HBB 2313: PHARMACEUTICAL BIOTECHNOLOGY

BIOTECHNOLOGY: It implies with the use of microorganisms, plants, animals or parts of them
for the production of useful compounds.
Pharmaceutical industry discovers, develops, produces, and markets drugs or pharmaceutical
drugs for use as medications to be administered (or self-administered) to patients, with the aim to
cure them, vaccinate them, or alleviate the symptoms
Pharmaceutical biotechnology
It is concerned as the biotechnological manufacturing of pharmaceutical products such as
Antibodies, Proteins and Recombinant DNA products
Protein engineering
Protein engineering is the design of new enzymes or proteins with new or desirable functions. It
is based on the use of recombinant DNA technology to change amino acid sequences. Protein
engineering process helps in developing useful or valuable proteins. It is a young discipline, with
much research taking place into the understanding of protein folding and recognition for protein
design principles.
There are two general strategies for protein engineering, 'rational' protein design and directed
evolution. These techniques are not mutually exclusive; researchers will often apply both. In the
future, more detailed knowledge of protein structure and function, as well as advancements in
high-throughput technology, may greatly expand the capabilities of protein engineering.
Eventually, even unnatural amino acids may be incorporated, thanks to a new method that allows
the inclusion of novel amino acids in the genetic code.

Protein engineering techniques

Rational design
In rational protein design, the scientist uses detailed knowledge of the structure and function of
the protein to make desired changes. In general, this has the advantage of being inexpensive and
technically easy, since site-directed mutagenesis techniques are well-developed. However, its
major drawback is that detailed structural knowledge of a protein is often unavailable, and, even
when it is available, it can be extremely difficult to predict the effects of various mutations.
Computational protein design algorithms seek to identify novel amino acid sequences that are
low in energy when folded to the pre-specified target structure. While the sequence-
conformation space that needs to be searched is large, the most challenging requirement for
computational protein design is a fast, yet accurate, energy function that can distinguish optimal
sequences from similar suboptimal ones.
Directed evolution
In directed evolution, random mutagenesis is applied to a protein, and a selection regime is used
to pick out variants that have the desired qualities. Further rounds of mutation and selection are
then applied. This method mimics natural evolution and, in general, produces superior results to
rational design. An additional technique known as DNA shuffling mixes and matches pieces of
successful variants in order to produce better results. This process mimics the recombination that
occurs naturally during sexual reproduction. The advantage of directed evolution is that it
requires neither prior structural knowledge of a protein, nor is it necessary to be able to predict
what effect a given mutation will have. Indeed, the results of directed evolution experiments are
often surprising in that desired changes are often caused by mutations that were not expected to
have that effect. The drawback is that they require high-throughput screening, which is not
feasible for all proteins. Large amounts of recombinant DNA must be mutated and the products
screened for desired qualities. The sheer number of variants often requires expensive robotic
equipment to automate the process. Furthermore, not all desired activities can be easily screened
for.
Examples of engineered proteins
Using computational methods, a protein with a novel fold has been designed, known as Top7, as
well as sensors for unnatural molecules.
The engineering of fusion proteins has yielded rilonacept, a pharmaceutical that has secured
FDA approval for the treatment of cryopyrin-associated periodic syndrome.
Another computational method, Iterative Protein Redesign and Optimization (IPRO),
successfully engineered the switching of cofactor specificity of Candida boidinii xylose
reductase.
Iterative Protein Redesign and Optimization (IPRO) redesigns proteins to increase or give
specificity to native or novel substrates and cofactors. This is done by repeatedly randomly
perturbing the structure of the proteins around specified design positions, identifying the lowest
energy combination of rotamers, and determining whether the new design has a lower binding
energy than previous ones.
Computation-aided design has also been used to engineer complex properties of a highly ordered
nano-protein assembly. A protein cage, E. coli bacterioferritin (EcBfr), which naturally shows
structural instability and an incomplete self-assembly behavior by populating two
oligomerization states, is the model protein in this study.
Enzyme engineering
Enzyme engineering is the application of modifying an enzyme's structure (and, thus, its
function) or modifying the catalytic activity of isolated enzymes to produce new metabolites, to
allow new (catalyzed) pathways for reactions to occur, or to convert from some certain
compounds into others (biotransformation). These products are useful as chemicals,
pharmaceuticals, fuel, food, or agricultural additives.
DNA TECHNOLOGY IN PHARMACEUTICAL INDUSTRY
Recombinant DNA Technology is playing very important role in revolutionizing medicine i.e.,
enabling mass production of safe, pure, more effective versions of biochemicals that human body
produces naturallyexamples includes a variety of products such as hormones, Therapeutic
proteins, Vaccines and various Enzymes and also to create new pharmaceuticals. The
commercial exploitation of recombinant DNA (rDNA) technology began in late 1970s by
biotechnological companies to produce proteins.There are around 400 different proteins being
produced by rDNAtechnologyand as of now around 30 have been approved for human use.
TYPES:
The pharmaceutical products of rDNA technology are broadly divided into following three types
1. Human protein replacements
2. Therapeutic agents for human diseases
3. Vaccines

1) HUMAN PROTEIN REPLACEMENTS

The synthesis of the cellular proteins is ultimately under the control of genes. Any defect in a
gene produces an incorrect protein or no protein at all. Thus, gene defects will result in inherited
or genetically linked diseases.
Identification of defective or deficient proteins in the causation of inherited diseases is very
important. The rDNA technology can be fruitfully employed to produce human proteins that can
be used for the treatment of genetically linked diseases. This is referred to as human protein
replacement strategy in biotechnology.
EXAMPLES:
INSULIN:
The hormone insulin is produced by the β-cells of islets of Langerhans of pancreas. Human
insulin contains 51 amino acids, arranged in two polypeptide chains. The chain A has 21 amino
acids while b has 30 amino acids. Both are held together by disulfide bonds.
Insulin is central to regulating carbohydrateand fat metabolism in the body. Insulin causes cells
in the liver, skeletal muscles, and fat tissue to absorb glucose from the blood. In the liver and
skeletal muscles, glucose is stored as glycogen, and in fat cells (adipocytes) it is stored as
triglycerides.
Insulin stops the use of fat as an energy source by inhibiting the release of glucagon. With the
exception of the metabolic disorder diabetes mellitus and metabolic syndrome, insulin is
provided within the body in a constant proportion to remove excess glucose from the blood,
which otherwise would be toxic. When blood glucose levels fall below a certain level, the body
begins to use stored sugar as an energy source through glycogenolysis, which breaks down the
glycogen stored in the liver and muscles into glucose, which can then be utilized as an energy
source. As a central metabolic control mechanism, its status is also used as a control signal to
other body systems (such as amino acid uptake by body cells). In addition, it has several other
anabolic effects throughout the body.
When control of insulin levels fails, diabetes mellitus can result. As a consequence, insulin is
used medically to treat some forms of diabetes mellitus. Patients with type 1 diabetes depend on
external insulin (most commonly injected subcutaneously) for their survival because the
hormone is no longer produced internally. Patients with type 2 diabetes are often insulin resistant
and, because of such resistance, may suffer from a "relative" insulin deficiency. Some patients
with type 2 diabetes may eventually require insulin if other medications fail to control blood
glucose levels adequately. Over 40% of those with Type 2 diabetes require insulin as part of their
diabetes management plan
Diabetes mellitus affects about 2-3% of the general population.it is a genetically linked disease
characterized by the increased blood glucose concentration (hyperglycemia). Insulin facilitates
the cellular uptake and utilization of glucose for release of energy. In the absence of insulin,
glucose accumulates in the blood stream at higher concentration, usually when the blood glucose
concentration exceeds about 180mg/dl, glucose is excreted into urine. The patients of diabetes
ate weak and tired since the production of energy (i,e ATP) is very much depressed.The more
serious complications of uncontrolled diabetis include kidney damage (neuropathy), nerve
diseases (neuropathy), and circulatory diseases (atheroscelerosis,stroke).
Production of recombinant insulin:
Attempts to produce insulin by rDNA technology started in late 1970s.the basic technique
consisted of inserting human insulin gene and the promoter gene of lac operon on to the plasmids
of E.coli. Recently the procedure employed for synthesis involves insertion of genes for insulin
A chain and B chain separately to the plasmids of differentE.colicultures.the lac operon system(
consisting of inducer gene, promoter gene, operator gene and structural gene Z for –
galactosidase)is used for expression of both genes.the presence of llactose in the culture
mediuminduces the synthesis of insulin A and B chains in separate cultures. The so formed
insulin chains can be isolated, purified and joined together to give a full-fledged human insulin.

Forms of human insulin:

Human insulin is available in two forms, a short acting (regular) form and an intermediate acting
(NPH) form. NPH (Neutral Protamine Hagedorn) insulin, also known as isophane insulin, is a
suspension meaning that the insulin vial should be rolled or repeatedly turned upside down to
ensure the solution is uniformly cloudy.
Some examples of human insulin:
  Regular (short acting): Humulin S, Actrapid, Insuman Rapid
 NPH (intermediate acting):Humulin I, Insuman basal, Insulatard
 Premixed human insulins:Humulin M2, M3 and M5, Insuman Comb 15, 25 and 50

Insulin vial
Biosynthetic "human" insulin is now manufactured for widespread clinical use using
recombinant DNA technology. More recently, researchers have succeeded in introducing the
gene for human insulin into plants and in producing insulin in them, to be specific safflower.
This technique is anticipated to reduce production costs.
Several of these slightly modified versions of human insulin, while having a clinical effect on
blood glucose levels as though they were exact copies, have been designed to have somewhat
different absorption or duration of action characteristics. They are usually referred to as "insulin
analogues". For instance, the first one available, Humalog(insulin lispro), does not exhibit a
delayed absorption effect found in regular insulin, and begins to have an effect in as little as 15
minutes.

Other rapid-acting analogues are NovoRapid and Apidra, with similar profiles. All are rapidly
absorbed due to a mutation in the sequence that prevents the insulin analogue from forming
dimers and hexamers. Instead, the insulin molecule is a monomer, which is more rapidly
absorbed. Using it, therefore, does not require the planning required for other insulins that begin
to take effect much later (up to many hours) after administration. Another type is extended-
release insulin; the first of these was Lantus(insulin glargine). These have a steady effect for the
entire time they are active, without the peak and drop off effect in other insulins; typically, they
continue to have an insulin effect for an extended period from 18 to 24 hours.

Likewise, another protracted insulin analogue (Levemir) is based on a fatty acid acylation
approach. A myristyric acid molecule is attached to this analogue, which in turn associates the
insulin molecule to the abundant serum albumin, which in turn extends the effect and reduces the
risk of hypoglycemia. Both protracted analogues need to be taken only once-daily, and are very
much used in the type 1 diabetes market as the basal insulin. A combination of a rapid acting and
a protracted insulin is also available for the patients, making it more likely for them to achieve an
insulin profile that mimics that of the body´s own insulin release.Insulin is usually taken as
subcutaneous injections by single-use syringes with needles, via an insulin pump, or by repeated-
use insulin pens with needles

CLOTTING FACTOR VIII

The clotting factor VIII is required for proper blood clotting process. A genetic defect in the
synthesis of factor VIII results in the disorder hemophilia A. This is a sex-linked disease
(incidence 1 in 10,000 males) transmitted by females affecting males. The victims have
prolonged clotting time and suffer from internal bleeding.
Traditional treatment for hemophilia A:
Clotting factor VIII was isolated from the whole blood and administered to the patients of
hemophilia A. This approach requires large quantities of blood. Another problem is the risk of
transmission of certain diseases like AIDS to the hemophiliacs.
Production of recombinant factor VIII:
The gene for the formation of factor VIII is located on X chromosome. It is a complex gene of
186 kb (i.e., 186,000 base pairs) in size, organized into 26 exons of varying length. In between
the exons, many introns are present. The introns vary in their size, starting from 200 base pairs to
as high as 32,000 base pairs.
Biotechnologists were able to isolate mature mRNA (containing only exons and no introns) that
is responsible for the synthesis of factor VIII. This mRNA contains 9,000 bases and synthesizes
the protein, factor VIII. Factor VIII contains 2332 amino acids, with carbohydrate molecules
attached at least at 25 sites.
DNA technologists synthesized the complementary DNA (cDNA) for mature mRNA of factor
VIII. This cDNA can be inserted into mammalian cells or hamster kidney cells for the production
of recombinant factor VIII. Since 1992, factor VIII is available in the market

GROWTH HORMONE
Growth hormone is used as a prescription drug in medicine to treat children's growth disorders
and adult growth hormone deficiency. In the United States, it is only available legally from
pharmacies, by prescription from a doctor. In recent years in the United States, some doctors
have started to prescribe growth hormone in GH-deficient older patients (but not on healthy
people) to increase vitality. While legal, the efficacy and safety of this use for HGH has not been
tested in a clinical trial.
Function
Effects of growth hormone on the tissues of the body can generally be described as anabolic
(building up). Like most other protein hormones, GH acts by interacting with a specific receptor
on the surface of cells.
Increased height during childhood is the most widely known effect of GH. Height appears to be
stimulated by at least two mechanisms:
1. Because polypeptide hormones are not fat-soluble, they cannot penetrate cell membranes.
Thus, GH exerts some of its effects by binding to receptors on target cells, where it activates the
MAPK/ERK pathway. Through this mechanism GH directly stimulates division and
multiplication of chondrocytes of cartilage.
2. GH also stimulates, through the JAK-STAT signaling pathway, the production of insulin-like
growth factor 1(IGF-1, formerly known as somatomedin C), a hormone homologous to
proinsulin The liver is a major target organ of GH for this process and is the principal site of
IGF-1 production. IGF-1 has growth-stimulating effects on a wide variety of tissues. Additional
IGF-1 is generated within target tissues, making it what appears to be both an endocrine and an
autocrine/paracrine hormone. IGF-1 also has stimulatory effects on osteoblast and chondrocyte
activity to promote bone growth.
In addition to increasing height in children and adolescents, growth hormone has many other
effects on the body:
* Increases calciumretention, and strengthens and increases the mineralization of bone
* Increases muscle mass through sarcomerehypertrophy
* Promotes lipolysis
* Increases protein synthesis
* Stimulates the growth of all internal organs excluding the brain
* Plays a role in homeostasis
* Reduces liveruptake of glucose
* Promotes gluconeogenesisin the liver
* Contributes to the maintenance and function of pancreatic islets
* Stimulates the immune system

Production of recombinant HGH

The technique adopted is quite comparable with that of insulin production. The procedure
essentially consists of inserting HGH gene into E.coli plasmid, culturing the cells and isolation of
the HGH from the extracellular medium.

Limitation in HGH production : The HGH is a protein comprised of 191 amino acids. During
the course of its natural synthesis in the body.,HGH is tagged with a single peptide (with 26
amino acids) The signal peptide is removed during secretion to release the active HGH for
biological functions. The entire process of HGH synthesis goes on in an orderly fashion in the
body. However, signal peptide interrupts HGH production by recombinant technology. The
complementary DNA (cDNA) synthesized from the mRNA encoding HGH is inserted into the
plasmid. The plasmid containing E.coli when cultured, produces full length HGH along with
signal peptide.ButE.coli cannot remove the signal peptide. Further, it is also quite difficultto get
rid of signal peptide by various other means. Theoretically, cDNA encoding signal peptide can
be cut to solve these problems. Unfortunately, there is no restriction endonuclease to do this job,
hence this is not possible.

A novel approach for HGH production:

Biotechnologists have resolved the problem of signal peptide interruption by a novel approach.
The base sequence in cDNA encoding signal peptide ( 26 amino acids ) plus the neighbouring 24
amino acids is cut by restriction endonuclease ECoRI. Now a gene (cDNA ) for 24 amino acid
sequence of HGH is freshly synthesized and ligated to the remaining HGHcDNA. The so
constituted cDNA , attached to a vector, is inserted into a bacterium such as E. coli for culture
and production of HGH. In this manner, the biologically functional HGH can be produced by
DNA technology.

THERAPEUTIC AGENTS FOR HUMAN DISEASES

Biotechnology is very useful for the production of several therapeutic products for treating
human diseases. A selected list of rDNAderived therapeutic agents along with trade names and
their uses in human are given below…..
rDNA Product Trade name Application / Uses
Insulin Humulin Diabetes
Growth hormone Protropin/Humatrope Pituitary dwarfism
Interferon Intron A Hairy cell leukemia
Recombinax HB/
Hepatitis B vaccine Hepatitis B
Engerix
TissuEplasminogen
Activase Myocardial infarction
activator
Factor vIII Kogenate/Recombinate Hemophilia
Dnase Pulmozyme Cystic fibrosis
Severe anemia with
Erythropoietin Epogen/rocrit
kidney damage

INTERFERONS:

Interferons(IFNs) are proteins made and released by host cells in response to the presence of
pathogenssuch as viruses, bacteria, parasites or tumor cells. They allow for communication
between cells to trigger the protective defenses of the immune system that eradicate pathogens or
tumors.
IFNs belong to the large class of glycoproteins known as cytokines. Interferons are named after
their ability to "interfere" with viral replication within host cells. IFNs have other functions: they
activate immune cells, such as natural killer cells and macrophages; they increase recognition of
infection or tumor cells by up-regulating antigen presentation to T lymphocytes; and they
increase the ability of uninfected host cells to resist new infection by virus. Certain symptoms,
such as aching muscles and fever, are related to the production of IFNs during infection.

Functions
All interferons share several common effects; they are antiviral agents and can fight tumors. As
an infected cell dies from a cytolytic virus, viral particles are released that can infect nearby
cells. However, the infected cell can warn neighboring cells of a viral presence by releasing
interferon. The neighboring cells, in response to interferon, produce large amounts of an
enzymeknown as protein kinase R(PKR). This enzyme phosphorylates a protein known as eIF-2
in response to new viral infections; the phosphorylated eIF-2 forms an inactive complex with
another protein, called eIF2B, to reduce protein synthesis within the cell. Another cellular
enzyme, RNAse L— also induced following PKR activation—destroys RNA within the cells to
further reduce protein synthesis of both viral and host genes. Inhibited protein synthesis destroys
both the virus and infected host cells. In addition, interferons induce production of hundreds of
other proteins—known collectively as interferon-stimulated genes (ISGs)—that have roles in
combating viruses. They also limit viral spread by increasing p53 activity, which kills virus-
infected cells by promoting apoptosis. The effect of IFN on p53 is also linked to its protective
role against certain cancers.
Another function of interferons is to upregulate major histocompatibility complex molecules,
MHC Iand MHC II, and increase immunoproteasomeactivity. Higher MHC I expression
increases presentation of viral peptides to cytotoxic T cells, while the immunoproteasome
processes viral peptides for loading onto the MHC I molecule, thereby increasing the recognition
and killing of infected cells. Higher MHC II expression increases presentation of viral peptides
to helper T cells; these cells release cytokines (such as more interferons and interleukins, among
others) that signal to and co-ordinate the activity of other immune cells.
Interferons, such as interferon gamma, directly activate other immune cells, such as macrophages
and natural killer cells. Interferons can inflame the tongue and cause dysfunction in taste bud
cells, restructuring or killing taste buds entirely.
Interferon therapy
The immune effects of interferons have been exploited to treat several diseases. Agents that
activate the immune system, such as small imidazoquinoline molecules that activate TLR7, can
induce IFN-α. Imidazoquinoline is the main ingredient of Aldara (Imiquimod) cream, a treatment
approved in the United States by the Food and Drug Administration (FDA) for actinic keratosis,
superficial basal cell carcinoma, papilloma and external genital warts. Synthetic IFNs are also
made, and administered as antiviral, antiseptic and anticarcinogenic drugs, and to treat some
autoimmune diseases.

New research has shown that imiquimod's anti-proliferative effect is totally independent of
immune system activation or function. Imiquimod exerts its effect by increasing levels of the
opioid growth factor receptor (OGFr). Blocking OGFr function with siRNA technology resulted
in loss of any antiproliferative effect of imiquimod.

Interferon beta-1a and interferon beta-1b are used to treat and control multiple sclerosis, an
autoimmune disorder. This treatment is effective for slowing disease progression and activity in
relapsing-remitting multiple sclerosis and reducing attacks in secondary progressive multiple
sclerosis.
Interferon therapy is used (in combination with chemotherapy and radiation) as a treatment for
many cancers. This treatment is most effective for treating hematological malignancy; leukemia
and lymphomas including hairy cell leukemia, chronic myeloid leukemia, nodular lymphoma,
cutaneous T-cell lymphoma

Patients with recurrent melanomas receive recombinant IFN-α2b. Type I IFNs have a therapeutic
potential for the treatment of a wide variety of leukemias and solid tumors due to their
antiproliferative and apoptotic effects, their anti-angiogenic effects and their ability to modulate
an immune response specifically activating dendritic cells, cytolytic T cells and NK cells.

Interferon a 2b is also being used for treatment of ocular surface squamous neoplasia (OSSN) in
the form of perilesional injection followed by topical interferon a 2b drops at Lahore General
Hospital Eye unit II.

Both hepatitis B and hepatitis C are treated with IFN-α, often in combination with other antiviral
drugs. Some of those treated with interferon have a sustained virological response and can
eliminate hepatitis virus. The most harmful strain—hepatitis C genotype I virus—can be treated
with a 60-80% success rate with the current standard-of-care treatment of interferon-α, ribavirin
and recently approved protease inhibitors such as Telaprevir (Incivek) or Boceprevir (Victrelis).
Biopsies of patients given the treatment show reductions in liver damage and cirrhosis. Some
evidence shows giving interferon immediately following infection can prevent chronic hepatitis
C, although diagnosis early in infection is difficult since physical symptoms are sparse in early
hepatitis C infection. Control of chronic hepatitis C by IFN is associated with reduced
hepatocellular carcinoma.
Administered intranasally in very low doses, interferon is extensively used in Eastern Europe and
Russia as a method to prevent and treat viral respiratory diseases such as cold and flu. However,
mechanisms of such action of interferon are not well understood; it is thought that doses must be
larger by several orders of magnitude to have any effect on the virus. Although most scientists
are skeptical of any claims of good efficacy recent findings suggest that interferon applied to
mucosa may act as an adjuvant against influenza virus, boosting the specific immune system
response against the virus. A flu vaccine that uses interferon as adjuvant is currently under
clinical trials in the US.
When used in the systemic therapy, IFNs are mostly administered by an intramuscular injection.
The injection of IFNs in the muscle, in the vein, or under skin is generally well tolerated. The
most frequent adverse effects are flu-like symptoms: increased body temperature, feeling ill,
fatigue, headache, muscle pain, convulsion, dizziness, hair thinning, and depression. Erythema,
pain and hardness on the spot of injection are also frequently observed. IFN therapy causes
immunosuppression, in particular through neutropenia and can result in some infections
manifesting in unusual ways
Drug formulations:
Generic name Trade name
Interferon alpha 2a Roferon A
Intron
Interferon alpha 2b
A/Reliferon/Uniferon
Human leukocyte Interferon-alpha (HuIFN-
Multiferon
alpha-Le)
Interferon beta 1a, liquid form Rebif
Interferon beta 1a, lyophilized Avonex
Interferon beta 1a, biogeneric (Iran) Cinnovex
Interferon beta 1b Betaseron/ Betaferon
Interferon gamma 1b Actimmune
PEGylated interferon alpha 2a Pegasys
PEGylated interferon alpha 2a(Egypt) Reiferon Retard
PEGylated interferon alpha 2b PegIntron
PEGylated interferon alpha 2bplus
Pegetron
ribavirin(Canada)

ERYTHROPOIETIN
It is also known as erythropoetin or erthropoyetin and or EPO, is a glycoprotein hormone that
controls erythropoiesis, or red blood cell production. It is a cytokine(protein signaling molecule)
for erythrocyte (red blood cell) precursors in the bone marrow. Human EPO has a molecular
weight of 34 kDa.

Also called hematopoietin or hemopoietin, it is produced by interstitial fibroblasts in the kidney

in close association with peritubular capillary and tubular epithelial cells. It is also produced in
perisinusoidal cells in the liver. While liver production predominates in the fetal and perinatal
period, renal production is predominant during adulthood. In addition to erythropoiesis,
erythropoietin also has other known biological functions.
For example, it plays an important role in the brain's response to neuronal injury. EPO is also
involved in the wound healing process.
When exogenous EPO is used as a performance-enhancing drug, it is classified as an
erythropoiesis-stimulating agent (ESA). Exogenous EPO can often be detected in blood, due to
slight differences from the endogenous protein, for example, in features of posttranslational
modification.

Mechanism of action:
Erythropoietin has been shown to exert its effects by binding to the erythropoietin receptor
(EpoR).
EPO is highly glycosylated (40% of total molecular weight), with half-life in blood around five
hours. EPO's half-life may vary between endogenous and various recombinant versions.
Additional glycosylation or other alterations of EPO via recombinant technology have led to the
increase of EPO's stability in blood (thus requiring less frequent injections). EPO binds to the
erythropoietin receptor on the red cell progenitor surface and activates a JAK2 signaling cascade.

Erythropoietin receptor expression is found in a number of tissues, such as bone marrow and
peripheral/central nervous tissue. In the bloodstream, red cells themselves do not express
erythropoietin receptor, so cannot respond to EPO. However, indirect dependence of red cell
longevity in the blood on plasma erythropoietin levels has been reported, a process termed
neocytolysis.

Pharmaceutical companies make human recombinanterythropoietin with recombinant

technology, in which genes are inserted to create a custom organism. In this particular case,
bacteria are modified with recombination so that they will produce human erythropoietin which
can be administered to patients. The same technology is used to produce a variety of other
human hormones. These hormones are as effective in the body as hormones of human or animal
origin, but they are easier and safer to produce.

There are side effects associated with human recombinanterythropoietin, especially in patients
who use it for a long time. It can increase the risk of heavy clotting and adverse cardiovascular
events, and it can also lead to iron deficiency and high blood pressure. In some young athletes,
unexpected death has been linked to EPO usage, which is one of the reasons sports authorities
are concerned about blood doping. Recombinant EPO is chemically slightly different from the
made in the body version, and this can be used on blood tests to determine whether or not an
athlete is doping.
3) VACCINES:
Vaccination is the phenomenon of preventive immunization. In the modern concept, vaccination
involves the administration (injection or oral) of an antigen to elicit an antibody response that
will protect the organism against the future infections.

Vaccine is a biological preparation that improves immunity to a particular disease. A vaccine

typically contains an agent that resembles a disease-causing microorganism, and is often made
from weakened or killed forms of the microbe, its toxins or one of its surface proteins. The agent
stimulates the body's immune system to recognize the agent as foreign, destroy it, and
"remember" it, so that the immune system can more easily recognize and destroy any of these
microorganisms that it later encounters.

Vaccines may be prophylactic (example: to prevent or ameliorate the effects of a future infection
by any natural or "wild" pathogen), or therapeutic (e.g. vaccines against cancer are also being
investigated; see cancer vaccine).
The term vaccine derives from Edward Jenner's 1796 use of cow pox (Latin variolavaccinia,
adapted from the Latin vacc?n-us, from vacca, cow), to inoculate humans, providing them
protection against smallpox.

Effectiveness
Vaccines do not guarantee complete protection from a disease. Sometimes, this is because the
host's immune system simply does not respond adequately or at all. This may be due to a
lowered immunity in general (diabetes, steroid use, HIV infection, age) or because the host's
immune system does not have a B cell capable of generating antibodies to that antigen.
Even if the host develops antibodies, the human immune system is not perfect and in any case
the immune system might still not be able to defeat the infection immediately. In this case, the
infection will be less severe and heal faster.
Adjuvants are typically used to boost immune response. Most often aluminium adjuvants are
used, but adjuvants like squalene are also used in some vaccines and more vaccines with
squalene and phosphate adjuvants are being tested. Larger doses are used in some cases for older
people (50–75 years and up), whose immune response to a given vaccine is not as strong.
Maurice Hilleman's measles vaccine is estimated to prevent 1 million deaths every year.
The efficacy or performance of the vaccine is dependent on a number of factors:
 the disease itself (for some diseases vaccination performs better than for other diseases)
 the strain of vaccine (some vaccinations are for different strains of the disease)
 whether one kept to the timetable for the vaccinations (see Vaccination schedule)
 some individuals are "non-responders" to certain vaccines, meaning that they do not
generate antibodies even after being vaccinated correctly other factors such as ethnicity,
age, or genetic predisposition.

Recombinant vaccines:
Biotechnology sector has also played its part in developing vaccines against certain diseases.
Such vaccine which makes use of recombinant DNA technology is known as recombinant
vaccines. It is also known as subunit vaccines.
Recombinant vaccines can be broadly grouped into two kinds:
(i) Recombinant protein vaccines: This is based on production of recombinant DNA which is
expressed to release the specific protein used in vaccine preparation
(ii) DNA vaccines: Here the gene encoding for immunogenic protein is isolated and used to
produce recombinant DNA which acts as vaccine to be injected into the individual
Steps involved:
Production of recombinant vaccines involves the following steps:
(i) The protein which is crucial to the growth and development of the causative organism
be identified.
(ii) The corresponding gene is then isolated applying various techniques. Further to this,
an extensive study of the gene explains the gene expression pattern involved in the
 production of corresponding protein.
(iii) This gene is then integrated into a suitable expression vector to produce a
recombinant DNA.
(iv) This rDNA is used as vaccines or is introduce into another host organism to produce
immunogenic proteins which acts as vaccines.
Recombinant protein vaccines:
A pathogen upon infection produces proteins, vital for its functions, which elicit an immune
response from the infected body. The gene encoding such a protein is isolated from the causative
organism and used to develop a recombinant DNA.

This DNA is expressed in another host organism, like genetically engineered microbes; animal
cells; plant cells; insect larvae etc, resulting in the release of the appropriate proteins which are
then isolated and purified.

These when injected into the body, causes immunogenic response to be active against the
corresponding disease providing immunity against future attack of the pathogen.
Based on the proteins involved in evoking immune response recombinant protein vaccines are of
two types:
Whole protein vaccines: The whole immunogenic protein is produced in another host organism
which is isolated and purified to act as vaccines.
Polypeptide vaccines: It is known that in the immunogenic protein produced, the actual
immunogenic property is limited to one or two polypeptides forming the protein. The other parts
of the protein may be successful in evoking an immune response but do not actually cause the
disease. For eg: in the case of cholera caused by Vibrio cholerae, consists of three polypeptide
chains like A1, A2, and B. The A polypeptides are toxic while B is non-toxic. Thus while
producing vaccines, the polypeptide B is produced by rDNA technology and used for
vaccination.

DNA vaccines:
It refers to the recombinant vaccines in which the DNA is used as a vaccine. The gene
responsible for the immunogenic protein is identified, isolated and cloned with corresponding
expression vector. Upon introduction into the individuals to be immunized, it produces a
recombinant DNA. This DNA when expressed triggers an immune response and the person
becomes successfully vaccinated. The mode of delivery of DNA vaccines include: direct injection
into muscle; use of vectors like adenovirus, retrovirus etc; invitro transfer of the gene into
autologous cells and reimplantation of the same and particle gun delivery of the DNA.
In certain cases, the responsible gene is integrated into live vectors which are introduced into
individuals as vaccines. This is known as live recombinant vaccines. Eg: vaccinia virus. Live
vaccinia virus vaccine (VV vaccine) with genes corresponding to several diseases, when
introduced into the body elicit an immune response but does not actually cause the diseases.
Advantages:
(i) Since it does not involve actual pathogen, recombinant vaccines is considered to be safe than
the conventional vaccines.
(ii) It induces both humoral and cellular immune response resulting in effective vaccination.
Risks involved:
(i) High cost of production.
(ii) Have to be stored at low temperature since heat destabilizes protein. Hence storage and
transportation is tedious.
(iii) Individuals with immunodeficiency may elicit poor immune response.
ANTIBODIES
Antibodies are host proteins that are produced by the immune system in response to foreign
molecules that enter the body. These foreign molecules are called antigens, and their molecular
recognition by the immune system results in selective production of antibodies that are able to
bind the specific antigen. Antibodies are made by B-lymphocytes and circulate throughout the
blood and lymph where they bind to their specific antigen, enabling it to be cleared from
circulation.

This ability of animal immune systems to produce antibodies capable of binding specifically to
antigens can be harnessed to manufacture probes for detection of molecules of interest in a
variety of research and diagnostic applications. Certainly, no other current technology allows
researchers to design and manufacture such highly specific molecular recognition tools.

Several important features besides their high specificity make antibodies particularly conducive
to development as probes. For example, except in those portions that determine antigen binding,
antibodies share a relatively uniform and well-characterized protein structure that enables them
to be purified, labeled and detected predictably and reproducibly by generalized methods.
Procedures for generating, purifying and modifying antibodies for use as antigen-specific probes
were developed during the 1970s and 1980s and have remained relatively unchanged since
Harlow and Lane published their classic Antibodies: A Laboratory Manual in 1988.

Antibody production
The term "antibody production" has both general and specific meanings. In the broad sense, it
refers to the entire process of creating a usable specific antibody, including steps of immunogen
preparation, immunization, hybridoma creation, collection, screening, isotyping, purification,
and labeling for direct use in a particular method. In the more restricted sense, antibody
production refers to the steps leading up to antibody generation but does not include various
forms of purifying and labeling the antibody for particular uses.
Antibody production involves preparation of antigen samples and their safe injection into
laboratory or farm animals so as to evoke high expression levels of antigen-specific antibodies in
the serum, which can then be recovered from the animal. Polyclonal antibodies are recovered
directly from serum (bleeds). Monoclonal antibodies are produced by fusing antibody-secreting
spleen cells from immunized mice with immortal myeloma cell to create monoclonal hybridoma
cell lines that express the specific antibody in cell culture supernatant.
Successful antibody production depends upon careful planning and implementation with respect
to several important steps and considerations:
 Synthesize or purify the target antigen (e.g., peptide or hapten)
 Choose an appropriate immunogenic carrier protein
 Conjugate the antigen and carrier protein to create the immunogen
 Immunize animals using appropriate schedule and adjuvant formula
 Screen serum (or hybridoma) for antibody titer and isotype (also called antibody
Antibody purification
Antibody purification involves isolation of antibody from serum (polyclonal antibody), ascites
fluid or culture supernatant of a hybridoma cell line (monoclonal antibody). Purification methods
range from very crude to highly specific:
 Crude—precipitation of a subset of total serum proteins that includes immunoglobulins
 General—affinity purification of certain antibody classes (e.g., IgG) without regard to
antigen specificity
 Specific—affinity purification of only those antibodies in a sample that bind to a
particular antigen molecule
Which level of purification is necessary to obtain usable antibody depends upon the intended
application(s) for the antibody.

Antibody characterization
Antibody characterization involves three kinds of activities that are usually performed at various
stages throughout an entire antibody production and purification project
 Screening—identifying antibody samples having antigen-binding specificity
 Titering—measuring antibody concentration and functional assay titer
 Isotyping—determining a monoclonal antibody class and subclass identity
Screening is first required during production to identify which animals and hybridoma clones are
producing a high level of antigen-specific antibody. This is usually accomplished using ELISA
techniques.
Antibody concentration can be estimated using either a general protein assay or a species- and
immunoglobulin-specific method, such as with specialized microagglutination assay kits.
Antibody titer is related to concentration but refers more specifically to the effective potency of a
given antibody sample. Measuring titer usually means determining the functional dilution of an
antibody sample necessary for detection in a given assay, such as ELISA.
Isotyping involves determining the class (e.g., IgG vs. IgM) and subclass (e.g., IgG1 vs. IgG2a)
of a monoclonal antibody. This is a critical step in antibody production, as it is necessary for
choosing an appropriate purification and modification method for the molecule. Isotyping is
most easily accomplished with commercial, ready-to-use antibody isotyping kits.

Antibody fragmentation
Purified antibodies can be modified for particular uses by several methods including
fragmentation into smaller antigen-binding units, conjugation with enzyme or other detectable
markers, and immobilization to solid supports. Most often antibodies are used in whole-molecule
form. However, the performance of some techniques and experiments can be improved by using
antibodies whose nonessential portions have been removed.
Antibody fragmentation refers to procedures for cleaving apart whole antibody molecules and
removing portions that are not necessary for binding antigen. Fab and F(ab)'2 are antibody
fragments of IgG that are most frequently created and utilized by researchers.
Antibody labeling and immobilization
Antibodies are produced and purified for use as antigen-specific probes. However, their utility in
any given technique (ELISA, western blotting, cellular imaging, immunohistochemistry)
depends upon having a mechanism to secondarily detect the antibody.
Techniques that utilize antibodies for immunoprecipitation or other form of affinity purification
depend upon mechanisms for attaching or immobilizing them to chromatography media (e.g.,
beaded agarose resin). Strategies for accomplishing this involve the same considerations and
chemical methods as antibody labelling

MONOCLONAL ANTIBODIES
Monoclonal Antibodies are cells derived by cell division from a single ancestral
cell.Monoclonals are a class of antibodies with identical offspring of a hybridoma and are very
specific for a particular location in the body derived from a single clone and can be grown
indefinitely. Monoclonal Antibodies recognize and bind to antigens in order to discriminate
between specific epitopes which provides protection against disease organisms.

Monoclonal antibodies target various proteins that influence cell activity such as receptors or
other proteins present on the surface of normal and cancer cells. The specificity of Monoclonal
Antibodies allows its binding to cancerous cells by coupling a cytotoxic agent such as a strong
radioactive which then seek outs to destroy the cancer cells while not harming the healthy ones.
Tumor cells that are able to replicate endlessly are fused with mammalian cells that produce a
specific antibody which result in fusion called hybridoma that continuously produce antibodies.
Those antibodies are named monoclonal because they come from only 1 type of cell, which is
the hybridoma cell. Antibodies that are produced by conventional methods and derived from
preparations containing many kinds of cells are called polyclonal Antibodies.

Monoclonal antibodies are artificially produced against a specific antigen in order to bind to their
target antigens. Laboratory production of monoclonal antibodies is produced from clones of only
1 cell which means that every monoclonal antibody produced by the cell is the same.
Fusion of cell culture myeloma cells with mammalian spleen cells antibodies result in hybrid
cells/hybridomas which produces large amounts of monoclonal antibody. The cell fusion resulted
in two different types of cells, one with the ability to grow continually, and the other with ability
to produce bulk amounts of purified antibody. Hybrid cells produce only 1 exact antibody that is
more pure than polyclonal antibodies produced by conventional techniques. Monoclonal
Antibodies are far more effective than conventional drugs since drugs attack the foreign
substance & the body's own cells that cause harsh side effects & the monoclonal antibody only
targets the foreign antigen/target molecule, without or only minor side effects.

The presence of a large amount of a specific monoclonal antibody in the blood means that there
is an abnormal protein. Typically this protein can be detected during a physical examination and
is identified using a screening blood test called ―protein electrophoresis. The source of abnormal
production of monoclonal antibody is a small population of plasma cells in the bone marrow.
Production of Monoclonal Antibodies:
Process by which bulk quantities of targeted antibodies against a specific antigen are
produced.Monoclonal antibodies are produced via multiple/identical copies of a certain cell
called a hybridoma.

To create Hybridoma cells the fusion of 2 cells are needed in order to combine the characteristics
of the 2 cells into 1 cell. 1 of the cells is a producing cell antibody which is a B-Lymphocyte
used from a laboratory mouse and the other is a tumor cell named myeloma. Tumor cells have
the ability to grow indefinitely and at an exceeding rate from normal cell growth. Laboratroy
produced Hybridoma cells replicate much faster than normal antibody producing cells, and the
individual hybridomas produce the specific antibodies for an indefinite period of time.

Hybridoma cells manufacture the specific monoclonal antibody that was originally produced by
the B-Lymphocyte cell. The original B-Lymphocyte cell will produce the Monoclonal antibody
depending on the kind of antigen that was injected into the mouse just prior to the harvesting of
the B-Lymphocyte cells. A small example is, if the mice were injected with a certain virus, the
mouse will have B-Lymphocytes that produce those specific virual antibodies. Fusion with a
tumor cell to make the hybridoma, result in the production of monoclonal antibodies against the
specific virus.

The hybridoma cells are placed into media that can help them grow and produce the bulk
quantities of monoclonal antibodies. There are 2 ways for growing monoclonal antibodies, 1 is to
grow them in laboratory flasks meaning In Vitro, and the other is to grow them in the stomach
lining of mice. Injecting the hybridomas into the mice is the familiar method of harvesting
monoclonal antibodies.

This method is done by mixing spleen cells from the mouse that has been immunized with the
desired antigen with myeloma cells.The myeloma cells need to have lost their ability to
synthesize HGPRT enzyme (hypoxanthine-guanine-phosphoribosyltransferase) which enables
the cells to synthesize purines using an extracellular source of hypoxanthine as a precursor. Cells
have another pathway that they synthesize purines so lack of HGPRT is not a problem for the
cell but when cells are exposed to aminopterin they are unable to use this other pathway and are
fully dependent on HGPRT for their survival. So to summarize, unfused myeloma cells can’t
grow since they lack HGPRT and unfused normal spleen cells can’t grow since they have a
limited life-span. Hybridoma cells are grow indefinitely since the spleen-cell copartner supplies
HGPRT and the myeloma partner is immortal.

The first step is transferring of the cell fusion mixture to HAT culture medium which contains
hypoxanthine, aminopterin & pyrimidine thymidine. The 2nd step is testing the supernatants from
each culture in order to locate the producing the desired antibody. One must isolate the single
cells from each antibody-positive culture and subculture them, this represents the clone which its
antibodies are monoclonal. Every single cell culture secretes a specific kind of antibody that is
directed against a certain determinant/selected antigen.
The 3rd step is scaling up the size of the cultures of the successful clones. Hybridoma cultures
can be grown indefinitely in vitro in culture vessels which yield 15-65 µg/ml and in vivo using
mouse, where the antibody concentration in the serum 0.5-15 mg/ml. In the past years, animal
welfare activists in worldwide are trying to limit the use of mice for the production of
monoclonal antibodies. When the monoclonal antibody is produced it can be used as a probe to
track down, bind to and purify the specific protein that induced its formation.

Clinical Uses of Monoclonal Antibodies

Since the most common methods for producing monoclonal antibodies use mouse cells, it is
necessary to create humanized monoclonal antibodies for human clinical use. Mouse
antibodies cannot be injected repeatedly into humans, because the immune system will recognize
them as being foreign and will respond to them with neutralizing antibodies.

This problem can be minimized by genetically engineering the antibody in the mouse B cell. The
variable regions of the mouse light and heavy chain genes are ligated to human constant
regions, and the chimeric gene is then transferred into a host cell. This allows production of a
mAb that is mostly ―human‖ with only the antigen-binding site being of mouse origin.
Humanized mAbs have been successfully used to treat cancer with minimal side effects. For
example, the humanized monoclonal antibody drug Herceptin has been helpful for the treatment
of some types of breast cancer.

There have also been a few preliminary trials of humanized mAb for the treatment of infectious
diseases, but none of these treatments are currently in use. In some cases, mAbs have proven too
specific to treat infectious diseases, because they recognize some serovars of a pathogen but not
others. Using a cocktail of multiple mAbs that target different strains of the pathogen can address
this problem.

However, the great cost associated with mAb production is another challenge that has prevented
mAbs from becoming practical for use in treating microbial infections.[1]
One promising technology for inexpensive mAbs is the use of genetically engineered plants to
produce antibodies (or plantibodies). This technology transforms plant cells into antibody
factories rather than relying on tissue culture cells, which are expensive and technically
demanding.

In some cases, it may even be possible to deliver these antibodies by having patients eat the
plants rather than by extracting and injecting the antibodies. For example, in 2013, a research
group cloned antibody genes into plants that had the ability to neutralize an important toxin from
bacteria that can cause severe gastrointestinal disease.Eating the plants could potentially deliver
the antibodies directly to the toxin.

PRODUCING POLYCLONAL ANTIBODIES

Antibodies used for research and diagnostic purposes are often obtained by injecting a lab animal
such as a rabbit or a goat with a specific antigen. Within a few weeks, the animal’s immune
system will produce high levels of antibodies specific for the antigen.

These antibodies can be harvested in an antiserum, which is whole serum collected from an
animal following exposure to an antigen. Because most antigens are complex structures with
multiple epitopes, they result in the production of multiple antibodies in the lab animal.

This so-called polyclonal antibody response is also typical of the response to infection by the
human immune system. Antiserum drawn from an animal will thus contain antibodies from
multiple clones of B cells, with each B cell responding to a specific epitope on the antigen
(Figure 2).
Figure 2. This diagram illustrates the process for harvesting polyclonal antibodies produced in
response to an antigen.

Lab animals are usually injected at least twice with antigen when being used to produce
antiserum. The second injection will activate memory cells that make class IgG antibodies
against the antigen. The memory cells also undergo affinity maturation, resulting in a pool of
antibodies with higher average affinity.

Affinity maturation occurs because of mutations in the immunoglobulin gene variable regions,
resulting in B cells with slightly altered antigen-binding sites. On re-exposure to the antigen,
those B cells capable of producing antibody with higher affinity antigen-binding sites will be
stimulated to proliferate and produce more antibody than their lower-affinity peers.

An adjuvant, which is a chemical that provokes a generalized activation of the immune system
that stimulates greater antibody production, is often mixed with the antigen prior to injection.
Antiserum obtained from animals will not only contain antibodies against the antigen artificially
introduced in the laboratory, but it will also contain antibodies to any other antigens to which the
animal has been exposed during its lifetime.

For this reason, antisera must first be ―purified‖ to remove other antibodies before using the
antibodies for research or diagnostic assays.

Clinical Uses of Polyclonal Antisera

Polyclonal antisera are used in many clinical tests that are designed to determine whether a
patient is producing antibodies in response to a particular pathogen. While these tests are
certainly powerful diagnostic tools, they have their limitations, because they are an indirect
means of determining whether a particular pathogen is present.
-Tests based on a polyclonal response can sometimes lead to a false-positive result—in other
words, a test that confirms the presence of an antigen that is, in fact, not present. Antibody-based
tests can also result in a false-negative result, which occurs when the test fails to detect an
antibody that is, in fact, present.

-The accuracy of antibody tests can be described in terms of test sensitivity and test specificity.
Test sensitivity is the probability of getting a positive test result when the patient is indeed
infected. If a test has high sensitivity, the probability of a false negative is low. Test specificity,
on the other hand, is the probability of getting a negative test result when the patient is not
infected. If a test has high specificity, the probability of a false positive is low.

-False positives often occur due to cross-reactivity, which can occur when epitopes from a
different pathogen are similar to those found on the pathogen being tested for. For this reason,
antibody-based tests are often used only as screening tests; if the results are positive; other
confirmatory tests are used to make sure that the results were not a false positive.

-For example, a blood sample from a patient suspected of having hepatitis C can be screened for
the virus using antibodies that bind to antigens on hepatitis C virus. If the patient is indeed
infected with hepatitis C virus, the antibodies will bind to the antigens, yielding a positive test
result. If the patient is not infected with hepatitic C virus, the antibodies will generally not bind
to anything and the test should be negative; however, a false positive may occur if the patient has
been previously infected by any of a variety of pathogens that elicit antibodies that cross-react
with the hepatitis C virus antigens.

-Antibody tests for hepatitis C have high sensitivity (a low probability of a false negative) but
low specificity (a high probability of a false positive). Thus, patients who test positive must have
a second, confirmatory test to rule out the possibility of a false positive.

-The confirmatory test is a more expensive and time-consuming test that directly tests for the
presence of hepatitis C viral RNA in the blood. Only after the confirmatory test comes back
positive can the patient be definitively diagnosed with a hepatitis C infection. Antibody-based
tests can result in a false negative if, for any reason, the patient’s immune system has not
produced detectable levels of antibodies.

-For some diseases, it may take several weeks following infection before the immune system
produces enough antibodies to cross the detection threshold of the assay. In
immunocompromised patients, the immune system may not be capable of producing a detectable
level of antibodies.

-Another limitation of using antibody production as an indicator of disease is that antibodies in

the blood will persist long after the infection has been cleared. Depending on the type of
infection, antibodies will be present for many months; sometimes, they may be present for the
remainder of the patient’s life. Thus, a positive antibody-based test only means that the patient
was infected at some point in time; it does not prove that the infection is active.
In addition to their role in diagnosis, polyclonal antisera can activate complement, detect the
presence of bacteria in clinical and food industry settings, and perform a wide array of
precipitation reactions that can detect and quantify serum proteins, viruses, or other antigens.
However, with the many specificities of antibody present in a polyclonal antiserum, there is a
significant likelihood that the antiserum will cross-react with antigens to which the individual
was never exposed. Therefore, we must always account for the possibility of false-positive
results when working with a polyclonal antiserum.

Table 1. Characteristics of Polyclonal and Monoclonal Antibodies

Monoclonal Antibodies Polyclonal Antibodies
Expensive production Inexpensive production
Long production time Rapid production
Large quantities of specific antibodies Large quantities of nonspecific
antibodies
Recognize a single epitope on an antigen Recognize multiple epitopes on an
antigen
Production is continuous and uniform once the hybridoma Different batches vary in composition
is made
DELIVERY OF BIOTECHNOLOGY PRODUCT
Drugs are introduced into the body by several routes. They may be
 Taken by mouth (orally)
 Given by injection into a vein (intravenously, IV), into a muscle (intramuscularly, IM),
into the space around the spinal cord (intrathecally), or beneath the skin (subcutaneously,
sc)
 Placed under the tongue (sublingually) or between the gums and cheek (buccally)
 Inserted in the rectum (rectally) or vagina (vaginally)
 Placed in the eye (by the ocular route) or the ear (by the otic route)
 Sprayed into the nose and absorbed through the nasal membranes (nasally)
 Breathed into the lungs, usually through the mouth (by inhalation) or mouth and nose (by
nebulization)
 Applied to the skin (cutaneously) for a local (topical) or bodywide (systemic) effect
 Delivered through the skin by a patch (transdermally) for a systemic effect
Each route has specific purposes, advantages, and disadvantages.

Oral route
Many drugs can be administered orally as liquids, capsules, tablets, or chewable tablets. Because
the oral route is the most convenient and usually the safest and least expensive, it is the one most
often used. However, it has limitations because of the way a drug typically moves through the
digestive tract. For drugs administered orally, absorption may begin in the mouth and stomach.
However, most drugs are usually absorbed from the small intestine.

The drug passes through the intestinal wall and travels to the liver before being transported via
the bloodstream to its target site. The intestinal wall and liver chemically alter (metabolize) many
drugs, decreasing the amount of drug reaching the bloodstream. Consequently, these drugs are
often given in smaller doses when injected intravenously to produce the same effect.
When a drug is taken orally, food and other drugs in the digestive tract may affect how much of
and how fast the drug is absorbed.

Thus, some drugs should be taken on an empty stomach, others should be taken with food, others
should not be taken with certain other drugs, and still others cannot be taken orally at all.
Some orally administered drugs irritate the digestive tract. For example, aspirin and most other
nonsteroidal anti-inflammatory drugs (NSAIDs) can harm the lining of the stomach and small
intestine to potentially cause or aggravate preexisting ulcers. Other drugs are absorbed poorly or
erratically in the digestive tract or are destroyed by the acid and digestive enzymes in the
stomach.
Other routes of administration are required when the oral route cannot be used, for example:
 When a person cannot take anything by mouth
 When a drug must be administered rapidly or in a precise or very high dose
 When a drug is poorly or erratically absorbed from the digestive tract

Injection routes
Administration by injection (parenteral administration) includes the following routes:
 Subcutaneous (under the skin)
 Intramuscular (in a muscle)
 Intravenous (in a vein)
 Intrathecal (around the spinal cord)
A drug product can be prepared or manufactured in ways that prolong drug absorption from the
injection site for hours, days, or longer. Such products do not need to be administered as often as
drug products with more rapid absorption
or the subcutaneous route, a needle is inserted into fatty tissue just beneath the skin. After a
drug is injected, it then moves into small blood vessels (capillaries) and is carried away by the
bloodstream. Alternatively, a drug reaches the bloodstream through the lymphatic vessels.

Protein drugs that are large in size, such as insulin, usually reach the bloodstream through the
lymphatic vessels because these drugs move slowly from the tissues into capillaries. The
subcutaneous route is used for many protein drugs because such drugs would be destroyed in the
digestive tract if they were taken orally.

Certain drugs (such as progestins used for hormonal birth control) may be given by inserting
plastic capsules under the skin (implantation). Although this route of administration is rarely
used, its main advantage is to provide a long-term therapeutic effect (for example, etonogestrel
that is implanted for contraception may last up to 3 years).

The intramuscular route is preferred to the subcutaneous route when larger volumes of a drug
product are needed. Because the muscles lie below the skin and fatty tissues, a longer needle is
used. Drugs are usually injected into the muscle of the upper arm, thigh, or buttock. How quickly
the drug is absorbed into the bloodstream depends, in part, on the blood supply to the muscle:
The sparser the blood supply, the longer it takes for the drug to be absorbed.
Intravenous route: a needle is inserted directly into a vein. A solution containing the drug may
be given in a single dose or by continuous infusion. For infusion, the solution is moved by
gravity (from a collapsible plastic bag) or, more commonly, by an infusion pump through thin
flexible tubing to a tube (catheter) inserted in a vein, usually in the forearm.

Intravenous administration is the best way to deliver a precise dose quickly and in a well-
controlled manner throughout the body. It is also used for irritating solutions, which would cause
pain and damage tissues if given by subcutaneous or intramuscular injection. An intravenous
injection can be more difficult to administer than a subcutaneous or intramuscular injection
because inserting a needle or catheter into a vein may be difficult, especially if the person is
obese.
When given intravenously, a drug is delivered immediately to the bloodstream and tends to take
effect more quickly than when given by any other route. Consequently, health care practitioners
closely monitor people who receive an intravenous injection for signs that the drug is working or
is causing undesired side effects. Also, the effect of a drug given by this route tends to last for a
shorter time. Therefore, some drugs must be given by continuous infusion to keep their effect
constant.

Intrathecal route: a needle is inserted between two vertebrae in the lower spine and into the
space around the spinal cord. The drug is then injected into the spinal canal. A small amount of
local anesthetic is often used to numb the injection site. This route is used when a drug is needed
to produce rapid or local effects on the brain, spinal cord, or the layers of tissue covering them
(meninges)—for example, to treat infections of these structures. Anesthetics and analgesics (such
as morphine) are sometimes given this way.

Sublingual and buccal routes: A few drugs are placed under the tongue (taken sublingually) or
between the gums and teeth (buccally) so that they can dissolve and be absorbed directly into the
small blood vessels that lie beneath the tongue. These drugs are not swallowed. The sublingual
route is especially good for nitroglycerin, which is used to relieve angina, because absorption is
rapid and the drug immediately enters the bloodstream without first passing through the
intestinal wall and liver. However, most drugs cannot be taken this way because they may be
absorbed incompletely or erratically.

Rectal route: Many drugs that are administered orally can also be administered rectally as a
suppository. In this form, a drug is mixed with a waxy substance that dissolves or liquefies after
it is inserted into the rectum. Because the rectum’s wall is thin and its blood supply rich, the drug
is readily absorbed. A suppository is prescribed for people who cannot take a drug orally because
they have nausea, cannot swallow, or have restrictions on eating, as is required before and after
many surgical operations. Drugs that can be administered rectally include acetaminophen (for
fever), diazepam (for seizures), and laxatives (for constipation). Drugs that are irritating in
suppository form may have to be given by injection.
Vaginal route: Some drugs may be administered vaginally to women as a solution, tablet,
cream, gel, suppository, or ring. The drug is slowly absorbed through the vaginal wall. This route
is often used to give estrogen to women during menopause to relieve vaginal symptoms such as
dryness, soreness, and redness.

Ocular route: Drugs used to treat eye disorders (such as glaucoma, conjunctivitis, and injuries)
can be mixed with inactive substances to make a liquid, gel, or ointment so that they can be
applied to the eye. Liquid eye drops are relatively easy to use but may run off the eye too quickly
to be absorbed well. Gel and ointment formulations keep the drug in contact with the eye surface
longer, but they may blur vision. Solid inserts, which release the drug continuously and slowly,
are also available, but they may be hard to put in and keep in place.
Ocular drugs are almost always used for their local effects. For example, artificial tears are used
to relieve dry eyes. Other drugs (for example, those used to treat glaucoma [see table Drugs Used
to Treat Glaucoma], such as acetazolamide and betaxolol, and those used to dilate pupils, such as
phenylephrine and tropicamide) produce a local effect (acting directly on the eyes) after they are
absorbed through the cornea and conjunctiva. Some of these drugs then enter the bloodstream
and may cause unwanted side effects on other parts of the body.
Otic route: Drugs used to treat ear inflammation and infection can be applied directly to the
affected ears. Ear drops containing solutions or suspensions are typically applied only to the
outer ear canal. Before applying ear drops, people should thoroughly clean the ear with a moist
cloth and dry it. Unless the drugs are used for a long time or used too much, little of the drugs
enter the bloodstream, so bodywide side effects are absent or minimal. Drugs that can be given
by the otic route include hydrocortisone (to relieve inflammation), ciprofloxacin (to treat
infection), and benzocaine (to numb the ear).

Nasal route: If a drug is to be breathed in and absorbed through the thin mucous membrane that
lines the nasal passages, it must be transformed into tiny droplets in air (atomized). Once
absorbed, the drug enters the bloodstream. Drugs administered by this route generally work
quickly. Some of them irritate the nasal passages. Drugs that can be administered by the nasal
route include nicotine (for smoking cessation), calcitonin (for osteoporosis), sumatriptan (for
migraine headaches), and corticosteroids (for allergies).

Inhalation route: Drugs administered by inhalation through the mouth must be atomized into
smaller droplets than those administered by the nasal route, so that the drugs can pass through
the windpipe (trachea) and into the lungs. How deeply into the lungs they go depends on the size
of the droplets. Smaller droplets go deeper, which increases the amount of drug absorbed. Inside
the lungs, they are absorbed into the bloodstream.

Relatively few drugs are administered this way because inhalation must be carefully monitored
to ensure that a person receives the right amount of drug within a specified time. In addition,
specialized equipment may be needed to give the drug by this route. Usually, this method is used
to administer drugs that act specifically on the lungs, such as aerosolized antiasthmatic drugs in
metered-dose containers (called inhalers), and to administer gases used for general anesthesia.
Nebulization route: Similar to the inhalation route, drugs given by nebulization must be
aerosolized into small particles to reach the lungs. Nebulization requires the use of special
devices, most commonly ultrasonic or jet nebulizer systems. Using the devices properly helps
maximize the amount of drug delivered to the lungs. Drugs that are nebulized include
tobramycin (for cystic fibrosis), pentamidine (for pneumonia caused by Pneumocystis jirovecii),
and albuterol (for asthma attacks).
side effects can include those that occur when the drug is deposited directly in the lungs (such as
cough, wheezing, shortness of breath, and lung irritation), spread of the drug into the
environment (possibly affecting people other than the one taking the drug), and contamination of
the device used for nebulization (particularly when the device is reused and inadequately
cleaned). Using the device properly helps prevent side effects.

Cutaneous route: Drugs applied to the skin are usually used for their local effects and thus are
most commonly used to treat superficial skin disorders, such as psoriasis, eczema, skin infections
(viral, bacterial, and fungal), itching, and dry skin. The drug is mixed with inactive substances.
Depending on the consistency of the inactive substances, the formulation may be an ointment,
cream, lotion, solution, powder, or gel

Transdermal route: Some drugs are delivered bodywide through a patch on the skin. These
drugs are sometimes mixed with a chemical (such as alcohol) that enhances penetration through
the skin into the bloodstream without any injection. Through a patch, the drug can be delivered
slowly and continuously for many hours or days or even longer. As a result, levels of a drug in
the blood can be kept relatively constant. Patches are particularly useful for drugs that are
quickly eliminated from the body because such drugs, if taken in other forms, would have to be
taken frequently. However, patches may irritate the skin of some people. In addition, patches are
limited by how quickly the drug can penetrate the skin. Only drugs to be given in relatively small
daily doses can be given through patches. Examples of such drugs include nitroglycerin (for
chest pain), scopolamine (for motion sickness), nicotine (for smoking cessation), clonidine (for
high blood pressure), and fentanyl (for pain relief).

PHARMACEUTICAL FORMULATION
Is the process of combining various chemical substances with the active drug to form a final
medicinal product, which is called a drug mixture or drug formulation.

A drug formulation can be given to the patient in various forms like solid, semisolid or
liquid. The type of the formulation given depends upon the patient’s age, sex, and health
condition and is specific for particular routes of administration.

Solid Formulations
 Tablets – a tablet is disc-shaped and prepared by compressing a granulated powder in a
die of suitable machinery. They are mostly coated with inert substances like starch to
help them disintegrate in the digestive tract of the patient. A binding agent, lubricating
material, and flavors are added to the tablets to make them palatable.
  Enteric Coated Tablets – are coated with a material that does not disintegrate in the
acidic medium of the stomach but in the alkaline medium of the intestine. They can’t be
chewed but consumed only by swallowing.
 Controlled Release Tablet – is designed to release the active ingredient of the drug in a
specific amount over a specified period of time. Here, the amount of drug released is
gradual over the day and doesn’t depend upon the pH of the digestive tract of the patient.
Thus, a uniform amount of drug is released at a uniform rate.
 Sustained release preparations – release a fixed amount of drug over an extended
period of time. Thus, they improve the treatment compliance by the patient.
 Capsules – can be hard or soft. Hard capsules contain the drug in solid form, which gets
dissolved easily in water. Soft capsules have the drug in liquid or semi-solid form, which
is non-soluble in water and soluble in propylene or glycol.

Liquid and Semi-Solid Formulations

They are more readily absorbed than the solid formulations and can be administered by various
routes like:
1. Oral preparations – Oral preparations are easier to swallow and administer medicines
to children and old-age patients. Flavourings and sugar are added to some liquids to
make them palatable. They are available as solutions, suspensions, or emulsions and
must be shaken well before use.
2. Topical Preparations – The application of a drug to an area of the body for direct
treatment is called topical application. It includes:
o Eye drops
o Ear drops
o Nasal drops
o Nebulisers and inhalers
o Creams and ointments for skin application
o Gels and lotions
o Pessaries for vaginal administration of the drug
3. Sublingual and Buccal Administration – It is useful for drugs which are active in very
low concentration in the blood. Such drugs are administered as tablets under the tongue
or between the cheek and the gum and allowed to dissolve. In this manner, the drug
directly enters the bloodstream, bypassing the digestive tract and acts faster.
4. Rectal Administration
o Suppositories: are used for drugs which are administered through the rectum.
The drug is absorbed by the rectal mucosa and directly enters the bloodstream.
The method is useful when a patient is unconscious, has nausea or difficulty in
swallowing.
o Enemas: are liquid preparations for rectal administration. They can be used for
topical or systemic treatment and also for a bowel movement.
5. Parental Drug Administration – is drug administration outside the GI tract of the
patient. Drugs can be inserted anywhere with the help of injections.
o Intradermal administration: where the drug is inserted into the dermis. g. Local
Anaesthesia
 o Subcutaneous injection: where the drug is inserted into the subcutaneous tissue
or under the skin. It is mainly for the drugs that cannot be given through the
mouth e.g. Insulin.
o Intramuscular injection: where the drug is inserted into the skeletal system or the
muscle. The system is highly vascular, and hence, drugs with low molecular
weight can easily pass through by direct diffusion into the bloodstream.
o Intravenous injection: is given directly in the vein and allows for a faster action
of the drug.

EXCIPIENTS IN FORMULATIONS

Excipients are addictive substances used in tablet formulation to improve bulkiness,

disintegration, dissolution rate and bioavailability of the drug.
The drug and excipient interaction study is carried using Infrared Spectrum to know the stability
of excipients and drug.
In the pharmaceutical industry it is a catch-all term which includes various sub-groups
comprising diluents or fillers, binders or adhesives, disintegrants, lubricants, glidant, flavors,
colors and sweeteners. All of these must meet certain criteria as follows
A) Physiologically inert.
B) Acceptable to regulatory agencies.
C) Physiologically and chemically stable.
D) Free from bacteria.
E) Should not interfere with the bioavailability of the drug.
F) Commercially available in the form and purity commensurate to pharmaceutical
standards.
G) Low cost, inexpensive.
H) Meet the standards of regulatory requirements.

EXAMPLES OF EXCIPIENTS
-Diluents: Diluents are fillers used to make up the volume of tablet if tablet is inadequate to
produce the volume. Diluents used as disintegrants in dispersible and orally disintegrating tablet.
Example: Lactose, Spray dried lactose, Micro crystalline cellulose, Sorbitol, Dibasic calcium
phosphate dehydrates, Calcium sulphate dehydrate etc.
-Binders: Binders are used as binding agent in tablets; it provides cohesive strength to powdered
materials. Binders are added in both dry and wet form to form granules.
Example: Gelatin, glucose, Lactose, cellulose derivatives-Methyl cellulose, Ethyl cellulose,
-Lubricants: Used to reduce the friction between die wall and tablet, prevent adhesion of tablet
to dies and punches. Helps in easy ejection of tablets from die cavity.
Classified in to 2 types:
Insoluble- Stearic acid, Magnesium stearate, Calcium stearate, Talc, Paraffin.
Soluble- Sodium lauryl sulphate, Sodium benzoate,
-Glidants: Helps in free flowing of granules from hopper to die cavity. Minimize friction
between particles.
Example: Colloidal Silicon dioxide (Aerosil), Cornstarch, Talc etc.
-Anti-adherents: These are added to prevent adhesion of tablet material to punches and dies.
Example: Talc
-Anti-adherent: Prevent sticking of tablet to dies and punches.
-Super disintegrants: When they come in contact with water in oral cavity/GIT break down in
to small particles.
Example: Croscarmellose sodium (Ac-di-sol),Crospovidone (Polyplasdone), and Sodium starch
glycollate, Starch etc.
Role of Super disintegrants in the manufacturing of tablets
Disintegrating agents are substances included in tablet formulations and in some hard shell
capsule formulations to promote moisture penetration and dispersion of the matrix of the dosage
form in dissolution fluids. An oral solid dosage form should ideally disperse into the primary
particles from which it was prepared. Although various compounds have been proposed and
evaluated as disintegrants, relatively few are in common usage today. Traditionally, starch has
been the disintegrant of choice in tablet formulations, and it is still widely used. For instance,
starch generally has to be present at levels greater than 5% to adversely affect compactibility,
especially in direct compression. Moreover, intra granular starch in wet granulations is not as
effective as dry starch
Characteristics of disintegrant
The ideal disintegrant should have the following characteristics:
• Poor solubility
• Poor gel formation
• Good hydration capacity
• Good compressibility and flow properties
• No tendency to form complexes with the drugs
Factors affecting action of disintegrants
• Percentage of disintegrants present in the tablets.
• Types of substances present in the tablets.
• Combination of disintegrants.
• Presence of surfactants.
• Hardness of the tablets.
• Nature of Drug substances.
• Mixing and Screening.

STABILITY OF A PHARMACEUTICAL PRODUCT

Stability of a pharmaceutical product means how long it can maintain its original form without
any visible changes under the influence various environmental factors like temperature,
humidity, light.
The physical, chemical and microbial properties of a pharmaceutical product may change under
extreme storage conditions.
1. Shelf-life determination: The quality of a pharmaceutical product varies with time under
temperature, humidity and light intensity. Stability testing studies; how long a
pharmaceutical product can be stored at normal and accelerated conditions without any
degradation. This study helps to determine the shelf-life of that product. As per the report
of the study, the expiry date of the product is fixed.
2. Storage condition recommendation: Different products require different storage
conditions. Instability lab, storage conditions and changes in the substances are recorded.
As per the stability study, storage condition is recommended for a particular product.
 3. Elimination of impurities: Instability testing, each ingredient has been analysed under
various environmental factors. So, it becomes easy to eliminate impurities.
4. Product Development: Stability testing is a reliable way to study the effectiveness of a
new product. This testing helps to assess physical, chemical, therapeutic stability of a
product.
5. Ensures Quality: Quality assurance is an integral part of the pharmaceutical industry.
The product is kept under the influence of high stresses and rate of decomposition is
observed. Stability testing assures the purity of ingredients and the quality of the final
product. The stability report ensures that the pharmaceutical product is fit for human
consumption. This gives the companies confidence to launch new product in the market.
The chances of product recall may decrease.
6. Packaging material selection: During stability testing, the pharmaceutical product is
exposed to humidity and temperature. As per the effect of water activity and temperature
on the product, packaging materials are chosen for the product. The ideal container must
tolerate the stresses. The packaging should maintain the quality of the product during
transportation and storage.
7. Legal approval: Stability testing of the pharmaceutical product is required for legal
approval. If a product failed to meet the quality standards WHO, the product will not get
approval for commercialization

FACTORS AFFECTING DRUG STABILITY:

1. Temperature: high temperature accelerate oxidation, reduction and hydrolysis reaction which
lead to drug degradation
2. pH:
• Acidic and alkaline pH influence the rate of decomposition of most drugs.
• Many drugs are stable between pH 4 and 8.
• Weekly acidic and basic drugs show good solubility when they are ionized and they also
decompose faster when they are ionized.
• So if the pH of a drug solution has to be adjusted to improve solubility and the resultant
pH leads to instability then a way out of this tricky problem is to introduce a watermiscible
solvent into the product. It will increase stability by:
- suppressing ionization
- reducing the extreme pH required to achieve solubility
- enhancing solubility and
- reducing the water activity by reducing the polarity of the solvent. For example, 20%
propylene glycol is placed in chlordiazepoxide injection for this purpose.
• Reactions catalyzed by pH are monitored by measuring degradation rates against pH,
keeping temperature, ionic strength and solvent concentration constant. Some buffers such
as acetate, citrate, lactate, phosphate and ascorbate buffers are utilized to prevent drastic
change in pH.
• Sometimes pH can have a very serious effect on decomposition. As little as 1 pH unit
change in pH can cause a change of ten fold in rate constant. So when we are formulating a
drug into a solution we should carefully prepare a pH – decomposition profile and then
formulate the solution at a pH which is acceptable physiologically and stability-wise also.

3. Moisture:
a. Water catalyses chemical reactions as oxidation, hydrolysis and reduction reaction
b. Water promotes microbial growth

4. Light: affects drug stability through its energy or thermal effect which lead to oxidation

5. Pharmaceutical dosage forms: solid dosage forms are more stable than liquid dosage forms
for presence of water.

6. Concentration: rate of drug degradation is constant for the solutions of the same drug with
different concentration. So, ratio of degraded part to total amount of drug in diluted solution is
bigger than of concentrated solution.
Stock solutions: are concentrated solutions which diluted by using (i.e. syrup 85%) at high
concentration the stability is high

7. Drug incompatibility: reactions between components of pharmaceutical dosage forms it self or

Between these components and cover of the container .

8. Oxygen: exposure of drug formulations to oxygen affects their stability

THREE STABILITIES OF DRUG MUST BE CONSIDERED

1. Physical stability
2. Chemical stability
3. Microbiologicall stability

A. PHYSICAL STABILITY:
Physical instabilities possibilities are:
1. Crystal formation in pharmaceutical preparations:
Causes:
a. Polymorphism phenomena: i.e. Chloramphenicol (change of amorphous to crystalline
form.
b. Saturated solution: by different temperature precipitation of solute may occur.
c. In suspension: when very fine powder is used a part of suspending agent will dissolve
then precipitate as crystal.
2. Loss of volatile substances from pharmaceutical dosage forms:
Examples:
a. Aromatic waters
b. Elixirs
c. Spirits
d. Some types of tablets which contain aromatic water (Nitroglycerin tablets)
3. Loss of water:
This can be seen in the following dosage forms:
a. Saturated solution: by loss of water they become supersaturated and precipitate as
crystals is formed
b. Emulsions: Loss of water lead to separation of the two phases and change to other type
c. Creams: especially oil/water, they become dry by loss of water
d. Pastes
 e. Ointments: especially aqueous base ointments
Humectants is added to the previous dosage forms which defined as hydrophilic
substances added
to aqueous phase to absorb water from atmosphere and prevent its loss from the dosage
forms.
Examples: Glycerin
4. Absorption of water:
This phenomenon can be seen in the following pharmaceutical forms:
a. Powders: Liquification and degradation may occur as a result of absorption of water
b. Suppositories which base made from hydrophilic substances as Glycerin, Gelatin, poly
ethylene glycol.
The consistency of these forms becomes jelly-like appearence
5. Change in crystalline form:
• Example: Cocoa butter which is capable of existing in four polymorphic forms.

B. Chemical stability:
Reaction between two or more substances which lead to change in chemical properties of
pharmaceutical dosage form
Chemical Incompatibilities is usually a result of chemical interaction taking place among the
ingredients of a prescription.
•Such interactions may take place immediately upon compounding when these are termed as
immediate in compatibilities and are evident as effervescence, precipitation or colour change.
•More often the interaction are not evident immediately on compounding but take place over a
period of time. Such interaction are termed delayed incompatibilities.

Provided the product is harmless the interaction falls into:

a. Tolerated-: the reaction is minimized by applying some suitable order of mixing or
mixing the solution in dilute form but no alteration is made in the active ingredients of
the preparation.
b. Adjusted-: the reaction is prevented by addition or substitution of one of the reacting
substances with another of equal therapeutic value but does not affect the medicinal of
the preparation (substitution of caffeine citrate with caffeine in sodiun salicylate and
caffeine citrate mixture)

1. Oxidation
Oxidation is defined as loss of electrons or gain of oxygen
Auto-oxidation: It is a reaction with oxygen of air which occur spontaneously without other
factors.
Pre-oxidants: are substances catalyze oxidation process i.e. metals, some impurities.
Certain prescription mixtures may oxidise on exposure to air, heat, light or due to change in PH
or reaction with trace metal ions

2. Hydrolysis
A chemical reaction in which water is used to break down a compound; this is achieved by
breaking a covalent bond in the compound by inserting a water molecule across the bond
Types of hydrolysis
 1. Ionic hydrolysis:
In which the compound is broken into ions by water.
2. Molecular hydrolysis:
In which the molecule it self is broken down.
Ex.: Acetylsalicylic acid Salicylic acid + Acetic acid

Chemical groups which undergo hydrolysis:

1. Esters: R-C-OR
Ex: Benzocaine, Procaine
2. Amides: R-C-NH-R
Ex: Chloramphenicol, Sulfonamide, Procainamide
3. Nitrites: (NO3, N2O, NO2)

3. Acid – Base Reaction

•Precipitation, gas formation, colour formation or change
• a. Precipitation
•Most medicaments in use are often salts of weak acids or bases.
• These salts have a very good water solubility whereas their corresponding unionised acids or
bases are practically insoluble in water.
• if a solution of a salt of a weakly acidic drug is acidified,, the free acid may be precipitated.
•Similarly, precipitation of free base may occur if a solution of a salt of weakly basic drug is
made alkaline.
4. Polymerization
• In polymerization, small repeating units called monomers are bonded to form a long chain
polymer.
• Ex:
Formaldehyde Paraformaldehyde (Polymer: white precipitate )
- To avoid this formaldehyde must be stored in suitable temperature and addition of methanol
15%.
Ampicillin in high temperature forms polymers which cause allergy.

5.Isomerization
• It means conversion of drug to its isomer
• Isomers have:
- Identical molecular formulas.
- A different arrangement of atoms

Types of isomerization

A- Optical isomerization:
Conversion of optical active drug into less active
- Ex:
a. L-Adrenaline is converted to d-adrenaline by change of pH or temperature
b. L-adrenaline is more therapeutically active than d-adrenaline, a although they have the
same physical properties but different arrangement of atoms.
c. This is not general for other drugs: d-tubocurarine is more active than l-type
B. Geometric isomerization:
- One type of isomers
- Expressed by cis or trans
Cis: means the groups A in the same direction: C C
Trans: means the group A in opposite direction: C C
-Cis is more therapeutically active than trans (ex.: Vitamin A)
6. Decarboxylation:
Ex.: NaHCO3>>> Na + CO2
All drugs contain bicarbonate are not sterilized in high temperature
7. CO2 – absorption:
- When some pharmaceutical dosage forms contain CO2, precipitate is formed:
Ex: Ca(OH)2 + CO2 >>>CaCO3
8. Combination:
- Take place when the pharmaceutical dosage form contain substances with different charges
Ex.: Surfactants with positive and negative charges
9. Formation of insoluble complexes:
Ex.: Tetracycline + heavy metals

C. Microbiological stability:
1. Contamination from microorganisms is a big problem for all formulations containing moisture
but it can be a bother in solid dosage forms also if some natural polymers are used because many
natural polymers are fertile sources of microorganisms.
2. In the type of hygienic manufacture carried out today where ―Quality Assurance‖ is a
prerequisite as per the GMP procedures, there are definite procedures to prevent microbial
contamination in all formulations.

Sources of Microbial Contamination:

1. Water
2. Air
3. Raw materials, containers and closures
4. Personnel
5. Instruments and apparatus