Bacterial cultivation

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Microbiological control of medicines in
pharmaceutical manufacturing and pharmaceutical
companies. Fundamentals of biotechnology and
genetic engineering.
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Microbiological control of medicines in
pharmaceutical manufacturing and pharmaceutical
companies.
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Microbial Control Considerations

• Product Development
• Routine Monitoring
• Water systems and Usage
• Active Ingredients
• Equipment Design and Use Conditions
• Personnel
• Manufacturing Environment

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• Guidance and Recommendations for performing a
  microbiological assessment a microbiological
  assessment considering a total program of
  facility, material and personnel management
• recommend a program of control for the
  manufacturing environment rather than control by
  direct environmental monitoring of the
  manufacturing area.

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The order of risk of pharmaceutical products
based on the invasiveness of the route of
administration

• Injectable products (sterile)
• Ophthalmic products (sterile)
• Inhalation solutions (sterile)
• Metered-dose dose and dry powder inhalants and
  dry powder inhalants
• Nasal sprays
• Otics
• Vaginal suppositories
• Topicals
• Oral liquids (aqueous)
• Oral liquids (non-aqueous)
• Rectal suppositories
• Liquid-filled capsules
• Oral tablets and powder-filled capsules

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Microbiological Samplings Methods

• Air Sampling
• Active
• Passive
• Surface Sampling
• Contact Plates
• Swabs
• Rinse Sampling

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Manufacturing facility

• Appropriate design and layout of the facility
  Crucial to the production of safe and effective
  medicines
• Commonly contains
• - Specific production of a target drug
• - Quality control, Storage areas, etc
• cf) Injectable bio-drugs Require unique
  facility design and operation ? safety of product
  
• - Clean room technology
• - Generation of ultra pure water (WFI
  water for injection)
• - Proper design and maintenance of
  non-critical
• areas storage, labeling, and packing
  areas

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Clean rooms

• Environmentally controlled areas for
  injectable/sterile biopharmaceutricals
  specifically designed to protect the product from
  contamination (microorganisms and particulate
  matters etc.)
• Designed in a way that allows tight control of
  entry of all substances and personnel (e.g.,
  equipment, in-process product, air etc..)
• A basic feature of design Installation of high
  efficiency particulate air (HEPA) filters in the
  ceilings

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• - Layers of high-density glass fiber Depth
  filter
• - Flow pattern of HEPA-filtered air
• - Air is pumped into the room via the
  filters, generating a constant downward sweeping
  motion
• Clean rooms with various levels of cleanliness
• - Classified based on the number of airborne
  particles
• and viable microorganisms in the room
• - Maximum permitted number of particles or
  microorganisms per m3 of clean room air

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• Europe
• 5 µm particle dia viable
  microorganisms
• Grade A 0
  lt 1
• B 0
  5
• C 2,000
  100
• D 20,000
  500
• USA
• class 100 (grade A/B),
• class 10,000(grade C),
• class 100,000 (grade D)

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Factors affecting the clean room condition

• Use of HEPA filters with high particulate-removing
  efficiency
• Generation of a unidirectional downward air
  distribution pattern (i.e. laminar flow)
• Additional elements critical to maintaining
  intended clean room conditions
• - All exposed surfaces a smooth, sealed
  impervious finish in order to minimize
  accumulation of dirt/microbial particles to
  facilitate effective cleaning procedures
• - Floors, walls, and ceilings coated with
  durable, chemical-resistance materials like epoxy
  resins, polyester, PVC coatings

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• - Fixtures (work benches, chairs, equipments
  etc..) designed and fabricated to facilitate
  cleaning processes
• - Air-lock systems buffer zone
• - prevention of contamination
• - entry of all substances/personnel into a
  clean room
• must occur via air-lock systems
• An interlocking system doors are never
  simultaneously open, precluding formation of a
  direct corridor between
• the uncontrolled area and clean area
• Generalized clean room design
• - Separated entries and exits for personnel,
  raw materials,
• and products

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• Personnel represent a major potential source of
  process contaminants required to wear
  specialized protective clothing when working in
  clean area
• Operators enter the clean area via a separated
  air-lock
• High standard of personnel hygiene
• Only the minimum number of personnel required
  should be present in the clean area at any given
  time

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Cleaning, decontamination, and sanitation (CDS)

• CDS regime essential to the production of a
  safe and effective biopharmaceuticals
• - Cleaning removal of dirt
  (organic/inorganic materials)
• - Decontamination inactivation and removal
  of undesirable
• substances, which generally exhibit some
  specific biological activity
• ex) endotoxins, viruses, prions
• - Sanitation destruction and removal of
  viable microorganisms
• Effective CDS procedures are routinely applied to
  
• - Surfaces are not direct contact with the
  product (e.g. clean room
• walls and floors)
• - Surfaces coming into direct contact with
  the product (e.g.
• manufacturing vessels, product filters,
  columns)

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• CDS of process equipment
• - surfaces/equipment in direct contact with
  the product special
• CDS requirement
• - no trace of the CDS reagents ? product
  contamination
• ? Final stage of CDS procedures involves
  exhaustive rinsing with
• highly pure water (water for injections
  (WFI))
• CDS of processing and holding vessels as well as
  equipment that is easily detachable/dismantled
  (e.g., homogenizer, centrifuge rotors etc.,) ?
  straightforward

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• Cleaning in place(CIP) large equipment/process
  fixtures due to the impracticality/undesirability
  of their dismantling
• ex) internal surfaces of fermentation
  equipment, fixed piping, large
  processing/storage tanks, process-scale
  chromatographic column
• - General procedure A detergent solution in
  WFI, passage of sterilizing live steam generated
  from WFI
• CDS of process-scale chromatography systems
  challenging
• ex) Processing of product derived from
  microbial sources contamination with lipid,
  endotoxins, nucleic acids, proteins

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Water for pharmaceutical processing

• Water One of the most important raw materials
  
• ? used as a basic ingredient
• - Cell culture media, buffers, solvent in
  extraction and
• purification, solvent in preparation of
  liquid form and
• freeze-dried products
• - used for ancillary processes cleaning
• - 30,000 liters of water production of 1
  kg of a
• recombinant biopharmaceutical produced in
  a
• microbial system
• ? Generation of water of suitable purity
  central to
• successful operation of facility

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• Two levels of water quality purified water and
  WFI
• - Outlined in international pharmacopoeias
• Use of purified water
• - Solvent in the manufacture of aqueous-based
  oral products (e.g., cough mixtures, )
• - Primary cleaning of some process
  equipment/clean room floors in class D or C area,
  
• - Generation of steam in the facilities,
  autoclaves
• - Cell culture media
• Water for injection (WFI)
• - Highest purity
• - Extensive use in biopharmaceutical
  manufacturing

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Generation of purified water and WFI

• Generated from potable water
• Potential impurities in potable water
• Multi-step purification steps for purified water
  and WFI
• Monitoring of each step continuous measurement
  of the resistivity of the water
• ex) Deionization anion/cation exchangers
• ?Increased resistivity with purity up to 1-
  10 MO
• Filters to remove microorganisms 0.22 µm, 0,45
  µm
• Reverse osmosis (RO) membrane Semi-permeable
  membrane (permeable to the solvent, water, but
  impermeable to solute, i.e., contaminants)

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General procedure for WFI

• Potable water
• ? depth filtration? organic trap (resin)
• ? activated charcoal
• ? Anion exchanger? Cation exchanger
• Deionization step monitored by measuring
  the
• water resistivity
• ? Filtration with membrane to remove
  microorganisms
• - purified water
• ? Distillation (or reverse osmosis)
• ? Water for injection(WFI)

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Sterility Testing

• Sterility test is a quality control test used as
  part of product release for product required to
  be sterile
• Has significant statistical limitations - will
  really only detect gross contamination
• Sampling
• No of containers and volume to be tested defined
  in Pharmacopoeia
• Samples from aseptically manufactured product
  should be taken from beginning, middle and end of
  batch fill and also after interventions and
  stoppages
• Samples from terminally sterilized product should
  be taken from previously identified cool spots
  within load
• Sampling should be sufficient to allow for
  retests if needed

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Sterility Testing

• Facilities
• Sterility testing should be carried out under the
  same conditions as aseptic manufacture
• In a Grade A laminar air flow cabinet in a Grade
  B background (may also be carried out in an
  isolator)
• Air supply through HEPA filters, pressures should
  be monitored and alarmed
• Access to area should be through airlocks
• Operators should be appropriately gowned is
  sterile garments
• Operators should be appropriately trained and
  validated
• Appropriate cleaning, sanitisation and
  disinfection procedures should be in place
• Environmental monitoring should be conducted

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Sterility Testing

• Methods are defined in Pharmacopoeia
• membrane filtration is the preferred method if
  product is filterable
• direction innoculation is alternative
• Media types
• Soybean Casein Digest medium (SCD), (also knows
  as Trypticase Soy Broth(TSB)) and Fluid
  Thioglycollate medium (FTM) is usually used (to
  detect aerobic and anaerobic organisms)
• validation studies should demonstrate that the
  media are capable of supporting growth of a range
  of low numbers of organisms in the presence of
  product. May need to incorporate inactivators
• growth should be evident after 3 days (bacteria),
  5 days (moulds)
• media may be purchased or made in-house using
  validated sterilization procedures

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Sterility Testing

• Media
• should be tested for growth promoting qualities
  prior to use (low number of organisms)
• should have batch number and shelf life assigned
• Incubation Period
• At least 14 days incubation
• 20-25C for SCD/TSB, 30-35C for FTM
• Test containers should be inspected at intervals
• temperatures should be monitored and temperature
  monitoring devices should be calibrated
• if product produces suspension, flocculation or
  deposit in media, suitable portions (2-5) should
  be transferred to fresh media, after 14 days, and
  incubated for a futher 7 days

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Sterility Testing

• Negative Contols
• media should be incubated for 14 days prior to
  use, either a portion or 100 of batch (may be
  done concurrently with test)
• negative product controls - items similar in type
  and packaging to actual product under test should
  be included in each test session
• facilitate interpretation of test results
• negative control contamination rate should be
  calculated and recorded

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Sterility Testing

• Positive Test Controls
• bactiostasis/fungistasis test
• should demonstrate that media are capable of
  supporting growth of a range of low numbers of
  organisms in the presence of product. May need to
  incorporate inactivators
• growth should be evident after 3 days (bacteria),
  5 days (moulds)

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Sterility Testing

• Positive Controls
• should be performed on all new products and when
  any changes are made.
• Should be repeated annually
• Stasis test recommended particularly for product
  with antibiotics or preservatives or slow release
  tested by direct innoculation
• demonstrates that media can support growth at the
  end of the incubation period and has not been
  affected by product
• Results
• Any growth should be identified (genotypic)
• Automated/Semi-automated systems used for
  identification should be periodically verified
  using reference strains

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Sterility Testing

• Interpretation and Repeat Tests
• No contaminated units should be found
• A test may only be repeated when it can be
  demonstrated that the test was invalid for causes
  unrelated to the product being examined

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Sterility Testing

• Interpretation and Repeat Tests
• No contaminated units should be found
• A test may only be repeated when it can be
  demonstrated that the test was invalid for causes
  unrelated to the product being examined
• European Pharmacopoeia criteria
• (a) the data of the micro monitoring of the
  sterility test facility show a fault
• (b) a review of the testing procedure used during
  the test in question reveals a fault
• (c) microbial growth is found in negative
  controls
• (d) after determination of the identity of the
  microorganisms isolated from the test, the growth
  of this species or these species may be ascribed
  unequivocally to faults with respect to the
  material and/or technique used in conducting the
  sterility test procedure

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Sterility Testing

• Interpretation and Repeat Testing
• When conditions (a), (b) or (c) apply the test
  should be aborted
• If a stasis test performed at the end of the test
  shows no growth of challenge organisms, this also
  invalidates the test
• For conditions (d) to apply must demonstrate that
  the orgamisms isolated from the sterility test is
  identical to an isolate from materials (e.g.
  media) and/or the environment
• must use genotypic identification methods
• Repeat test is carried out with same number of
  samples as first test
• Any contamination detected in repeat test,
  product does not comply

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Other Microbiological Laboratory Issues

• Testing of Biological Indicators
• if tested in-house the method should include a
  heat-shock step (this verifies that the
  indicators do actually contain spores and not
  vegetative organisms)
• BIs should occasionally be tested in house to
  verify the suppliers count

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Other Microbiological Laboratory Issues

• Endotoxin Testing
• Parenteral products should be free from endotoxin
• Endotoxin is a lipopolysaccharide present in the
  cell wall of gram negative bacteria which can
  cause fever if introduced into the body
• Raw materials, WFI used in manufacture and some
  finished product must be tested for endotoxin

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Other Microbiological Laboratory Issues

• Endotoxin Testing (2)
• LAL (Limulus Amebocyte Lysate) test is used for
  detecting endotoxin (previously a rabbit test)
• based on clotting reaction of horseshoe crab
  blood to endotoxin
• Types of LAL test
• Gel Clot
• Turbidimetric
• Colorimetric
• Equipment used in test must be endotoxin free
• Validation of accuracy and reliability of the
  method for each product is essential

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Other Microbiological Laboratory Issues
Endotoxin Testing (3)

• Gel Clot Method
• Original method
• The official referee test
• The specimen is incubated with LAL of a known
  senstivity. Formation of a gel clot is positive
  for endotoxin.

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Other Microbiological Laboratory Issues
Endotoxin Testing (4)

• Turbidimetric Method
• A kinetic method

• The specimen is incubated with LAL and either the
  rate of increase in turbidity or the time taken
  to reach a particular turbidity is measured
  spectrophotometrically and compared to a standard
  curve.

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Other Microbiological Laboratory Issues
Endotoxin Testing (5)

• Colorimetric Method
• Endotoxin catalyzes the activation of a proenzyme
  in LAL which will cleave a colorless substrate to
  produce a colored endproduct which can be
  measured spectrophotmetrically and compared to a
  standard curve.
• Can be kinetic or endpoint

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Other Microbiological Laboratory Issues
Endotoxin Testing (6)
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Fundamentals of biotechnology and genetic
engineering.
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What is Biotechnology?

• The use of microbial, animal or plant cells or
  enzymes to synthesize, break down or transform
  materials.

It mainly depends upon the expertise of
biological systems in recognition and catalysis.
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The Biotechnology Tree
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Biotechnology and Genetic Engineering

• Genes are the fundamental basis of all life.
• They determine the properties of all living
  forms.
• Genes are de?ned segments of DNA.
• DNA structure and composition in all living forms
  is essentially the same.
• Any technology that can isolate, change or
  reproduce a gene is likely to have an impact on
  almost every aspect of society.
• Genetic recombination, as occurs during normal
  sexual reproduction, consists of the breakage and
  rejoining of the DNA molecules of the
  chromosomes, and is of fundamental importance to
  living organisms for their assortment of genetic
  material.

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The flow of genetic material
RNA and DNA
Bacterial chromosome and plasmid
Bacteriophage
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• Historical Development of Biotechnology
• Sumarians and Babylonians were drinking beer by
  6000 BC, they were the ?rst to apply direct
  fermentation to product development.
• Egyptians were baking leavened bread by 4000 BC
  wine was known in the Near East by the time of
  the book of Genesis.
• Microorganisms were first seen in the seventeenth
  century by Anton van Leeuwenhoek who developed
  the simple microscope
• The fermentative ability of microorganisms was
  demonstrated between 1857 and 1876 by Pasteur
   the father of Microbiology/Biotechnology 
• Cheese production has ancient origins, as does
  mushroom cultivation.
• Biotechnological processes initially developed
  under non-sterile conditions
• Ethanol, acetic acid, butanol and acetone were
  produced by the end of the nineteenth century by
  open microbial fermentation processes.

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• Historical Development of Biotechnology
• Waste-water treatment and municipal composting of
  solid wastes represents the largest fermentation
  capacity practiced throughout the world.
• Introduction of sterility to biotechnological
  processes In the l940s complicated engineering
  techniques were introduced to the mass production
  of microorganisms to exclude contaminating
  microorganisms.
• Examples include the production of antibiotics,
  amino acids, organic acids, enzymes, steroids,
  polysaccharides, vaccines and monoclonal
  antibodies.
• Applied genetics and recombinant DNA technology
• Traditional strain improvement of important
  industrial organisms has long been practiced
  recombinant DNA techniques together with
  protoplast fusion allow new programming of the
  biological properties of organisms.

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Recent developments in Biotechnology
Examples Category
- Production of antibiotics, steroids, monoclonal antibodies, vaccines, gene therapy, recombinant DNA technology drugs and improving diagnosis by enzymes and enzyme sensors. Plant tissue culture, protoplast fusion, introduction of foreign genes into plants and nitrogen fixation. Organic acids (citric, gluconic), mineral extraction. Improvement of waste treatment, replacement of chemical insecticides by biological ones and biodegradation of xenobiotics. Single cell protein (SCP), use of enzymes in food processing and food preservation. - Use of enzymes in detergent industry, textile and energy production 1- Medicine 2- Agriculture 3- Chemicals 4- Environment 5- Food 6- Industry
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• Genetic engineering
• The formation of new combinations of heritable
  material by the insertion of nucleic acid
  molecules, produced by whatever means outside the
  cell, into any virus, bacterial plasmid or other
  vector system so as to allow their incorporation
  into a host organism in which they do not
  naturally occur.
• Princple
• DNA can be isolated from cells of plants, animals
  or microorganisms (the donors) and can be
  fragmented into groups of one or more genes.
• Passenger DNA fragments can then be coupled to
  another piece of DNA (the vector ) and then
  passed into the host or recipient cell.
• The host cell can then be propagated in mass to
  form novel genetic properties and chemical
  abilities that were unattainable by conventional
  ways of selective breeding or mutation.

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Steps

1. DNA is isolated from the cells and purified.
2. Restriction enzymes are used to cut the DNA for
   cloning.
3. Ligases are the used to join the DNA fragments
   together.
4. The new cloned plasmid is the transformed into
   competent cells (Cells treated chemically to
   allow passage of foreign DNA).

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Overview of a Biotechnological Process
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Applications in Genetic Engineering

• 1- Therapeutic proteins and peptides
• A- Insulin production

Insulin protein 2 polypeptide chains A chain
30 amino acids B chain 21 amino acids
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B- Interferons

• Interferons are proteins produced by eukaryotic
  cells in response to viral infection.
• They prevent replication of the infecting virus
  in adjacent cells.
• There are several kinds of interferons each made
  by a different cell type
• a-Interferon is produced by leukocytes.
• ?-interferon is produced by fibroblasts.
• ?-interferon is produced by sensitized T cells.
• Interferon can be produced (commercially) by two
  methods
• 1- Cultures of human diploid fibroblasts attached
  too a solid support.
• 2- Bacteria in which the interferon gene is
  cloned and expressed , the interferon is then
  purified.
• Used for treatment of Hepatitis B and C and many
  other Cancer and autoimmune diseases.
• PEGylated interferons are interferons conjugated
  with PEG to allow for slow release inside the
  body, injected once a week.

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• C- Human-growth hormone
• Human growth hormone is another pharmaceutical
  product made more efficiently by a genetically
  engineered bacterium.
• Previously the hormone was obtained only in
  extremely small quantities by extracting it from
  the pituitary glands of the animals.
• The genetically engineered product is being used
  to treat children pituitary dwarfism and other
  conditions related to growth hormone deficiency.

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D- Hepatitis B vaccine

• Production of certain vaccines such as hepatitis
  B, has been difficult because the virus was
  unable to grow in cell cultures and the extreme
  hazards of working with large quantities of the
  virus which can be obtained from the blood of
  humans and experimentally infected chimpanzees.
• Using DNA from HBV, it was possible to clone the
  gene for hepatitis B surface antigen (HBs
  antigen) into cells of the yeast Saccharomyces
  cerevisiae.
• The yeast expressed the gene and made HBs antigen
  particles that could be extracted after the cells
  were broken.
• Since yeast cells are easy to propagate, it was
  possible to obtain-unlimited quantities of HBs
  antigen particles.
• This was the first vaccine against a human
  disease produced with genetic engineering methods.

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2- Chemicals Indigo dye

• The dye indigo is a plant product but was
  manufactured chemically to reduce the cost.
• However, it was possible to clone naphthalene
  oxidase gene from Pseudomonas sp. into E. coli.
• The modified E. coli produced indigo, as the
  naphthalene oxidase enzyme oxidized indole of E.
  coli to 2-3 dihydrodiol which spontaneously
  oxidize and dimerize to indigo resulting in blue
  E. coli.
• It is the blue of blue genes that is why the
  commercial importance.

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3- Construction of new microbes Ice-minus
Pseudomonas syringae

• An interesting ecological relationship between
  bacteria and plants involves the role of
  Pseudomonas syringae which produce a surface
  protein initiating ice crystals formation, which
  results in frost damage to the plant.
• These bacteria are conditional plant- pathogens,
  causing death due to frost damage only at
  temperatures that can initiate the freezing
  process.
• A genetically engineered ice-minus strain (with
  the surface protein deleted) is sprayed to
  replace the indigenous population and protect the
  crop.
• The release of genetically engineered raised
  environment questions.

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4- Improvement of performance and productivity
The key control gene for an important product
can be identified and manipulated to be
insensitive to repression. The manipulated gene
is cloned and reintroduced at a high copy
number. Ex The genes of antibiotic-producing
organisms.
5- Protein engineering
Knowledge of the tertiary structure of an enzyme
with knowledge of its DNA sequence can enable the
rational modification of the molecule to produce
the desired change such as substrate specificity
and temperature stability. Substitution of
certain amino acid at a specific position can be
achieved by site-directed mutation in the cloned
gene.
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6- Modification of macroscopic animals

• Transgenic animals Transgenesis is the use of
  gene manipulation to permanently modifying germ
  cells of animals.
• For example the production of super mice was a
  result of the over-production of human growth
  hormone.
• Over-expression of growth hormone has also been
  tried in order to increase the rate of growth of
  livestock, poultry and fish.
• Production of foreign proteins in transgenic farm
  animals find a more significant progress.
• For example a1-antitrypsin, a protein used as
  replacement therapy for genetically-deficient
  individuals at risk from emphysema, have been
  produced in transgenic sheep. The compound is
  excreted in their milk.

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7- Plant biotechnology Introduction of genes into
plant that enables the plant to degrade or
detoxify the herbicide
Herbicide tolerant crops To allow the use of
non-selective herbicides to remove all weeds in
a single and quick application. Advantages Less
spraying, less traffic on the field, and lower
operating costs.
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Genetically Modified Products Genetically
engineered Tomatoes with reduced
polygalacturonase enzyme. This enzyme is involved
in softening and over ripening of
tomatoes. Advantages Faster growth, better
yield ,quality and longer shelf life)
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Gene Therapy Any treatment strategy that
involves the introduction of genes or genetic
material into human cells to alleviate or
eliminate disease. The aim of gene therapy is to
replace or repress defective genes with sequences
of DNA that encode a speci?c genetic message.
Within the cells, the DNA molecules may provide
new genetic instructions to correct the host
phenotype.
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Ex Vivo gene therapy
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What factors have kept gene therapy from becoming
an effective treatment for genetic disease? 1-
Short-lived nature of gene therapy Problems with
integrating therapeutic DNA into the genome and
the rapidly dividing nature of many cells prevent
gene therapy from achieving any long-term
benefits. Patients will have to undergo multiple
rounds of gene therapy.2- Immune response
Anytime a foreign object is introduced into human
tissues, the immune system is designed to attack
the invader. The risk of stimulating the immune
system in a way that reduces gene therapy
effectiveness is always a potential risk. 3-
Problems with viral vectors Viruses, while the
carrier of choice in most gene therapy studies,
present a variety of potential problems to the
patient --toxicity, immune and inflammatory
responses, and gene control and targeting issues.
In addition, there is always the fear that the
viral vector, once inside the patient, may
recover its ability to cause disease.
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4- Multigene disorders Conditions or disorders
that arise from mutations in a single gene are
the best candidates for gene therapy.
Unfortunately, some the most commonly occurring
disorders, such as heart disease, high blood
pressure, Alzheimer's disease, arthritis, and
diabetes, are caused by the combined effects of
variations in many genes. Multigene or
multifactorial disorders such as these would be
especially difficult to treat effectively using
gene therapy