Designing Drug Delivery Systems for Biotech Products Stability

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Designingof DrugDeliverySystemforBiotechnologyProductsConsideringStabilityAspectsand
MonitoringMethodsof ImprovingStability
Summery:
"There where is life, there is DNA, where there is DNA, there is “biotechnology". 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 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.

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1. Introduction:
The approach of biotechnological techniques for the fabrication of drugs brought a mutiny to the
pharmaceutical field. Biotechnology allows bespoke production of biopharmaceuticals and
biotechnological drugs. However, many of them require special formulation technologies to
overcome drug-associated problems. Such probable challenges to solve are as follows: poor
solubility, limited chemical stability in vitro and in vivo after administration (i.e., short half-life),
poor bioavailability, and potentially strong side effects requiring drug enrichment at the site of
action. 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 also will have implications for the development and success of new therapeutic
strategies. On the long way from the clinic to market, however, several issues will have to be
addressed, including suitable scientific development, specific financial support as a result of
altered scientific policy, government regulations, and market forces. Medication delivery
systems that concentrate medications only where needed and used could reduce the destruction
of surrounding tissues while minimizing side effects. The benefits of such systems in the
treatment of both acute and chronic conditions are clear. Because research demonstrates that
patient adherence is improved when side effects are minimized, it is imperative that drug
delivery systems efficiently and precisely deliver medications in a manner that the patient finds
acceptable and tolerable. Patients themselves are demanding drug delivery systems that are
convenient, easy to use, and affordable. 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].
2. Biotechnology:
Biotechnology, as the word suggests, is combination of biology and technology. Biotechnology
is the use of technology to use, modify or upgrade the part or whole of biological system for
industrial and human welfare. Biotechnology is the technological application which utilizes
biological entities, living organisms or biological derivatives [Raju, 2016].

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Biotechnology is the use of living things especially cells and bacteria for production of various
products for benefiting human beings. It is a combination of various technologies, applied
together to living cells, including not only biology, but also subjects like mathematics, physics,
chemistry and engineering. Its application ranges from agriculture (Animal Husbandry, Cropping
system, Soil science and Soil Conservation, Plant Physiology, Seed Technology etc. and Crop
Management) to industry (food, pharmaceutical, chemical, byproducts, textiles etc.), medicine,
nutrition, environmental conservation, Cell Biology, making it one of the fastest growing fields.
Biotechnology is to modify genetic structure in animals and plants to improve them in desired
way for getting beneficial products [Raju, 2016].
2.1 History of Biotechnology:
The origin of biotechnology was not novel. The origin of biotechnology arose in ancient age.
The ancient Egypt and China were the countries that used biotechnology in the form of food
fermentation. The concept of biotechnology bound in a wide range of procedures for modifying
living organisms based on the need of human activities. If it can be considered a trade, can be
traced many centuries back, when wine making, production of vinegar and distilling were
important human skills. The history of biotechnology as an industry begins in the early 19th
century [Raju, 2016].
After the discovery of Leeuwenhoek’s microscope microorganisms could be seen, in 1865 only
after 200 years, Pasteur has given scientific description for fermentation process. At that time
another achievement was done, at a session of the Hungarian Society of Natural Sciences on 13th
November, 1861, a Hungarian chemist, M. Preysz, reported on a procedure for the preservation
of wine by heat treatment. His method was published, however, only in 1865, after Pasteur’s
famous publication, the discovery has not given legal priority. It is generally not known that the
term “biotechnology” was first used by a Hungarian expert, K. Ereky, in his book published in
1919: "The Biotechnology of Meat, Fat, and Milk Production in the Agricultural Plant". Since
ancient times Hungarians were interested in life related problems to resolve them. Humankind
was interested in biology since the close relationship with nature and adopted the attitude of
observing the field of science. The American Chemical Society defines biotechnology as the
application of biological organisms, systems, or processes by various industries to learn about
the science of life and the improvement of the value of materials and organisms such as

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pharmaceuticals, crops, and livestock. As per European Federation of Biotechnology,
biotechnology is the integration of natural science and organisms, cells, parts thereof, and
molecular analogues for products and services [Raju, 2016].
Basically biotechnology classified in to four major categories including crop production and
agriculture, health care (medical), Environmental and industrial biotechnologies.
 Green biotechnology: It is the technology applied to agricultural processes.
 Red biotechnology: It is the technology used in medical applications.
 Blue biotechnology: Blue biotechnology is the term used to describe aquatic and marine
applications of biotechnology.
 White biotechnology: It is the technology used to industrial processes [Raju, 2016].
2.2 Modern Biotechnology:
The Second World War became a major impediment in scientific discoveries. After the end of
the second world war some, very crucial discoveries were reported, which paved the path for
modern biotechnology and to its current status. In 1953, JD Watson and FHC Crick for the first
time cleared the mysteries around the DNA as a genetic material, by giving a structural model of
DNA, popularly known as, ‘Double Helix Model of DNA’. This model was able to explain
various phenomena related to DNA replication, and its role in inheritance. Dr. Hargobind
Khorana was able to synthesize the DNA in test tube, while Karl Mullis added value to
Khorana's discovery by amplifying DNA in a test tube, thousand times more than the original
amount of DNA. Using this technological advancement, other scientists were able to insert a
foreign DNA into another host and were even able to monitor the transfer of a foreign DNA into
the next generation. In 1997, Ian Wilmut an Irish scientist, was successful to clone a sheep and
named the cloned sheep as ‘Dolly’. In 2003, the Human Genome Project completes sequencing
of the human genome. In 1978, Boyer was able to isolate a gene for insulin (a hormone to
regulate blood sugar levels) from human genome using biotechnology. He then inserted it into
bacteria, which allowed the gene to reproduce a larger quantity of insulin for diabetics. Structure
of DNA by Watson and Crick (1953) Modern biotechnology provides breakthrough products and
technologies to combat rare diseases, reduce our environmental footprint, feed the hungry, use
less and cleaner energy, and have safer, cleaner and more efficient industrial manufacturing
processes. Currently, there are: More than 250 biotechnology health care products and vaccines

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available to patients, many for previously untreatable diseases. More than 13.3 million farmers
around the world use agricultural biotechnology to increase yields, prevent damage from pests
and reduce farming's impact on the environment. More than 50 biorefineries are being built
across North America to test and refine technologies to produce biofuels and chemicals from
renewable biomass, which can help reduce greenhouse gas emissions [Raju, 2016].
2.3 Fields of Biotechnology:
2.3.1 Plant Biotechnology:
Plant biotechnology is the technique which is used to manipulate the plants for specific needs or
requirement. In basic agricultural practices we generally wait for natural production of offspring
that will have basic quality. But in plant biotechnology we select the desired quality of a trait to
clump with other quality to produce multiple qualitative traits in one offspring. For that plant
biotechnology applies genetics, tissue culture, genetic engineering and transgenic crops. Plant
tissue culture is a part of plant biotechnology which is the collection of many techniques that is
used to maintain and grow plant, plant cells, plant tissues under controlled sterile conditions over
the nutrient medium [Raju, 2016].
2.3.2 Healthcare biotechnology:
Healthcare biotechnology refers to a vaccine or diagnostic or medicinal that consists of or has
been produced by living organisms through recombinant DNA technology. This biotechnological
application has major impact on patients to meet their needs. This application not only
encompasses diagnostics and medicines by biotechnological process and also helps in gene,
tissue and cell therapies [Raju, 2016].
2.3.3 Marine Biotechnology:
Marine Biotechnology is one of the new field of study, emerged in the past few years. It began in
1998 when scientists from the Scripps Institution of Oceanography and various departments of
the University of California, San Diego, came together and formed the Centre for Marine
Biotechnology and Biomedicine. The intention of Marine Biotechnology is to host scientific
contributions in marine science that are based on the enormous biodiversity of marine
ecosystems and the genetic uniqueness of marine organisms to develop useful products and
applications [Raju, 2016].

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2.3.4 Environmental Biotechnology:
Environmental biotechnology is biotechnology that is applied to and used to study the natural
environment. Environmental biotechnology could also imply that one tries to harness biological
process for commercial uses and exploitation. The International Society for Environmental
Biotechnology defines environmental biotechnology as "the development, use and regulation of
biological systems for remediation of contaminated environments land, air, water for
environment-friendly processes (green manufacturing technologies and sustainable development)
[Raju, 2016].
3. Different Routes of Drug Delivery System for Biotech Products:
3.1 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 bioadhesives has been studied to promote the penetration
of drugs through and between intestinal cells. Polymers such as polyanhydrides 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 bioadhesives, owing to their non-toxicity and special
binding properties, which simulate a ligand–receptor interaction [Orive et al., 2003].
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 [Orive et al., 2003].
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

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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 [Orive et al., 2003].
3.2Nasal 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 [Orive et al., 2003].
3.3 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 [Orive et al., 2003].
3.4 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

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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
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 [Akers,
2013].
3.5 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 nanoparticulate 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
[Purohit et al., 2018].
3.6 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 [Panda et al., 2013].

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3.7 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,
gender specificity, local irritation and influence of sexual intercourse, need to be addressed
during the design of a vaginal formulation [Wassen et al., 1996].
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
[Wassen et al., 1996].
4. Designing of Drug Delivery System for Biotechnology Products:
Numerous techniques are used to create biotechnology products. These include rDNA
technology, MAb technology, polymerase chain reaction, gene therapy, nucleotide blockade or
antisense nucleic acids, and peptide technology etc [Ansel et al., 2005].
4.1 Recombinant DNA:
DNA, deoxyribonucleic acid, has been called the substance of life. It is DNA that constitutes
genes, allowing cells to reproduce and maintain life.The ability to hydrolyze selectively a
population of DNA molecules with a number of endonucleases promoted a technique for joining
two different DNA molecules: recombinant DNA, or rDNA. This technique uses other
techniques (replication, separation, identification) that permit production of large quantities of
purified DNA fragments. These combined techniques, referred to as rDNA technology, allow the

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removal of a specific piece of DNA out of a larger, more complex molecule. Consequently,
rDNAs have been prepared with DNA fragments from bacteria combined with fragments from
humans, viruses with viruses, and so forth. The ability to join two different pieces of DNA at
specific sites within the molecules is achieved with two enzymes: a restriction endonuclease and
a DNA ligase [Ansel et al., 2005].
DNA probe technology is being used to diagnose disease. It uses small pieces of DNA to search
a cell for viral infection or for genetic defects. DNA probes have application in testing for
infectious disease, cancer, genetic defects, and susceptibility to disease. Using DNA probes,
scientists can locate a disease-causing gene, which in turn can lead to the development of
replacement therapies. In producing a DNA probe, the initial step is synthesis of the specific
strand of DNA with the sequence of nucleotides that matches those of the gene being
investigated[Figure:01]. For instance, to test for a particular virus, first the DNA strand is
developed to be identical to one in the virus. The second step is to tagthe synthetic gene with a
dye or radioactive isotope. When introduced into a specimen, the synthetic strand of DNA acts as
a probe, searching for a matching or complementary strand. When one is found, the two
hybridize or join together. When the probe is bound to the virus, the dye reveals the location of
the viral gene. If the synthetic DNA strand carries a radionuclide isotope, it will bind to the viral
strand of DNA and reveal the virus through gamma ray scanning [Ansel et al., 2005].
Figure 1: DNA Probe Technology

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4.2.1 Anticoagulant Drug: Lepirudin (Refludan):
Lepirudin (rDNA), a recombinant hirudin derived from yeast cells, is a highly specific direct
inhibitor of thrombin. It is the first of the hirudin class of anticoagulants. Natural hirudin is
produced in trace quantity as a family of highly homologous isopolypeptides by the leech Hirudo
medicinalis. Biosynthetic lepirudin is identical to natural hirudin except for substitution of a
leucine molecule for isoleucine at the N-terminal end of the molecule and the absence of a
sulfate group on the tyrosine molecule at position 63 [Ansel et al., 2005].
Lepirudin is indicated for heparin-induced thrombocytopenia (HIT) and associated
thromboembolic disease to prevent further thromboembolic complications. The formation of
antihirudin antibodies have been observed in approximately 40% of HIT patients treated with the
drug. This ultimately may increase the anticoagulant effect of the lepirudin because of delayed
renal elimination of active lepirudin–antihirudin complexes [Ansel et al., 2005].
Figure 2: Refludan
4.2.2 Recombinant Alteplase (Activase):
Alteplase, a tPA produced by rDNA, is used in the management of acute myocardialzinfarction
(AMI), acute ischemic stroke, and pulmonary embolism (PE). It is a sterile, purified glycoprotein
of 527 amino acids. It is synthesized using the complementary DNA for natural human tissue-
type plasminogen activator obtained from a human melanoma cell line [Ansel et al., 2005].
4.3 Monoclonal Antibodies:
When a foreign body or antigen molecule enters the body, an immune response begins. This
molecule may contain several different epitopes, and lines of beta lymphocytes will proliferate,
each secreting an immunoglobulin (antibody) molecule that fits a single epitope. By contrast,

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MAbs are produced as a result of perpetuating the expression of a single beta lymphocyte.
Consequently, all of the antibody molecules secreted by a series of daughter cells derived from a
single dividing parent beta lymphocyte are genetically identical. Through thedevelopment of
hybridoma technology emanating from Kohler and Milstein’s research, it became possible to
produce identical monospecific antibodies in almost unlimited quantities. These are constructed
by the fusion of beta lymphocytes, stimulated with a specific antigen, with immortal myeloma
cells. The resultant hybridomas can be maintained in cultures and produce large amounts of
antibodies. From these hybrid cells, a specific cell line or clone producing monospecific
immunoglobulins can be selected [Vermeij and Blok, 1996].
Figure 2: Monoclonal Antibody Technology
4.3.1 Adalimumab (Humira):
Adalimumab was approved by the FDA in early 2003 for reducing signs and symptoms in
rheumatoid arthritis patients who have not responded to previous treatments with methotrexate
and other DMARDs. Administered subcutaneously every 2 weeks, this drug now offers an
attractive alternative for patients who require TNF-a blocker therapy. TNF-a is responsible for
much of the pain and inflammation associated with rheumatoid arthritis. Adalimumab is also
indicated for psoriatic arthritis, ankylosing spondylitis, and Crohn disease.Adalimumab is
administered subcutaneously as an easy-to-use, single-use, prefilled syringe or disposable,
single-use pens for subcutaneous injection (40 mg/0.8 mL) [Yang et al., 2003].

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4.3.2 Daclizumab (Zenapax):
Daclizumab is an immunosuppressive humanized IgG1 MAb produced by rDNA technology that
binds specifically to the alpha unit (Tac subunit) of the human high-affinity IL-2 receptor that is
expressed on the surface of activated lymphocytes. Daclizumab is a composite of human (90%)
and murine (10%) antibody sequences. Daclizumab is indicated for the prophylaxis of acute
organ rejection in patients receiving renal transplants. It is used as part of an immunosuppressive
regimen that includes cyclosporine and corticosteroids. The recommended dose is 1 mg/kg
intravenously as part of an immunosuppressive regimen [Ansel et al., 2005].
4.4 Gene Therapy:
In gene therapy, a virus is used as a vector to deliver corrective and active genes into the cells of
a patient. Gene therapy is designed to introduce genetic material into cells to compensate for
abnormal genes or to make a beneficial protein. If a mutated gene causes a necessary protein to
be faulty or missing, gene therapy may be able to introduce a normal copy of the gene to restore
the function of the protein [Khan et al., 2016].
A gene that is inserted directly into a cell usually does not function. Instead, a carrier called a
vector is genetically engineered to deliver the gene. Certain viruses are often used as vectors
because they can deliver the new gene by infecting the cell. The viruses are modified so they
can't cause disease when used in people. Some types of virus, such as retroviruses, integrate their
genetic material (including the new gene) into a chromosome in the human cell. Other viruses,
such as adenoviruses, introduce their DNA into the nucleus of the cell, but the DNA is not
integrated into a chromosome [Khan et al., 2016].
The vector can be injected or given intravenously (by IV) directly into a specific tissue in the
body, where it is taken up by individual cells. Alternately, a sample of the patient's cells can be
removed and exposed to the vector in a laboratory setting. The cells containing the vector are
then returned to the patient. If the treatment is successful, the new gene delivered by the vector
will make a functioning protein [Khan et al., 2016].

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Figure 3: Mechanism of Gene Therapy
Many different cancers including lung, gynecological, skin, urological, neurological, and
gastrointestinal tumors, as well as hematological malignancies and pediatric tumors, have been
targeted through gene therapy. Inserting tumor suppressor genes to immunotherapy, oncolytic
virotherapy and gene directed enzyme prodrug therapy is different strategies that have been used
to treat different types of cancers. The p53, a commonly transferred tumor suppressor gene, is a
key player in cancer treating efforts [Khan et al., 2016].
Treatment of cardiovascular diseases by gene therapy is an important strategy in health care
science. In cardiovascular field, gene therapy will provide a new avenue for therapeutic
angiogenesis, myocardial protection, regeneration and repair, prevention of restenosis following
angioplasty, prevention of bypass graft failure, and risk-factor management [Khan et al., 2016].
High density lipoprotein gene ABCA1 mutation in cells can make the cells be differentiated into
macrophages. Gene knockouts in embryonic stem cells enhance the capability of cells to be
differentiated into macrophages and specifically target the desired pathogens. The allele

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replacements in this case will assist in studying protein coding changes and regulatory variants
involved in alteration of mRNA transcription and stability in macrophages [Khan et al., 2016].
4.5Polymerase Chain Reaction:
Polymerase chain reaction is a biotechnologic process whereby there is substantial amplification
(more than 100,000-fold) of a target nucleic acid sequence (a gene). This enzymatic reaction
occurs in repeated cycles of a three-step process. First, DNA is denatured to separate the two
strands. Next, a nucleic acid primer is hybridized to each DNA strand at a specific location
within the nucleic acid sequence. Finally, a DNA polymerase enzyme is added for extension of
the primer along the DNA strand to copy the target nucleic acid sequence. Each cycle duplicates
the DNA molecules. This cycle is repeated until sufficient DNA sequence material is copied. For
example, 20 cycles with a 90% success rate will yield 375,000 amplification of a DNA sequence
[Ansel et al., 2005].
Figure 4: Polymerase Chain Reaction Technology
4.6 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 [Ansel et al., 2005].

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4.7Antisense Technology:
Antisense agents are valuable tools to inhibit the expression of a target gene in a sequence-
specific manner,and may be used for functional genomics,target validation and therapeutic
purposes. Three types of anti-mRNA strategies can be distinguished. Firstly,the use of single
stranded antisenseoligonucleotides; secondly,the triggering of RNA cleavage through
catalytically active oligonucleotides referred to as ribozymes; and thirdly,RNA interference
induced by small interfering RNA molecules [Kurreck, 2003].
The difference between antisense approaches and conventional drugs,most of which bind to
proteins and thereby modulate their function. In contrast, antisense agents act at the mRNA
level,preventing its translation into protein. Antisense-oligonucleotides (AS-ONs) pair with their
complementary mRNA,whereas ribozymes and DNA enzymes are catalytically active ONs that
not only bind,but can also cleave,their target RNA. In recent years,considerable progress has
been made through the development of novel chemical modifications to stabilize ONs against
nucleolytic degradation and enhance their target affinity. In addition,RNA interference has been
established as a third,highly efficient method of suppressing gene expression in mammalian cells
by the use of 21–23-mer small interfering RNA (siRNA) molecules [Kurreck, 2003].
Figure 5: Antisense Technology

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4.7.1 Efavirenz (Sustiva):
Efavirenz is a nonnucleoside reverse transcriptase inhibitor and the first anti-HIV drug to be
approved by the FDA for once-daily dosing in combination with other anti-HIV drugs. Clinical
trials demonstrated that efavirenz reduces plasma viral RNA to below quantifiable levels in a
majority of HIV-1-infected naïve and treatment-experienced individuals in two, three, and four-
drug combinations. Efavirenz is available as an oral capsule and can be taken once a day on an
empty stomach, preferably at bedtime to improve any nervous system symptoms. However, if
taken with food, it is advised that it should not be administered with high-fat meals, because this
interaction may increase the drug’s systemic absorption [Ansel et al., 2005].
5. Current Biotechnology Products:
5.1 Vaccine:
Genetically engineered vaccines use a synthetic copy of the protein coat of a virus to fool the
body’s immune system into mounting a protective response. This avenue avoids the use of live
viruses and minimizes the risk of causing the disease the vaccine was intended to prevent.
Further, these vaccines will all but eliminate concern about the natural vaccine, which could be
derived from blood donor carriers who may harbor the AIDS virus. The first genetically
engineered vaccine for use in the United States was approved by the FDA in 1986 for hepatitis
B, a widespread liver infection. This vaccine has now replaced the plasma-derived vaccine
5.2 Hepatitis B Vaccine Recombinant (Engerix-B, Recombivax HB):
The plasma-derived hepatitis B vaccine is no longer being produced in the United States, and its
use is limited to hemodialysis patients,other immunocompromised patients, and persons with
known allergies to yeast. Recombinant hepatitis B vaccine has demonstrated an ability to induce
antibody to hepatitis B surface antigen (anti-HBs) that is biochemically and immunologically
comparable to antibody induced by the plasma-derived hepatitis B vaccine.
Hepatitis B recombinant vaccine is indicated for immunization of persons of all ages against
infection caused by all types of hepatitis B virus. A dialysis formulation (Recombivax HB
Dialysis Formulation) is indicated for immunization of adult predialysis and dialysis patients.
The vaccine should be administered by intramuscular injection into the deltoid muscle (outer

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aspect of the upper arm) for immunization of adults and older children. The anterolateral thigh is
recommended for infants and younger children. For patients with a risk of hemorrhage following
intramuscular injection, the vaccine may be administered subcutaneously, although the
subsequent antibody titer may be lower and there may be an increased risk of a local reaction
5.3 Haemophilus B Conjugate Vaccine (HibTITER, Liquid PedvaxHIB, ActHIB):
Prior to the introduction of Haemophilus B conjugate vaccines, Haemophilus influenzae type B
(HIB) was the most frequent cause of bacterial meningitis and leading cause of serious systemic
bacterial disease among children worldwide.HIB conjugate vaccines use a new technology,
covalent bonding of the capsularpolysaccharide of HIB to diphtheria toxoid, diphtheria CRM197
protein, or an outer membrane protein complex (OMPC) of Neisseria meningitidis, to produce an
antigen that is postulated to convert the T-independent antigen to a T-dependent antigen. The
protein carries both its own antigenic determinants and those of the covalently bound
polysaccharide. Thus, the polysaccharide is theorized to be presented as a T-dependent antigen,
resulting in both an enhanced antibody response and an immunologic memory [Ansel et al.,
2005].
5.4 Hormone:
In 1982, the FDA approved the first dosage form obtained through biotechnological processes,
recombinant human insulin for the treatment of patients with diabetes, using recombinant DNA
techniques in the bacteria E. coli (Humulin®, Novolin®, Velosulin®). Today, recombinant
human insulin is available in different concentrations under different forms of therapeutic action
(insulin lispro, insulin aspart, insulin glargine - respectively, very fast, fast, long acting) and for
different applications (intramuscular, sub-cutaneous, etc.). The recombinant human growth
hormone improved the long-term treatment of children whose body was not producing enough
growth hormone. Somatropin is a recombinant human growth hormone, marketed under different
brand names such as Saizen®, Nutropin®, Humatrope® and Serostin® [Almeida et al., 2011].
5.5 Systemic Growth Hormone (Humatrope, Protropin):
Somatrem (Protropin) is a biosynthetic single polypeptide chain of 192 amino acids produced by
rDNA in E. coli. This drug has one more amino acid (methionine) than natural hGH. Somatropin

19
recombinant (Humatrope), biosynthetically produced by another rDNA process, possesses amino
acid sequencing identical to the naturally occurring hGH (191 amino acids). This hormone
stimulates linear growth by affecting the cartilaginous growth areas of long bones. It also
stimulates growth by increasing the number and size of skeletal muscle cells, influencing the size
of organs, and increasing red cell mass through erythropoietin stimulation. Somatrem for
injection is initially administered intramuscularly or subcutaneously [Ansel et al., 2005].
5.6 Antibiotics:
Antibiotics are the largest group in terms of economic importance among the products obtained
by fermentation. Some examples of antibiotics whose synthesis involved microorganisms
include penicillin produced from Penicillium notatum; cephalosporins (usually semisynthetic
process) from the genus Streptomyces; chloramphenicol from Streptomyces venezuelae;
streptomycin from Streptomyces griseus; cycloserine from Streptomyces orchidaceus;
clindamycin from Streptomyces lincolnensis; vancomycin isolated from cultures of Streptomyces
orientalis (Nocardia orientalis); teicoplanin from Actinmoplanes teichomyceticus and mupirocin
from Pseudomonas fluoresces [Almeida et al., 2011].
5.7 Blood Factors:
Even with identical causes, two types of hemophilia can be distinguished, namely, hemophilia A
(the deficient or abnormal element is Factor VIII or antihemophilic factor A) and hemophilia B
(the deficient or abnormal element is Factor IX or antihemophilic Factor B). These two blood
clotting factors are produced by recombinant techniques. The recombinant Factor VIII produced
in CHO cells, containing 1438 a.a. is used in the treatment of hemophilia A (a hereditary disease
characterized by slow blood clotting and difficulty controlling blood loss). Another example is
the Factor IX produced in CHO cells, containing 415 a.a. used in the treatment of hemophilia B.
The gene that produces this factor was cloned in a sheep by a Scottish laboratory in 1997, and
this sheepsubsequently produced milk that contained this factor [Almeida et al., 2011].
5.7.1 Atryn® (antithrombin recombinant):
In 2009, the FDA approved Atryn® (antithrombin recombinant), the first medicine produced
using genetically engineered animals. This protein with anticoagulant and anti-inflammatory
properties is produced in the milk of goats that have been genetically modified. Atryn® is used

20
for the prevention of peri-operative and peri-partum thromboembolic events in hereditary
antithrombin deficient patients. The European Medicines Agency (EMEA) also announced
approval of the first drug produced in an animal bioreactor: Atryn from GTC Biotherapeutics
[Almeida et al., 2011].
5.7.2 Recombinant Factor VIII (ReFacto):
Approved for clinical use in March 2000, recombinant factor VIII is indicated for control and
prevention of bleeding episodes and surgical prophylaxis to reduce the frequency of spontaneous
bleeding episodes. This product is the only factor VIII product indicated for short-term routine
prophylaxis. Recombinant technology allows preparation of clotting factors without human
blood or plasma products. This eliminates the risk of blood-borne viral contamination associated
with nonrecombinant factor VIII products prepared from pooled human blood. Also, ReFacto
does not contain human serum albumin, whereas previously approved recombinant products
(e.g., Kogenate, Bayer) add albumin during the cell culture phase and during the final product
formulation. This procedure theoretically increases the possibility of viral contamination in the
final product [Ansel et al., 2005].
5.8 Cytokines:
Cytokines are molecules that activate the immune cells (e.g. lymphocytes and macrophages),
regulate growth and differentiation of immune cells, also important messengers in cells,
influencing the response in inflammation, response immune and tissue repair [Mahmoud, 2007].
5.8.1 Interleukins:
Originally, ILs were thought to oversee interactions among white blood cells, key components of
the immune system. Now, however, it is known that these substances affect a wider variety of
cell types. Most clinical interest centers on IL-1, secreted primarily by the monocyte-macrophage
that activates T cells and B cells, and IL-2, secreted by the T cell that supports growthand
differentiation of T cells and B cells [Ansel et al., 2005].
Interleukins are molecules that act as leukocytes messengers, for example the interleukin-2
stimulates T lymphocytes. IL-2 recombinant interleukin, approved by FDA, produced by E. coli,
which differs from the natural interleukin by the alanine absence on the N-terminal and by the
fact that serine is replaced by cysteine at 125 amino acid 125, as exemplified in aldesleukin

21
(Proleucina®). This drug is used in the treatment of renal cell cancer, and its effect is
proportional to the amount of recombinant drug administered. There are other drugs that block
interleukin, for example, Arcalyst® (rilonacept) used for the treatment of CAPS - Cryopyrin
Associated Periodic Syndromes. This drug blocks a chemical messenger called interleukin-1-
beta and interleukin- 1-alpha [Bhopal and Nanda, 2005].
5.8.2 Interferons:
Interferons are a part of the large immune regulatory network within the body that includes
lymphokines, monokines, growth factors, and peptide hormones. Interferons are classified into
two types: type I, alpha and beta, which share the same molecular receptor, and type II, gamma
or immune, which have a different receptor [Ansel et al., 2005].
The recombinant interferons (potent cytokines that act against viruses and against uncontrolled
proliferation of cells) exist in three forms: alpha, beta and gamma, and feature a wide variety of
applications. The α recombinant interferon is used in patients with Kaposi’s sarcoma, hepatitis B,
hepatitis C and renal cell cancer. The β recombinant interferon (produced by E. coli containing
165 a.a.) is used in patients with secondary progressive sclerosis, because it inhibits the
production of Th1 cytokines and activates the monocytes involved in the immune response
Examples of α recombinant interferons are Intron-A®, Roferon-A® and Actimmume® whereas
β recombinant interferons include Avonex®, Rebif® and Betaseron®. Finally, γ recombinant
interferon (produced by E. coli containing 139 a.a.) is used in patients with infections associated
with chronic granulomatous disease [Almeida et al., 2011].
5.9Enzymes:
 Recombinant dornase alpha (formulated in the form of an aerosol - Pulmozyme®) is an
enzyme produced by CHO cells, used in the treatment of patients with cystic fibrosis, a
genetic disorder marked by excessive mucous secretions and frequent lung infections
 Another example of a recombinant enzyme is a plasminogen activator, known as
alteplase (Activase®), used to dissolve blood clots formed in the circulatory system,
which can cause heart attacks, pulmonary embolisms and strokes [Almeida et al., 2011].

22
 On the other hand, Naglazyme® (Galsulfase) is a form of recombinant enzyme used for
the treatment of patients with mucopolysaccharidosis VI (MPS VI or Maroteaux-Lamy).
This disease is caused by the lack of an enzyme called B arylsulfatase, required in the
degradation of substances, known as glycosaminoglycans (GAGs). If the enzyme is not
present, the GAG cannot be degraded and accumulates in cells, causing large head and
movement difficulties [Almeida et al., 2011].
 Elaprase® (idursulfase) is another enzyme produced by biotechnological processes used
in the treatment of patients with Hunter syndrome (patients are not able to degrade
glycosaminoglycans, which gradually accumulates in cells, affecting most organs,
causing difficulty breathing and walking) [Okuyama et al., 2010].
 Another case of using biotechnology to produce drugs is the production of essential
enzymes in patients with Gaucher syndrome type 1 and 3 (a disease characterized by
deficiency of the beta-glucosidase enzyme). This disease is usually characterized by a
neurological disorder that includes mental degeneration and seizures. There are a few
effective therapies for treatment including VPRIV® (velaglucerase alpha - a human cell
line derived enzyme replacement therapy - for the long-term treatment of type 1 Gaucher
disease), the Protalix Biotherapeutics (taliglucerase alpha - a plant cell-expressed
recombinant glucocerebrosidase enzyme), Cerezyme® (imiglucerase - produced by
recombinant DNA technology using mammalian cell culture, CHO) and Zavesca®
(miglustat -reduces the harmful buildup of fatty substances throughout the body by
reducing the amount of glycosphingolipids produced by the body - used in patients who
cannot be treated with enzyme replacement therapy) [Almeida et al., 2011].
 A different enzyme produced using human cell lines is alfagalsidase (Replagal®). This
enzyme is a copy of the human enzyme used in enzyme replacement therapy for Fabry’s
disease (chronic and progressive genetic diseases caused by absence or deficiency of an
enzyme called alpha-galactosidase A, responsible for the decomposition of lipids in the
body, consequently the lipids accumulate in vital organs causing serious problems)

23
5.10 Growth Factors:
Many Hematopoietic Growth Factors (HGFs) have been isolated, and the understanding of their
clinical potential continues to grow. HGFs have had a significant impact on the prevention of
infections associated with chemotherapy-induced neutropenia, chemotherapy induced
thrombocytopenia, and chemotherapy-induced anemia. Patients with HIV/AIDS can also been
helped by the administration of recombinant HGFs. Erythropoietin, a hormone produced by the
kidneys, stimulates the bone marrow to produce red blood cells. The recombinant human
erythropoietin (Procrit®, Epogen®, Eprex®, NeoRecormon®) may appear in different forms:
alpha (produced in CHO), beta (produced in CHO) and gamma (produced in BHK). This
recombinant growth factor is used in the treatment of anemia associated with renal failure, HIV
infections, surgery, etc. Erythropoietin alpha is targeted for the treatment of anemia due to
chronic renal failure, HIV infection and cancer [Bhopale and Nanda, 2005].
5.10.1 Mircera® (Beta methoxypolyethyleneglycol-Epoetin):
Mircera® (beta methoxypolyethyleneglycol-epoetin) is used for the treatment of anemia
associated with chronic renal failure. On the other hand, Palifermin (Kepivance®) is very similar
to a natural growth factor that exists in the human body, known as keratinocyte growth factor
(KGF). Kepivance® stimulates the growth of cells, helping to reduce the incidence, severity and
duration of oral mucositis in cancer patients subjected to intensive care [Almeida et al., 2011].
6. Emerging Delivery Methods:
6.1 RNAi Technologies:
RNAi technologies are considered the medical treatments of the future. In fact, RNAi was
announced as the scientific breakthrough of 2002. The hopes from these technologies have been
partially dashed [Orive et al., 2003].
RNA interference is a powerful and rapid technique to knockout the gene expression by
introducing either short interfering RNA or double stranded RNA fragments into targeted
host. These RNA fragments degraded specific homologous mRNA. RNAi technology used as an
essential tool in several model organisms such as invertebrates, mouse and fly to explore the
role of individual gene. In recent era, RNAi is directly used in therapy of various
infectious and non-infectious diseases. Variety of human diseases like cancer, viral and

24
neuromuscular has been controlled through RNAi. Some vectors and inducible systems are
available now to treat numerous disorders [Andleeb and Ali, 2016].
6.2.1 RNAi Mechanism:
Mechanism of RNAi can be divided into three steps. The first step is the breakdown of long
double-stranded RNA (dsDNA: 200-500 nucleotides) into short-interfering RNAs (siRNA:
21–26 nt) by a Ribonuclease III-like enzyme/ endoribonuclease or helicase called Dicer.
Highly conserved Dicers have been found in yeastDrosophila, C. elegans, mice, plants and
humans. They suggested that similar mechanism of RNAi pathways was shared by these
organisms. These small fragments of 21 to 24 nucleotides are also called small interfering
(siRNAs) and micro RNA (miRNAs). These molecules play role in gene expression
regulation and cell growth control. In second step, these duplex siRNAs companion with
RNA-induced silencing complex (RISC) protein of ~160 kDa. RISC comprises of
Argonaute (Ago) proteins illustrated that eight members of Ago family were originate in
humans, but only Ago2 possessed cleavage activity due to an active catalytic domain.
Within the RISC these duplexes unwind and the one strand is degraded and removed by
nucleases. Thirdly, the loaded ssRNA called guided strand, directs the RISC to the target
mRNA. Argonaute slices the phosphodiester bond and releases fragments of mRNA that
are lastly degraded resulting in the gene silencing [Andleeb and Ali, 2016].
Figure 7: Mechanism of RNAi

25
6.2.2 Strategies for RNA Interference:
Two types of strategies for RNAi reagents delivery into mammalian cells can be used i.e.
stable/inducible RNAi and transient RNAi . Various deliverymethods i.e. transfection,
transduction, andbacterial transformation were used to deliver gene in host for RNAi.
Different proteins (Dicers, RISC, AGOs) and promoters (RNA Polymerase III promoters i.e.,
H1, U6, and tRNA promoters) used to derive the RNAi. The viral based vectors like
retroviruses, adenoviruses and adeno-associated viral vectors have also been demonstrated for
high-efficiency gene delivery [Andleeb and Ali, 2016].
6.3 Viral Vectors:
Viral vectors are optimal vehicles for gene transfer because of their ability to efficiently infect
host cells. The removal of the replicative and pathogenic ability of viruses, combined with their
capacity to carry the therapeutic transgene and an ability to efficiently infect a variety of
mammalian cell types makes them amenable for use in gene therapy (Figure 8). However, the
immune system has evolved to fight off invading pathogens, which makes viral vectors subject
to immune responses that have to be blocked or avoided to achieve therapeutic transgene
expression. Administration of viral vectors can lead to the initiation of innate and adaptive
immune responses against the viral particles and gene products, leading to decreased efficiency
of gene transfer or elimination of the transduced cells over time. Recent research has
concentrated on various immune modulatory regimens utilizing 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
therapeutic transgene product, which may represent a neo-antigen owing to the type of gene
mutation present, rendering patients with e.g. null mutations, susceptible to recognizing the
transgene product as a foreign antigen. While 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, 2011].

26
Figure 8: Viral Vectors
6.4 Non-viral Gene Therapy (Gene Gun):
Viral vectors have limitations and often it is more appropriate to use non-viral alternatives
because they have less size limitations and are virtually non-immunogenic. Generally, non-viral
vectors are molecules of circular DNA. Sizeable attention is being focused towards the
development of gene delivery using non-viral methods, through the use of molecular engineering
to produce vectors capable of efficient gene delivery. An ideal non-viral vector must provide
protection in the extracellular matrix against gene degradation by nuclease, assimilate plasma
membranes, escape from the endosome and unpackage the gene whilst avoiding any detrimental
effects. Generally, non-viral vectors are easy to produce, have low immunogenicity, cheaper, and
have no size limitations of gene that can be delivered. Non-viral vectors can be further split into
two categories: physical and chemical methods of delivery [Gao et al., 2007].

27
Figure 9: Nonviral Gene Therapy
6.5 Liposomes:
Liposomes are defined as structure consisting of one or more concentric spheres of lipid bilayers
separated by water or aqueous buffer compartments. (OR) Liposomes are simple microscopic
vesicles in which aqueous volume is entirely enclosed by a membrane composed of lipid
bilayers. They can encapsulate and effectively deliver both hydrophilic and lipophilic substances,
and may be used as a non-toxic vehicle forinsoluble drug, because lipids are amphiphatic(both
hydrophilic and hydrophobic)in aqueous media, their thermodynamic phase properties and self-
assembling characteristics evoke entropically driven sequestration of their hydrophobic regions
into spherical bilayers are referred as lamellar. Liposomes vary in charge and size depending on
the method of preparation and the lipids used [Thulasiramaraju et al., 2012].
6.5.1 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

28
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 [Rafe and Ahmed, 2017].
6.5.2 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 [Rafe
and Ahmed, 2017].
Figure 10: Structural Features of Liposomal Drug Delivery Systems with Entrapped
Molecule

29
6.5.3 Liposome in cancer therapy:
The main problem with the anticancer drugs is their low therapeutic index because of low
therapeutic index normal dose of these which is needed for intended effect causes toxicity to
normal cells. Targeted delivery of drugs to the tumor cells by liposome have been changed the
pattern of cancer treatment. Due to the targeted delivery of toxic anticancer drugs, its toxicity has
been reduced greatly than delivery of free anticancer drugs. Entrapment of anticancer drugs
greatly increased its lifetime, decreased its degradation rate, increased deposition in the tumor
cells, and decreased uptake to the normal cells. Liposome with passively targeted tumor cells can
increase vascular permeability. Doxil, Caelyx, and Myocet are some commonly used liposomal
formulation used in cancer treatment [Rafe and Ahmed, 2017].
6.6 Microfabricated Systems:
Microfabricated systems combine the principles of microtechnology and biology to provide
sophisticated drug delivery systems that could provide advantages over existing technologies.
Micromachining presents the opportunity to create multiple reservoirs of desired size to contain
not just one, but many drugs or biomolecules of interest. The wide range of possibilities include
implanted microchips for localized drug delivery and nanoporous immuno-isolating devices for
cell immobilization that are surrounded by microfabricated membranes with perfectly defined
monodisperse pores in the nanometer scale [Orive et al., 2003].
6.7 Encapsulation Methods:
The inclusion of therapeutic active molecules in microparticulate delivery systems represents
another way to protect and transport the medication to exactly the right place. Examples of these
systems include polymer-based microparticules, micelles [Orive et al., 2003].
6.7.1 Micelles:
Recently, reported the development of tiny micelles built from two types of polymer. After
loading the molecular ‘globs’ with drugs they showed that these biocompatible nanocontainers
could pass through the wall of a rat cell. Although they did not reach the cell nucleus, they were
able to access the mitochondria and Golgi apparatus, which constitute important targets for drug
delivery [Orive et al., 2003].

30
Figure 11: Micelles
6.7.2 Microencapsulation:
Using the technology of microencapsulation, researchers have investigating the possibility of
introducing cells that would work as ‘factories’ secreting therapeutic molecules. To succeed,
microcapsules must be coated with a semipermeable immunobarrier that would exert a double
protective function: immunoisolating the transplanted tissue from the host’s immune response
and protecting the host from any biological risk. Cell encapsulation technology presents several
advantages over the encapsulation of peptides, including the secretion of de novo produced
therapeutic proteins and the possibility to regulate peptide delivery as a function of physiological
requirements. As a result, a wide range of encapsulated cells have been developed for a variety
of applications and to treat a number of diseases [Orive et al., 2003].
These include the development of a bioartificial pancreas and liver, the treatment of classical
Mendelian disorders caused by an enzymatic or gene product deficiency, and the treatment of
cancer and central nervous system diseases [Orive et al., 2003].
6.7.3 Nanotechnology:
Recently, cells secreting ciliary neurotrophic factor have been encapsulated and administered in
dogs suffering from retinitis pigmentosa (a disease characterized by the degeneration of
photoreceptor cells). Results showed that seven weeks after implantation an increased survival of
the photoreceptor cells was achieved and the implant showed no adverse effects. When the size
of the particles used for encapsulation is reduced to less than 100 nm in size, the result is
nanotechnology [Orive et al., 2003].

31
6.7.4 Nanocomposites:
Nanocomposites include nanocapsules, micellar systems, conjugates and nanoparticles. One of
the main advantages of these submicron systems is that they present a higher intracellular uptake
than microsized particles. This has special implications for gene delivery, as DNA can be easily
encapsulated, protected from lysosomal enzymes and transfected with high efficiency [Orive et
al., 2003].
6.7.5 Nanosurgery:
Recently, a hybrid nanodevice composed of oligonucleotide DNA covalently attached to
titanium dioxide nanoparticles with the ability to target, bind and cleave DNA has open the door
to novel strategies for drug delivery and nanosurgery [Orive et al., 2003].
7. Standard Stability Aspects for Biotech Product:
Biotechnological products are meanwhile one of the most important parts for modern medicinal
therapy concepts. They are much more complex than other chemically defined active ingredients
in terms of molecular mass and higher order structure and are produced by genetically modified
organisms. However, stability testing is a requirement for establishing shelf life of all therapeutic
products independent from their chemical nature. Environmental conditions such as temperature
and humidity can affect the integrity of the product, so it is mandatory to assure safety and
efficacy until end of shelf life [Muthu and Feng, 2009].
For this reason, pharmaceutical stability testing is a major investigation studying the changes in
the quality of any biotech drug product with respect to time under the influence of environmental
factors, such as temperature, humidity and light. Stability testing is generally recommended
during the product development of new drugs in order to establish a shelf-life for the drug
product and to recommend a suitable storage condition. For all new drug products, including
nanomedicines, stability testing should include the testing of all parameters that are susceptible
to change during transportation and storage and are likely to influence the safety, efficacy and
quality of these products [Ohshima et al,. 2009].

32
7.1Environmental conditions:
7.1.2 Temperature:
Since most finished biotechnological product need precisely defined storage temperatures, the
storage conditions for the real-time/real-temperature stability studies may be confined to the
proposed storage temperature [Ammann, 2011].
7.1.3 Humidity:
Biotechnological products are generally distributed in containers protecting them against
humidity. Therefore, where it can be demonstrated that the proposed afford sufficient protection
against high and low humidity, stability tests at different relative humidities can usually be
omitted. Where humidity-protecting containers are not used, appropriate stability data should be
provided [Ammann, 2011].
7.1.4 Light:
Applicants should consult the appropriate regulatory authorities on a case-by-case basis to
determine guidance for testing [Ammann, 2011].
7.2Suitable Storage Condition:
7.2.1 Container/Closure:
Changes in the quality of the product may occur due to the interactions between the formulated
biotechnological/biological product and container/closure. Where the lack of interactions cannot
be excluded in liquid products (other than sealed ampoules), stability studies should include
samples maintained in the inverted or horizontal position (i.e., in contact with the closure), as
well as in the upright position, to determine the effects of the closure on product quality.In
addition to the standard data necessary for a conventional single-use vial, the applicant should
demonstrate that the closure used with a multiple-dose vial is capable of withstanding the
conditions of repeated insertions and withdrawals so that the product retains its full potency,
purity, and quality for the maximum periodspecified in the instructions-for-use on containers,
packages, and/or package inserts. Such labelling should be in accordance with relevant
national/regional requirements [Ammann, 2011].

33
7.3Selection of Batches:
7.3.1 Drug Substance (Bulk Material):
Where bulk material is to be stored after manufacture but prior to formulation and final
manufacturing, stability data should be provided on at least 3 batches for which manufacture and
storage are representative of the manufacturing scale of production. A minimum of 6 months
stability data at the time of submission should be submitted in cases where storage periods
greater than 6 months are requested. For drug substances with storage periods of less than 6
months, the minimum amount of stability data in the initial submission should be determined on
a case-by-case basis. Data from pilot-plant scale batches of drug substance produced at a reduced
scale of fermentation and purification may be provided at the time the dossier is submitted to the
regulatory agencies with a commitment to place the first 3 manufacturing scale batches into the
long-term stability program after approval.The quality of the batches of drug substance placed
into the stability program should be representative of the quality of the material used in
preclinical and clinical studies and of the quality of the material to be made at manufacturing
scale. In addition, the drug substance (bulk material) made at pilot-plant scale should be
produced by a process and stored under conditions representative of that used for the
manufacturing scale. The drug substance entered into the stability program should be stored in
containers which properly represent the actual holding containers used during manufacture.
Containers of reduced size may be acceptable for drug substance stability testing provided that
they are constructed of the same material and use the same type of container/closure system that
is intended to be used during manufacture [Ammann, 2011].
7.3.2 Intermediates:
During manufacture of biotechnological/biological products, the quality and control of certain
intermediates may be critical to the production of the final product. In general, the manufacturer
should identify intermediates and generate in-house data and process limits that assure their
stability within the bounds of the developed process. While the use of pilot-plant scale data is
permissible, the manufacturer should establish the suitability of such data using the
manufacturing scale process [Ammann, 2011].

34
7.3.3 Sample Selection:
Where one product is distributed in batches differing in fill volume (e.g., 1 milliliter (ml), 2 ml,
or 10 ml), unitage (e.g., 10 units, 20 units, or 50 units), or mass (e.g., 1 milligram (mg), 2 mg, or
5 mg) samples to be entered into the stability program may be selected on the basis of a matrix
system and/or by bracketing [Ammann, 2011].
Matrixing, i.e., the statistical design of a stability study in which different fractions of samples
are tested at different sampling points, should only be applied when appropriate documentation is
provided that confirms that the stability of the samples tested represents the stability of all
samples. The differences in the samples for the same drug product should be identified as, for
example, covering different batches, different strengths, different sizes of the same closure and
possibly, in some cases, different container/closure systems. Matrixing should not be applied to
samples with differences that may affect stability, such as different strengths and different
containers/closures, where it cannot be confirmed that the products respond similarly under
storage conditions [Ammann, 2011].
Where the same strength and exact container/closure system is used for 3 or more fill contents,
the manufacturer may elect to place only the smallest and largest container size into the stability
program, i.e., bracketing. The design of a protocol that incorporates bracketing assumes that the
stability of the intermediate condition samples are represented by those at the extremes. In
certain cases, data may be needed to demonstrate that all samples are properly represented by
data collected for the extremes [Ammann, 2011].
7.4 Chemical Stability:
There are different reactions of proteins and polypeptides, which lead to chemical
instability. They are-
 Deamidation
 Oxidation
 Racemization
 Disulfide exchange
 Proteolysis

35
7.4.1 Deamidation:
It involves hydrolysis of a side chain amide linkage of an amino acid recidue to form a
carboxylic acid. Some common protein which undergo in vitro deamidation, are human growth
hormone, insulin and prolactine. Glutamine and asparagin are the amino acids, which undergo
deamidation. The rate of amidation is increased by an increase in pH, temperature and ionic
strength. The tertiary structure of proteins resists deamidation. Example:tertiary structure of
trypsin prevents deamidation. Deamidation reduces biological activity. Example: the activity of
ACTH (adreno corticotropic hormone) is reduces by deamidation [Ammann, 2011].
7.4.2 Oxidation:
Oxidation occurs in the side chain of histidine, methionine, lysine, tyrocine and tryptophan
residues in proteins.It are common during synthesis during synthesis, isolation and storage.
Example: atmospheric oxygen oxidize methionine under acidic condition. Oxidizing agents like
hydrogen peroxide iodine and diethyl sulfoxide are also responsible for oxidation. Oxidation also
reduces biological activity. Example:oxidation of methionin in gastrin and corticotrophin results
in loss of activity [Ammann, 2011].
7.4.3 Racemization:
Except glycine, all other amino acids are chiral at carbon bearing the side chain and hence are
susceptible to racemization. This racemization may convert the protein non- metabolizable,
because the recimic peptide bonds are inaccessible to proteolytic enzymes and can reduce
biological activity [Ammann, 2011].
7.4.4 Disulfide Exchange:
Breaking and incorrect reformation of disulfide bonds mayalter the three-dimensional structure
of a protein and hence can change or alter biological activity. The reaction is catalyzed by thiols
which arise as a result of hydrolytic cleavage of disulfide [Ammann, 2011].
7.4.5 Proteolysis:
Proteolysis is a cleavage of protein molecule by the breakage of peptide bond. This depends on
the residues involved. For example: asparagines residues, particularly the bond between
asparagines and proline is highly susceptible to cleavage. But the peptide bonds in general, are

36
stable at natural pH and room temperature. Proteolysis occurs upon heating. Like, heating at 90-
100℃ inactivates Lysosomes [Ammann, 2011].
7.5 Others:
7.5.1 Stability after Reconstitution of Freeze-Dried Product:
The stability of freeze-dried products after their reconstitution should be demonstrated for the
conditions and the maximum storage period specified on containers, packages, and/or package
inserts. Such labelling should be in accordance with relevant national/regional requirements
[Ammann, 2011].
7.5.2 Shelf-Lives of Biotechnological Products:
The shelf-lives of biotechnological/biological products may vary from days to several years.
Thus, it is difficult to draft uniform guidelines regarding the stability study duration and testing
frequency that would be applicable to all types of biotechnological/biological products. With
only a few exceptions, however, the shelf-lives for existing products and potential future
products will be within the range of 0.5 to 5 years. Therefore, the guidance is based upon
expected shelf-lives in that range. This takes into account the fact that degradation of
biotechnological/biological products may not be governed by the same factors during different
intervals of a long storage period [Ammann, 2011].
When shelf-lives of 1 year or less are proposed, the real-time stability studies should be
conducted monthly for the first 3 months and at 3 month intervals thereafter [Ammann, 2011].
For products with proposed shelf-lives of greater than 1 year, the studies should be conducted
every 3 months during the first year of storage, every 6 months during the second year, and
annually thereafter [Ammann, 2011].
7.6 Labeling:
For most biotechnological/biological drug substances and drug products, precisely defined
storage temperatures are recommended. Specific recommendations should be stated, particularly
for drug substances and drug products that cannot tolerate freezing. These conditions, and where
appropriate, recommendations for protection against light and/or humidity, should appear on

37
containers, packages, and/or package inserts. Such labeling should be in accordance with
relevant national/regional requirements [Ammann, 2011].
7.6.1 Conjugated Product:
A conjugated product is made up of an active ingredient (for example, peptide, carbohydrate)
bound covalently or noncovalently to a carrier (for example, protein, peptide, inorganic mineral)
with the objective of improving the efficacy or stability of the product [Ammann, 2011].
7.6.2 Degradation Product:
A molecule resulting from a change in the drug substance (bulk material) brought about over
time. For the purpose of stability testing of the products such changes could occur as a result of
processing or storage (e.g., by deamidation, oxidation, aggregation, proteolysis). For
biotechnological/biological products some degradation products may be active [Ammann, 2011].
7.6.3 Impurity:
Any component of the drug substance (bulk material) or drug product (final container product)
which is not the chemical entity defined as the drug substance, an excipient, or other additives to
the drug product [Ammann, 2011].
7.6.4 Intermediate:
For biotechnological/biological products, a material produced during a manufacturing process
which is not the drug substance or the drug product but whose manufacture is critical to the
successful production of the drug substance or the drug product. Generally, an intermediate will
be quantifiable and specifications will be established to determine the successful completion of
the manufacturing step prior to continuation of the manufacturing process. This includes material
which may undergo further molecular modification or be held for an extended period of time
prior to further processing [Ammann, 2011].
7.7 Manufacturing Scale Production:
Manufacture at the scale typically encountered in a facility intended for product production for
marketing [Ammann, 2011].

38
7.7.1 Pilot-Plant Scale:
The production of the drug substance or drug product by a procedure fully representative of and
simulating that to be applied at manufacturing scale. The methods of cell expansion, harvest, and
product purification should be identical except for the scale of production [Ammann, 2011].
8. Methods of Improving Stability:
Over the past two decades, biotechnology medicines are using to treat human diseases. One of
the primary challenges to the broader adoption of therapeutic proteins is the need to increase the
stability of recombinant proteins to improve on their formulation and shelf lives, while
maintaining their activities or efficacies. [Carlsson et al., 2018].
Efforts to increase or enhance the stability of biological molecules are limited by the molecular
tools provided by nature. Many approaches to stabilize proteins have been developed, including
directed evolution with the canonical amino acids,applying principles learned from proteins of
extremophiles or attaching proteins to a matrix material, such as surfaces or polymers [Carlsson
et al., 2018].
While these approaches have all seen some success, it is rare that adding an electrostatic
interaction would stabilize a protein by significantly more than 1 kcal/mol.The advent of
methods for incorporating noncanonical building blocks into proteins has helped to overcome
some limitations but continues to be constrained by the standard menu of noncovalent
interactions that dictate molecular folding [Carlsson et al., 2018].
8.1 PEGylation:
PEGylation is a Recent Advancement of Protein and Peptide Drug Delivery systems, PEGylation
is a process of attaching the strands of the polymer PEG to most typical peptides fragments that
can help to meet the protein and challenges of improving the safety and efficiency of many
therapeutic macromolecules such as Protein and Peptides. It is widely used for the modification
of proteins and peptides, antibody fragments and oligonucleotides. PEG are the Non-toxic. And
non –immunogenic, it is having a specified Hydrophilicity and it is having high Flexibility.
PEGylation is important to increases the Bioavailability, it is applicable for the optimized

39
Pharmacokinetics, it is important for Decreasing Immunogenicity, It is important to decreases the
frequency of administration. The PEGylation is important Mechanism for increasing the
molecular weight of the molecules, it can increases the drug solubility and it is applicable for the
protection against Proteolytic degradations, it is having an important mechanism to reducing the
dosing frequency and maintain therapeutic activity [Savale, 2016].
8.2 Stochastic approaches:
Stochastic laboratory techniques have been developed to produce enzymes with improved
functional properties including stability. These methods are commonly referred to as directed
evolution. The classical version of directed evolution implies that mutations are introduced
completely at random at any position in a protein structure and does not employ any
computational or bioinformatic techniques.To increase the chance of success and reduce the
number of variants to be screened, the bioinformatic analysis of sequences and structures has
been incorporated into evolutionary methods. A strategy to increase protein thermostability has
been proposed based on iterative saturation mutagenesis and structural analysis. Amino acids
were selected as hotspots for mutagenesis if characterized by a high B-factor an atomic
displacement parameter which corresponds to thermal motion and flexibility. This strategy is
based on the observation that structures of thermophilic enzymes are more rigid compared to
mesophilic ones. The approach was used to enhance thermostability of mesophilic lipase A from
Bacillus subtilis. Another two commonly used strategies to improve protein stability consider
multiple sequence alignments to guide the generation of variants: the consensus approach and the
ancestral mutation method. In the backto-consensus approach the most frequently occurring
amino acids are identified at each column of a multiple alignment. These are considered as the
stabilizing residue types favored by natural evolution and are used to propose the consensus
mutations [Suplatov et al., 2014].
8.3 Empirical rational design:
Empirical rational design of enzymes is becoming more and more popular with the development
of computational tools for sequence and structure analysis. A study usually begins with a visual
expert inspection of sequence or structural data. Results of this expert analysis are then used, for
example, to introduce new interactions in an attempt to stabilize protein folding. Expert analysis
may involve the comparison of a less stable protein with a more stable homolog. In a proof of

40
concept study, two cold shock proteins were compared that differed by only 12 positions (out of
67 residues) but showed a significant difference in thermostability. Site-directed mutagenesis
revealed that two residues were responsible for this diversity and facilitated the construction of a
highly thermostable variant from a mesophilic protein. These empirical studies have made an
important contribution to the development of more rational ways of engineering stability, but
share a general weakness, the choice of the particular hotspots for mutation is very subjective.
These rationalized strategies can reduce the experimental evaluation to a smaller number of
mutants [Suplatov et al., 2014].

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