An Assignment of Biotechnology(Pharm 453)

1. Designing of Drug Delivery System for
Biotechnology Products Considering
Stability Aspects and Monitoring Methods
of Improving Stability
An assignment submitted to Mohammad Shahriar, Associate Professor,
Department of Pharmacy , University of Asia Pacific in partial fulfillment of the
requirement for the completement of the course of Pharmaceutical
Biotechnology (Pharm 453).
Submitted By
Registration No.: 15203002
15203014
15203015
15203021
15203038
Submission Semester: Spring 2019
Submission Date: 9
th
July 2019
Department of Pharmacy
University of Asia Pacific

2. Summary
Our world is built on biology and once we begin to understand it, it then becomes a technology.
The discovery of insulin in 1922 marked the beginning of research and development to improve
the means of delivering biotechnology products. From that period forward, investigators have
contemplated every possible route of delivering biotechnology products. In recent years, the
pharmaceutical industry has used different technologies to obtain new and promising
biotechnology products as exemplified by the gene therapy, recombinant DNA technique,
monoclonal antibodies, polymerase chain reaction, peptide technology, antisense technology and
so on. The advancement in recombinant DNA technology has made a great impact on the success
of the development of these products. This technology allows for manipulation of DNA
fragments from different sources, such as inserting a human gene into a bacterial plasmid. This
ability to manipulate DNA fragments along with the ability to insert the recombinant DNA into
different cells can be used for the production of therapeutic proteins and peptides such as insulin
hormone and monoclonal antibodies. The final aim of pharmacy and medicine is the delivery of
biotech products at the right time in a safe and reproducible manner to a specific target at the
required level. Gene therapy and RNAi technologies are considered the medical treatments of the
future. Furthermore, novel, harmless viral vectors and non-viral gene therapy systems such as the
‗gene gun‘, liposomes, microfabricated systems combine the principles of microtechnology and
biology are also under investigation. In addition most of the biotechnology products are usually
protein and enzyme based. So, the ability of proteins and enzymes to maintain a functionally
active conformation under adverse environmental conditions is the most crucial factor. In our
topic, we also tried to discuss bioinformatic-driven strategies that are used to predict structural
changes that can be applied to wild type proteins in order to produce more stable variants. The
most commonly employed techniques PEGylation, stochastic approaches, empirical or
systematic rational design strategies. Finally, we want to say, overcoming the current obstacles,
including government regulations, financial support, and large-scale production and
manufacturing will lead us to a day where all biotechnology products are delivered in a targeted
and safer manner.
i| P a g e

3. Table of Contents
Sl. No. Topic Page
1.0 Abstract i
2.0 Table of Contents ii
3.0 List of Tables vi
4.0 List of Figures vi
Chapter 1- Introduction
Sl. No. Topic Page
1. Introduction 2
1.1 General Concept 2
1.2 Biotechnology 3
1.3 Branches of Biotechnology 4
1.4 History of Biotechnology 4
1.4.1 Ancient Technology 4
1.4.2 Classical Biotechnology 5
1.4.3 Modern Biotechnology 5
1.5 Routes of Drug Delivery System 6
1.5.1 Oral Drug Delivery 6
1.5.2 Nasal Drug Delivery 6
1.5.3 Transdermal Drug Delivery 7
1.5.4 Parenteral Drug Delivery 7
1.5.5 Rectal Drug Delivery 8
1.5.6 Buccal Drug Delivery 8
1.5.7 Vaginal Drug Delivery 8
ii

4. Chapter-2 Production Processes for Biotechnology Based Products
Sl. No. Topic Page
2. Designing of Drug Delivery 11
System
2.1 Recombinant DNA Technology 12
2.1.1 Steps of Constructing a 12
Recombinant DNA
2.1.2 Applications of r-DNA 15
Technology
2.2 Monoclonal Antibodies: 17
2.2.1 Available Monoclonal Antibody 19
(Drug Product)
2.3 Gene Therapy and Types 20
2.3.1 Somatic Gene Therapy 20
2.3.2 Gene Therapy Clinical Trials: 23
The US Scenario (Accomplished
Clinical Trials)
2.4 The PCR ( Polymerase Chain 23
Reaction ) Technology and DNA
Sequencing
2.4.1 Applications of Polymerase 26
Chain Reaction (Pcr)
2.5 Antisense Technology Based 27
Available Product
2.5.1 Antisense Oligonucleotides: 27
Molecular Mechanisms
2.5.2 Antisense Technology based 31
Available Product
2.6 Peptide Technology 32
iii

5. Sl. No. Topic Page
2.7 Emerging Delivery Methods 32
2.7.1 Viral Vectors 32
2.7.1.1 Importance of Vectors in Gene 34
Therapy
2.7.2 RNAi Technologies 36
2.7.2.1 RNAi Mechanism 37
2.7.2.2 Applications of RNAi 38
Technology
2.7.3 Non-viral Gene Therapy 39
2.7.3.1 Physical Methods of Non Viral 40
Gene Therapy
2.7.4 Liposomes 41
2.7.4.1 Liposomal Vaccine and Antigen 42
Delivery System
2.7.4.2 Liposome in Combination 42
Therapy
2.7.4.3 Liposome in Delivery of Nucleic 43
Acids
2.7.5 Encapsulation Techniques 43
2.7.5.1 Microencapsulation 44
Chapter-3 Current Biotechnology Products
Sl. No. Topic Page
3. Current Biotechnology Products 46
3.1 Antibiotics 46
3.2 Hormones 47
3.3 Enzymes 49
3.4 Blood Clotting Factors 50
iv

6. Sl. No. Topic Page
3.5 Cytokines 51
3.6 Interferon‘s 52
3.7 Interleukins 53
3.8 Monoclonal Antibodies 54
3.9 Vaccines 54
3.10 A Summary of Commercially 56
Available Leading Biotechnology
Based Products
Chapter-4
Formulation and Characterization of Biotech Products: Considering
Stability Aspects and Monitoring
Sl. No. Topic Page
4. Standard Stability Aspects for 59
Biotech Product
4.1 Environmental conditions 59
4.2 Problems Associated 63
With Biotechnology Products
4.2.1 Chemical Degradation 64
4.2.2 Physical Degradation 64
4.3 Formulation and Delivery 65
Approaches to Overcome
Instability
4.3.1 Protein and Peptide Drugs 65
4.3.1.1 Methods of Improving 65
Stability
for Protein and Peptide Drugs
4.3.2 Nucleic Acid-based Drugs 69
v

7. Sl. No. Topic Page
4.3.2.1 Methods of Improving 71
Stability for Nucleic Acid
based Drugs
Chapter-5 Summary and Conclusions
Sl. No. Topic Page
5. Summary and Conclusions 74
5.1 References 75
List of Tables
Sl. No. Title Page
1.0 Viral Vectors Applied For 33
Gene Therapy
2.0 Important Biotechnology 57
Based Pharmaceutical
Products that are Approved for
Medical Applications
List of Figures
Sl. No. Title Page
1.0 Making of Recombinant 13
DNA
2.0 Making of Recombinant 13
DNA Product
3.0 Basic Concept of Genetic 14
Transformation
vi

8. Sl. No. Topic Page
4.0 Making of Human Insulin by Recombinant 16
DNA Technology
5.0 Pharmacologic Mechanisms of Action for 18
mAbs. Panel A, Inhibition of Cell
6.0 Gene Therapy 21
7.0 Ex Vivo and In Vivo Gene Therapy 22
8.0 The Polymerase Chain Reaction 24
9.0 PCR Temperature Cycling Profile 25
10. Antisense Oligonucleotide (ASO): Molecular 28
Mechanism
11. Occupancy-Only Antisense Mechanisms 30
12. RNA Cleavage Antisense Mechanisms Promote 31
Degradation of the Targeted RNA
13. Immune Response to Viral Vector 34
14. Gene Therapy by Using Adenovirus and 36
Retrovirus Vectors
15. Gene Therapy by Using An Adenovirus 36
16. RNAi Technology 37
17. Mechanism of RNAi Technology 38
18. Non Viral Gene Therapy 40
19. 2D Structure of Liposome 43
20. Structural Features of Liposomal Drug Delivery 44
Systems With Entrapped Molecule
21. General Chemical Structures of Penicillins and 47
Cephalosporins
22. The General Structure of an Antibody 55
23. The General Principle of Hybridoma 56
Technology
vii

9. Chapter-1
Introduction
1

10. 1. Introduction
1.1 General Concept
Drug delivery is becoming a whole interdisciplinary and independent field of research and is
gaining the attention of pharmaceutical makers, medical doctors and industry. A targeted and
safe drug delivery could improve the performance of some classical medicines already on the
market and, moreover, will have implications for the development and success of new
therapeutic strategies, such as peptide and protein delivery, glycoprotein administration, gene
therapy and RNA interference. Many innovative technologies for effective drug delivery have
been developed, including implants, nanotechnology, cell and peptide encapsulation, micro-
fabrication, chemical modification and others [Orive et al., 2003].
A major focus of drug-related research has long been the synthesis and discovery of potent,
pharmacologically active agents to manage, treat, or cure disease. Globally, the market for
pharmaceutical spending is expected to surpass $1.3 trillion by 2018. However, it is now
apparent that the therapeutic benefit and potency of a drug are not directly correlated; rather it is
linked to the method of drug formulation and delivery within the body. The mode of delivery
affects numerous factors that contribute to therapeutic efficacy, including pharmacokinetics,
distribution, cellular uptake and metabolism, excretion and clearance, as well as toxicity.
Furthermore, drugs can lose their pharmacological activity due to changes in environmental
factors such as moisture, temperature, and pH, which can occur in the body or during storage. As
the biotechnology industry continues to develop new classes of biopharmaceuticals, improved
fundamental understanding of how drug delivery affects safety and efficacy, along with new
delivery technologies, are needed [Fenton et al., 2018].
Conventional forms of drug administration generally rely on pills, eye drops, ointments, and
intravenous solutions. Recently, a number of novel drug delivery approaches have been
developed. Many drugs, both old pharmaceutical products and new molecular entities, can be
administered in ways that not only improve safety and efficacy but, in some cases, permit new
therapies. Newer and complex drugs such as proteins are becoming available through genetic
engineering; the delivery of these drugs is often more complicated than that of more
conventional drugs, necessitating novel delivery systems. These techniques have already led to
2

11. delivery systems that improve human health, and continued research may revolutionize the way
many drugs are delivered [Langer, 1990].
Advances in materials science and biotechnology are permitting the development of new
physical and chemical methods of drug delivery. Progress in the development of novel drug
delivery systems is bringing researchers and clinicians closer to meeting the goals of maximum
efficacy with minimal toxicity and inconvenience. Interest and investments in this area will
continue to provide contemporary and profound medical applications. Therefore,
biotechnological drugs swathe all drugs created by a biotechnological procedure [Saini et al.,
2011].
1.2 Biotechnology
The simplest way to define biotechnology is to split this word into its two constituent parts
(biotechnology = biology + technology). By considering these two key words we can define
biotechnology as a set of techniques that are employed to manipulate living organisms, or utilize
biological agents or their components, to produce useful products/services. The vast nature of
biotechnology has frequently made a detailed definition of the subject rather difficult [Bhatia,
2018]
Some definitions of biotechnology are as follows:
 ‗Biotechnology means any scientific application that uses biological systems, living
organisms or derivatives thereof, to produce or alter products or processes for particular
use‘

 ‗The utilization of living organisms, systems or processes constitutes biotechnology‘

 Based on the Collins English Dictionary definition, biotechnology is the employment of
living organisms, their parts or processes, to develop active and useful products and to
provide services e.g. waste treatment. The term signifies a broad range of processes, from
the use of earthworms as a source of protein to the genetic modification of bacteria to
offer human gene products, e.g. growth hormones.
It is obvious from the above definitions that biotechnology includes different technologies that
rely on information gained by modern discoveries in biochemistry, cell biology and molecular
3

12. biology. These technologies are already having a huge impact on diverse areas of life, including
agriculture, food processing, medical technology and waste treatment[Bhatia, 2018]
1.3 Branches of Biotechnology
The definition of biotechnology can be further divided into different areas known as red, green
blue and white [Bhatia, 2018].
Red biotechnology: This area includes medical procedures such as utilizing organisms for the
production of novel drugs or employing stem cells to replace/regenerate injured tissues and
possibly regenerate whole organs. It could simply be called medical biotechnology.
Green biotechnology: Green biotechnology applies to agriculture and involves such processes
as the development of pest-resistant grains and the accelerated evolution of disease-resistant
animals.
Blue biotechnology: Blue biotechnology, rarely mentioned, encompasses processes in the
marine and aquatic environments, such as controlling the proliferation of noxious water-borne
organisms.
White biotechnology: White (also called gray) biotechnology involves industrial processes such
as the production of new chemicals or the development of new fuels for vehicles.
1.4 History of Biotechnology
1.4.1 Ancient Technology
In the period before the year 1800, some events that were based on common observations about
nature can be categorized as biotechnological developments. Three important basic needs of
human civilization are food, clothes and shelter. The ancient Egyptians, for example, used yeast
to brew beer and to bake bread. Some 7,000 years ago in Mesopotamia people used bacteria to
convert wine into vinegar. And ancient civilizations exploited tiny organisms that live in the
earth by rotating crops in the field to increase crop yields. They didn't know why it worked:
Theophrastus - an ancient Greek who lived 2,300 years ago - swore that broad beans left magic
in the soil. It took another 2,200 years before a French chemist suggested in 1885 that some soil
organisms might be able to 'fix' atmospheric nitrogen into a form that plants could use as
fertilizer [Bhatia, 2018].
4

13. 1.4.2 Classical Biotechnology
Classical biotechnology is the second phase of the development of biotechnology. This stage
existed from 1800 to almost the middle of the twentieth century. In the classical era different
observations started pouring in, supported by scientific evidence. These observations made it
possible to solve the puzzles of biotechnology. Each and every observation has made its own
contribution in furthering the exploration of new discoveries. The fundamental idea of the
transfer of genetic information from one generation to another forms the core of biotechnology.
Information on the transfer of genetic information was first deciphered by Gregor John Mendel
(1822–1884), an Austrian Augustinian monk [Bhatia, 2018].
1.4.3 Modern Biotechnology
A major obstacle to scientific discoveries was the Second World War. After the war, some
essential discoveries were explored. These discoveries form the basis for modern biotechnology
and have brought this field to its current status.
Modern Biotechnology is a new technology and basically refers to cell and tissue culture and
genetic engineering. According to Cartagena protocol ―Modern Biotechnology‖ is defined as the
application of:
 In-vitro nucleic acid techniques including recombinant DNA and direct injection of
nucleic acid in to cells or organelles, or

 Fusion of cells beyond the taxonomic family, that over comes natural physiological
reproductive or recombination barriers and that are not techniques used in traditional
breeding and selection.
Recombinant DNA technology is the foundation of Modern Biotechnology. During the 1970s
scientists developed new methods for precise recombination of portions of deoxyribonucleic acid
(DNA), the biochemical material in all living cells that governs inherited characteristics, and for
transferring portions of DNA from one organism to another. This set of enabling techniques is
referred to as rDNA technology or genetic engineering [Bhatia, 2018].
5

14. 1.5 Different Routes of Drug Delivery System
Different types of routes of drug delivery system foe biotechnology are given below [[Orive et
al., 2003; Akers, 2013; Purohit et al., 2018; Irfan et al., 2016; Neves et al., 2010; Wassen et al.,
1996]:
1.5.1 Oral Drug Delivery
There is a great need in oral delivery of protein and peptide drugs, suitable devices for delivering
the therapeutic agent incorporated microspheres selectively in the intestine. Many research
groups are investigating new ways to improve the protection and absorption of peptides after oral
administration. For instance, the use of bio-adhesives has been studied to promote the
penetration of drugs through and between intestinal cells. Polymers such as poly-anhydrides bind
to the gut and cross the intestinal mucosa, leading to improved bioavailability of the drug.
Lectins have been deemed as a second generation of bio-adhesives, owing to their non-toxicity
and special binding properties, which simulate a ligand–receptor interaction.
Other researchers have been working on the blockade of protease inhibitors and cellular pump
systems, which could prevent effective absorption of certain drugs and thus reduce their
therapeutic effectiveness. In this regard, Glytech technology, designed to temporarily inhibit
and/or block the p-glycoprotein pump system, has been developed by Eurand. Results obtained
with this system in animal models show improved absorption profiles of several therapeutically
active compounds. Peptidic drugs can be also conjugated to a macromolecular carrier, such as a
polymer or protein. At present, polyethylene glycol is the most widely used polymer for the
modification of proteins with therapeutic potential, because of its low toxicity and cost and the
commercial availability of many molecular weight variants.
Using a similar approach, Nobex corporation attached low molecular weight polymers to specific
sites on drug molecules to create drug–polymer conjugates. Nobex is using this technology to
make a form of insulin that can be given orally. In fact, according to a Phase II clinical trial,
which involved more than 150 patients, a rapid, dose-dependent absorption of the oral insulin
and a lowering of fasted blood glucose levels (i.e. morning levels before food) have been
achieved with no safety concerns.
6

15. 1.5.2 Nasal Drug Delivery
The use of nasal routes for drug delivery has created much interest in the pharmaceutical
industry in recent years. Different absorption enhancers have been studied to improve the
absorption of polar drugs. For example, formulations based on chitosan powder have been tested
for the nasal administration of insulin and morphine. Furthermore, the use of cyclodextrins, poly-
L-arginine and lipids as absorption enhancers is also under investigation. A range of companies
working in novel nasal delivery systems has come to the fore, for example, Aradigm has
developed a disposable nozzle-containing element to ensure superior aerosol performance each
and every time the patient inhales medication. The precision of this technology is currently being
studied in clinical trials for different drugs, including testosterone, insulin, morphine and
interferon a-2b.
1.5.3 Transdermal Drug Delivery
The transdermal administration of drugs is a relatively direct route to the bloodstream. As
recently reviewed by Langer, two different physical mechanisms (iontophoresis and ultrasound)
are being applied to circumvent the physical barrier of the skin. Using iontophoresis, Iomed Inc.
has developed Phoresor1 for the administration of iontocaine for local dermal anesthesis.
Another approach to transdermal drug delivery is the development of microneedles, which create
microscale pathways across the skin improving its permeability.
1.5.4 Parenteral Drug Delivery
Parenteral dosage forms differ from all other drug dosage forms, because they are injected
directly into body tissue through the primary protective systems of the human body, the skin, and
mucous membranes. They must be exceptionally pure and free from physical, chemical, and
biological contaminants. These requirements place a heavy responsibility on the pharmaceutical
industry to practice current good manufacturing practices (cGMPs) in the manufacture of
parenteral dosage forms and on pharmacists and other health care professionals to practice good
aseptic practices (GAPs) in dispensing parenteral dosage forms for administration to patients.
Certain pharmaceutical agents, particularly peptides, proteins, and many chemotherapeutic
agents, can only be given parenterally, because they are inactivated in the gastrointestinal tract
when given by mouth. Parenterally administered drugs are relatively unstable and generally
7

16. highly potent drugs that require strict control of administration to the patient. Due to the advent
of biotechnology, parenteral products have grown in number and usage around the world.
1.5.5 Rectal Drug Delivery
Drug delivery via the rectum is a useful alternative route of administration to the oral route for
patients who cannot swallow. Traditional rectal dosage forms have been historically used for
localized treatments including delivery of laxatives, treatment of hemorrhoids and for delivery of
antipyretics. However, the recent trend is showing an increase in the development of novel rectal
delivery systems to deliver drug directly into the systemic circulation by taking advantage of
porto-systemic shunting. Novel rectal drug delivery systems including hollow-type suppositories,
thermo-responsive and muco-adhesive liquid suppositories, and nano-particulate systems
incorporated into an appropriate vehicle have offered more control over delivery of drug
molecules for local or systemic actions. In addition, various methods for in vitro–in vivo
evaluation of rectal drug delivery systems are covered which is as important as the formulation,
and must be carried out using appropriate methodology. Continuous research and development in
this field of drug delivery may unleash the hidden potential of the rectal drug delivery systems.
1.5.6 Buccal Drug Delivery
The buccal mucoadhesive formulations are to be an alternative to the conventional oral small
amount of medicaments as they can be readily attached to the buccal cavity retained for a longer
period of time and removed at any time. The epithelium of the mouth is accessible with small
surface area approximately 100 cm2. Buccal adhesive drug delivery systems using matrix tablets,
films, layered systems, discs, microspheres, ointments and hydrogel systems have been studied
and reported by several research groups. However, limited studies exist on novel devices that are
superior to those of conventional buccal adhesive systems for the delivery of therapeutic agents
through buccal mucosa.
1.5.7 Vaginal Drug Delivery
The main advantages of vaginal drug delivery over conventional drug delivery are the ability to
by-pass first pass metabolism, ease of administration and high permeability for low molecular
weight drugs. However, several drawbacks, including cultural sensitivity, personal hygiene,
8

17. gender specificity, local irritation and influence of sexual intercourse, need to be addressed
during the design of a vaginal formulation [Neves et al., 2010].
The vaginal route offers a favorable alternative to the parenteral route for some drugs such as
bromocriptine, oxytocin, misoprostol, calcitonin, LHRH agonists, human growth hormone and
insulin. For systemic delivery, insulin suspended in a poly (acrylic acid) gel base was observed
to facilitate the rate of vaginal absorption in diabetic rats and rabbits. Plasma insulin reached a
peak and hypoglycaemic effects were observed [Wassen et al., 1996].
In recent years, there have been several reports of successful immunization with DNA vaccines
administered via various mucosal routes including the vaginal route. A recent study demonstrates
the formulation and application of plasmid DNA vaccine to mucosal inductive tissues, including
the vagina. The female genital tract has the capacity to produce humoral and cellular immune
responses against locally encountered antigens. Intravaginal delivery of cholera vaccine showed
a greater mucosal response in female genital tract compared to oral administration of the vaccine
[Neves et al., 2010].
9

18. Chapter-2
Production Processes for
Biotechnology Based
Products
10

19. 2. Designing of Drug Delivery System
The term biotechnology encompasses any technique which uses living organisms (e.g.,
microorganisms) in the production or modification of products. The production process of
biotechnology-based products can be divided mainly into two stages: upstream and downstream
processing. Upstream processing is the stage where the targeted compound such as a protein is
synthesized and increased quantitatively by the host cells such as bacterial cells. The next stage
is the downstream process, which is concerned with the isolation and purification of the targeted
compound synthesized by the host cells.
A. Upstream Process
The upstream process is the stage that is involved in the synthesis and production of the targeted
compound inside the host cells. For the upstream process to be efficient, a suitable host cell
should be selected that can synthesize the targeted compound in proper amounts. In cases where
the targeted compound is a protein or peptide molecule, the gene of that compound should be
isolated and cloned [Kayser and Warzecha, 2012]. There is a variety of efficient technologies
available for this purpose. The cloned gene is then inserted into a vector molecule, which is
required to allow the gene to replicate in the host cell as well as to be expressed at an efficient
rate. There are different types of vector molecules; in the case of bacterial cells, plasmids are the
most common vectors. The vector that contains the gene of interest is inserted in the host cells,
which will express the gene and produce the targeted molecule. After the targeted compound is
produced at the desired amount, the upstream process is over and the culture of cells is then
harvested for the downstream process [Nagaich, 2015; Doherty and Suh, 2000].
B. Downstream Process
Following the upstream process in which the host cells synthesize the compound of interest, the
downstream process is conducted. The downstream process is concerned with the isolation and
purification of the synthesized compounds from the host cells or the biological medium in which
it is present. The downstream process can be complex and usually consists of many steps that
involve the use of various separation methods. This is because the biological medium in which
the protein of interest resides contains a large number of other molecules (contaminants) that
11

20. belong to the cell or the culture medium. These contaminants can have various degrees of
similarity with the protein of interest in terms of physicochemical properties. Naturally, the more
similar the contaminants are with the protein of interest, the more difficult the separation of the
protein of interest from these contaminants [Gottschalk, 2012; Straathof, 2011].
The classic example of biotechnologic drugs was proteins obtained from recombinant DNA
(rDNA) technology. However, biotechnology now encompasses the use of tissue culture, living
cells, or cell enzymes to make a defined product. rDNA and monoclonal antibody (MAb)
technologies have provided exciting opportunities for development of more pharmaceuticals and
approaches to the diagnosis, treatment, and prevention of disease [ Allen et al., 2009].
2.1 Recombinant DNA technology
The process of r-DNA technology begins with the isolation of a gene of interest, which is then
inserted into a vector; these vectors are further cloned into multiple copies. A vector is basically
a piece of DNA that is capable of independent growth; bacterial plasmids and viral phages are
the commonly used vectors. When the gene of interest (foreign DNA) is integrated into the
plasmid or phage, this process is generally referred to as r-DNA and it shown in Figure 1. The
next step in r-DNA technology is to introduce the vector containing the foreign DNA into host
cells so that the cells can express the desirable proteins. In order to get sufficient amounts of
protein, the vectors must be cloned to produce large quantities of the DNA. Once the vector is
isolated in large quantities, it can be introduced into the desired host cells, which include
mammalian, yeast, or special bacterial cells. Finally, the host cells can synthesize the foreign
protein from the r-DNA. When the cells are grown in vast quantities in the bioreactor or
fermenter, the recombinant protein can be isolated in large amounts and the entire process is
commonly referred as r-DNA technology. There are three different methods available through
which r-DNA products are being developed: (1) transformation, (2) phage introduction, and (3)
nonbacterial transformation and it given below in Figure 2 [Khan, 2014].
2.1.1 Steps of constructing a recombinant DNA
The making of r-DNA has been briefly described below in a stepwise manner [Khan, 2014].
12

21. Figure-1: Making of Recombinant DNA
Figure-2: Making of Recombinant DNA Product
13

22. A. Transformation
The first step in constructing an r-DNA is to transform a select piece of DNA into a vector. The
next step is to cut that piece of DNA with a restriction enzyme known as endonucleases and then
ligate the DNA insert into the vector using DNA ligase. After that, the DNA insert can be
visualized by a selectable marker, which permits the identification of r-DNA molecules. It has
been reported that an antibiotic marker is often used to tag host cells without affecting the vector.
The process of inserting a vector into a host cell is called transformation. During the making of
an r-DNA product, Escherichia coli is found be the most widely used host organism and it has
been reported that E. coli can easily take foreign DNA. It has been suggested that different
vectors have different characteristics to make them suitable to different applications and it shown
in Figure 3 [Khan, 2014]:
Figure-3: Basic Concept of Genetic Transformation
B. Nonbacterial transformation
This is a process very similar to transformation. The only difference between the two is that in
nonbacterial transformation, there is no need for bacteria such as E. coli for the host cells. There
are various ways through which DNA nonbacterial transformation can be achieved, which
include DNA microinjection. In this process, the DNA is inserted straight into the nucleus of the
cell, which is being transformed. In another method called biolistic transformation, the host cells
are bombarded with a high velocity of gold or tungsten particles that are coated with DNA
[Khan, 2014].
14

23. C. Phage introduction
Phage introduction is the process of DNA transfection, which is equivalent to the transformation
method. The only difference is that instead of bacteria, phage is used. It has been reported that
during phage introduction, various types of phages such as lambda or MI3 phages have been
used to produce phage plaques that contain recombinants. The recombinants that are produced by
phage introduction can be easily identified by differences in the recombinant and non-
recombinant DNA using various selection approaches [Khan, 2014].
2.1.2 Applications of r-DNA technology
r-DNA technology is not only an important tool in scientific research but also a useful tool in the
diagnosis and treatment of various diseases and genetic disorders. We have discussed its various
applications especially in the medical field [Khan, 2014].
A. Therapeutic proteins (Recombinant human insulin)
One of the main discoveries in r-DNA technology was the production of biosynthetic human
insulin, which was the first biomolecule made through r-DNA technology. Later on, this
bioengineered insulin became the first biotechnology product approved by the U.S. Food and
Drug Administration (FDA). Therapeutic proteins are those proteins that are either removed
from human cells or engineered in the laboratory for pharmaceutical use. In addition to human
insulin, other human proteins such as follicle-stimulating hormone, plasminogen, erythropoietin,
and growth hormones have been created by using r-DNA technology and it shown in Figure 4
[Khan, 2014].
There are many proteins essential to for the normal function of the human body and
unfortunately some people fail to produce sufficient amounts of these proteins in their body,
which may lead to the development of functional deformities and genetic defects. These essential
proteins include various blood-clotting factors causing hemophilia, insulin, growth hormone, and
other proteins. Patients who do not produce sufficient amounts of these proteins need to take
these proteins externally either in the form of a medicine or an injection. These proteins can be
synthesized outside of the human body by using recombinant technology. It has been reported
that the majority of therapeutic proteins are recombinant human proteins manufactured using
nonhuman mammalian cell lines that are engineered to express certain human genetic sequences
15

24. to produce precise proteins of human use. Over the past decade, recombinant proteins are
extensively used to replace deficiencies and to strengthen the immune system to fight cancer and
infectious disease [Khan, 2014].
Figure-4: Making of Human Insulin by Recombinant DNA Technology
B. Drotrecogin Alfa (activated) (Xigris)
Drotrecogin alfa (activated) is recombinant human activated protein C. Produced naturally in the
liver, protein C is converted to activated protein C (APC) through interaction with the thrombin–
thrombomodulin complex. APC demonstrates antithrombotic activity through inhibition of
factors Va and VIIIa (15). Approved by the FDA in November 2001, drotrecogin alfa is
indicated for a reduction of mortality in patients with severe sepsis associated with acute organ
system dysfunction (Fig. 19.8). Sepsis remains a significant cause of death in patients who are
critically ill. Drotrecogin alfa (activated) should be administered by continuous intravenous
16

25. infusion at 24 μg/kg/hour for 96 hours. Because compatibility data are sparse, it should be
administered via a dedicated line or dedicated lumen of a multilumen central venous catheter.
Administration must be conducted within 12 hours of reconstitution. Periods during which the
infusion is interrupted for procedures with an inherent risk of bleeding do not count toward the
96-hour duration of therapy. Bleeding is the most common adverse effect associated with
drotrecogin alfa. The incidence is approximately 3.5% (compared to 2.0% in placebo controls).
Therefore, it is contraindicated when bleeding may be associated with a high risk of death (e.g.,
active internal bleeding, hemorrhagic stroke in the past 3 months, intracranial or intraspinal
surgery, or severe head trauma within the past 2 months). When evaluating the patient‘s ability
to handle drotrecogin alfa (activated) therapy, the potential benefi ts and risks must be evaluated
and carefully considered, especially if one of the following situations is present: concurrent
therapeutic heparin therapy, international normalized ratio (INR) > 3.0, platelet count below
30,000 × 106/L even if the platelet count is increased after transfusions, ischemic stroke in the
past 3 months, aspirin more than 650 mg per day, or other platelet inhibitors within the past 7
days. Drotrecogin alfa (activated) therapy must be considered very carefully, especially in light
of the inclusion and exclusion patient criteria, the stability and unique duration of infusion, and
the cost of treatment [Allen et al., 2009].
2.2 Monoclonal Antibodies
MAbs are purified antibodies produced by a single source or clone of cells. These substances are
engineered to recognize and bind to a single specific antigen. Thus, a MAb will target a
particular protein or cell having the specific matching antigenic feature. When coupled with a
drug molecule, radioactive isotope, or toxin, a MAb theoretically can target the desired cells or
tissues with great precision. Specificity for the target antigen is the primary characteristic for the
MAb and reflects affinity and strength of binding for the target antigen and cross reactivity with
normal cells[ Allen et al., 2009]. Therapeutic responses to mAbs may be mediated through either
the Fab or Fc region of the antibody. Key pharmacodynamic mechanisms for mAbs in oncology
include: inhibition of cell signaling, induction of apoptosis, antibody-dependent cellular
cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and targeting a toxic payload
to tumor cells and it shown in Figure-5. Additionally, there has been some interest in the
development of mAbs known as ‗superagonists‘ that stimulate immune function to accelerate
17

26. immune clearance of tumor cells. It is important to note that a single mAb may act through a
combination of mechanisms to achieve anti-tumor effects [Glassman and Balthasar, 2014].
Figure-5: Pharmacologic Mechanisms of Action for mAbs
Here, Panel A, Inhibition of Cell Signaling via Binding to Soluble Targe; Panel B, Inhibition of
Cell Signaling via Binding to Membrane-Bound Receptor; Panel C, Direct Induction of
Apoptosis; Panel D, Antibody-Dependent Cellular Cytotoxicity; Panel E, Complement-
Dependent Cytotoxicity; Panel F, CD28 Superagonist; Panel G, Delivery of Toxic Payload
(Antibody-Drug Conjugate, Immunotoxin, Radio-immunoconjugate).
Treatments using monoclonal antibodies are being used now-a-days against numerous forms of
cancer. In this type of treatment, synthetic monoclonal antibody is attached with the cancer cell
followed by killing the cancerous cells in different ways. The monoclonal antibody Cetuximab
blocks the growth signals produced by the cancer cells resulting in the halting of the cells growth
and thus colon cancer is treated. Gemtuzumab combined with strong chemotherapeuticals are
administered into the cancer cell where they become active and minimize damage to adjacent
normal tissues, help in treating acute myelogenous leukaemia (AML). Rituximab facilitates the
cancer cell to be more visible to the immune system to get destroyed. This antibody is useful in
18

27. treating non-Hodgkin‘s lymphoma. Herceptin is a monoclonal antibody to treat breast cancer
cells in women expressing the protein HER2. Herceptin specially binds to those cancer cells and
discontinue their proliferation. A radioactive particle is combined with Ibritumomab monoclonal
antibody to deliver the radiation directly to the cancer cells which does not harm the
neighbouring normal tissues. Non-Hodgkin‘s lymphoma is treated in this way also other than
Rituximab antibody [ Modak and Biradar,2015].
Diagnostically, the specificity of MAbs helps to detect the presence of endogenous hormones
(e.g., luteinizing hormone [LH], human chorionic gonadotropin) in the urine to establish the test
results. They are also used to detect allergies, anemia, and heart disease, and commercial MAb
diagnostic kits are available for drug assays, tissue and blood typing, and infectious diseases
including hepatitis, AIDS-related CMV, streptococcal infections, gonorrhea, syphilis, herpes,
and chlamydia. When covalently linked with radioisotopes, contrast agents, or anticancer drugs,
MAbs can be used to diagnoseand treat malignant tumors [Allen et al., 2009].
2.2.1 Available Monoclonal Antibody (Drug Product)
Different types of monoclonal antibody which are available are given below [[Glassman and
Balthasar, 2014]:
A. Rituximab (Rituxim)
Rituximab (Rituxan) is an anti-CD20 mAb indicated as a therapy for treatment of NHL and CLL
and was the first mAb approved by the FDA for use in oncology. In clinical trials for NHL,
rituximab was found to have non-stationary pharmacokinetics, with clearance decreasing from
38.2 mL/h after the first dose to 9.2 mL/h after the fourth dose70. This observation may be due
to a reduction in TMDD caused by wipeout of CD20-positive cells after the initial infusion. In
clinical trials, addition of rituximab to the standard CHOP-21 chemotherapy regimen was
associated with an improvement in 3-year progression-free survival (85% vs. 68%)17. In a
different study, lymphoma patients treated with rituximab alone had an overall response rate of
50%, with a median duration of response of 8.6 months].
B. Trastuzumab (Herceptin)
Trastuzumab (Herceptin) is an anti-HER2 mAb approved for the treatment of breast cancer,
metastatic gastric cancer, and metastatic gastroesophageal junction adenocarcinoma. The half-
19

28. life of trastuzumab has been observed to range from 1.1 days (10 mg dose) to 23 days (500 mg
dose) in clinical trials75. Additionally, population pharmacokinetic modeling has suggested that
clearance of trastuzumab is directly related to shed extracellular domain of HER2 and has a
weaker association with the number of tumor metastases75. In mice, tumor distribution was
found to be more uniform at higher doses and at later time points, suggesting that saturation of
the binding site barrier may be crucial in optimizing the efficacy of trastuzumab.
Phase III clinical trials investigated the potential benefits of adding trastuzumab to standard
chemotherapy in previously untreated breast cancer patients with HER2-overexpressing tumors.
The trial results indicated that addition of trastuzumab was associated with a 4.8-month increase
in overall survival (20.3-25.1 months) and a 2.8-month increase in progression-free survival (4.6-
7.4 months).
2.3 Gene Therapy and Types
One of the most sought after techniques to treat genetic disorders is gene therapy. In this
technique, the absent or faulty gene can be replaced by a working or normal gene and it shown in
Figure-6, so that the human body can make the correct protein and consequently eliminate the
root cause of the disease. This type of gene therapy generally involves somatic cells, whereas
gene therapy can also use germline cells which contribute to the genetic heritage of the offspring.
It has been reported that gene therapy in germline cells has the probability to affect not only the
individual being treated, but also his or her children as well. The gene therapy can be classified
into two major types: somatic gene therapy and germline gene therapy [Khan, 2014].
2.3.1 Somatic Gene Therapy
By using somatic gene therapy, somatic cells can be treated by inserting a vector loaded with the
correct gene into a person‘s body. The somatic cells are cells that form in the body and cannot
produce progenies. Gene therapy, in its present stage only treats somatic cells in humans. There
are two types of somatic gene therapy, ex vivo and in vivo. In ex vivo gene therapy genes or cells
are modified outside the body and then transplanted back into the body, whereas in vivo gene
therapy, cells are modified or treated within the patient‘s body. It has been suggested that
somatic gene therapy does not affect any descendants of the person being treated. It has been
reported that scientists used gene therapies on genetic diseases which include hemophilia,
muscular dystrophy, sickle cell anemia, and cystic fibrosis [Khan, 2014].
20

29. Figure-6: Gene therapy
A. Ex vivo somatic gene therapy
Ex vivo somatic gene therapy involves the introduction of vectors directly into the body of the
person, most usually into the afflicted tissue. For example, if the aim was to treat skin cancer, the
vectors would be introduced into the melanoma itself. Eventually, there is hope that vectors will
be found that can be introduced directly into the bloodstream, however, there are difficulties with
the immune system response that have slowed development in this area to date. One might recall
that the body is programmed to mount an immune response if ever a foreign cell should be
introduced. Thus, because in vivo somatic gene therapy involves the introduction of thousands of
what amount to being viruses into the human body, it has been a particularly difficult field to
master and it shown in Figure-7 [Khan, 2014].
B. In vivo somatic gene therapy
The process by which the genetic makeup of cells is altered to produce a therapeutic effect that
prevents or treats diseases in the patients is called in vivo somatic gene therapy. Defective or
missing hereditary material DNA in the nucleus of the patient‘s cells is altered or replaced by
healthy genes. Specially modified viruses act as the carriers of the new genetic material,
delivering it to the patient‘s targeted cells or tissues. The transfer of genetic material takes place
within the patient‘s body during in vivo gene therapy. The process of in vivo gene therapy is
21

30. differentiated from ex vivo gene therapy in that the latter procedure takes cells from the patient‘s
body, inserting genes and culturing the cells in the laboratory rather than inside the patient‘s
body. This treatment generally requires extraction and replacement of the patient‘s bone marrow
in two separate surgeries [Khan, 2014].
Figure-7: Ex Vivo and In Vivo Gene Therapy
One of the biggest challenges of in vivo-based gene therapy is the insertion of genes into
respective sites in the cells. The vector which carries the gene has a challenging task to complete
as they have to deliver the genes to all affected cells for results and at the same time these
vectors remain undetected by the body‘s immune system to avoid immune rejection. The use of
virus as a vector to deliver the gene inside the cells is primarily owing to the fact that viruses are
known to deliver genetic information from cell to cell, this is what viruses normally do to insert
their genes into host cells so that their host cells can replicate them. It has been reported that
through millions of years of evolution, viruses have developed very sophisticated ways of
transforming genetic information. To make gene therapy effective, there are two classes of
viruses—retroviruses and adenoviruses—found to be critical in gene delivery [Khan, 2014].
22

31. 2.3.2 Gene therapy clinical trials: The US scenario (Accomplished clinical trials)
Clinical trials of gene therapy are given below [Khan, 2014]:
A. Liver transplantation with ADV-TK gene therapy
The liver cancer disease with advanced hepatocellular carcinoma (HCC) can be treated with
ADV-TK gene therapy highlight its potentiality as adjuvant treatment for HCC patients after
liver transplant. It has been reported as an improved method outcome of liver transplant with the
combined treatment of ADV-TK gene therapy in patients with intermediate or advanced HCC.
The overall survival in the liver transplant with ADV-TK gene therapy group was found around
55% at 3 years. The patient with nonvascular invasion condition, treated with liver transplant
plus ADV-TK therapy should 100% survival and recurrence-free survival than those with
vascular invasion subgroup.
B. Gene therapy in patients with severe angina pectoris
The aim of this study was to evaluate the mobilization of nonhematopoietic mesenchymal and
hematopoietic stem cells from the bone marrow with granulocyte colony stimulating factor (G-
CSF) treatment alone and in combination with vascular endothelial growth factor (VEGF) gene
therapy in patients with severe chronic occlusive coronary artery disease. In recent clinical trials,
VEGF delivered as plasmid DNA percutaneously by a catheter-based, intra-myocardial
approach, have been demonstrated to be safe and are associated with a reduction in angina and
an increase in exercise time or an improvement in regional wall motion in ―no-option patients‖
with chronic myocardial ischemia. It has been demonstrated, that BM-derived stem cells
mobilized by cytokines as G-CSF were capable of regenerating the myocardial tissue, leading to
improve the survival and cardiac function after myocardial infarction. These data suggested that
a combination therapy with exogenous administration of gene vascular growth factor combined
with G-CSF mobilization of bone marrow stem cells might induce both angiogenesis and
vasculogenesis in ischemic myocardium.
2.4 The PCR (Polymerase Chain Reaction) Technology and DNA Sequencing
Two molecular biology techniques in recent years have revolutionised the availability of DNA
data, the polymerase chain reaction (PCR) and the development of automated DNA sequencing.
The polymerase chain reaction is basically a technique that allows the selective amplification of
23

32. any fragment of DNA (of about 0.2 and 40 kbp (kilo base pairs) in size) provided that the DNA
sequences flanking the fragment are known , described as a technique that finds a needle in a
haystack and then produces a haystack of needles by specific amplification. The inventor of
PCR, Kary Mullis, shared the Nobel prize in Chemistry in 1993 [Smith, 2009].
The polymerase chain reaction process relies on the sequence of ‗basepairs‘ along the length of
the two strands that make the complete DNA molecule. In DNA there are four deoxynucleotides
derived from the four bases, adenosine (A), thymidine (T), guanine (G) and cytidine (C). The
strands or polymers that comprise the DNA molecule are held to each other by hydrogen bonds
between the base pairs. In this arrangement A only binds to T while G only binds to C, and this
unique system folds the entire molecule into the now well recognised double-helix structure. The
polymerase chain reaction involves three processing steps – denaturation, annealing and then
extension by DNA polymerase and it given below in Figure 8 and Figure 9 [Smith, 2009].
Figure-8: The Polymerase Chain Reaction
24

33. Here, the double-stranded DNA is heated and separates into two single strands. The synthetic
oligonucleotide primers then bind to their complementary sequence and are extended in the
direction of the arrows giving a new strand of DNA identical to the template‘s original partner.
Figure-9: PCR Temperature Cycling Profile
In Step 1, the double-stranded DNA is heated (95–98 ◦C) and separates into two
complementary single strands. In Step 2 (60 ◦C) the synthetic oligonucleotide primers
(chemically synthesised short-chain nucleotides), short sequences of nucleotides (usually about
20 nucleotide base pairs long), are added and bind to the single strands in places where the
strand‘s DNA complements their own. In Step 3 (37 ◦C) the primers are extended by DNA
polymerase in the presence of all four deoxynucleoside triphosphates resulting in the synthesis of
new DNA strands complementary to the template strands. The completion of the three steps
comprises a cycle and the real power of PCR is that with 25–30 cycles this experimental
25

34. synthesis leads to massive amplification of DNA, which can then be used for analytical purposes.
A major recent advance has been the development of automated thermal cyclers (PCR machines)
that allow the entire PCR to be performed automatically in several hours [Smith, 2009].
2.4 1 Applications of Polymerase Chain Reaction (Pcr)
The PCR has made an enormous impact in both basic and diagnostic aspects of molecular
biology since the few years since its discovery. Like the PCR, the number of applications has
been accumulating exponentially and will most probably continue to do so in the near future
[Atawodi et al., 2010]. Broadly, the applications of PCR in the biological sciences may be
divided into:
1. Medical applications
2. Research applications
A. Medical Application of PCR
Polymerase chain reaction has helped in the realization of the potential of clinical DNA-based
diagnoses by producing enough of the target sequence, so that simple, rapid and robust methods
for identifying it could be employed. Specific applications of PCR in the medical sciences are
given below [Atawodi et al., 2010]:
 Diagnosis of Monogenic Diseases

 Diagnosis of Mutation Diseases DNA Typing,

 Evolutionary Trends and Disease Susceptibility Studies.

 PCR and Forensic Science

 Detection of ras Oncogenes

 Detection of Human Infectious Diseases

 PCR and DNA Vaccine Production.

B. Research Applications of PCR
The ability to synthesize large amount of a specific DNA fragment from a complex template has
significantly facilitated subsequent analysis. The nucleotide sequence of amplified DNA
fragments can be determined directly without molecular cloning and preparation of template by
26

35. growth of the host and biochemical purification of the vector [Atawodi et al., 2010]. Most
examples of applications of PCR in scientific research may be summarized as follows:
 Direct sequencing of in vitro amplified DNA.

 Engineering DNA to meet specific needs.

 Detection of mutation.

 Detection of gene expression.

 Specific amplification of a DNA specie.

 Geometric amplification of unknown DNA sequence through inverse PCR .

 Analysis of DNA sequences in individual gametes.

 Evolutionary analysis.
2.5 Antisense Technology
2.5.1 Antisense Oligonucleotides: Molecular Mechanisms
Antisense oligonucleotides (ASOs) are defined as chemically synthesized oligonucleotides,
generally 12–30 nucleotides in length, that are designed to bind to RNA by Watson-Crick base
pairing rules and it shown in Figure-12. The length of ASOs in part contributes to their
specificity, as oligonucleotides that are 16–20 nucleotides long are capable of uniquely binding
to only one target RNA. Following binding to the targeted RNA, the oligonucleotide modulates
RNA function by several different mechanisms. These can be broadly categorized as
mechanisms promoting RNA cleavage and degradation or occupancy-only mechanisms,
sometimes referred to as steric blocking (Figure-13). The mechanism(s) by which the ASO
modulates the RNA is dependent on the ASO chemistry and design, the position on the RNA
where the ASO is designed to bind, and the function of the RNA. Based on the chemical and
positional requirements for the different mechanisms, it is possible to rationally design ASOs to
modulate the target RNA, although some screening is still required for optimal activity and
tolerability [Bennett, 2019].
27

36. Figure-10: Antisense oligonucleotide (ASO)
Here, Antisense oligonucleotide (ASO) binding to the targeted RNA. (a) The RNA polymerase
transcribes the RNA from the DNA template. The synthetic ASO (red) binds to the RNA (blue)
bywatson-Crick base pairing rules, e.g., adenine binds to uracil and cytosine binds to guanine.
(b) The modified bases thymine and 5-methylcytosine are frequently used in ASO drugs.
A. Occupancy-Only Mechanisms
Several antisense mechanisms do not result in direct degradation of the target RNA and it shown
in Figure 13. Paul Zamecnik is credited for first introducing the concept that synthetic
oligonucleotides could be developed therapeutically to block protein translation . His seminal
papers demonstrated that a synthetic oligodeoxynucleotide designed to bind to the Rous sarcoma
virus RNA blocked translation of the viral RNA and subsequently blocked virus replication .
Once the technology for chemical synthesis of oligonucleotides was developed , interest in using
oligonucleotides as a therapeutic platform expanded. Blocking protein translation remains a
viable antisense mechanism, but it is not broadly used as a therapeutic strategy. More recently,
28

37. several approaches to increase protein translation have been published. MicroRNAs are short
RNAs (approximately 21 to 23 nucleotides) that repress translation of multiple mRNAs targets,
resulting in control of gene networks . ASOs designed to bind to microRNAs block their ability
to bind to targeted RNA sequences, resulting in de-repression of translation of the microRNA
targets . Because microRNAs block translation of multiple targets, often in a tissue- or cell-
specific manner, blocking a single microRNA results in increased expression of numerous
proteins. A more specific approach to increase protein production is to design an ASO to bind to
a regulatory sequence in the 5_-untranslated region of a mRNA that represses protein translation,
such as an upstream open reading frame or stem-loop structure . Most mammalian protein coding
RNAs undergo a complex set of processing events that includes adding a 5_-cap structure,
removing large segments of RNA sequence and splicing the RNA back together, and adding a
polyadenylate (polyA) tail to the 3-end of the RNA. Each of these steps can be selectively
modulated by ASOs (Figure-13), with modulation of RNA splicing by ASOs being the most
broadly utilized. ASOs can be designed to cause exon skipping, as is the case with Eteplirsen , or
to promote exon inclusion, as is the case for Nusinersen , two recently approved antisense drugs.
Additional therapeutic applications for modulation of RNA splicing are being explored in the
laboratory and early clinical trials .Many transcripts have two or more alternate poly-A sites that
may be preferentially utilized in a disease state such as cancer . In addition, poly-A site selection
can mediate sub-cellular localization of an RNA transcript. ASOs have been shown to redirect
which poly-A site is utilized. Yet another example of using ASOs to modulate gene expression in
cells is preventing long noncoding RNAs from interacting with their sites on chromatin, resulting
in increased transcription of a repressed gene, as has been described for a long noncoding RNA
that inhibits SMN2 gene transcription through recruitment of the PRC2 complex . As we
enhance our understanding of the different regulatory roles RNAs play in health and disease,
there will likely be additional mechanistic insights for the application of ASOs [Bennett, 2019].
29

38. Figure-11: Occupancy-Only Antisense Mechanisms
Here, occupancy-only antisense mechanisms do not result in degradation of the targeted RNA.
Abbreviation: uorf, upstream open reading frame.
B. RNA Degradation Mechanisms
The majority of ASOs in development are designed to promote RNA cleavage by either RNase
H1 or argonaute 2 (Ago2) and it given below in Figure-14. . RNase H1 is an endogenous
nuclease present in most,
If not all, cells, which promotes cleavage of the RNA in an RNA-DNA heteroduplex .In
mammalian cells, RNase H1 is found in the nucleus, mitochondria, and cytoplasm, where it
serves several functions, including removing the RNA present in the Okazaki fragment, DNA
repair, and resolution of R loops .Oligonucleotides that are designed to utilize RNase H1 as their
mechanism of action must contain a minimum of 5 consecutive DNA nucleotides, with 7– 10
being optimal. Oligonucleotides designed to degrade target RNA by the RNase mechanism are
widely used as experimental tools and are being developed for a number of therapeutic
indications. ASOs designed to work through the RNA interference pathway, e.g., small
interfering RNAs (siRNAs), are also broadly used as experimental tools to selectively reduce the
expression of a target RNA and also as potential therapeutic agents, with an increasing number
30

39. of drugs entering clinical development . ASOs that work through the RNAinterference pathway
are generally delivered to the cell or organism as a duplex of two RNAs or modified RNAs, with
one strand designed to bind to the target RNA and the second, or passenger, strand ultimately
degraded .Once inside the cytoplasm, the duplex binds to the nuclease Ago2 and releases the
passenger strand . The mechanisms by which Ago2 determines which strand to bind appear to
be, in part, mediated by the 5-end of the oligonucleotide, as well as the end with the least stable
base pairing .Like RNase H1, Ago2 has specific structural requirements for the oligonucleotide,
limiting the types of chemical modifications that can be used [Bennett, 2019].
A key difference between these two mechanisms is that oligonucleotides that work through the
RNase H1 mechanism bind to the target RNA before the enzyme is recruited, while siRNAs bind
to the enzyme first and then the enzyme-oligonucleotide duplex binds to the RNA, although the
former has not been conclusively proven[Bennett, 2019].
Figure-12: RNA cleavage antisense mechanisms promote degradation of the targeted RNA
2.5.2 Antisense Technology based Available Product
A. Inotersen
Inotersen is a second-generation antisense drug that prevents production of the transthyretin
(TTR) protein by an RNase H1 dependent mechanism (48). TTR protein, which is primarily
31

40. produced in the liver, forms a tetramer that binds retinal binding protein 4 (RBP4)-retinal
complex, preventing renal clearance as well as serving as one of several thyroid hormone
transport proteins . Autosomal dominant mutations in the transthyretin gene cause the tetrameric
form of the protein to become less stable. The monomers form aggregates that deposit in
multiple tissues including peripheral nerves, cardiac tissue, and kidney.The aggregates usually
lead to a peripheral neuropathy, severe gastrointestinal dysfunction, and in some cases
cardiomyopathy. The average life expectancy of individuals with hereditary TTR (hTTR) is
typically 3–15 years from symptom onset. Inotersen is a gapmer design with five 2-MOE
nucleotides on the 5_- and 3_-ends of the oligonucleotide and ten DNA nucleotides in the middle
to support the RNase H1 mechanism. Inotersen produces a dose-dependent reduction of TTR
mRNA and protein in cultured cells and in transgenic mice . In the transgenic mice, a single dose
of inotersen produced effects that lasted 2–3 weeks. In cynomolgus monkeys, the inotersen
binding site is complementary to the TTR sequence, allowing measurement of pharmacology.
Monkeys administered inotersen demonstrated 90% reduction of TTR RNA expression in the
liver (the main source of circulating TTR protein) and 80% reduction in circulating TTR protein
(48). Treatment was well tolerated, with no deleterious liver or kidney effects observed [Bennett,
2019].
2.6 Peptide Technology
Peptide technology entails screening for polypeptide molecules that can mimic larger proteins.
This is intended to afford relatively simple products that can be stable and easy to produce.
These peptides can serve as either protein receptor agonists or antagonists [ Allen et al., 2009].
2.7 Emerging Delivery Methods
2.7.1 Viral Vectors
Viral vector is the most effective means of gene transfer to modify specific cell type or tissue and
can be manipulated to express therapeutic genes. Several virus types are currently being
investigated for use to deliver genes to cells to provide either transient or permanent transgene
expression. These include adenoviruses (Ads), retroviruses (g-retroviruses and lentiviruses),
poxviruses, adeno-associated viruses, baculoviruses, and herpes simplex viruses [Warnock et al.,
2011].
32

41. The spectrum of viral vectors is very broad including both delivery vehicles developed for
transient short-term and permanent long-term expression. Moreover, the types of vectors are
represented by both RNA and DNA viruses with either single-stranded (ss) or double-stranded
(ds) genomes. The main groups of viral vectors applied for gene therapy are summarized below
in Table 1 [Lundstrom, 2018]:
Table 1: Examples of Viral Vectors Applied For Gene Therapy
Virus Genome Insert capacity Features
Adenoviruses dsDNA <7.5kb Broad host range
Transient expression
Strong immunogenicity
Herpes dsDNA >30kb Broad host range
simplex Latent infection, long-term
expression
Low toxicity, large insert capacity
Retroviruses ssRNA 8kb Transduces only dividing cells
Long-term expression
So, Viral vectors are optimal vehicles for gene transfer and removal of the replicative and
pathogenic ability of viruses, combined with their capacity to carry the therapeutic transgene and
an ability to efficiently infect various mammalian cell types makes them amenable for use in
gene therapy. The immune system has evolved to fight off invading pathogens, which makes
viral vectors subject to immune responses that have to be either blocked or avoided to achieve
therapeutic transgene expression. . Administration of viral vectors can lead to the initiation of
innate and adaptive immune responses against viral particles and gene product. Recent research
has concentrated on various immune modulatory regimens using immune-suppressive drugs in
combination with gene therapy, modification of viral capsids or choice of viral envelope.
Immunogenicity of viral gene transfer can also provoke an immune response against the
33

42. therapeutic transgene product, which may represent a neoantigen owing to the type of gene
mutation present, rendering patients with, for example, null mutations, susceptible to recognizing
the transgene product as a foreign antigen. Although there are similarities in immunity to
different viruses, each vector contains its own set of activation signals, which are further
modified by the environment of a specific tissue [Nayak and Herzog, 2009]. Immune response to
viral vectors is shown below by Figure-15:
Figure-13: Immune Response to Viral Vector
2.7.1.1 Importance of vectors in gene therapy
Most viruses attack their hosts to insert their genetic material into the DNA of the host and this
DNA contains instructions to produce viruses in large numbers. Considering these capabilities,
scientist thought of using these viruses as a vehicle to deliver genetic materials to the host cells
in order to treat genetic diseases. Currently the most common vector is used as a vehicle virus
that has been genetically altered to carry normal human DNA. [ Khan, 2014].
A. Retroviruses
It has been reported that a retrovirus is a RNA virus that can be replicated in a host cell through
the enzyme reverse transcriptase to produce DNA from its RNA genome. Later, the DNA is 34

43. integrated into the host‘s genome by using an integrase enzyme. The virus subsequently
duplicates as part of the host cell‘s DNA. It has been reported that retroviruses are generally
enveloped from outside and belong to the viral family Retroviridae. Moreover, virions of
retroviruses consist of enveloped particles about 100 nm in diameter and it also contains two
identical single-stranded RNA molecules. Even though virions of different retroviruses do not
have the same morphology or biology, all the virion components are very comparable. Moreover,
the key brain component is the envelope which is composed of a protein capsid (Figure-8).
Retroviruses have better capabilities to integrate efficiently into the genomic DNA of animal
cells and can be replicated and transmitted to all of the progeny of these cells. Moreover, severe
oncogenic retroviruses often arise as the result of attaining of sequences derived from cellular
protooncogenes provided an additional stimulus [Khan, 2014].
B. Adenoviruses
Adenoviruses are viruses that contain their genetic material in the form of DNA when these
viruses infect host cells and they introduce their DNA into the host cells for replications. Genetic
material of the adenoviruses is not integrated into the host cells genetic material and the
adenovirus DNA molecule is left free in the nucleus of the host cell,. The genetic messages in
this extra DNA molecule are transcribed just like any other gene and the only difference is that
these extra DNA does not get replicated when the host cell is about to undergo cell division.
Therefore, the progenies of that cell will not have the extra DNA in their nucleus and it is shown
in Figures 14 [Khan, 2014].
35

44. Figure-14: Gene Therapy by Using Adenovirus and Retrovirus Vectors
Figure-15: Gene therapy by using an adenovirus.
2.7.2 RNAi Technologies
RNAi is an evolutionarily conserved defence mechanism occurring naturally against double-
stranded RNA (dsRNA) that can target cellular and viral mRNAs. In this biological process
small RNA interferes with the translation of target mRNA transcript eventually suppressing the
gene expression. The small non-coding RNAs are the cleavage product of dsRNA called
microRNA (miRNA) and small interfering RNA (siRNA). The cleavage is carried out by a
ribonuclease called DICER or Dicer-like enzyme. The small non-coding RNAs in association
with RNA-induced silencing complex (RISC), Argonaute (AGO) and other effector proteins lead
to the phenomenon called RNAi illustrated in Figure-16. The discovery of this phenomenon has
transformed it into a powerful tool of genetic engineering and functional genomics. The
improvement of crop plants by alteration of traits using traditional plant breeding programme is
time consuming and labour intensive. Since last two decades the researchers are switching
towards biotechnological approaches for crop improvement. The manipulations in gene
expression for quality traits in crop can now easily be achieved by RNAi. It can be employed by
36

45. identifying the target gene(s) developing vectors as an RNAi construct, transforming plant and
finally screening and evaluating the traits [Saurabh et al., 2014].
Figure-16: RNAi Technology
2.7.2.1 RNAi Mechanism
Sciencetist are discovered, the mechanism of RNAi is emerging more clearly. In the last few
years, important insights have been gained in elucidating the mechanism of RNAi. A
combination of results obtained from several in vivo and in vitro experiments have gelled into a
two-step mechanistic model for RNAi/PTGS. The first step, referred to as the RNAi initiating
step, involves binding of the RNA nucleases to a large dsRNA and its cleavage into discrete 21-
to 25-nucleotide RNA fragments (siRNA). In the second step, these siRNAs join a multinuclease
complex, RISC,which degrades the homologous singlestranded mRNAs. At present, little is
known about the RNAi intermediates, RNA-protein complexes, and mechanisms of formation of
different complexes during RNAi. In addition to several missing links in the process of RNAi,
the molecular basis of its systemic spread is also largely unknown [Agrawal et al., 2003].
Mechanism of RNAi technologies are given below in Figure-17:
37

46. Figure-17: Mechanism of RNAi Technology
2.7.2.2 Applications of RNAi Technology
Different types of application of RNAi technologies are given below [Singh et al., 2016]:
A. Discovery of RNAi in Plants and Fungi
R. Jorgensen and his colleagues identified a novel mechanism of post-transcriptional gene
silencing in Petunia. They were attempting to introduce a chalcone synthase gene under a strong
promoter to deepen the purple color of Petunia flowers; however, instead of getting a stronger
purple color flower they observed that most flowers lost their color. Thus, they observed
diminished expression of both the homologous endogenous gene and the exogenously introduced
transgenic copy of the gene and termed the phenomenon as co suppression. Although the exact
mechanism of this phenomenon remained undeciphered at that time, the posttranscriptional
nature of gene silencing was still appreciated. The suppression of endogenous
38

47. gene expression by transformation of exogenous homologous sequences was later termed as
quelling in Neurospora crassa.
B. RNAi Technology in Drosophila
Specific gene silencing has been achieved in the embryo extracts and cultured cells of
Drosophila flies by utilizing the RNAi tool. Zamore and colleagues utilized Drosophila
melanogaster 22 RNA Interference embryo lysates to demonstrate the cleavage of long dsRNA
strands into short interfering dsRNA fragments (siRNA) of ~22 nucleotides (nt). Later Elbashir
and colleagues demonstrated that chemically synthesized 21- or 22-nt-long dsRNA carrying 3′
overhangs could induce efficient RNA cleavage in embryo extracts from Drosophila.
C. RNAi in Mammalian Systems
A global nonspecific inhibition of protein synthesis was observed in mammalian cells by
exposing them to dsRNAs that were greater than 30 base pairs (bp) in length. RNAdependent
protein kinase (PKR), and 2′, 5′ oligoadenylate synthetase (2′, 5′-OAS) were responsible for the
nonspecific silencing. PKR phosphorylates eIF-2α, a translation initiation factor,to shut down
global protein synthesis.A synthesis produc to fenzyme2′,5′-OASactivates RNase L, which
induces nonspecific degradation of all mRNAs in a mammalian cell. Long dsRNAs induce
interferon response that activates both of these enzymes in mammalian cells. The nonspecific
interference pathways represent the mammalian cell response to viral infection or other
stressRNA interference could be directly mediated by small interference RNA (siRNA) in
cultured mammalian cells. However, because siRNA does not integrate into the genome, the
RNAi response from siRNA is only transient. In order to induce stable gene suppression in
mammalian cells, Hannon and his colleagues utilized RNA PolIII promoter-driven (e.g., U6 or
H1) expression of short hairpin RNAs (shRNAs). Various approaches have since been developed
for mammalian cells to obtain successful gene silencing.
2.7.3 Non-viral Gene Therapy
The nonviral gene delivery method, use synthetic or natural compounds or physical forces to
deliver a piece of DNA into a cell. The materials used are generally less toxic and immunogenic
than the viral counterparts. In addition, cell or tissue specificity can be achieved by harnessing
cell-specific functionality in the design of chemical or biological vectors, while physical
39

48. procedures can provide spatial precision. Nonviral methods are generally viewed as less
efficacious than the viral methods, and in many cases, the gene expression is short-lived.
However, recent developments suggest that gene delivery by some physical methods has reached
the efficiency and expression duration that is clinically meaningful [Al-Dosari et al., 2009]. Non
viral gene therapy is shown following in Figure-18:
Figure-18: Non Viral Gene Therapy
2.7.3.1 Physical methods of non viral gene therapy
Physical methods of non viral gene technologies are given below [Al-Dosari et al., 2009]:
A. Hydrodynamic Gene Transfer
The hydrodynamic procedure was reported in 1999. When rapid injection of large volume of
DNA solution into a mouse via the tail vein was performed, efficient transfection in liver, lung,
kidney, and heart was achieved. The hydrodynamic method employs the high pressure as a
driving force for gene transfer. The injection of large DNA volume, 8–12% of body weight in
short time (3–5 s), leads to a reversible permeability change in the endothelial lining and the
generation of transient pores in hepatocyte membranes allowing the DNA molecules to diffuse
internally. Up to 30–40% of the hepatocytes can be efficiently transfected. Currently, this
method is considered to be the most efficient nonviral gene transfer method for in vivo gene
delivery in rodents. Using this method, it was possible to provide levels of transgene expression
40

49. close to average levels of physiological gene expression. By using catheter-assisted perfusion,
efficient gene transfer can also be achieved in kidney, muscle, or a specific lobe in the liver. The
simplicity and safety of the hydrodynamic gene delivery allows a wide range of use of this
technique for in vivo transfection of hepatocytes to study promoter function, gene function, and
therapeutic effects of liver-generated secreted proteins in established disease models.
B. Electroporation
The use of an electric field to alter the cell permeability was known since 1960s. However, the
first in vitro and in vivo attempts to utilize electroporation in gene transfer were demonstrated in
1982 and 1991, respectively. In vivo electroporation depends on electric pulses to drive gene
transfer. These pulses generated transient pores in cell membranes followed by intracellular
electrophoretic DNA movement. in vivo electroporation is conducted by first injecting DNA to
the target tissue followed by electric pulses, with varied voltage, pulse duration, and number of
cycles, from two electrodes applied. In vivo electroporation technique is generally safe, efficient,
and can produce good reproducibility compared to other nonviral methods. When parameters are
optimized, this method can generate transfection efficiency equal to that achieved by viral
vectors.
C. Gene Gun
Gene gun delivery, also called ballistic DNA transfer or DNA-coated particle bombardment, was
first used in 1987 for gene transfer in plants. This method depends on the impact of heavy metal
particles on target tissues and delivery of coated DNA on particles in passing. The particles are
accelerated to sufficient velocity by highly pressurized inert gas, usually helium. Macroparticles
made of gold,tungsten, or silver have been used for gene delivery through gene gun. Gas
pressure, particle size, and dosing frequency are critical factors that determine penetration
efficiency to the tissues, the degree of tissue injury, and overall gene transfer levels. Gene gun-
based gene transfer has been extensively tested for intramuscular, intradermal, and intratumor
genetic immunization. It was demonstrated that this approach is able to produce more immune
response with lower doses comparing to needle injection in large animal models and in clinical
human trials.
2.7.4 Liposomes
41

50. Liposomes are colloidal, vesicular structures composed of one or more lipid bilayers surrounding
an equal numbers of aqueous compartments. The sphere like shell encapsulated a liquid interior
which cotain a substance such as peptides and protein, hormone, enzyme, antibiotic, antifungal
and anticancer agents. A free durg injected in blood stream typically achieves therapeutic level
for short duration due to metabolism and excreation. Drug encapsulated by liposomes achieve
therapeutic level for long duration as drug must first be release from liposome before metabolism
and excreation [Shashi et al 2012].
2.7.4.1 Liposomal vaccine and antigen delivery system
The safety of the liposomal drug delivery system makes it a smart choice for mesenchymal stem
cell-based therapy to deliver the viral gene. This is a preferred drug delivery for the vaccine and
antigen because it has a lack of immunogenicity, minimal toxicity and can entrap large gene for
delivery. At a variety of diseases are treated with liposomal antigen delivery system. Vaccine and
protein entrapped in liposome use various combinations of components like lipids surfactants
and other solvent. Liposomal drug delivery of vaccine is prepared by mixing various compounds
like microbes to be vaccinated, antigen in soluble form, and cytokines from DNA and liposome.
Antigens are usually covalently bonded to liposomal membrane. Liposome in immunological
therapy was first used for diphtheria toxoid to enhance immune response.
Structure of liposome is given below in Figure-19:
Figure-19: 2D Structure of Liposome
42

51. 2.7.4.2 Liposome in combination therapy
Combination therapy is used for the treatment to reduce toxic side effects of a single drug as well
as to increase therapeutic efficacy of the combinations than individual drugs. As mainly the
highly toxic drugs are used in combination so it will be a potential approach to deliver those
drugs on targeted sites of action by liposome or nanomedicine.
2.7.4.3 Liposome in delivery of nucleic acids
Gene expression is observed in local than systemic area, although there are a number of cationic
lipids have been synthesized recently, and after administering them, there were significant toxic
side effects were observed. It is evident that DNA or other nucleic acids conjugated with ligand-
bearing liposome have shown significant increase in gene expression than non-targeted delivery
of nucleic acids. There are two types of vectors used in liposomal DNA delivery which are LPD-
I and LPD-II (liposomeentrapped, polycation-condensed DNA). These two vectors are greatly
versatile and safe vectors than other. Most recent applications of liposome are DNA vaccination
and gene therapy to treat diseases caused by genetic deficiencies [Rafe and Ahmed, 2018].
Figure-20: Structural Features of Liposomal Drug Delivery Systems with Entrapped Molecule
2.7.5 Encapsulation Techniques
43

52. The encapsulation technique of choice depends on the type and physical properties of the core
and shell material. The chosen encapsulation technique should give a high encapsulation
efficiency and loading capacity of actives, capsules should not exhibit aggregation or adherence,
capsules should have a narrow particle size distribution without tails, threads, or dents on the
surface, and the process should be suitable for industrial scale production [Vladisavljevic, 2015]
2.7.5.1 Microencapsulation
Microencapsulation is described as a process of enclosing micron-sized particles of solids or
droplets of liquids or gasses in an inert shell, which in turn isolates and protects them from the
external environment. The products obtained by this process are called microparticles,
microcapsules and microspheres which differentiate in morphology and internal structure. When
the particle size is below 1 mm they are known as nanoparticles, nanocapsules, nanospheres,
respectively, and particles having diameter between 3–800 mm are known as microparticles,
microcapsules or microspheres. Particles larger than 1000 mm are known as macroparticles.
Microencapsulation can be done (i) to protect the sensitive substances from the external
environment, (ii) to mask the organoleptic properties like colour, taste, odour of the substance,
(iii) to obtain controlled release of the drug substance, (iv) for safe handling of the toxic
materials, (v) to get targeted release of the drug and (vi) to avoid adverse effects like gastric
irritation of the drug, e.g. aspirin is the first drug which is used to avoid gastric irritation [Jyothi
et al., 2010].
44

53. Chapter-3
Current
Biotechnology
Products
45

54. 3.Current Biotechnology Products
As previously mentioned, there are various classes of biotechnology-based products that are
produced for the treatment or prevention of different pathological conditions. In the following
sections, various classes of biotechnology-based products are discussed along with their
production process and therapeutic applications.
3.1 Antibiotics
Antibiotics are molecules that have the ability to inhibit the growth or killing of microorganisms.
Various antibiotics have been discovered that can be used against a wide range of pathogenic
microorganisms such as bacteria and fungi [Clardy et al., 2009]. Therefore large-scale
production of antibiotics is an important part of biotechnology-based products. An example of a
class of antibiotics produced by the fermentation process is the betalactam antibiotics class,
which includes the penicillins and the cephalosporins. The general structure of penicillin and
cephalosporin is shown in Figure 21. The beta-lactam antibiotics are one of the most clinically
used antibiotics for the treatment of bacterial infections. Their mechanism of action involves the
inhibition of a peptidoglycan transpeptidase enzyme that is required by the bacterial cell for
completing the synthesis of the cell wall. Therefore inhibition of this enzyme by the beta-lactams
prevents the completion of the cell wall synthesis and without an intact cell wall, the bacterial
cell will not be able to survive [Silverman and Hollady, 2014; Purohit et al., 2007]. The
biosynthesis of penicillins and cephalosporins has been well demonstrated metabolically and the
precursors are the amino acids L-cysteine, L-valine, and L-aminoadipic acid.
Figure-21: General Chemical Structures of Penicillins and Cephalosporins with The Beta-
Lactam Ring
46

55. Penicillins G and V are produced by a fed-batch fermentation process that is conducted under
aseptic conditions in tank reactors made of stainless steel. The different factors such as the pH,
the temperature, and the gases are controlled using computers. The carbon source is usually
carbohydrates such as glucose and most of the carbon is used for cellular maintenance and
growth while only about 10% of the carbon is actually used for penicillin synthesis. Corn steep
liquor, ammonium sulfate, and ammonia can be used as nitrogen sources in the medium
[Elander, 2003].
The semisynthetic penicillins are molecules that have the 6-amino penicillanic acid (6-APA)
scaffold. Production of 6-APA by the fermentation process is usually inefficient, therefore, it is
more common to obtain 6-APA by removing the side chain of penicilli G or V. Removing the
side chain of Penicillin G can be done by treating it with the enzyme penicillin G acylase, which
catalyzes the conversion of Penicillin G to 6-APA. The resulting 6-APA is then treated with acid
chlorides to obtain various semisynthetic penicillins [Arroyo et al., 2003].
3.2 Hormones
There are therapeutically important hormones that are proteins or peptides in nature, such as
insulin and human growth hormone (hGH). Therefore these hormones can be produced by
recombinant DNA technology. The insulin hormone, which is used for the treatment of diabetes
(mainly type I) is a notable example of the success of biotechnologybased products [Voet and
Voet, 2011].
A. Insulin Hormone
Human insulin is a 51-amino acid nonglycosylated peptide hormone that consists of two
polypeptide chains, namely chains A and B. Insulin is synthesized in the beta cells of the
pancreas, however, the initial form of insulin is a precursor molecule called preproinsulin that
consists of 86 amino acids. This precursor molecule is converted to another insulin precursor
called proinsulin via cleavage of the N-terminal signal peptide by proteolytic enzymes. The
proinsulin is further cleaved internally to give the polypeptide chains A and B of insulin and the
C peptide. Disulfide bonds between the polypeptide chains A and B are formed to give the final
form of the insulin, which is stored in the beta cells of the pancreas for secretion [Luzhetskyy et
al., 2012].
47

56. Human insulin hormone has various effects on different metabolic processes, as it has a
significant role in the regulation of carbohydrates metabolism and hence blood glucose levels.
Additionally, insulin also has various effects on the metabolism of proteins and lipids. The
inability to produce sufficient insulin hormone gives rise to diabetes mellitus type 1, which is a
metabolic disease in which the glucose levels are elevated in the blood as well as in the urine.
Other metabolic abnormalities include high rates of ketogenesis, beta-oxidation of fatty acids,
and gluconeogenesis [Voet and Voet, 2011]. Diabetes is a prevalent condition that affects a wide
range of population with the possible progression of the disease to different complications or
even death. The causes of the insulin deficiency include the progressive destruction of the beta
cells that synthesize and secrete insulin by autoimmune conditions or viral infections. Treating
diabetes type 1 can be achieved through the administration of a proper insulin formulation to
compensate for the deficient endogenous insulin and lower the blood glucose levels [Walsh,
2014a].
In the past, the majority of the insulin preparations contained insulin extracted from the pancreas
of animals such as pigs. However, this method suffered from several issues such as
immunogenicity of the extracted insulin as well as the possible contaminants presents in the
pancreatic extracts that could cause harmful effects on the patient. Recombinant DNA
technology can be used to produce human insulin with better properties as well as in larger
quantities. Insulin can be prepared by the expression of the nucleotide sequence that codes for
the proinsulin polypeptide in a suitable host, usually E. coli cells. The synthesized proinsulin
molecules are converted to insulin by the action of proteolytic enzymes. It is also possible to use
separate systems for the production of the polypeptide chains A and B, then to isolate and purify
them from the different systems. Finally, the two chains are joined by the disulfide bonds to give
the final product [Luzhetskyy et al., 2012]. To ensure the purity of insulin preparations from any
impurities that can arise from the production process, various methods such as HPLC can be
employed to remove these impurities and give high purity insulin preparations [Walsh, 2014b].
B. Human Growth Hormone
hGH, also known as somatotropin, is a 191-amino acid nonglycosylated peptide hormone with a
molecular weight of 22,000 Da. This hormone is synthesized in the cells of the anterior pituitary;
however, this initially synthesized form contains an additional peptide sequence that is cleaved
48

57. later to give the hGH that circulate in the blood. This hormone is essential for proper human
growth and development processes, and any deficiencies can result in growth abnormalities
[Jamil, 2007; Simpson et al., 2002]. The effects of the hGH include metabolic regulation of
various pathways such as increasing the synthesis of proteins, decreasing glucose metabolism,
and increasing lipolysis. The hGH products have been shown to be successful in treating various
conditions such as hGH deficiency, Prader_Willi syndrome, and Turner syndrome. In addition to
the effectiveness of the hGH in treating these conditions, it also has good safety profile as no
serious side effects are present [Iglesias and Diez, 1999; Takeda et al., 2010].
There are several pharmaceutical companies that produce recombinant hGH using different
methods in the production, isolation, and purification of the hormone. E. coli cells can be used as
the host cells because the hGH is nonglycosylated. For example, Genotropin is produced into the
host system as a fusion protein, where an attached signal sequence (enterotoxin II signal
sequence) allows the protein to be secreted into the periplasmic space. Once the fusion protein is
at the periplasmic space, a peptidase enzyme cleaves the fusion protein into the active form of
hGH with all residues except the N-terminal methionine residue. After the hormone has been
expressed in the cells, the cells are harvested and the content is released by freezing and thawing
process. Several chromatographic methods are then required for the purification of the protein
[Luzhetskyy et al., 2012]. The available formulations of the recombinant hGH are usually
supplied in a lyophilized form [Beale, 2011].
3.3 Enzymes
Enzymes are proteins that are used for the catalysis of chemical reactions in the cells and are
responsible for the synthesis and degradation of various biological molecules. Therefore
enzymes can also play roles in pathological conditions, as the deficiency of enzymes can cause
various diseases depending on the function of the deficient enzyme. For example, if an enzyme
that is responsible for the degradation of a certain compound is deficient, then the accumulation
of that compound can lead to metabolic abnormalities and harmful effects [Silverman and
Hollady, 2014]. Deficiencies of enzymes mainly arise because of hereditary conditions. Because
of these various roles of enzymes in pathological conditions, the production of enzymes as drugs
for the treatment of different diseases is an attractive approach. Various biotechnology-based
49

58. enzymes have been introduced into the market and have been shown to be successful for the
treatment of the targeted diseases [Yari et al., 2017; Kunamneni et al., 2018].
For example, Gaucher‘s disease is caused by the deficiency of the enzyme
betaglucocerebrosidase, and is a hereditary condition. This enzyme is responsible for the
catalysis of the hydrolysis of glucocerebroside to give the corresponding ceramide and glucose.
A deficiency of a properly functioning beta-glucocerebrosidase in the cells will lead to the
accumulation of the substrate glucocerebroside, which is a glycolipid. The glucocerebroside
accumulates mainly in the macrophages, which are called Gaucher‘s cells in this case. These
Gaucher‘s cells can accumulate in other body organs and cause further complications such as
anemia. The spleen, liver, and bone marrow are the main sites of accumulation, although it is
possible for the accumulation to occur in other organs such as the kidney [Smith et al., 2017;
Beale, 2011]. Recombinant beta-glucocerebrosidase produced by CHO cells can be provided as
an enzyme therapy for the treatment of this Gaucher‘s disease by catalyzing the hydrolysis of the
glucocerebroside and thus normalizing the metabolic pathway [Beale, 2011].
Another example of using enzyme therapy to treat pathological conditions is in chronic
pancreatitis treatment. In this condition, different enzymes are used including lipase and
amylases, which are pancreatic enzymes for the treatment of chronic pancreatitis [Inatomi et al.,
2016]
3.4 Blood Clotting Factors
The blood clotting process involves a series of various plasma proteins that function with each
other to properly carry out the clotting process. In cases where any of these factors is absent or
deficient, the clotting process will not proceed properly leading to serious pathological
conditions such as hemophilia A and B [Palta et al., 2014; Zimmerman and Valentino, 2013].
Deficiencies of clotting factors can be caused by genetic disorders, although nongenetic blood
clotting disorders can occur because of liver dysfunction or deficiency in vitamin K, which plays
an important role in the coagulation process. Normally, treating the conditions related to blood
clotting factors deficiency is achieved through administration of the deficient blood clotting
factor. The sources of the blood clotting factors can be the blood of healthy human donors or by
recombinant DNA technology_based production (Sutor et al., 1999; Zimmerman and Valentino,
50

59. 2013). Blood clotting factors from the blood of a healthy human donor are obtained by treating
several purification steps as well as sterilization [Di Minno et al., 2016].
Purification of blood clotting factors can be done through fractionation techniques although
chromatographic techniques can also be employed in purifying some clotting factors.
Sterilization of the produced form is done by filtration. The final preparation is freeze-dried and
usually, an anticoagulant agent is added to the preparation. The major drawback of using blood
clotting factors obtained from donors is the possibility of the presence of viral contaminants or
other pathogens, which will be transferred to the patient receiving the treatment and thus
resulting in infectious diseases. Screening of blood donations, as well as the addition of antiviral
agents to the final preparation, can help in preventing the possible viral transmission to the
patients receiving the treatment [Franchini, 2013; Walsh, 2014c].
Another method for the production of blood clotting factors is by using recombinant DNA
technology. In this case, the recombinant blood clotting factors preparation has the advantage of
being devoid of the possible viral and pathogenic contaminants that are associated with obtaining
the clotting factors from blood donors. Since the majority of blood clotting factors are
glycosylated, in addition to having other posttranslational modifications, it is necessary to use a
eukaryotic cell as the host. The used cells for production include CHO cells as well as BHK
cells. The final form is purified using different chromatographic methods and the final product is
usually supplied in a lyophilized form [Pipe, 2008; Lusher, 2000; Lee, 1999].
3.5 Cytokines
Cytokines are a group of protein molecules produced mainly by the leukocytes that regulate the
immunological and inflammatory response in addition to carrying out various other functions
such as controlling the growth and differentiation of cells. Cytokines act through activation of
the receptors on the target‘s cell surface. Although cytokines are secreted mainly by the
leukocytes, certain other cells in the body can also produce them [Vilˇcek and Feldmann, 2004].
There are different classes of cytokines, including interferons and interleukins, which are of a
particular interest in the therapeutic applications of cytokines. Because of the diverse functions
of cytokines, their use as therapeutic agents has great potential for treating various conditions
such as cancer and viral infections [Tayaland Kalra, 2008].
51

60. 3.6 Interferon’s
Interferons are a class of cytokines that have antiviral activity as well as potential anticancer
effects. Interferons can be classified into two types, the type I consists of alphainterferon and
beta-interferons, while type II consists of gamma-interferon. Type I interferons are produced by
different cells in response to various stimuli [Vilˇcek and Feldmann, 2004].
The cells include specialized dendritic cells and macrophages. The stimulus can be viruses or
certain molecules such as double-stranded RNA. On the other hand, type II interferon is
produced mainly by the natural killer cells as well as the T cells and secreted in response to
different stimuli. All the interferons act by binding to different heterodimeric receptors on the
targeted cell‘s surface and transduce the signal through the activation of the Janus-activated
kinase as well as signal transducer and activator of transcription. This leads to the expression of
different genes to give the required biological response [Darnell et al., 1994; Vilˇcek and
Feldmann, 2004].
All the three mentioned interferons can be used clinically for treating various conditions that are
related to viral infections as well as cancer. Available alpha-interferon preparations include
recombinant interferon-2a (Rofereon-A, Roche), which is produced as nonglycosylated protein
that consists of 165 amino acids. This product is indicated mainlfor the treatment of hepatitis B
and C as well as Kaposi‘s sarcoma [El-Baky and Redwan, 2015].
Interferon-beta1b (betaferon, Schering) is a human beta-interferon-based product produced for
treating relapsing/remitting multiple sclerosis. The host cells used for expression are E. coli cells
with identical amino acid sequence except for one cysteine residue that is replaced by a serine
residue for improving the stability during the synthesis process inside the cells ([Rojas et al.,
2014; Zvonova et al., 2017].
Therapeutic preparations of gamma-interferon are also available; for example,
gammainterferon1b is a polypeptide chain that is composed of 140 amino acids. Although the
gamma-interferon produced naturally in the cells is a glycosylated polypeptide, the commercial
form is nonglycosylated as the host used is E. coli cells. Indications of this product include
severe malignant osteoporosis as well as chronic granulomatous [Watson, 2011].
52