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BINGHAM UNIVERSITY, KARU

FACULTY OF PHARMACEUTICAL SCIENCES
DEPARTMENT OF PHARMACEUTICAL MICROBIOLOGY
NASARAWA STATE

BTG 402

BIOTECHNOLOGY

Application of Pharmaceutical Biotechnology

Pharmaceutical Immunology and Biopharmaceuticals

Lecture notes by Prof. Adebola A. Onanuga

SPECIFIC AREAS OF CONCENTRATION

-Engineering antibodies for therapy-predicting monoclonal antibodies

-Recombinant antibodies
-Antibody fragment
-Gene therapy
-Biotechnology in vaccine development – DNA vaccines, vaccine production by recombinant DNA for
protection of viral and bacterial infection.

Introduction to Molecular Immunology

Some key definitions:

Pathogen: microbe that causes disease

Antigen: material (from a pathogen) that induces an immune response

Innate (natural) immunity: rapid, non specific immune response

Adaptive (acquired) immunity: slower, specific immune response

Leukocytes: blood cells

Lymphocytes: specialized blood cells that mediate adaptive immunity (e.g. T and B cells)

Thymus: primary lymphoid organ for T cell development

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Bone marrow: primary lymphoid organ for B cell development

Lymph nodes: collect antigens from tissues

Spleen: collects antigens from blood stream

The cells of the immune system circulate through the body via lymph and blood. Pathogens and their
antigens are transported from tissues via lymphatic vessels to the lymph nodes where they encounter immune
cells. The cells of the immune system spend much of their time in lymphoid organs. They develop (arise) in
primary lymphoid organs, and they interact with antigens in secondary lymphoid organs.

BONE MARROW

SOURCE OF CELLS
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Many different types of blood cells participate in the immune response to microbes:

Innate immune cells: They are ―phagocytes‖ which are macrophage, neutrophils, dendritic cells

Adaptive immune cells: They are ―lymphocytes‖ which are T cells, B cells

Most blood cells act to fight infection.

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Lymphocytes of the adaptive immune system are:

T – Lymphocytes originate in bone marrow, mature in thymus and enter circulation. They are T – helper
and killer cells.
i. T - helper cells - regulate other immune cells
ii. T - cytotoxic (killer) cells - kill infected cells
B – Lymphocytes are B – cells which originate in bone marrow, mature in spleen and enter circulation.
They become antibody producing ‗plasma‘ cells and have specific receptors for antigens.
B cells - produce antibodies (immunoglobulin)
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Dendritic cells and macrophage - directly kill microbes by phagocytosis and other mechanisms. They also
help to activate T cells which are the connection between innate and adaptive immunity.
NK cells are lymphocytes that have characteristics of innate and adaptive immunity.

Immune Effector Mechanisms:

Cell-mediated immunity:
i. Phagocytosis (cellular eating)
ii. Cytotoxcity (cellular killing)
Humoral immunity:
i. Complement – is a group of serum proteins that can directly kill pathogens.
ii. Antibodies (also called immunoglobulin) are proteins secreted by B cells that bind directly and
specifically to pathogens. Antibodies target pathogens by marking them for destruction by other
components of the immune system.

Antigen
Antigens are chemicals mostly proteins or large polysaccharides from a foreign organism (Microbes –
capsules, cell walls, toxins, viral capsids, flagella; Non-microbes – pollen, red blood cell surface molecules,
serum proteins, egg white and surface molecules from transplanted tissue) that create immune response.

Epitope is a small unique part of the antigen with 10 – 12 amino acids that is recognized by an antibody. Any given
antigen may have several epitopes and these epitopes bind with their antibody in a highly specific interaction.

Antibody
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Antibody is an immunoglobulin, a specialized immune protein, produced when an antigen is introduced into
the body and it possesses the remarkable ability to combine with the very antigen that triggered its
production.

The production of antibodies is a major function of the immune system and is carried out by a type of white
blood cell called a B cell (B lymphocyte). Antibodies can be triggered by and directed at foreign proteins,
microorganisms, or toxins. Some antibodies are autoantibodies and they are against our own tissues.

Structure of antibody

Each antibody consists of four polypeptides (protein chains):

- Two identical heavy chains and

- Two identical light chains joined to form a "Y" shaped molecule.

The amino acid sequence in the two sections at the tips of the ―Y‖ varies greatly among different antibodies
and it contains the antigen binding sites called the variable regions. This variable region is composed of
110-130 amino acids which give the antibody its specificity for binding antigen. The variable region includes
the ends of the light and heavy chains. Treating the antibody with a protease can cleave this region,
producing fragment antigen binding (Fab) that include the variable ends of an antibody. The lower parts of
the ―Y‖ arms is called the constant regions which is the stem of monomer and it is important in binding to
complement or cells.
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The basic structure of the heterotetrameric Immunoglobulin gamma (IgG)

Antibody Fragment
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Fab fragment

The 'F' stands for fragment and the 'ab' stands for antigen binding. This is the region where antigen binding
takes place. The Fab portion of an antibody is made up of the N-terminal domains of both the heavy and light
chains. This domain of the antibody is called the variable domain on the heavy chain (VH) and on the light
chain (VL) because there is such high variability between antibodies in this area. These domains allow
recognition of thousands of different antigens.

F(ab')2 fragment

F(ab')2 fragment is the two antigen binding fragments linked by cysteine bonds and it is formed when Pepsin
cleaves a whole immunoglobulin. The 'prime ( ' )' is included in F(ab')2 because it contains more amino acids
than the F(ab) alone.

Fc fragment

The 'F' stands for fragment and the 'c' stands for crystallizable or constant. The Fc portion of the antibody is
limited in variability and is responsible for the biological activity of the antibody (as opposed to antigen
binding). The Fc portion varies between antibody classes (and subclasses) but is identical within that class.
The C-terminal end of the heavy chains forms the Fc region. The Fc region plays an important role as a
receptor binding portion. The Fc portion of antibodies will bind to Fc receptors on phagocytic cells (like
macrophages) thereby inducing phagocytosis.

How Do B Cells Produce Antibodies?

B cells develop from stem cells in the bone marrow of adults (liver of fetuses). After maturation B cells
migrate to lymphoid organs (lymph node or spleen).

Clonal Selection: When a B cell encounters an antigen it recognizes, it is stimulated and divides into many
clones called plasma cells, which actively secrete antibodies.

Each B cell in an organism synthesizes only one kind of antibody. In an organism, there is an entire
population of different types of B cells and their respective antibodies that were produced in response to the
various antigens that the organism had been exposed to.

Specific Immunity is achieved through:

• T and B lymphocytes
• Specific response to a single antigen
• Memory for that response
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Different kinds of antibodies

There are five different isotypes of antibody depending on the difference in their heavy chain.

IgG: Heavy chain γ

This is the principle antibody found in blood and body fluids. Around 75% of the antibody circulating in the
blood is IgG, and it is this isotype which provides the majority of the antibody-mediated protection against
infection. IgG is rarely produced during an initial response to a given pathogen and it is not produced until
around one month initial B cell activation.

IgM: Heavy chian μ

The IgM isotype is expressed on the surface of B cells, but it is also secreted by cells. IgM is typically
involved early in an immune response, before B cells have had time to begin secreting large quantities of
IgG. IgM is a more efficient complement trigger because a single IgM-antigen complex can trigger the
cascade, whereas multiple IgG-antigen complexes are required.

IgA: Heavy chain α

The IgA is involved in mucosal immunity, preventing colonization by bacteria in areas which are otherwise
unprotected from pathogenic attack. This antibody is found in mucosal areas
In addition IgA is secreted in tears, saliva, and breast milk. In breast milk, IgA provides nursing infants with
passive immunity against pathogens the mother has encountered.

IgE: Heavy chain ε

This IgE isotype plays an important role in immune responses to certain parasites particularly parasitic
worms. IgE is also strongly associated with allergic reactions, due to its ability to trigger granulocytes to
release their toxic chemicals when the antibody comes into contact with its specific antigen.

IgD: Heavy chain δ

The function of IgD is not particularly well-defined. This antibody appears as an antigen receptor on naive B
cells (those which have not yet been activated by antigen), and provides a signal to B cells at the end of their
maturation process in the spleen.
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Functions of antibodies

A. Antigen binding - Immunoglobulins bind specifically to one or a few closely related antigens. Each
immunoglobulin actually binds to a specific antigenic determinant. Antigen binding by antibodies is the
primary function of antibodies and can result in protection of the host.

B. Effector Functions - The consequence of the binding of an antibody to an antigen has the significant
biological effects of the secondary "effector functions" of antibodies which include:

1. Fixation of complement - This result in lysis of cells and release of biologically active molecules

2. Binding to various cell types - The binding of antibodies to the receptors on cells like Phagocytic cells,
lymphocytes, platelets, mast cells, and basophils can activate the cells to perform some function. Some
immunoglobulins also bind to receptors on placental trophoblasts, which results in transfer of the
immunoglobulin across the placenta to provide immunity to the fetus and newborn.

Antibody Production

Antibody Production with the use of animals

Monoclonal antibodies are produced from a single clone of a B-cell and are derived from a single genetic
code. These homogeneous populations of highly specific antibodies recognize only a single epitope and will
not recognize other epitopes that may be present in the same antigen.

Monoclonals detect a single epitope; hence mono-clonal. They have High specificity and low avidity.

Polyclonal antibodies are mixtures of antibodies generated from more than one clone of B-cells as a
response to specific antigen(s) when an animal is immunized. The antibodies are isolated from the normal
serum as a group, which is the fluid component that separates from clotted blood and each antibody recognizes a
different epitope of the antigen(s) but have different degrees of specificity. The antibodies obtained from the
blood of an immunized animal are called polyclonal antibodies.

Polyclonals detect several epitopes; hence poly-clonal. High avidity means a polyvalent reagent; may be
specific, although higher chance of sensitivity.
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Monoclonal antibodies

Monoclonal antibodies (mAb or moAb) are mono-specific antibodies that are identical because they are
produced by one type of B lymphcytes that are all clones of a single parent cell. It is possible to create
monoclonal antibodies for a given substance that specifically bind to that substance; and they can then serve
to detect or purify that substance.

These antibodies that come from a single immune cell called monoclonal antibodies (mAbs). mAbs have
become ubiquitous tools in biomedical science and medicine. Some uses are:

a) They have been used to diagnose and combat diseases even viral diseases considered ‗untreatable‘
because mAbs have the ability to bind to cell-specific antigens

b) Researchers use monoclonal antibodies to identify and to trace specific cells or molecules in an
organism, e.g. Monoclonal antibodies have been used to identify which proteins are responsible for
cell differentiation in the respiratory system.

c) Antibodies are used in several diagnostic tests to detect small amounts of drugs, toxins or hormones,
e.g. monoclonal antibodies to human chorionic gonadotropin (HCG) are used in pregnancy test kits.
Another diagnostic uses of antibodies is the diagnosis of AIDS by the ELISA test.

d) Monoclonal antibodies can be used to classify strains of a single pathogen, e.g. Neisseria
gonorrhoeae can be typed using monoclonal antibodies.

e) mAbs have emerged as effective therapeutic treatments for cancer, various auto-immune disorders,
and other diseases.

f) In the area of toxicological research, mAbs are frequently used as capture reagents to detect and
measure protein and drug levels in biological fluids
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Production of monoclonal antibodies

The production of monoclonal antibodies was pioneered by Georges Kohler and Cesar Milstein in 1975.

Details of Production

(1) Immunization of a mouse

(2) Isolation of B cells from the spleen
(3) Cultivation of myeloma cells
(4) Fusion of myeloma and B cells
(5) Separation of cell lines
(6) Screening of suitable cell lines
(7) in vitro (a) or in vivo (b) multiplication
(8) Harvesting
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In order to isolate a B lymphocyte producing a certain antibody, the production of such a B cell in an
organism will have to be induced. For example, if we need an antibody for avian SERCA2 protein, we would
inject the protein into a mouse (immunizing a mouse). This is typically done in two doses, an initial
"priming" dose and a second "booster" dose 10 days later. Then the mouse immune system recognizes the
foreign protein and some of its B cells would begin the production of the antibody to avian SERCA2.

A sample of B cells is extracted from the spleen of the mouse and added to a culture of myeloma cells
(cancer cells). The fusion of a B cell and a myeloma cell results is the formation of hybridomas cells. This
fusion is done by using polyethylene glycol, a virus or by electroporation.

The next step is the selection of the hybridomas. The myeloma cells are HGPRT- and the B cells are
HGPRT+. HGPRT is hypoxanthine-guanine phosphoribosyl transferase, an enzyme involved in the
synthesis of nucleotides from hypoxanthine (an amino acid). The culture is grown in HAT (hypoxanthine-
aminopterin-thymine) medium, which can sustain only HGPRT+ cells. The myeloma cells that fuse with
another myeloma cell or do not fuse at all die in the HAT medium since they are HGPRT-. The B cells that
fuse with another B cell or do not fuse at all die because they do not have the capacity to divide indefinitely.
Only hybridomas between B cells and myeloma cells survive, being both HGPRT+ and cancerous.

The fusion creates hybridoma cells that secrete antibodies into fluid that surrounds them as they grow. The
hybridomas are either injected into the abdomen of a second mouse (the ascites method –the hybridoma cells
is injected into the peritoneum, which serves as a growth chamber for the cells. The hybridoma cells grow to
high densities and continue to secrete the antibody of interest,) or cultured in flasks or bioreactors (the in
vitro methods) to produce large amounts of mAb-containing fluid.
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Purification of monoclonal antibodies

There is need to purify the antibodies since in the in vitro case the cell culture sample would contain
contaminants which are primarily the media components such as growth factors, hormones, and transferrins.
However, the in vivo sample is likely to have host antibodies, proteases, nucleases, nucleic acids, and
viruses. In both cases, other secretions by the hybridomas such as cytokines may be present.

The purification methods are simply:

1. Affinity purification
2. Antibody precipitation
3. Using a chromatogram to identify the impurities
It is well established that the ascites method of mAb production causes discomfort, distress, and pain to the
animals involved, hence developed countries have effectively banned this mouse method.
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Humanized Antibodies

Humanized antibodies are antibodies from non-human species whose protein sequences have been modified
to increase their similarity to the naturally produced antibody variants in humans.

The process of "humanization" is usually applied to monoclonal antibodies developed for administration to
humans (for example, antibodies developed as anti-cancer drugs). Humanization can be necessary when the
process of developing a specific antibody involves generation in a non-human immune system (such as that
in mice).

The protein sequences of antibodies produced in this way are partially distinct from homologous antibodies
occurring naturally in humans, and are therefore potentially immunogenic when administered to human
patients.
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LIMITATIONS OF THIS METHOD OF MONOCLONAL PRODUCTION OF ANTIBODIES

1. These in vitro methods are also laborious, slow, and produce antibodies that often cause immune
reactions in patients, limiting their use as clinical therapies.
2. Mouse and other animal-derived antibodies must be altered, or ―humanized‖, before they can be
administered to humans.
3. Humanization of antibodies is an imperfect process that is fraught with uncertainty.
4. There is no guarantee that a humanized antibody will perform the same as its animal-derived predecessor
and
5. The immune reactions varying in degree from skin rashes and hypersensitivity to reactions that are more
sever may still occur.
Fortunately, an alternative to the animal-based methods of monoclonal antibody production exists that can
completely replace the use of animals.
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The synthetic antibodies called recombinant antibodies (rAbs) can be created using antibody genes made in a
laboratory or taken from human cells, eliminating hybridomas and animals from the antibody-production
process. Recombinant antibodies can be used in all applications in which traditional mAbs are used and have
advantages of their own.

Examples of therapeutic monoclonal antibodies

Main category Type Application Mechanism/Target Mode

 rheumatoid arthritis
 Crohn's disease
infliximab  Ulcerative Colitis inhibits TNF-α chimeric
 ankylosing spondylitis

 rheumatoid arthritis
 Crohn's disease
Anti- adalimumab  Ulcerative Colitis inhibits TNF-α human
inflammatory  ankylosing spondylitis

 Acute rejection of
inhibits IL-2 on activated
basiliximab kidney transplants chimeric
T cells

 moderate-to-severe
inhibits human
omalizumab allergic asthma humanized
immunoglobulin E (IgE)

 B cell leukemia targets an antigen CD52

alemtuzumab humanized
on T- and B-lymphocytes
 non-Hodgkin's
lymphoma targets phosphoprotein
rituximab chimeric
 rheumatoid arthritis CD20 on B lymphocytes

Anti-cancer  Approved in squamous

cell carcinomas,
cetuximab EGFR inhibitor chimeric
colorectal carcinoma

 Anti-angiogenic cancer
bevacizumab &
therapy inhibits VEGF humanized
ranibizumab

 cancer, hepatitis C
Anti-cancer and immunotherapy, targets
bavituximab[39] infection chimeric
anti-viral phosphatidylserine
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Recombinant Antibodies

Recombinant antibody engineering involves the use of viruses or yeast to create antibodies, rather than using
mice. Advances in molecular biology (recombinant DNA technology) have lead to the ability to synthesize
antibodies de novo in vitro – completely without the use of animals.

The term recombinant DNA regards to an artificial methods of producing DNA (synthetic DNA and
proteins). The manufacturing technique makes use of bacteria which are genetically modified in such a way
that a specific DNA segment (gene) is artificially combined with their original DNA and then inserted back
to the bacteria by transformation. The bacteria are nourished and kept in ideal conditions for reproduction
and after a relative short period of time of reproduction, these specific segments of DNA which have now
been multiplied are extracted out of the original DNA bacteria.

Using recombinant antibody has significant advantages compared with the conventional antibody and its use
is therefore becoming more popular now days. The fact that no animals are needed in the manufacturing
procedure of the recombinant antibodies, in addition, the manufacturing time is relatively short compared
with the conventional method. Moreover, the quality of the final product is higher that these of the non
recombinant method.

Production of Recombinant Antibodies

The advent of recombinant DNA technology laid the foundation for recombinant antibody production by
enabling the combination of genetic material from two or more sources.

These techniques rely on rapid cloning of immunoglobulin gene segments to create libraries of antibodies
with slightly different amino acid sequences from which antibodies with desired specificities can be
selected. Recombinant antibodies are translated from recombinant DNA and displayed on the surfaces of
cells or phage particles. Hence, the production of recombinant monoclonal antibodies involves technologies
referred to as repertoire cloning or phage display/yeast display.

In 1990, John McCafferty demonstrated that antibody fragments (variable regions from antibodies) could be
displayed on viruses that infect bacteria called bacteriophages or phages, by introducing antibody DNA into
phage genomes via vectors. Then, researchers have created libraries of antibody genes to display on phages
and developed methods to successfully isolate individual antibodies from the large phage-displayed libraries.
This method has become known as antibody phage display.
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Since 1990, researchers have expanded antibody display technology to microorganisms (yeast, bacteria)
other than phage. Koder and Wittrup in 1997 documented the display of antibody fragments on
Saccharomyces cerevisiae yeast.

General production methods for the manufacture of non-animal recombinant antibodies can be broken down
into five general steps:

1. creation of an antibody gene library;

2. display of the library on phage or cell surfaces;

3. isolation of antibodies against the antigen of interest;

4. modification of the isolated antibodies; and

5. scaled up production of selected antibodies in a cell culture expression system:

I. Antibody Gene Libraries: Antibody gene libraries consist of the collection of microorganisms
(bacteria) that have been transformed with the genes encoding the variable regions of different
antibodies. [The variable region genes can either be synthesized in vitro or amplified from the genetic material in
human antibody-producing B cells. Variable region genes are used instead of genes for whole antibody molecules because
fragments of antibodies are more easily assembled in microorganisms than whole antibody molecules and the variable
regions of an antibody are the most important fragments in terms of function.] Each variable region gene is spliced
(joined) into a vector (vehicles that transfer foreign genetic material into another cell), and the vector
(phage –bacterial virus) is taken up and integrated into the genome of a microorganism.

II. Library Display: Genes encoding antigen-binding variable domains of antibodies are fused to phage
genes that encode coat protein, thereby allowing the phage coat to express, or ―display‖, the antibody
fusion protein. A collection of recombinant phage that displays unique antigen binding domains on
their surfaces is known as a phage display library. Display on the cell or phage surface of the
cloned antibody sequence allows for rapid, high throughput (volume) selection of recombinant
antibodies that bind a specific antigen.

III. Antibody Isolation: Once the rAbs are displayed, tools such as paramagnetic beads, fluorescence-
activated cell sorting (FACS), and/or Enzyme-Linked Immunosorbent Assays (ELISAs) can be used
to isolate individual antibodies that bind to a specific antigen target. Library members (antibody-
displaying microorganisms) are incubated with the target molecule (antigen of interest) and unbound
library members are washed away. Bound rAbs are removed from the antigen and screened for
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desirable characteristics. The result is a library enriched for target-binders. This process can be
repeated as needed to achieve the desired specificity.

IV. Modification: Bacteria, yeast or phage encoding the selected antibodies are grown in greater
quantities and are put through the selection process again to enrich for the strongest binding (highest
specificity) candidates. If the affinities of the lead candidates are not strong enough, antibodies with
higher specificity can be generated through "maturation" by random or rational mutagenesis.

V. Antibody Expression: Once an antibody is selected, the genes for that antibody are transferred via
expression vectors into an expression system—bacteria, yeast, or mammalian cell lines specially
designed for the expression of foreign proteins. The choice of vector and expression system depends
on the type of antibody that is to be produced.

It is possible to perform all of these techniques in a standard molecular biology lab or the process can be
outsourced to a variety of facilities specializing in the generation of rAbs.

Uses of recombinant antibodies

These include:

i. Diagnostic test that discriminates between closely related pathogens or antigens

ii. Production of binders to difficult agents especially in areas where conventional antibodies often
fail
iii. Highly pathogenic agents and emerging diseases
iv. Conserved mammalian epitopes
v. Small and less immunogenic haptens
vi. Detection of non-protein moieties, for example, toxins
vii. Range of highly specific reagents for use in multiplex assays

Advantages of Recombinant Antibody Use

1. Non-animal technology: Recombinant antibodies derived from synthetic or human antibody libraries
are an entirely non-animal technology. This alleviates animal welfare concerns associated with
traditional monoclonal antibody production.
2. Speed: Once an antibody library is established, a researcher skilled in the art of recombinant antibody
production can furnish an antigen-specific antibody suitable for research purposes in as little as 8
weeks. This is significantly shorter than the 4 or more months required for hybridoma technology.
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3. Control of production process: Recombinant antibody production gives researchers control over the
state of the antigen to which they are making antibodies against. With traditional mAb technology
researchers lose control after injecting the antigen into an animal. Inside the animal an antigen could
be processed into something different or cut into pieces and antibodies may be made against these
altered versions of the antigen instead. Animal-derived antibodies must be tested extensively after
they are created in the hope that an antibody against the intended antigen has been made.
4. Isotype Conversion: Once a desirable antibody fragment is found it can be easily converted into any
antibody isotype (e.g. IgA, IgM IgG etc.) from any species by adding the appropriate constant
domain.
5. Concern on antigen purity: Recombinant antibodies from antibody gene libraries require less
purified antigen to produce and eliminate constraints on the types of antigens that can be used.
6. Creation of varied types of antibodies: Antibodies to highly toxic or non-immunogenic antigens can
be created using library methods, unlike animal immunization technologies.
7. Increased affinity of antibodies: The affinity of antibodies derived from libraries can be increased to
levels unobtainable by an animal‘s natural immune system.
8. High volume of production: Recombinant antibodies from antibody gene libraries are also amenable
to high-throughput production,—a fact that makes them attractive to the field of proteomics since the
antibody gene sequences used are derived from human or synthetic genes,
9. Lack of immunological reactions: Recombinant antibodies do not trigger the intense immunogenic
reactions in patients that animal-derived antibodies do, making them ideal for clinical use.

Impediments to Recombinant Antibody Use

1. Intellectual property: Recombinant antibodies represent the fastest growing segment of today‘s
pharmaceutical industry. As a consequence, commercial interests have restricted access to improved
methods of generating rAbs to protect their intellectual property.
2. Initial Investment: Recombinant antibody technology requires a substantial investment of time and
energy in order to implement; laboratories needing only a few antibodies may be unwilling to make
the investment to learn and implement this new technique.
3. Technical Expertise and Robotics: The library design step can be bypassed by acquiring aliquots of
a proven, pre-made library. However, sorting through the immense libraries may also present an
obstacle to embracing this technology.
4. Maturation and Structure Conversion: Upon repeated exposure to an antigen, an animal will create
progressively stronger antibodies against it. This process, called affinity maturation, may repeat itself
several times until strong antibodies that can neutralize an antigen are generated.
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INSULIN PRODUCTION

The Insulin:

Insulin is a peptide hormone mainly used in treatment of diabetes mellitus to control elevated
blood glucose level. It is secreted by the β-cells of islets of Langerhans of the pancreas. It was
discovered by sir Edward Sharpey Schafer in 1916 while studying Islets of Langerhans but
was first isolated from dog pancreas in 1921 by Frederick Banting and Charles Herbert
Best.

Structure of Human Insulin:

Frederick Sanger sequenced the amino acid structure in 1951, which made insulin the first
protein to be fully sequenced. The crystal structure of insulin in the solid state was
determined by Dorothy Hodgkin in 1969. The Human insulin is a small, simple protein
composed of 51 amino acids sequences and has a molecular weight of 5808 Da. It is a dimer
of a A- chain and a B-chain linked/joined together by two inter-chain and one intra-chain
disulphide bond between two cysteine residue
Insulin consists of two polypeptide chain,
- Chain A- 21 amino acids long
- ChainB- 30 amino acids long
Both chains are joined together by disulphide bond

Insulin produced inside pancreas:

Pancreatic β-cells first synthesize inactive pre-pro-insulin, which is a 109 amino acids long
polypeptide and among these amino acids are 23 amino acids which are signal molecules that
allow the pre-pro-insulin to pass through cell membrane. Upon entering inside cell, it
becomes 86 amino acids long pro-insulin, also inactive. Some Proteolytic enzymes then cut
and expose the active site of pro-insulin converting it into active form of insulin of 51 amino
acids long.
Originally insulin was first identified from dog pancreas which was commercially produced
from various sources like foetal calf pancreas obtained from slaughter houses.

Genetic engineering in the production of Human insulin

Insulin produced by recombinant DNA technology.

Now, human insulin protein is mass-produced through genetic engineering processes.

Recombinant DNA technology has been a great enabler in producing human insulin outside
the body for being used as a therapeutic. Insulin is the first human hormone (protein) to be
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chemically synthesized and produced in bacteria by DNA recombinant technology to be

tested in humans for medical purposes.

There are many methods for the production of recombinant human insulin in both bacteria
and yeast.

Manufacturing of insulin using microbes as a cell factory involves the following general
steps:

1. Isolation of Insulin gene: The gene for producing human insulin protein is isolated
from β-cell
2. Preparation of suitable vector (target DNA): Circular piece of DNA called plasmid
is obtained from bacteria E. coli and then it is cut open by restriction endonuclease
enzyme.
3. Insertion of DNA (Insulin coding gene) into plasmid: The gene for insulin is
inserted into the plasmid construct. The human insulin gene is now recombined with
bacterial DNA (plasmid) by DNA ligase enzyme.
4. Recombined Plasmid insertion: The bacterial plasmid having insulin gene is inserted
back into suitable bacteria (E. coli).
5. Plasmid multiplication: The bacterial cells having insulin gene are allowed to grow
and multiply and during this process bacterial cells start to produce recombinant
insulin hormone. During division newly synthesized copy of cell are produced.
6. Purification of Human insulin: The hormone produced by bacteria is purified.
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There are two typical ways of producing human Insulin by genetic engineering. One of the
methods is:

1. The chemically synthesize DNA sequence of insulin for two chains A and B and
separately inserted into two PBR322 plasmid vector.
2. These genes are inserted by the side of β-galactosidase gene of the plasmid.
3. The recombinant plasmids were then separately transformed into E. coli host.
4. The recombinant host produced pro-insulin chains i.e. fused β-galactosidase-A chain
and β-galactosidase-B-chain separately.
5. These pro-insulin chains A and B were separated from β-galactosidase by treating the
bacterium DNA with cyanogen bromide, a reagent that splits protein chains at the
methionine residues. This separates the insulin chains from the rest of the DNA.
6. After detachment, the two chains A and B chains are then mixed together and joined in
vitro by disulphide bonds through redox reaction to reconstitute the naïve insulin by
sulphonating the peptide chains with sodium disulphonate and sodium sulphite.
7. The DNA mixture is then purified so that only the insulin chains remain.
Manufacturers can purify the mixture through several chromatography, or separation,
and the procedures used include an ion-exchange column, reverse-phase high
performance liquid chromatography, and a gel filtration chromatography column.

Diagrammatic representation of insulin production by genetic engineering

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Another method of insulin production by recombinant DNA technology is designed by

Gilbert and Villokomaroff. In this method, direct precursor to insulin gene, proinsulin is
being used. Many of the steps are the same as when producing insulin with A and B chains
except:
1. mRNA for pre-pro-insulin is isolated from islets of Langerhans cell
2. mRNA is reverse transcribed to form DNA and then it is inserted into PBR 322
plasmid in the middle of the gene for penicillinase.
3. Then the recombinant plasmid is transformed into suitable host i.e. non-pathogenic E.
coli cell. The bacteria go through the fermentation process where it reproduces and
produces proinsulin.
4. The host produced penicillinase + pre-pro insulin
5. Insulin is later separated by trypsin treatment
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GENE THERAPY

What is Gene?

Genes are the basic unit of heredity and are carried on a chromosome in the nucleus of a cell. They are
specific sequences bases that encode instructions to make proteins (DNARNA proteins). Although
genes get a lot of attentions but it is the proteins that perform most life functions. When genes are altered,
encoded proteins are unable to carry out their normal functions, resulting in genetic disorders.

A mutation in the gene will change the codon (the nucleotides that specifies a particular amino acid) which
will change amino acids which in turn change conformation of the protein and this change the functions in
the protein. Hence, alteration in genes causes dysfunction of a protein.

What is Gene Therapy?

Gene therapy is the use of genes to correct defective genes that are responsible for disease development
(genetic disorder). The defective genes can be corrected using the following four approaches:

1. A normal gene could be inserted into a nonspecific location within the genome to replace the
nonfunctional gene (most common).

2. An abnormal gene could be swapped for a normal gene through homologous recombination

3. An abnormal gene could be repaired through selective reverse mutation, which returns the gene to its
normal function.

4. Change the regulation (the degree to which a gene is turned on or off) of a particular gene pairs.

To design and carry out a gene therapy treatment, a researcher must:

1. Identify the gene(s) responsible for the disorder.

2. Make copies of the normal gene.

3. Insert the copies into vectors.

4. ―Infect‖ the affected cells with the vectors.

5. Activate the gene so that transcription and translation take place.

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The Flow chart shows gene therapy

Historical Background of Gene Therapy

In the 1980s, Scientists began to look into gene therapy.

 They would insert human genes into a bacteria cell.

 Then the bacteria cell would transcribe and translate the information into a protein
 Then they would introduce the protein into human cells

The first gene therapy was performed on September 14th, 1990

 Ashanti DeSilva was treated for Severe combined immunodeficiency (SCID

 Doctors removed her white blood cells, inserted the missing gene into the WBC, and then put them
back into her blood stream.
 This strengthened her immune system and
 Only worked for a few months
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How Gene Therapy works

 Modified gene is inserted into the vector (virus)

 A vector delivers the therapeutic gene into a patient‘s target cell
 The target cells become infected with the viral vector
 The vector‘s genetic material is inserted into the target cell
 Functional proteins are created from the therapeutic gene causing the cell to return to a normal state

Viruses as Vectors

 Replicate by inserting their DNA into a host cell

 Gene therapy can use this to insert genes that encode for a desired protein to create the desired trait

 Four different types

 Adenovirus

 Adeno-Associated Virus (AAV)

 Retrovirus

 Herpes Simplex Virus (HSV)

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Vector Advantages and Disadvantages

• Adenovirus – a double stranded DNA genome that cause respiratory, intestinal and eye infections in
humans
+ Infects many cell types
- Does not integrate into host genome and can be lost.
• Retrovirus – created double stranded DNA copies from RNA genome
+ Integrates into host genome and cannot be lost
- Integrates into host genome at anywhere and can cause cancer
• Adeno-Associated Virus (AAV) – small, single stranded DNA that insert genetic material at a
specific point on chromosome 19. It causes no known disease.
+ Integrates into host genome, does not trigger patient immune response and cannot be lost
- Difficult to work with (low information capacity)
• Herpes Simplex Virus (HSV) – double stranded DNA virus that infect neurons
+ DNA stays in nucleus without integrating into host genome.
- Only infects cells of the nervous system.
Gene Therapy Disappointments
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• In 1999 a boy died due to an immune response to an adenovirus gene therapy vector.

• Four children have developed cancer due to a retrovirus gene therapy vector

Problem of Gene therapies

One problem with gene therapy is that one does not have control over where the gene will be inserted into
the genome. The location of a gene in the genome is of importance for the degree of expression of the gene
and for the regulation of the gene (the so-called "position effect"), and thus the gene regulatory aspects are
always uncertain after gene therapy

Current Status

 FDA hasn‘t approved any human gene therapy product for sale

Reasons:

1. In 1999, 18-year-old Jesse Gelsinger died from multiple organ failure 4 days after treatment for
omithine transcarboxylase deficiency.

 Death was triggered by severe immune response to adenovirus carrier

2. January 2003, There was a halt to using retrovirus vectors in blood stem cells because children
developed leukemia-like condition after successful treatment for X-linked severe combined
immunodeficiency disease

Gene Therapy Successes

Although no gene therapies have been approved by the FDA for sale, some diseases have been
experimentally successful:

– Melanoma (skin cancer)

– Severe Combined Immunodeficiencies (SCID)

– Hereditary Blindness

– Sickle Cell Anemia

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BIOTECHNOLOGY IN VACCINES DEVELOPMENT

What is a vaccine?

A vaccine is a biological preparation that improves immunity to a particular disease in the body of the host.
A vaccine contains a disease-causing microorganism which have been weakened or killed but have the
ability to induce specific immune system of the body by producing antibodies against those agents in the body
without creating any disease in the host.

Biotechnology is used in three different ways in the development of vaccine:

1. Separation of a pure antigen using a specific monoclonal antibody.

2. Synthesis of an antigen with the help of a cloned gene.
3. Synthesis of peptides to be used as vaccines.

DNA VACCINES

DNA vaccination is a novel technique for protecting an animal against disease by injecting it with
genetically engineered DNA and the cells directly produce an antigen thereby resulting in a protective
immunological response. This vaccination efficiently stimulates humoral and cellular immune responses to
protein antigens.

Several DNA vaccines have been released for veterinary use, and there has been promising research using
the vaccines for viral, bacterial and parasitic diseases, as well as to several tumor types. Although only one
DNA vaccine has been approved for human use,

What is DNA Vaccine?

 DNA vaccine is DNA sequence used as a vaccine which code for antigenic protein of pathogen.

 The DNA sequence when inserted into the cells, get translated to form antigenic protein which is
foreign to cells.

 This then stimulates immune response against the protein.

 In this way, DNA vaccine provides immunity against that pathogen.

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HISTORY

 In 1990, University of Wisconsin, Jon Wolff found that injection of DNA plasmids produce a protein
response in mice.

 In 1993, Merck Research Laboratories, Dr. Margaret Liu found that intramuscular injection of DNA
from influenzae virus into mice produced complete immune response

COMPARISON OF DNA VACCINES AND TRADITIONAL VACCINES

DNA VACCINES TRADITIONAL VACCINES

1. Uses only the DNA from infectious Uses weakened or killed form of infectious
organisms. organism.
2. Avoid the risk of using actual infectious Create possible risk of the vaccine being
organism. fatal.
3. Provide both Humoral & Cell mediated Provide primarily Humoral immunity
immunity
4. Refrigeration is not required Usually requires Refrigeration.

HOW DNA VACCINE IS MADE

1. The DNA sequence which code for antigenic protein of a pathogen is removed and inserted into an
expression plasmid.
2. The plasmid with foreign gene is transformed into a bacterium for multiplication (amplification) and
then purified.
3. Immunize the host or human with immunogen expressing plasmid

METHODS OF DELIVERY

1. Syringe delivery: Either intramuscularly or intrademally

2. Gene gun delivery: Adsorbed plasmid DNA into gold particles and it is ballastically accelerated into
the body with gene gun.
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ADVANTAGES
 Elicit both Humoral & cell mediated immunity
 Subunit vaccination with no risk for infection
 Immune response focused only on Antigen of interest
 Ease of development and production
 Long term persistence of immunogen (immunity)
 Refrigeration is not required
 Stable of vaccine for storage and shipping
 Cost effectiveness
DISADVANTAGES
 Limited to protein immunogen only (not useful for non protein based antigens
 Risk of affecting genes controlling cell growth
 Possibility of inducing antibody production against DNA
 Extended immunostimulation leads to chronic inflammation
 Some antigen require processing which sometime does not occur