throat lozenges, nasal ointments and cosmetic creams. Prior to 1955, its sale was not controlled,
Textbook of Pharmaceutical Biotechnology
170
so anyone could buy it over the counter without a prescription. This excessive and uncontrolled
use led to the overgrowth of resistant bacteria, and the damage had been done. Resistance had
become a major problem, and epidemics of staphylococcus-resistant infections began to emerge in
hospitals.
A systematic search of antibiotic-producing microorganisms from soil was undertaken. In 1939,
Dubos isolated from soil a culture of B. brevis and produced two valuable antibacterial substances,
now known as gramicidin and tyrocidin that killed many gram-positive bacteria.
The study of soil bacteria and the reasons why they are not more capable of causing disease
was the lifelong work of Selman Waksman, a research scientist at Rutgers University in New
Jersey. In 1939, Merck and Company provided him with financial assistance to mount a search
for antibiotics in soil microorganisms. This search culminated in the isolation of streptomycin,
the first antibiotic to offer hope to patients with TB. This antibiotic is still used today in the
treatment of TB.
In 1947, the antibiotic chloramphenicol was used in a clinical trial to treat an epidemic of
typhus in Bolivia. Its success in curbing the epidemic led to its use on the other side of the world–
treating scrub typhus in Malaysia.
Modifications of the basic cephalosporin chemical structure led to the development of a whole
range of these antibiotics for clinical use. Research into the development of new cephalosporins
continues today.
In 1947, chlortetracycline was isolated from a Missouri River mud sample by Benjamin M.
Duggar. Chlortetracycline was the first tetracycline introduced. Duggar’s discovery has led to the
isolation and subsequent development of a large number of very powerful antibiotics, which now
rank second only to the penicillins in their use worldwide.
They are active against a broad range of bacteria and are relatively inexpensive to produce.
The tetracyclines quickly gained favour and are now used to treat a long list of infections.
However, they are also known to cause a number of toxic side effects. The tetracyclines form
calcium complexes in growing bone, which may lead to lifelong discolouration and enamel
defects in teeth, as well as, reduced bone growth. They are prohibited in the treatment of
children below the age of 7, as they may inhibit small children’s growth. Tetracyclines also
cross the placenta and have a greater toxicity in the fetus. Therefore, they are not administered
to pregnant women.
The tetracycline antibiotics form complexes with calcium, magnesium and iron. Therefore,
they should not be taken with dairy products or any mineral and vitamin supplements containing
calcium, magnesium or iron.
Further research took place during the 1960s that led to the development of the second
generation of antibiotics. Among these was methicillin, a semisynthetic derivative of penicillin
produced specifically to overcome the problem of penicillin resistance. Methicillin was hailed as a
major breakthrough in the fight against bacterial resistance to penicillin, and scientists believed that
they could now win this battle. Unfortunately, bacteria had the last word, and we now have bacteria
that are resistant to methicillin.
Antibiotics
171
Amoxicillin is another widely used penicillin derivative. Like ampicillin, it has a broad range
of activity, as it can treat both gram-positive and gram-negative bacteria.
Ampicillin is also a derivative of penicillin. It was developed to broaden the range of infections
that penicillin could treat and has now replaced penicillin to a great extent. It is often the first choice
in the treatment of a whole range of infections, including respiratory and urinary tract infections.
Gentamicin is in the same family of antibiotics as streptomycin. It is generally reserved for
serious infections, as it can have severe toxic side effects on the ears and kidneys.
Recently, a new family of antibiotics called the fluoroquinolones has been developed by
pharmaceutical laboratories. In addition to being effective against a broad range of bacteria, these
antibiotics can reach a high concentration in the bloodstream when taken orally. This means that
many more infections that may once have required a hospital stay can now be treated at home. The
fluoroquinolones are often used for cases in which long courses of antibiotics (weeks to months) are
required. A whole range is now available, and is proving effective against bacteria that were once
difficult to treat, such as the leprosy bacteria.
The search for new and more effective drugs, which began with Florey, Chain and Waksman,
continues today. The pace, however, has slowed remarkably, as it is now much more difficult for
pharmaceutical companies to get approval for new drugs. The time gap between the discovery of
an antibiotic in the laboratory and the approval to produce it commercially is so great that it has led
many companies to abandon the efforts completely. The resistance offered by bacterial strains is
also another reason for discontinuation of research undertaken by drug companies.
ANTIMICROBIAL SPECTRUM
Antibacterial spectrum refers to the range of bacteria susceptible to a particular antimicrobial or
class of antimicrobials.
Spectrum of activity: A specific antibiotic has a range of microorganisms whose growth it is
capable of inhibiting.
1. Broad spectrum: The term broad-spectrum antibiotic refers to an antibiotic with activity
against a wide range of disease-causing bacteria. It acts against both gram-positive and gram-
negative bacteria, e.g. levofloxacin, tetracycline.
2. Narrow spectrum: An antibiotic that affects only limited number of microorganisms is said
to have a narrow spectrum of activity. The spectrum of activity might include most gram-
positive bacteria, but few, if any, gram-negative bacteria, e.g. penicillin.
The advantage with broad-spectrum antibiotic is that there is less of a need (as compared
with narrow-spectrum antibiotic) to identify the infecting pathogen with real certainty before
commencing treatment. On the other hand, a broad-spectrum antibiotic will have a more profound
effect on your normal flora, thus interfering with microbial antagonism.
Site of action and organisms from which antibiotics are produced are given in Table 12.1.
Textbook of Pharmaceutical Biotechnology
172
STANDARDIZATION OF ANTIBIOTICS
Antibiotics are not equally effective against all types of microorganisms. Some antibiotics are
inhibitory to many species ( broad spectrum) while others are inhibitory to only a few species of
microorganisms ( narrow spectrum). Sometimes, a resistant strain of a microbial species arises as a
result of overexposure to an antibiotic. To determine the effectiveness of an antibiotic, sensitivity
test is performed against different microbial species. These tests are commonly done by placing
small filter paper discs containing antibiotics on the surface of an agar plate heavily seeded with test
organisms. The plate is incubated. A zone of inhibition surrounding the filter paper disc indicates
that the test organism is sensitive to the antibiotic.
The assay and standardization of antibiotic is done in a similar fashion. An agar plate is seeded
with a standard test organism. Measured volume of an antibiotic is placed in glass or metal cylinders
on the surface of the inoculated agar. The diameter of the zone of inhibition is a measure of the
antibiotic concentration in any given cylinder.
Minimal Inhibitory Concentration (MIC)
Minimal inhibitory concentration (MIC) is defined as the lowest concentration of antimicrobial
agent required to inhibit growth of the organism. The agar plates, tubes or microtitre trays with
two-fold dilutions of antibiotics are inoculated with a standardized inoculum of the bacterium and
incubated under standardized conditions following National Committee for Clinical Laboratory
Standards (NCCLS) guidelines. The next day, the MIC is recorded as the lowest concentration
of antimicrobial agent with no visible growth. The MIC provides information about degree of
resistance and might provide some important information about the resistance mechanism and the
resistance genes involved. MIC determination performed as agar dilution is regarded as the gold
standard for susceptibility testing.
Table 12.1 Some clinically important antibiotics
Antibiotic Producer organism Site or mode of action Activity against
Amphoterecin B
Streptomyces nodosus
Cell membrane Fungi
Bacitracin
Bacillus subtilis
Wall synthesis Gram-positive bacteria
Cephalosporin
Cephalosporium acremonium
Wall synthesis Broad-spectrum
Erythromycin
Streptomyces erythreus
Protein synthesis Gram-positive bacteria
Gentamicin
Micromonospora purpurea
Protein synthesis Braod-spectrum
Griseofulvin
Penicillium griseofulvum
Microtubules Dermatophytic fungi
Neomycin
Streptomyces fradiae
Protein synthesis Broad-spectrum
Penicillin
Penicillium chrysogenum
Wall synthesis Gram-positive bacteria
Polymyxin B
Bacillus polymyxa
Cell membrane Gram-negative bacteria
Streptomycin
Streptomyces griseus
Protein synthesis Gram-negative bacteria
Tetracycline
Streptomyces rimosus
Protein synthesis Broad-spectrum
Vancomycin
Streptomyces orientalis
Protein synthesis Gram-positive bacteria
Antibiotics
173
Agar Diffusion Tests
It is often used as a qualitative method to determine whether a bacterium is resistant, intermediately
resistant or susceptible to an antibiotic. However, the agar diffusion method can be used for
determination of MIC values provided. Moreover, necessary reference curves for conversion of
inhibition zones into MIC values are available. After an agar plate is inoculated with the bacterium,
a tablet, disk or paper strip with the antimicrobial agent is placed on the surface. During incubation
the antimicrobial agent diffuses into the medium and inhibits growth of the bacterium if susceptible.
Diffusion tests are cheaper in comparison to most MIC-determination methods. Although E-test is
a diffusion test, it has been developed to give an approximate MIC value.
The agar diffusion methods are also influenced by factors like depth of agar medium, diffusion
rate of the antimicrobial agent and growth rate of specific bacterium.
The MIC-determination and disk diffusion methods described are in accordance with the
international recommendations given by the National Committee for Clinical Laboratory Standards
(NCCLS). The NCCLS describes how to perform the tests and sets international guidelines for
interpretation of the results. It may be noted that the WHO does not prescribe any specific method
for performance and interpretation of susceptibility tests.
Internal quality control should be regularly performed as recommended by NCCLS.
MIC Determination by Broth Dilution (Using Sensititre)
1. Materials used
a. McFarland standard 0.5
b. Disposable loops (1 and 10 L)
c. Microtitre trays with dehydrated antibiotics in two-fold concentrations
d. Sensititre plates from Trek Diagnostic System Ltd., England
e. Microtitration reader with mirror
f. Multichannel pipette
g. Disposable reservoir for reagents
h. Graduated pipettes (20–1000 L)
i. Nephelometer or white paper with black lines
2. Media
a. Nutrient agar plates for purity control of inoculum suspensions
b. Sterile normal saline, 4 mL volumes in tubes for nephelometer
c. 10 mL cation adjusted Mueller-Hinton II broth in sensititre tubes
3. Bacterial strains
a. Salmonella strains on nonselective agar
b. Four strains for quality control: S. aureus ATCC 29213, Pseudomonas aeruginosa ATCC
27853, Enterococcus faecalis ATCC 29212 and Escherichia coli ATCC 25922
4. Safety: The procedures are carried out in accordance with the local codes of safe practice.
5. Standardization of inoculum: The material is picked up from at least 3 to 4 colonies. It is totally
suspended in 4 mL saline in tubes and mixed. It is adjusted to McFarland 0.5 (nephelometer).
Textbook of Pharmaceutical Biotechnology
174
The nephelometer is calibrated before use and gently the suspensions are turned upside down
before measuring. The turbidity of inoculum is adjusted to match that of standard.
If a nephelometer is not available, it is compared visually with the McFarland 0.5 standard
using white paper with black lines as background. The McFarland 0.5 suspension is diluted as
follows for the species tested at this course:
a. Gram-negative: 10 L McFarl. 0.5 into 10 mL broth
b. Gram-positive: 50 L McFarl. 0.5 into 10 mL broth
The suspension should be used for inoculation within 15 min.
6. Inoculation and incubation: The microtitre trays are inoculated with 50 L of the inoculum
suspension using a multichannel pipette or Sensititre autoinoculator. The plates are sealed and
incubated at 37°C for 18–22 h. Do not stack plates more than two.
Purity control: About 10 L of the inoculation suspension is spread on a nutrient agar plate and
incubated at 35°C overnight. The quality control strains are run in parallel to the test strains.
This is done to avoid picking bacteria that have lost their resistance. The standardization of
inoculum is essential because the interpretation of the results is based on a certain inoculum.
The dilution procedure varies with the bacterium species and has to be calculated for each.
NCCLS recommends that each well contain approximately 5 u 10
5
CFU/mL after inoculation.
Many gram-negative bacterial species grow faster than gram-positive species.
Each well is inoculated with approximately 55 u 10
3
gram-negative cells or 2.5 u 10
4
Gram-positive cells.
The incubation time is extremely important to obtain reliable endpoints.
7. Reading MIC/interpretation of results: The purity of inoculum suspension is checked. It
has to be satisfactory for favourable interpretation. The plates are read as follows:
a. The record sheet is used for orientation of the plates.
b. The growth is checked in three positive control wells.
c. The MIC is read as the lowest concentration without visible growth.
The antibiotic trimethoprim and the sulphonamides allow growth of the bacteria for several
generations before inhibition occurs. In case of trimethoprim and sulphonamides, the MIC is
recorded as the lowest concentration where a growth reduction of 80–90% can be seen. Further
interpretation of the MIC is done according to the NCCLS recommendations. The breakpoints are
also shown on the record sheet. The acceptable MIC ranges for the quality control strains are as
recommended by the NCCLS. Strange patterns of growth in the microtitre trays are often caused
by contamination. The MIC is determined from two-fold dilutions of the antimicrobial agent. The
true MIC could be anywhere between the observed MIC and the dilution step below. The NCCLS
standard does not include breakpoint recommendations for all the compounds tested.
SCREENING OF SOIL FOR ORGANISMS PRODUCING ANTIBIOTICS
The common sources for isolation of microorganism are soils, lakes and river muds. The common
techniques used for isolation of industrially useful microorganisms include the following:
1. Direct sponge of the soil