Pharmaceutical Biotechnology

BY Selam Melese

Wolkite University
2021
 Introduction

Objectives of the lesson

At the end of this lesson students will be able to:

• Recognize histrocial overview of bipharmacuticals.

• Explain Pharmaceutical biotechnology and

pharmaceutical products.

• Identify sources of pharmaceutical products.

 Essentially, Biotechnology refers to the use of biological
systems (e.g. cells or tissues) or biological molecules (e.g.
enzymes or antibodies) for/in the manufacture of commercial
products.

 Terms such as ‘biologic’, ‘biopharmaceutical’ and ‘products

of pharmaceutical biotechnology’ or ‘biotechnology
medicines’ have now become an accepted part of the
pharmaceutical literature
 Biotechnology now encompasses the use of;
 tissue culture,

 living cells or

 cell enzymes to make a defined product

 The classic example of biotechnological drugs was proteins

obtained from recombinant DNA technology.

 Recombinant DNA (rDNA) and monoclonal antibody (MAb) are

providing exciting opportunities for
 new pharmaceuticals development as well as

 new approaches to drug delivery.

 The term ‘biopharmaceutical’ was first used in the 1980s and
came to describe a class of therapeutic proteins produced by
modern biotechnological techniques, specifically via; genetic
engineering or, in the case of monoclonal antibodies, by
hybridoma technology.
 Biopharmaceuticals may be produced from:
 microbial cells (e.g., recombinant E. coli or yeast cultures),
 mammalian cell lines ( cell culture),

 plant cell cultures (plant tissue culture) and

 moss plants in bioreactors of various configurations, including photo-
bioreactors.
Examples of biopharmaceuticals include
 vaccines
 blood or blood components
 allergenics
 somatic cells
 gene therapies
 tissues
 recombinant therapeutic protein and
 living cells.
Techniques used to produce biopharmaceuticals

 Recombinant DNA (rDNA) technology

 Monoclonal antibodies

 Cell therapy

 Gene therapy

 Polymerase chain Reaction

7
 Sources of Biopharmaceuticals

• The bulk of biopharmaceuticals currently on the market are produced

by genetic engineering using various recombinant expression
systems.

• Most of the recombinant proteins that have gained marketing

approval to date are produced either in recombinant Eschericia coli
or in recombinant mammalian cell lines

• Such recombinant systems are invariably constructed by the

introduction of a gene or cDNA coding for the protein of interest into
a well characterized strain of the chosen producer cell.
 E. coli as a source of recombinant, therapeutic proteins

• The expression of recombinant proteins in cells in which they

do not naturally occur is termed ‘heterologous protein
production’.

• By far the most common microbial species used to produce

heterologous proteins of therapeutic interest is Eschericia coli.

• The first biopharmaceutical produced by genetic engineering

to gain marketing approval (in 1982) was recombinant human
insulin (trade name Humulin), produced in E. coli.
 Advantages of using E. coli

 E. coli has long served as the model system for studies relating
to prokaryotic genetics. Its molecular biology is thus well
characterized.

 High levels of expression of heterologous proteins can be

achieved in recombinant E. coli.

 E. coli cells grow rapidly on relatively simple and inexpensive

media, and the appropriate fermentation technology is well
established.
 Disadvantages of using E.coli

 Heterologous proteins accumulate intracellularly

 Inability to undertake post-translational modifications

(particularly glycosylation) of proteins;

 The presence of lipopolysaccharide (LPS) on its

surface.
 Animal cell culture system

Objectives of the lesson:

• At the end of this lesson students will be able to:

• Explain animal cell culture system and insect cell

based system.

• Describe transgenic animals and transgenic plant.

 Animal cell culture system

• Technical advances facilitating genetic manipulation of animal

cells now allow routine production of therapeutic proteins in
such systems.

• The major advantage of these systems is their ability to carry

out post-translational modification of the protein product.

• Cell culture- removed cells from tissue or whole animals, then

continue to grow if supplied with nutrient and growth factors.
 Con…

 Animal cell culture was successfully established in 1907.

 First, cell culture development were for antibiotics

development.

 Important to produce therapeutic protein and tissue

plasminogen activator.
 Advantages and Disadvantages
 Advantages
– Consistency and reproducibility of results
– Toxicological testing procedures are less expensive
– Ease of sterilization
– Reduced change of contamination

 Disadvantages
– Cell characteristic can change after a period of continuous
growth
– Cells may need to adapt with the environment
 Insect cell based system

• The approach generally entails the infection of cultured insect

cells with an engineered baculovirus (a viral family that
naturally infects insects) carrying the gene coding for the
desired protein, placed under the influence of a powerful viral
promoter.

• Amongst the systems most commonly employed are:

 Con…

• the silkworm virus Bombyx mori nuclear polyhedrovirus

(BmNPV), in conjunction with cultured silkworm cells (i.e.
Bombyx mori cells) or;

• the virus Autographa californica nuclear polyhedrovirus

(AcNPV), in conjunction with cultured armyworm cells
(Spodoptera frugiperda cells).

 Baculovirus are noninfectious to vertebrates and their

promoters are inactive in mammalian cells.
 Advantages of working with Baclo system

 High expression levels using the polyhedrin or p10 promoter

 Supports post-translational modifications

 High-level intracellular recombinant protein

 Insect cells can be cultured more rapidly and using less

expensive media.

 Human pathogens (e.g. HIV) do not generally infect insect cell

lines.
 Disadvantages
 Targeted extracellular recombinant production generally
results in low-level extracellular accumulation of the desired
protein (often in the milligram per litre range).

 Post-translational modifications, in particular glycosylation

patterns, can be incomplete and/or can differ very significantly
from patterns associated with native human glycoproteins.
 Transgenic Animals

• A transgenic animal is an animal that possess the DNA of

different organism that is introduced artificially.

• The generation of transgenic animals is most often undertaken

by directly microinjecting exogenous DNA into an egg cell.

• A transgenic animal harbouring a gene coding for a

pharmaceutically useful protein could become a live
bioreactor, producing the protein of interest on an ongoing
basis.
 Con…

• In order to render such a system practically useful, the

recombinant protein must be easily removable from the
animal.

• A simple way of achieving this is to target protein production

to the mammary gland.

• Mammary-specific expression can be achieved by fusing the

gene of interest with the promoter-containing regulatory
sequence of a gene coding for a milk-specific protein.
 Con…
Regulatory sequences of

 the whey acid protein (WAP)

 β-casein and

 α- and β-lactoglobulin genes have all been used to date to

promote production of various pharmaceutical proteins in the
milk of transgenic animals.

• One of the earliest successes in this regard entailed the

production of human tissue plasminogen activator (tPA) in the
milk of transgenic mice.
 Con…
• More practical from a production point of view was the
subsequent production of tPA in the milk of transgenic goats,
again using the murine WAP gene regulatory sequence to drive
expression.

• General characteristics may be cited which render attractive

the production of pharmaceutical proteins in the milk of
transgenic farm animals. These include:
Con…
 Ease of harvesting of crude product, which simply requires the
animal to be milked.

 Low capital investments (i.e. relatively low-cost animals replace

high-cost traditional fermentation equipment) and low running
costs.

 High expression levels of proteins are potentially attained.

 Ongoing supply of product is guaranteed (by breeding).

 Milk is biochemically well characterized, and the

physicochemical properties of the major native milk proteins of

various species are well known.

The production and purification of tPA from
the milk of transgenic goats
 Transgenic Plants

• The introduction of foreign genes into plant species can be

undertaken by a number of means of which Agrobacterium-
based vector-mediated gene transfer is most commonly
employed.
• Agrobacterium tumefaciens and A. rhizogenes are soil-based
plant pathogens.
• Upon infection, a portion of Agrobacterium Ti plasmid is
translocated to the plant cell and is integrated into the plant
cell genome.
 Con…

• Plants are regarded as potentially attractive recombinant protein

producers for a number of reasons, including:

 cost of plant cultivation is low

 harvest equipment/methodologies are inexpensive and well

established

 ease of scale-up

 proteins expressed in seeds are generally stable in the seed for

prolonged periods of time (often years)

 plant-based systems are free of human pathogens (e.g. HIV).

 Con…

• However, a number of potential disadvantages are also associated

with the use of plant-based expression systems, including:

 variable/low expression levels sometimes achieved;

 potential occurrence of post-translational gene silencing (a

sequence-specific mRNA degradation mechanism);

 glycosylation patterns achieved differ significantly from native

human protein glycosylation patterns;

 seasonal/geographical nature of plant growth.

 Production process

Objectives of the lesson:

• At the end of this lesson students will be able to:

• Recognize upstream production process

• Describe microbial cell fermentation and

mammalian cell culture process.

• Explain downstream processing

 Production Process

• Production can be divided into ‘upstream’ and ‘downstream’

processing.

• Upstream processing- the initial fermentation process, which

results in the initial generation of product.

• Downstream processing- the actual purification of the protein

product and generation of finished product format, followed by
sealing of the final product containers.

• Subsequent labelling and packaging steps represent the very

final steps of finished product manufacture.
Overview of Pharmaceutical products Production
Process
 Cell Banking System

• Recombinant biopharmaceutical production cell lines are most

often initially constructed by the introduction into these cells
of a plasmid housing a nucleotide sequence coding for the
protein of interest.

• The cell bank’s construction design is normally two-tiered,

consisting of a ‘master cell bank’ and a ‘working cell bank’.

• The master cell bank is constructed first, directly from a

culture of the newly constructed production cell line
A master cell bank :
 Constructed first from a culture of the newly constructed production cell
line
 Several hundred individually stored ampoules
 Used to generate a working cell bank
A working cell bank :
 A working cell bank is constructed from a single ampoule from a master
cell bank

 Directly used for a single production run

 Each ampoule is thawed and used for a new batch

 When all the vials of first working cell bank are exhausted, a second vial of
the master cell bank is used to generate a second working cell bank
 Con…

Preparation of cell banking system:

 Initial cultivation of production cell line.

 The resulting cell line is aliquoted into small volume in

ampoules, immersed in liquid nitrogen.

 The content of all the ampoules is identical, and the cells

are effectively preserved for indefinite periods.
The master cell bank/working cell bank system.
 Upstream Processing
Initial generation of a target product :
 A single ampoule of the working cell bank
 Inoculation for seed culture in a small volume of sterile growth
medium : lab-scale starter culture of the producer cell line
 Starter culture is used to inoculate several liters/ tens of liters of
growth medium in a small bioreactor
 Production-scale starter culture is used to inoculate the
production-scale bioreactor of several thousands/tens of
thousands liters
 Harvest of the crude product
 Downstream processing
Choice of media:

 Exact nutrient requirements of producer cell line to

maximize cell growth and product

 Economics (total media cost)

 Extracellular or intracellular nature of product

 In the case of extracellular product, the less complex media

composition, the better for subsequent product purification
process.
Optimal culture conditions :

 temp, pH, Dissolved oxygen level etc.

Cell culture process :

 Industrial scale microbial culture process :
 Bioreactor configuration,
 oxygen transfer,
 mode of operation (batch, fed-batch),
 heat removal (microbial metabolism and mechanical friction),
 mixing/agitation etc..
 Mammalian cell culture system

 More technically complex and expensive

 Long culture time due to slow growth rate

 Only used for production of therapeutic proteins requiring

extensive post-translational modifications like
glycosylation

 Outline of the upstream processing stages involved in the

production of a single batch of product :

- Gradual scale-up : 100 mL  5 L  30 L  1,000L

 Downstream Process

• Normally undertaken under clean room conditions, with the final

steps (e.g. sterile filtration and aseptic filling into final product
containers) being undertaken under Grade A laminar flow
conditions.

• In general, animal cell culture-derived biopharmaceutical products

are secreted into the media (i.e. are produced as extracellular
proteins), whereas the product accumulates intracellularly in many
recombinant prokaryotic producer cell types.
 Con….

• In the case of intracellular proteins, fermentation is followed

by harvesting of the cells.

• This is normally achieved by centrifugation or sometimes

filtration.

• Recovery of cells is followed by their disruption (using

homogenizer) in order to release their intracellular contents,
including the protein of interest.
 Con…

• A suspension of cells is forced under high pressure through an

orifice of narrow internal diameter  High shear force  High
pressure drop at the outlet to atmospheric pressure  Rupture
of most microbial cells.
• Centrifugation to remove cell debris : generation of crude
(unpurified protein product ) protein solution.
• concentration of the crude protein may be achieved by inducing
product precipitation using, for example, salts such as
ammonium sulphate or solvents such as ethanol.
• However, ultrafiltration is the more usual method employed
 Con…

Ultrafiltration is a popular method of concentration because:

 high product recovery rates may be attained (typically of the

order of 99%);

 processing times are rapid;

 process-scale ultrafiltration equipment is readily available, and

running costs are relatively modest.

• After concentration, high-resolution chromatographic

purification is usually undertaken.
 Final product Formulation

• This generally involves:

 addition of various excipients

 filtration of the final product through a 0.22 mm absolute filter in order

to generate sterile product, followed by its aseptic filling into final
product containers;

 freeze-drying (lyophilization) if the product is to be marketed in a

powdered format.
 Final product fill

 Final bulk product is then passed through a (sterilizing) 0.22 µm filter.

 The sterile product is housed (temporarily) in a sterile-product holding
tank, from where it is aseptically filled into pre-sterile final product
containers (usually glass vials).
 The filling process normally employs highly automated liquid filling
systems.
 All items of equipment, pipe work, etc. with which the sterilized
product comes into direct contact must obviously themselves be sterile.
 Most such equipment items may be sterilized by autoclaving, and be
aseptically assembled prior to the filling operation (which is undertaken
under Grade A laminar flow conditions).
 Labelling and packing

 After the product has been filled (and sealed) in its final
product container carry out tests to ensure conformance to
final product specification.
 The most important specifications will relate to product
 potency,
 sterility and
 final volume fill,
 as well as the absence of endotoxin or other potentially toxic
substances.
 Information presented on a label should normally include:

 name and strength/potency of the product;

 specific batch number of the product;

 date of manufacture and expiry date;

 storage conditions required.

 Product Analysis

• All pharmaceutical finished products undergo rigorous QC

testing, in order to confirm their conformance to pre-
determined specifications.

• Potency testing is of obvious importance, ensuring that the

drug will be efficacious when administered to the patient.

• A prominent aspect of safety testing entails analysis of product

for the presence of various potential contaminants.
Protein-based contaminants

 Most of the chromatographic steps undertaken during

downstream processing are specifically included to separate
the protein of interest from additional contaminant proteins.

 Proteins may be introduced during upstream or downstream

processing.

 For example, upstream: animal cell culture media are

typically supplemented with;
 bovine serum

 defined cocktail of various regulatory proteins required to maintain and

stimulate growth of these cells.
 Downstream;

 intracellular microbial proteins

- requires the addition of endonucleases to the cell homogenate to
degrade the large quantity of DNA liberated upon cellular disruption.

 The clinical significance of protein-based impurities relates to

(a) their potential biological activities and

(b) their antigenicity.

 Some contaminants may display no undesirable biological activity.

 Others may exhibit activities deleterious to either the product itself

(e.g. proteases that could modify/degrade the product) or the recipient
patient (e.g. the presence of contaminating toxins)
 Detection of protein-based product impurities

 Polyacrylamide gel electrophoresis (SDS-PAGE)

 Commonly used analytical technique in the assessment of final
product purity.
 It provides high-resolution separation of polypeptides on the
basis of their molecular mass.
 Bands containing as little as 100 ng of protein can be visualized
by staining the gel with dyes such as Coomassie blue.
 Capillary electrophoresis

 High-performance liquid chromatography

 Mass spectrometry
Endotoxin and other pyrogenic contaminants

 Pyrogens: are substances that, when they enter the blood

stream, influence hypothalamic regulation of ;
 body temperature, usually resulting in fever and

 in severe cases results in patient death.

 Pyrogens represent a diverse group of substances:-

 including various chemicals,

 particulate matter and endotoxin (LPS), a molecule derived from

the outer membrane of Gram-negative bacteria.
 In many instances the influence of pyrogens on body temperature is
indirect.
 For example, entry of endotoxin into the bloodstream stimulates the production
of IL-1 by macrophages.

 It is the IL-1 that directly initiates the fever response (hence its alternative name,
‘endogenous pyrogen’).

 Effective implementation of GMP (good manufacturing practice)

minimizes the likelihood of product contamination by pyrogens.

 For example, GMP dictates that chemical reagents used in the

manufacture of process buffers be extremely pure.
 Furthermore, GMP encourages filtration of virtually all
parenteral products through;
 a 0.45 or 0.22 μm filter at points during processing and prior to
filling in final product containers (even if the product can
subsequently be sterilized by autoclaving).

 As an additional safeguard, the final product will usually be

subject to a particulate matter test by quality control (QC)
before final product release.
Contamination of the final product with endotoxin is more difficult
to control because:-
1. Many recombinant biopharmaceuticals are produced in
Gram-negative bacterial systems; thus, the product source is
also a source of endotoxin..
2. Most biopharmaceutical preparations will be contaminated
with low levels of Gram-negative bacteria at some stage of
manufacture.
 This is one of many reasons why GMP dictates that the level of
bioburden in the product stream should be minimized at all stages
of manufacture.
3. The heat stability exhibited by endotoxin means that
autoclaving of process equipment will not destroy
endotoxin present on such equipment.

4. Adverse medical reactions caused by endotoxin are

witnessed in humans at dosage rates as low as 0.5 ng per
kilogram body weight.
Microbial and viral contaminants

 Finished-product biopharmaceuticals, along with other

pharmaceuticals intended for parenteral administration, must
be sterile (the one exception being live bacterial vaccines).
The presence of microorganisms in the final product
is unacceptable for a number of reasons:
1. Parenteral administration of contaminated product would
likely lead to the establishment of a severe infection in the
recipient patient.
2. Microorganisms may be capable of metabolizing the
product itself, thus reducing its potency.

3. Microbial-derived substances secreted into the product

could adversely affect the recipient’s health.
 Examples include endotoxin secreted from Gram-negative
bacteria.
 Biopharmaceutical products are also subjected to screening for
the presence of viral particles prior to final product release.

 Although viruses could be introduced,

 for example, via infected personnel during downstream processing,

 proper implementation of GMP minimizes such risk.

 Any viral particles found in the finished product are most

likely derived from raw material sources.
Removal of Viruses:

1. Gel-filtration chromatography, for example, effectively separates

viral particles from most proteins on the basis of differences in
size.
2. Filtration through a 0.22 μm filter effectively removes microbial
agents from the product stream, but fails to remove most viral
types.
– Repeat filtration through a 0.1 μm filter is more effective in this regard.

3. Alternatively, incorporation of an ultrafiltration step (preferably at

the terminal stages of downstream processing) also proves
effective.
4. Heating the product to between 40 and 60°C for several hours
inactivates a broad range of viruses.

– Many biopharmaceuticals can be heated to such

temperatures without being denatured themselves.

– Such an approach has been used extensively to inactivate

blood-borne viruses in blood products.

5. Exposure of product to controlled levels of UV radiation can

also be quite effective, while having no adverse effect on the
product itself.
Viral assays:

 Viral assays currently available will detect only a

specific virus, or at best a family of closely related
viruses.
 Current viral assays fall into one of three categories:
1. immunoassays;
2. assays based on viral DNA probes;
3. bioassays.
1. Immunoassays capable of detecting a wide range of viruses are
available commercially.

– The sensitivity, ease, speed and relative inexpensiveness of

these assays render them particularly attractive.

2. An alternative assay format entails the use of virus-specific

DNA probes.

– These can be used to screen the biopharmaceutical product

for the presence of viral DNA.
3. Viral bioassays: different formats have also been
developed.
 One format entails incubation of the final product with cell
lines sensitive to a range of viruses.
 The cells are subsequently monitored for cytopathic effects
or other obvious signs of viral infection.
 These bioassays entail administration of the product to a test
animal.
 Any viral agents present will elicit production of antiviral
antibodies in that animal.
Miscellaneous contaminants

 Could include buffer components, precipitants (ethanol or other

solvents, salts, etc.), proteolytic inhibitors, glycerol, anti-foam
agents, etc.
 In addition to these, other contaminants may enter the product
during downstream processing in a less controlled way.
 Examples could include metal ions leached from product-
holding tanks/pipework, or breakdown products leaking from
chromatographic media.
 For this reason, high-quality glass vials are often used.
 In some instances it may be necessary to demonstrate that all traces
of specific contaminants have been removed prior to final product
filling.

 This would be true, for example, of many proteolytic inhibitors

added during the initial stages of downstream processing to prevent
proteolysis by endogenous proteases.

 Some such inhibitors may be inherently toxic, and many could

(inappropriately) inhibit endogenous proteases of the recipient
patient.

 Various chemical-coupling methods may be used to attach affinity

ligands to the chromatographic support material.
 Biopharmaceutical Products

Cytokines and Growth Factors

Objectives of the lesson

At the end of this lesson students will be able

to:

• Describe the use of cytokines, interferons and

growth factors as biopharmacuticals.
 Cytokines as a Biopharmaceuticals

• Cytokines / immunocytokines (Greek , cyto =‘cell’ & kinos

=‘movement’) are low molecular weight regulatory proteins or
glycoproteins secreted by white blood cells and various other
cells in the body in response to a number of stimuli.

• They act as chemical communicators between various cells,

inducing their effect by binding to specific cell surface
receptors, thereby triggering various intracellular signal
transduction events.
 Con…

 Cytokines, in many ways, constitute most important group of

biopharmaceutical substances.

 As coordinators of the immune and inflammatory response,

manipulation of cytokine activity can have a major influence on
the body’s response to a variety of medical conditions.
 Administration of certain cytokines can enhance the immune
response against a wide range of infectious agents and cancer cells.
 EPO has proven effective in stimulating red blood cell production
in anaemic persons.
 Growth factors have obvious potential in promoting wound healing.
 Con…

• A better understanding of the molecular principles underlining

cytokine biology may also provide new knowledge-based
strategies aimed at defeating certain viral pathogens.

• These pathogens appear to successfully establish an infection,

at least in part, by producing specific proteins which thwart the
normal cytokine-based immunological response.
 The cowpox virus, for example, produces an IL-1-binding protein,

 the shope fibroma virus produces a TNF-binding protein.

 The Epstein–Barr virus, on the other hand, produces a protein

homologous to IL-10.
 Con…

• A variety of pro-inflammatory cytokines, including IL-6 and

IL-8 as well as TNF, have been implicated in the pathogenesis
of both septic shock and rheumatoid arthritis.
• Inhibiting the biological activity of such cytokines may
provide effective therapies for such conditions.
• This may be achieved by administration of monoclonal
antibodies raised against the target cytokine, or administration
of soluble forms of its receptor which will compete with cell
surface receptors for cytokine binding.
 Nomenclatures
• Interleukins - that act as mediators between leukocytes. The vast
majority of these are produced by T-helper cells.

• Lymphokines - produced by lymphocytes.

• Monokines - produced exclusively by monocytes.

• Interferons - involved in antiviral responses.

• Colony Stimulating Factors - support the growth of cells blood cell .

• Chemokines - mediate chemoattraction (chemotaxis) between cells.

 The Interferons

• Interferons play an important role in the first line of defense

against viral infections.

• They are part of the non specific immune system and are
induced at an early stage in viral infection before the specific
immune system has had time to respond.

• In 1957, researchers observed that susceptible animal cells, if

they were exposed to a colonizing virus, immediately became
resistant to attack by other viruses.
 Con…
• This resistance was induced by a substance secreted by virally
infected cells which was named interferon.
• The anti-viral and anti-proliferative activity of IFNs, as well as
their ability to modulate the immune and inflammatory response,
renders obvious their potential medical application.
• Interferons are made by cells in response to an appropriate
stimulus, and are released into the surrounding medium; they
then bind to receptors on target cells and induce transcription of
approximately 20-30 genes in the target cells, and this results in
an anti-viral state in the target cells.
 Con…

• Interferons produce clinical benefits for disease states such as;

 hepatitis,

 various cancers,

 multiple sclerosis, and

 many other diseases.

• Structurally, they are part of the helical cytokine family which

are characterized by an amino acid chain that is 145-166 amino
acids long.
 Different Interferon Drugs
Interferons are broken down into recombinant versions of a specific
interferon subtype and purified blends of natural human interferon.

 Many of these are in clinical use and are given intramuscularly or

subcutaneously.

 Recombinant forms of alpha interferon include:

Alpha-2a drug name Roferon

Alpha-2b drug name Intron A
Alpha-n1 drug name Wellferon
Alpha-n3 drug name AlferonN
Alpha-con1 drug name Infergen
 Recombinant forms of beta interferon include:
 Beta-1a drug name Avonex
 Beta-1b drug name Betaseron
Recombinant forms of gamma interferon include:
Gamma-1b drug name Ac immune
 Alpha Interferon-2a (Roferon A)

 Protein chain that is 165 amino acids long

 Roferon-A is used to treat people with

 hepatitis C,

 hairy cell leukemia and

 Philadelphia chromosome positive chronic myelogenous leukemia (CML).

 Roferon-A can cause some serious side effects that may cause death in rare
cases

 Short half life, short terminal elimination of half life, a large volume of
distribution, and a larger reduction in renal clearance.
 Production of Roferon A

 Roferon-A (Interferon alfa-2a, recombinant) is a sterile protein

manufactured by recombinant DNA technology

 Genetically engineered Escherichia coli containing DNA that codes

for the human protein.

 Fermentation is carried out in a defined nutrient medium containing

the antibiotic tetracycline hydrochloride, 5 mg/L.

 However, the presence of the antibiotic is not detectable in the final

product. Roferon-A (interferon alfa-2a, recombinant) is supplied in
prefilled syringes.

 Each glass syringe barrel contains 0.5 mL of product. In addition,

there is a needle, which is ½ inch in length.
 Interferon Beta-2a (Avonex)
 FDA approval on May 17 1996 for Relapsing
Remitting multiple sclerosis (MS)

 Clinical trials showed that it slowed MS

progression and had an extra benefit of slowing or
preventing the development of MS-related brain
atropy.

Structurally IFNβ-2a is a 166 amino acid

glycoprotein.

 Produced by recombinant DNA technology

using genetically engineered mammalian cells
which the human beta gene has been used.
 Additional Interferons

 In the last few years additional members of the interferon family has
been discovered.
 Amino acid sequence analysis of a protein called trophoblastin
(which is found in many ruminants) revealed it was closely related
to IFN-α.
 This result was surprising because, in sheep and several other
ruminants, the primary function (and until recently the only known
function) of trophoblastin is to sustain the corpus luteum during the
early stages of pregnancy.
 Interleukins

• The interleukins (ILs) represent another large family of

cytokines, with at least 25 different constituent members (IL-1
to IL-25) having been characterized thus far.

• Most of these polypeptide regulatory factors are glycosylated

(a notable exception being IL-1) and display a molecular mass
in the range 15–30 kDa.

• A few interleukins display a higher molecular mass, e.g. the

heavily glycosylated, 40kDa, IL-9.
 Con…

• Nearly all ILs are soluble molecules (one form of IL-1 is cell-
associated).
• They promote their biological response by binding to specific
receptors on the surface of target cells.
• Most ILs exhibit paracrine activity (i.e. the target cells are in
the immediate vicinity of the producer cells), while some
display autocrine activity (e.g. IL-2 can stimulate the growth
and differentiation of the cells that produce it).
• Other ILs display more systematic endocrine effects (e.g. some
activities of IL-1).
 Con…

• The sum total of biological responses induced by the ILs is

large, varied and exceedingly complex.

• These cytokines regulate a variety of physiological and

pathological conditions, including:

 normal and malignant cell growth;

 all aspects of the immune response;

 regulation of inflammation.
 Con…

• The advent of recombinant DNA technology facilitates

production of these molecules in quantities sufficient to meet
actual/potential medical needs.

• The first IL to be approved for medical use was IL-2, approved

in 1992 by the FDA for the treatment of renal cell carcinoma.
Three-dimensional structure of IL-2
 Production of IL2

• IL-2, also known as T cell growth factor, represents the most studied
member of the IL family.

• IL-2 induces its characteristic biological activities by binding a specific

receptor on the surface of sensitive cells.

• Large-scale IL-2 production was made possible by rDNA technologies.

• While the IL-2 gene/cDNA has now been expressed in a wide variety
of host systems, it was initially expressed in E. coli, and most products
being clinically evaluated are obtained from that source.

• the absence of glycosylation on the recombinant product does not alter

its biological activity.
 Con…

• Proleukin is the trade name given to the recombinant IL-2

preparation manufactured by Chiron and approved for the
treatment of certain cancers.

• It is produced in engineered E. coli and differs from native

human IL-2 in that it is non-glycosylated, lacks an N-terminal
alanine residue and cysteine 125 has been replaced by a serine
residue.
 Tumor Necrosis Factors (TNF)

• The tumour necrosis factor (TNF) family of cytokines

essentially consists of two related regulatory factors: TNF-α
(cachectin) and TNF-β (lymphotoxin).
• Although both molecules bind the same receptor and induce
very similar biological activities, they display limited sequence
homology.
• The human TNF- α and - β genes are located adjacent to each
other on chromosome 6, being separated by only 1100 base
pairs.
 Con…
• The initial interest in utilizing TNF as a general anti-cancer agent has
diminished, largely due to the realization that:
 many tumours are not susceptible to destruction mediated by TNF (indeed,
some tumours produce TNF as an autocrine growth factor);

 tumour cell necrosis is not TNF’s major biological activity;

 severe side-effects usually accompany systemic administration of

therapeutically relevant doses of this cytokine.
• One such product has, however, been approved for general medical use.
• Beromun - TNF-α based product produced by recombinant means in E.
coli which is indicated for the treatment of soft tissue sarcoma in the
limbs.
 Growth Factors

 The differentiation, growth and division of eukaryotic cells is

modulated by various influences, of which growth factors are
amongst the most important for many cell types.

 A wide range of polypeptide growth factors have been

identified and more, undoubtedly, remain to be characterized.

 Factors that inhibit cell growth also exist. For example,

interferons and TNF inhibit proliferation of various cell types.
 They also have the potential to differentiate, thereby yielding
the range of cells normally found in blood.

 This process, by which a fraction of stem cells is continually

‘deciding’ to differentiate (thus continually producing new
blood cells and platelets to replace aged cells), is known as
haemopoiesis.
 Con…

Growth factors:

 are important to growing eukaryotic cells

 can cause growth or inhibition of growth in cells

 mitogenic effect - promotes cell growth

 Biopharmaceutical Products

Objectives of the lesson

At the end of this lesson students will be able

to:

• Explain the different biopharmacutical

products.
 Therapeutic hormones
 Hormones are amongst the most important group of regulatory

molecules produced by the body.

 Originally, the term hormone was defined as a substance

synthesized and released from a specific gland in the body.

 Hormones travel to the target cell via the circulatory system.

 This describes what is now termed a true endocrine hormone.

 Insuline

• Insulin is a polypeptide hormone produced by the B cells of

the pancreatic islets of Langerhans.

• It plays a central role in regulating blood glucose levels,

generally keeping it within narrow defined limits (3.5–8.0
mmol/l), irrespective of the nutritional status of the animal.

• Insulin orchestrates a suitable metabolic response to the

absorption of glucose and other nutrients in a number of ways:
 Con…

• it stimulates glucose transport (and transport of amino acids, K+

ions and other nutrients) into cells, thus reducing their blood
concentration;

• it stimulates (or helps to stimulate) intracellular biosynthetic

(anabolic) pathways, such as glycogen synthesis which helps to
convert the nutrients into a storage form in the cells;

• it inhibits (or helps to inhibit) catabolic pathways, such as

glycogenolysis;

• it stimulates protein and DNA synthesis (which underlies insulin’s

growth-promoting activity).
 Con…
• Failure of the body to synthesize sufficient insulin results in
the development of insulin dependent diabetes mellitus
(IDDM).

• This is also known as type 1 diabetes or juvenile-onset

diabetes.

• It is caused by an autoimmune-mediated destruction of the

insulin-producing pancreatic B cells.

• It is thus characterized by the absence or near-absence of

insulin in the blood, even at elevated blood glucose levels.
 Con…
• Non-insulin dependent diabetes mellitus (NIDDM) (maturity
onset or type II diabetes mellitus)- insulin is present in the
blood at normal (or even elevated) levels, but fails to promote
any of its characteristic effects.
• A number of factors can contribute to such insulin resistance,
including:
 reduced numbers of insulin receptors on sensitive cells;
 reduced receptor affinity for insulin;
 total/partial failure of insulin binding to initiate an intracellular
response.
 Production of Human Insulin

• production of genetically engineered human insulin was one of

the first breakthroughs of biotechnology in the pharmaceutical
industry.
• Insulin was first produced in Escherichia coli through
recombinant DNA technology in 1978
• Prior to the development of this technique, insulin was extracted
from the pancreas glands of cattle, pigs, and other farm animals.
• While generally efficacious in the treatment of diabetes, animal-
derived insulin is not indistinguishable from human insulin, and
may therefore produce allergic reactions.
 Con…

• Recombinant DNA technology facilitates not only production

of human insulin in microbial systems, but also facilitates
generation of insulins of modified amino acid sequences.

• The major aims of generating such engineered insulin

analogues include:
 identification of insulins with altered pharmacokinetic properties, such
as faster-acting or slower-acting insulins;

 identification of super-potent insulin forms (insulins with higher

receptor affinities).
 Con…

Production of Human Insulin:

It involves essentially the following stages:

(i) Isolation of Donor or DNA segment:

• A useful DNA segment is isolated from the donor organism.

(ii) Formation of Recombinant DNA (rDNA):

• Both the vector and donor DNA segments are cut in the presence of
restriction endonuclease. In the presence of ligase DNA segments of
both are joined to form rDNA.

(iii) Production of Multiple Copies of rDNA:

• Next step in the process is production of multiple copies of this

recombinant DNA.
 Con…

(iv) Introduction of rDNA in the recipient organism:

• This rDNA is inserted into a recipient organism.

(v) Screening of the transformed cells:

• The recipient (host) cells are screened in the presence of rDNA and
the product of donor gene

• The transformed cells are separated and multiplied, an economical

method for its mass production. The various steps and their sequence
for the production of human insulin

• In 1982 insulin (Eli Lilly’s Humulin) was the first product made
genetically engineered bacteria to be approved for use in Britain and
the U.S.A
 Glucagon

• Glucagon is a single-chain polypeptide of 29 amino acid

residues and a molecular mass of 3500 Da.
• It is synthesized by the A cells of the Islets of Langerhans, and
also by related cells found in the digestive tract.
• The major biological actions of glucagon tend to oppose those
of insulin, particularly with regard to regulation of
metabolism.
• Glucagon has an overall catabolic effect, stimulating the
breakdown of glycogen, lipid and protein.
 Con…

• A prominent metabolic effect is to increase blood glucose levels (i.e.

it is a hyperglycaemic hormone).

• The major physiological function of glucagon is to prevent

hypoglycaemia.

• Hypoglycaemia remains the most frequent complication of insulin

administration to diabetics.

• Glucagon is also used medically as a diagnostic aid during certain

radiological examinations of the stomach, small and large intestine
where decreased intestinal motility is advantageous
 Con…

‘GlucaGen’ is the trade name given to a product produced via

recombinant means , produced by Novo Nordisk using an engineered
Saccharomyces cerevisiae strain.

 Upstream processing (aerobic batch-fed fermentation) is followed

by an upward adjustment of media pH in order to dissolve
precipitated product (glucagon is insoluble in aqueous-based media
between pH 3–9.5).

 This facilitates subsequent removal of the yeast by centrifugation.

 Glucagon is then recovered and purified from the media by a series

of further precipitation.
 Human Growth Hormone

• Growth hormone is produced by the pituitary gland.

• It promotes normal body growth and lactation and influences

various aspects of cellular metabolism.

• Growth hormone stimulates overall body growth by

increasing the cellular uptake of amino acids, and protein
synthesis, and promoting the use of fat as body fuel.
 Con…

• Insufficient human growth hormone (hGH) in young children

results in retarded growth, clinically referred to as pituitary
dwarfism.
• The child usually is less than four feet in height, and has
chubby face and abundant fat around the waist.
• The successful bacterial production of hGH will enable the
production of sufficient quantities of hGH to treat all those
hypopituitary patients who could benefit from its
administration.
 Somatotropin

• Somatotropin, the hGH, is secreted by the anterior lobe of

pituitary glands which consists of 191 amino acid units.

• Its secretion is regulated by two other hormones (somatostatin

and growth hormone releasing hormone) produced by

hypothalamus.

• Deficiency of somatotropin in about 3% cases is of hereditary.

It has been estimated to about 1 child in 5,000

 Con…

• Turner's syndrome is one of the most common chromosome

disorders in girls and it is characterized by short stature and non-

functioning of ovaries affecting approximately 1 in 2,500 live

female birth.

• The extraction of somatotropin pharmaceutically from the pituitary

glands could not meet annual demand of this hormone.

• Biosynthesis of somatotropin was achieved through gene cloning

procedures.
 Production of recombinant hGH:

• Biotechnologists can now produce hGH by genetic

engineering.

• 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

• About 100,000 molecules of hormone per cell of E. coli have

been produced (Newmark, 1979).
 Somatostatin

• Somatostatin was first discovered in hypothalamic extracts and

identified as a hormone that inhibited secretion of growth
hormone.

• Subsequently, somatostatin was found to be secreted by a

broad range of tissues, including pancreas, intestinal tract and
regions of the central nervous system outside the
hypothalamus.
 Con…

• Somatostatin has two active forms produced by alternative

cleavage of a single pre-proprotein: one of 14 amino acids , the
other of 28 amino acids which is the short form with another
14 amino acids at one end.

• Somatostatin acts by both endocrine and paracrine pathways to

affect its target cells.

• A majority of the circulating somatostatin appears to come

from the pancreas and gastrointestinal tract.
 Pharmacologic Uses of Somatostatin

• Somatostatin and its synthetic analogs are used clinically to

treat a variety of neoplasms.

• It is also used in to treat gigantism and acromegaly, due to its

ability to inhibit growth hormone secretion.
 The gonadoterophines
 The gonadotrophins are a family of hormones for which the
gonads represent their primary target

 They directly and indirectly regulate;

 reproductive function and,

 in some cases, the development of secondary sexual characteristics.

 Insufficient endogenous production of this family will

affects reproductive function.
 Recombinant gonadoterophines
 Gonadoterophines also can produced by rDNA
technology.

 The gene coding for gonadoterophines from several

species have been identified and expressed in various
recombinant host system
 Particularly mammalian cell lines
 Recombinant blood products

Objectives of the lesson

At the end of this lesson students will be able

to:

• Recognise different recombinant blood

products.
 Blood and blood products constitute a major group of

traditional biologics.

 The main components of blood are

 red and white blood cells,

 platelets and the plasma.

 A variety of therapeutically important blood proteins

purified
 clotting factors and
 immunoglobulin.
Blood-related proteins produced by genetic
engineering are;
recombinant coagulation factors,

anticoagulants (such as hirudin) and

thrombolytics (such as tPA).

 Factor VIII

 Haemophilia is an X-linked recessive disorder caused

by a deficiency of factor VIII.

 Von Willebrand disease is a related disorder, also caused

by a defect in the factor VIII complex.

 It also can associate with platelets at the site of vascular

damage and it can participate in the coagulation cascade.
 Production of recombinant factor VIII

 Production of recombinant factor VIII has ended dependence

on blood as the only source of this product, and eliminated the
possibility of transmitting blood-borne diseases specifically
derived from infected blood.

 In the past, over 60 per cent of haemophiliacs were likely to be

accidentally infected via contaminated products at some stage
of their life.
 Native factor VIII: is traditionally purified from blood donations
first screened for evidence of the presence of viruses such as
hepatitis B and HIV.

A variety of fractionation procedures (initially mainly

precipitation procedures) have been used to produce a factor VIII
product.
 The final product is filter-sterilized and filled into containers.

 The product is then freeze-dried and the containers are subsequently sealed
under vacuum, or are flushed with an inert gas (e.g. N2) before sealing.

 No preservative is added.

 The freeze-dried product is then stored below 8 0C until shortly before its use.
 Recombinant factor VIII: cDNA coding for human factor

VIII:C in a variety of eukaryotic production systems (human

VIII:C contains 25 potential glycosylation sites).

 Both clinical and preclinical studies have shown that

administration of this product to patients suffering from

haemophilia A is equally as effective as administering blood-

derived factor VIII complex.

 Factors IX, VIIa and XIII
Factor IX
 Individuals who display a deficiency of factor IX develop
haemophilia B, also known as Christmas disease.

 Its clinical consequences are very similar to deficiency of

factor VIII, its general incidence in the population is far lower.

 Persons suffering from haemophilia B are treated by i.v.

administration of a concentrate of factor IX.

 This was traditionally obtained by fractionation of human

blood.
Factor VIIa
 Factor VIIa could directly activate the final common steps of
the coagulation cascade, independently of either factor VIII or
IX.

 A recombinant form of factor VIIa (called ‘NovoSeven’ or

‘eptacog alfa-activated’) is marketed by Novo-Nordisk.

 The recombinant molecule is produced in a BHK cell line, and

the final product differs only slightly (in its glycosylation
pattern only) from the native molecule.
Factor XIII
 A (very rare) genetic deficiency in the production of factor
XIII also results in impaired clotting efficacy in affected
persons.

 In this case, covalent links that normally characterize

transformation of a soft clot into a hard clot are not formed.

 Factor XIII preparations, partially purified from human blood,

are used to treat individuals with this condition; to date, no
recombinant version of the product has been commercialized.
 Anticoagulants
 Blood clot formation is essential to maintaining haemostasis,
inappropriate clotting can give rise to serious, sometimes fatal
medical conditions.
 Anticoagulants prevent blood from clotting and, therapeutic use
in cases where a high risk of coagulation is diagnosed.
 They are often administered to patients with coronary heart
disease and to patients who have experienced a heart attack or
stroke.

 The formation of a blood clot (a thrombus) occurs

inappropriately within diseased blood vessels.
 Thrombolytic Agents

 Thrombosis functions to plug a damaged blood vessel,

 maintaining haemostasis until the damaged vessel can be repaired.

 Subsequent to this repair, the clot is removed via an enzymatic

degradative process known as fibrinolysis.

 In situations where inappropriate clot formation results in the blockage

of a blood vessel and protect blood flow.

 Rapid removal of the clot can often minimize the severity of tissue
damage.

 Thus, several thrombolytic (clot-degrading) agents have found medical

application.
 Enzymes of therapeutic value

Objectives of the lesson

At the end of this lesson students will be able

to:

• Identify enzymes used for theraputic

purpose
 Asparaginase

 Asparaginase is an enzyme capable of catalysing the hydrolysis

of L-asparagine, yielding aspartic acid and ammonia.
 In the late 1970s, researchers illustrated that serum transferred
from healthy guinea pigs into mice suffering from leukaemia
contained some agent capable of inhibiting the proliferation of
the leukaemic cells. A search revealed the agent to be
asparaginase.
 Most healthy (untransformed) mammalian cells are capable of
directly synthesizing asparagine from glutamine.
 Although asparaginase therapy has proven effective, a
number of side effects have been associated with
initiation of therapy.
 These have included
 severe nausea,
 vomiting and diarrhoea,
 as well as compromised liver and kidney function.
 Con…

• The role of enzymes in medicine as markers, diagnostic and

prognostic applications are widely known.

• These enzymes are now considered as new biological drug in

the treatment of certain disorders and hence emerged as an
offshoot of therapy.

• Therapeutic enzymes are those enzymes which can be used

medically isolated with other therapies with the purpose of
treatment of various diseases safely.
 DNase
 Recombinant DNase preparations have been used in the
treatment of cystic fibrosis since the end of 1993.
 This genetic disorder is common, particularly in ethnic
groups of northern European extraction, where the
frequency of occurrence can be as high as 1 in 2500 live
births.
 A higher than average incidence has also been recorded in
southern Europe, as well as in some Jewish and African-
American populations.
 Con…

• The role of DNase I can hydrolyse long polymeric DNA

chains into shorter oligonucleotides and the purified enzyme
can be delivered in an aerosol mist to the lungs of CF patients
to prevent respiratory distress.

• The enzyme could decrease the mucus viscosity in the lungs

and allow patients for easy breathing, thus reducing the
severity and pain of the patient.
 The advent of genetic engineering and improvements in
chromatographic methodology facilitated the production of
highly purified recombinant human DNase (rhDNase)
preparations.

 This enzyme was approved for use by the US FDA in 1994.

 Debriding Agents

 Debridement refers to the process of cleaning a wound by

removal of foreign material and dead tissue.

 Cleansing of the wound facilitates rapid healing and minimizes

the risk of infection due to the presence of bacteria at the
wound surface.

 The formation of a clot, followed by a scab, on a wound

surface can trap bacteria, which then multiply (usually
evidenced by the production of pus), slowing the healing
process.
 Debridement may be undertaken by physical means (e.g.
cutting away dead tissue, washing/cleaning the wound),
proteolytic enzymes are also often used to facilitate this
process.

 Enzyme is formulated in an aqueous-based cream, and in

others it is impregnated (soaked) into special bandages.
 protease-containing maggot saliva

 Trypsin,

 papain,

 collagenase and various microbial enzymes

 Vaccine technology
 The application of vaccine technology forms a core element of
modern medicinal endeavour.

 It plays a central role in

 both human and veterinary medicine and

 represents the only common approach undertaken to control many

infectious diseases.

 Immunization programmes, incidence of many killer/disabling

diseases, such as
 smallpox,
 polio and
 tuberculosis.
 Vaccination seeks to exploit the natural defence mechanisms
conferred upon us by our immune system.

 A vaccine contains a preparation of antigenic components

consisting of, derived from or related to a pathogen.

 In most instances upon vaccine administration, both the

humoral and cell-mediated arms of the immune system are
activated.
Attenuated, dead or inactivated bacteria

 Attenuation (bacterial or viral) represents the process of elimination or

greatly reducing the virulence of a pathogen.
 This is traditionally achieved by,
 chemical treatment or heat,
 growing under adverse conditions
 propagation in an unnatural host.

 Live attenuated bacteria

 bacillus calmette – tuberculosis

 Dead or inactivated bacteria

 Cholera and pertussis vaccines
 Killing or inactivation of pathogenic bacteria usually renders
them suitable as vaccines.

 This is usually achieved by:

 heat treatment;

 treatment with formaldehyde or acetone;

 treatment with phenol or phenol and heat;

 treatment with propiolactone.

 Attenuated and inactivated viral vaccines
 Many of the more prominent vaccine preparations in current
medical use consist of attenuated viral particles.
 Mumps vaccines consist of live attenuated strains of
Paramyxovirus parotitidis.
 In many world regions, it is used routinely to vaccinate
children, often a part of a combined
 measles,
 mumps and
 rubella vaccine.
 Genetic engineering on vaccine technology

 recombinant DNA technology has rendered possible the

large-scale production of polypeptides normally present
on the surface of virtually any pathogen.
 These polypeptides, when purified from the producer
organism
 (e.g. E. coli, Saccharomyces cerevisiae) can then be used as
‘subunit’ vaccines.
 This method of vaccine production exhibits several advantages
over conventional vaccine production methodologies.

 These include:

 Production of a clinically safe product

 Production of subunit vaccine in an unlimited supply.

 Consistent production of a defined product that would thus

be less likely to cause unexpected side effects.
 DNA vaccines
 DNA vaccine is DNA sequence used as a vaccine.

 This DNA Sequence code for antigenic protein of pathogen.

 As this DNA inserted into cells it is translated to form

antigenic protein.

 As this protein is foreign to cells, so immune response raised

against this protein.

 In this way, DNA vaccine provide immunity against that

pathogen.
 DNA vaccines Vs Traditional vaccines

DNA vaccines Traditional vaccines

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

Viral gene

Recombinant DNA
Technology
Expression
plasmid

Plasmid with foreign gene

 Transform into
bacterial cell

Plasmid
DNA
Bacterial cell
Plasmid DNA get
Amplified
Plasmid DNA
Purified

Ready to use
 Subunit vaccines

•Do NOT use entire virus or bacteria (pathogenic agent)

•Use components of pathogenic organism instead of

whole organism

•Advantage: no extraneous pathogenic particles i.e DNA

•Disadvantage: Is rprotein same as in situ?

Cost
 Adjuvant ADJUVANTS

An adjuvant is a substance that, when added to a vaccine,

greatly enhances its protection against infection.
Help in:
• Decreasing the amount of antigen required for activation
• Safe
• Effective
• Reduce of vaccine doses
 WHY USE ADJUVANTS?

Adjuvants traditionally used to increase the magnitude of an

adaptive response to a vaccine.

The targets, therefore, are to:

(1)increase the response to a vaccine

(2)increase seroconversion rates in populations with reduced

responsiveness because of age (both infants and the elderly).

- the use on the MF59 adjuvant to enhance the response of

older subjects to influenza vaccine;
3) facilitate the use of smaller doses of antigen

 permit comparable responses with substantially lower

amounts of antigen.

important in circumstances in which large-scale vaccination

is urgent and production facilities limiting, as in the
emergence of a pandemic influenza strain.
 Examples of Adjuvants

i. Aluminum-potassium sulfate (alum)

ii. Freund’s incomplete adjuvant

iii. Freund’s complete adjuvant

Antibodies
 Antibodies

• Antibodies are proteins that recognise and bind to specific

antigens.

• Antibodies have important uses beyond fighting infections in

the body.

• Production of long-lasting monoclonal antibodies is a recent

invention and it is used in both medicine and research.

• Monoclonal Antibody: a stable antibody which can be used

over a period of time
 Monoclonal Antibody
• Monoclonal Antibody Production technology was developed
in 1975.

• Since its development it has been very important in the

modern medical science with the diagnosis, therapy, research
and even basic science today.

• It is still largely dependent upon animal testing however.

Because it requires immunization of mice in order for them to
create the antibodies to be grown.
 Con…
• Monoclonal Antibody Production or mAb is produced by cell
lines or clones obtained from the immunized animals with the
substance to be studied.

• Cell lines are produced by fusing B cells from the immunized

animal with myeloma cells.
 Con…

• To produce the desired mAb, the cells must be grown in either

of two ways:
 by injection into the peritoneal cavity of a suitably prepared mouse (the
in vivo, or mouse ascites, method) or

 by in vitro tissue culture.

• The in vitro tissue culture is the method used when the cells
are places in culture outside the mouse's body in a flask.
 Production of monoclonal antibodies

1. Inject a mouse with a specific antigen to stimulate its

immune system to produce necessary antibodies.
2. Extract mouse spleen cells (containing B-lymphocytes) and
culture them in the lab.
3. Extract mouse tumour cells, which grow continuously, and
culture them in the lab.
4. Mix spleen cells and tumour cells on the same plate and
culture.
 Con..

5. Add polyethylene glycol – this causes some B-lymphocytes

to fuse with tumour cells to produce a hybrid cell called a
hybridoma.
6. Grow the cells under conditions that allow only hybridoma
cells to survive.
7. Extract the cells, culture them separately and test the medium
around each cell for the specific antibody of interest.
8. Culture the cells making the desired antibody and use as
needed.
 Treatment of Cancer

• Cancer cells carry specific tumour-associated antigens (TAA)

on their plasma membrane.

• Monoclonal anti-TAA antibodies have been produced.

• Drugs which kill tumour cells or inhibit key proteins in tumour

cells are attached to monoclonal anti-TAA antibodies.

• Cancer cells are specifically targeted, avoiding damage to

healthy host cells.
 Problems

• Many patients develop immune response to monoclonal

antibodies produced in mice, as these are foreign proteins.

• Genetically engineered antibodies are being perfected to avoid

triggering immune response.
Polyclonal antibody

 Antigens possess multiple epitopes

 Serum antibodies are heterogeneous,

 Complement-mediated lysis of antigen

 Somatic gene therapy and cell/tissue
based therapy

Objectives of the lesson

At the end of this lesson students will be able to:

• Explain the use of gene therapy for the treatment of

diseases

• Recognize viral and non viral vectors used in gene

therapy.

• Explain stem cell therapy

 What is Gene Therapy?
 Gene therapy is a treatment or
cure for disorders caused by
mutated genes.
 It involves adding a normally
functioning copy of the gene(s) to
enough affected cells to restore
normal function.
 Types of gene therapy;
 Germline and
 somatic cell gene therapy
 Germline gene therapy would be the permanent transfer of
a gene into sperm or egg cells.
 Future generations would be “cured”
 Somatic cell (body cell) gene therapy is ideally only the
transfer of genes to the affected cells.
 Gene Therapy Successes
 Although no gene therapies have been approved by
the FDA for sale, some diseases have been
experimentally successful:
Melanoma (skin cancer)
Severe Combined Immunodeficiencies
Hereditary Blindness
Sickle Cell Anemia
 How is it done?
Viral Vector Carrying Healthy Gene

Cell with mutated Vector inserts New gene in the

gene(s) healthy gene into cell along with
cell original genes
Functional proteins are created from the therapeutic gene
causing the cell to return to a state.
 Gene Therapy
To design and carry out a gene therapy treatment, a researcher
must:
1. Identify the gene(s) responsible for the disorder.
2. Make copies of the normal gene.
3. Insert the copies into vectors.
4. “Infect” the affected cells with the vectors.
5. Activate the gene so that transcription and translation take
place.
 Viruses as Vectors
 Replicate by inserting their DNA into a host cell
 Gene therapy can use this to insert genes that encode
for a desired protein to create the desired trait.
 Four different types
 Adenovirus
 Adeno-Associated Virus (AAV)
 Retrovirus
 Herpes Simplex Virus (HSV)
 Retroviruses
 Created double stranded DNA copies from RNA genome
 The retrovirus goes through reverse transcription using reverse
transcriptase and RNA.

 the double stranded viral genome integrates into the human

genome using integrase.
• integrase inserts the gene anywhere because it has no specific site
• May cause insertional mutagenesis
– One gene disrupts another gene’s code (disrupted cell
division causes cancer from uncontrolled cell division)

 Vectors used are derived from the human immunodeficiency

virus (HIV) and are being evaluated for safety.
 Adenoviruses
 Adenoviruses also display potential as vaccine vectors.
 Are double stranded DNA genome that cause respiratory,
intestinal, and eye infections in humans.
 Live adenovirus strains that cause asymptomatic infection and
which have proven to be very safe and effective adenovirus vaccines
have been isolated.
 Not replicated though 
 Has to be reinserted when more cells divide
 Ex. Common cold
 Herpes Simplex Viruses
 Double stranded DNA viruses that infect neurons

 Ex. Herpes simplex virus type 1

 Non-viral Options
 Direct introduction of therapeutic DNA
– But only with certain tissue
– Requires a lot of DNA
 Creation of artificial lipid sphere with aqueous core, liposome
– Carries therapeutic DNA through membrane
 Chemically linking DNA to molecule that will bind to special
cell receptors
– DNA is engulfed by cell membrane
– Less effective 
 Trying to introduce a 47th chromosome
– Exist alongside the 46 others
– Could carry a lot of information
– But how to get the big molecule through membranes?
 Current Status
 FDA hasn’t approved any human gene therapy product for sale

Reasons !!!
 In 1999, 18-year-old Jesse Gelsinger died from multiple organ
failure 4 days after treatment for omithine transcarboxylase
deficiency.
– Death was triggered by severe immune response to adenovirus carrier
 January 2003, halt to using retrovirus vectors in blood stem cells
because children developed leukemia-like condition after successful
treatment for X-linked severe combined immunodeficiency disease.
 Problems with Gene Therapy
• Short Lived
– Hard to rapidly integrate therapeutic DNA into genome and
rapidly dividing nature of cells prevent gene therapy from long
time
– Would have to have multiple rounds of therapy
• Immune Response
– new things introduced leads to immune response
– increased response when a repeat offender enters
• Viral Vectors
– patient could have toxic, immune, inflammatory response
– also may cause disease once inside
• Multigene Disorders
– Heart disease, high blood pressure, Alzheimer’s, arthritis and
diabetes are hard to treat because you need to introduce more
than one gene
• May induce a tumor if integrated in a tumor suppressor gene
because insertional mutagenesis
 Unsuccessful Gene therapies
 Jesse Gelsinger, a gene therapy patient who lacked ornithine
transcribe amylase activity, died in 1999.

 Within hours after doctors shot the normal gene attached to a

therapeutic virus into his liver, Jesse developed a high fever. His
immune system began raging out of control, his blood began
clotting, ammonia levels climbed, his liver hemorrhaged and a flood
of white blood cells shut down his lungs.

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

• A stem cell is a cell that has the potential to become any cell
type in the human body.

• The easiest place to get stem cells is from an embryo.

• Stem cells are introduced into a damaged area of the body

where, under the right conditions, will replace the damaged
area.
 Con…

• Principles
Stem cells are introduced into a damaged area of the body where,
under the right conditions, will replace the damaged area.
• Application
The main areas where stem cells have proven their worth is in
bone marrow transplants, replacing damaged heart tissue after a
heart attack and replacing damaged nerve tissue which gives hope
to anyone who has had a spinal cord injury.
• Process
Often times stem cells are grown in a lab first to ensure the right
conditions and then placed into a sick person.
 Kinds of Stem Cells
Based on their ability to differentiate

Stem cell
type Description Examples

Each cell can develop into Cells from early (1-3

Totipotent
a new individual days) embryos

Some cells of
Cells can form any (over
Pluripotent blastocyst (5 to 14
200) cell types
days)
Cells differentiated, but Fetal tissue, cord
Multipotent can form a number of other blood, and adult
tissues stem cells
 Stem Cell Applications

Tissue repair
- nerve, heart, muscle, organ, skin
• Cancers
• Autoimmune diseases
- diabetes, rheumatoid arthritis, MS
 Bone marrow transplant:
Example of adult stem cell-based therapy
Somatic Cell Nuclear Transfer (SCNT)
Cloning of embryonic stem cells
Xenotransplantation
 Xenotransplantation

Objectives of the lesson

At the end of this lesson students will be able to:

• Idntify the role of pharmaceutical biotechnology

in tissue transplantation.

• Explain the methods used in

xenotransplantation.
 Types of transplants

Autograft

Allograft

Isograft

Xenograft and Xenotransplantation

 Autograft
 A transplant of tissue from one to oneself.
 Sometimes this is done with surplus tissue, or tissue that can
regenerate, or tissues more desperately needed elsewhere
(examples include skin grafts)
 Sometimes this is done to remove the tissue and then treat it
or the person, before returning it (examples include stem-
cell autograft and storing blood in advance of surgery).
 Allograft
 An allograft is a transplanted organ or tissue from a
genetically non-identical member of the same species.

 Most human tissue and organ transplants are

allograft.
 Isograft
 A subset of allografts in which organs or tissues are
transplanted from a donor to a genetically identical recipient
(such as an identical twin).

 Isografts are differentiated from other types of transplants

because while they are anatomically identical to allografts,
they are closer to autografts in terms of the recipient's immune
response.
Xenograft and Xenotransplantation

 A transplant of organs or tissue from one species to

another.
 Xenotransplantion is often an extremely dangerous
type of transplant.
 Examples include porcine heart valves, which are
quite common and successful, a baboon-to-human
heart (failed)
 Major organs and tissues transplanted
Thoracic organs
 Heart (Deceased-donor only)
 Lung(Deceased-donor and Living-Donor)
 En bloc Heart/Lung (Deceased-donor and Domino transplant)
Other organs
 Kidney (Deceased-donor and Living-Donor)
 Liver (Deceased-donor and Living-Donor)
 Pancreas (Deceased-donor only)
Tissues, cells, fluids
 Hand (Deceased-donor only
 Cornea (Deceased-donor only Skin graft including Face transplant (almost
always autograft)
 Penis (Deceased-donor only)
 Islets of Langerhans (Pancreas Islet Cells) (Deceased-donor and Living-Donor)
 Bone marrow/Adult stem cell (Living-Donor and Autograft)
 Blood transfusion/Blood Parts Transfusion (Living-Donor and Autograft)
 Blood vessels (Autograft and Deceased-Donor)
 Heart valve (Deceased-Donor, Living-Donor and Xenograft [Porcine/bovine])
 Bone (Deceased-Donor, Living-Donor, and Autograft)
 Skin(Deceased-Donor, Living-Donor, and Autograft)
 Factors affecting organ transplantation

• Size – differences in organ size limit the range of potential

recipients of xenotransplant.
• Hormone and protein differences- some proteins will be
molecularly incompatible, which could cause malfunction of
important regulatory processes.
• Longevity- life span of most pigs is roughly 15 years,
currently it is unknown whether or not a xenograft may be able
to last longer than that.
 Con…

• Environment – for example pig hearts work in a different

anatomical size and under different hydrostatic pressure than
in human.
 Ethical Issues

Religion
• Jewish – Forbidden to eat any part of a pig
• Heart is the seat of the soul
• No hybridization of man with any other
species.

Animal rights
• Physical discomfort
• Psychological discomfort
• Why should animals suffer for humans?
• The right to life
 Timeline of successful transplants
• 1905: First successful cornea transplant by Eduard Zirm
• 1954: First successful kidney transplant by Joseph Murray (Boston, U.S.A.)
• 1966: First successful pancreas transplant by Richard Lillehei and William Kelly
(Minnesota, U.S.A.)
• 1967: First successful liver transplant by Thomas Starzl (Denver, U.S.A.)
• 1967: First successful heart transplant by Christiaan Barnard (Cape Town, South Africa)
• 1970: First successful monkey head transplant by Robert White (Cleveland, U.S.A.)
• 1981: First successful heart/lung transplant by Bruce Reitz (Stanford, U.S.A.)
• 1983: First successful lung lobe transplant by Joel Cooper (Toronto, Canada)
• 1986: First successful double-lung transplant (Ann Harrison) by Joel Cooper (Toronto,
Canada)
• 1987: First successful whole lung transplant by Joel Cooper (St. Louis, U.S.A.)
• 1995: First successful laparoscopic live-donor nephrectomy by Lloyd Ratner and Louis
Kavoussi (Baltimore, U.S.A.)
• 1998: First successful live-donor partial pancreas transplant by David Sutherland
(Minnesota, U.S.A.)
• 1998: First successful hand transplant (France)
• 2005: First successful partial face transplant (France)
• 2006: First successful penis transplant (China)
Pharmacokinetics and pharmacodynamics
 Pharmacokinetics and pharmacodynamics

Objectives of the lesson

At the end of this lesson students will be able to:

• Recognise protein pharmacokinetics

• Explain protein mode of action and

pharmacodynamics.
 Pharmacokinetics

• Pharmacokinetics is the quantitative study of drug movement

in, through and out of the body.
• Intensity of effect is related to concentration of the drug at the
site of action, which depends on its pharmacokinetic
properties.
• Pharmacokinetic properties of particular drug is important to
determine the route of administration, dose, onset of action,
peak action time, duration of action and frequency of dosing
 Con…

• Pharmacokinetic properties of particular drug is important to

determine
 route of administration

 dose

 onset of action

 peak action time

 duration of action and frequency of dosing

 Con….

 Four Processes
– Absorption
– Distribution
– Metabolism
– Excretion
 Drug concentration at sites of action influenced by several
factors, such as:
– Route of administration
– Dose
– Characteristics of drug molecules (e.g., lipid solubility)
 Processes
Absorption- is the transfer of a drug from its site of
administration to the blood stream.

• Most of drugs are absorbed by the way of passive transport.

• Intravenous administration has no absorption.

• Fraction of administered dose and rate of absorption are

important.
 Drug Absorption
• Routes of Drug Administration
– Oral
– Inhalation
• vapors, gases, smoke
– Mucous membranes
• intranasal (sniffing)
• sublingual
• rectal suppositories
– Injection (parenteral)
• intravenous (IV)
• intramuscular (IM)
• subcutaneous (SC)
 Routes of Drug Administration

• Oral Drug Administration

– Advantages:
• relatively safe, economical, convenient, practical

– Disadvantages:
• Blood levels are difficult to predict due to multiple factors
that limit absorption.
• Some drugs are destroyed by stomach acids.
• Some drugs irritate the GI system.
 Advantages of Injection Routes

– Absorption is more rapid than with oral administration.

– Rate of absorption depends on blood flow to particular

tissue site (I.V. > I.M. > S.C.).

 Advantages specific to I.V. injection

– No absorption involved (inject directly into blood).

– A more accurate prediction of dose is obtained.

 Disadvantages/Risks of Injection

– A rapid onset of action can be dangerous if overdosing

occurs.

– If administered too fast, heart and respiratory function

could collapse.

– Drugs insoluble in water or dissolved in oily liquids can

not be given I.V.

– Sterile techniques are necessary to avoid the risk of

infection.
 Distribution of Drugs
• It is the passage of drug from the circulation to the tissue and
site of its action.

• The extent of distribution of drug depends on its lipid

solubility, ionization at physiological pH , extent of binding to
plasma and tissue proteins and differences in regional blood
flow.
 Con…

• Blood brain barrier (BBB): includes the capillary endothelial

cells (which have tight junctions and lack large intracellular
pores) and an investment of glial tissue, over the capillaries.

• A similar barrier is located in the choroid plexus.

• BBB is lipoidal and limits the entry of non-lipid soluble drugs

(amikacin, gentamicin, neostigmine etc.).
 Con…

• Placental Transfer

• Only lipid soluble Drugs can penetrate – limitation of

hydrophillic drugs.

• Plasma protein binding (PPB): Most drugs possess

physicochemical affinity for plasma proteins.

• Acidic drugs bind to plasma albumin and basic drugs to α1-

glycoprotein.
 Metabolism (Biotransformation)
• Chemical alteration of the drug in the body

• Aim: to convert non-polar lipid soluble compounds to polar

lipid insoluble compounds to avoid reabsorption in renal
tubules.

• Most hydrophilic drugs are less biotransformed and excreted

unchanged (streptomycin, neostigmine and pancuronium etc.)

• Biotransformation is required for protection of body from

toxic metabolites.
 Execration
• A transport procedure which the prototype drug (or parent
drug) or other metabolic products are excreted through
excretion organ or secretion organ

• Hydrophilic compounds can be easily excreted.

• Routes of drug excretion

 Kidney
 Biliary excretion
 Sweat and saliva
 Milk
 Protein pharmacokinetics

 A prerequisite to pharmacokinetic/pharmacodynamic studies is;

 selective and sensitive assay.

 The assay quantifying the therapeutic protein

 Additional analytical approaches used – chromatography

 Whole-body distribution studies are undertaken mainly in order to

assess tissue targeting and to identify the major elimination routes.

 Distribution volume usually subsequently increases, as the protein is

taken up into tissue during its metabolism/elimination.
 Pharmacokinetic and indeed pharmacodynamic characteristics
of therapeutic proteins can be rendered (even more)
complicated by a number of factors, including:
 The presence of serum-binding proteins
 Immunogenicity
 Sugar profile of glycoproteins

 The metabolism/elimination of therapeutic proteins occurs via

processes identical to those pertaining to native endogenous
proteins.
 proteolytic degradation, with amino acid residues released
 Clearance of protein drugs from systemic circulation
begin with passage across the capillary endothelia.

 The rate of passage depends upon the protein’s

physicochemical properties (e.g. mass and charge).
Tailoring of pharmacokinetic profile

 A number of different approaches may be used in order to alter

a protein’s pharmacokinetic profile.

 This can be desirable in order to achieve a predefined

therapeutic goal, such as;
 generating a faster or slower-acting product,

 lengthening a product’s serum half-life or

 altering a product’s tissue distribution profile

 The approach taken usually relies upon protein
engineering;
 alteration of amino acid sequence,

 alteration of a native post-translational modification

(usually glycosylation) or

 the attachment of a chemical moiety to the protein’s

backbone (often the attachment of PEG, i.e. PEGylation).
 Pharmacodynamics

• the study of the biochemical and physiologic effects of

drugs.

– Studies of drug mechanisms of action at the molecular

level provides basis for rational therapeutic uses and the
design of new, superior therapeutic agents.