Download 10 Microbiological control

Microbiological control of
medicines in pharmaceutical
manufacturing and pharmaceutical
companies.
Fundamentals of biotechnology
and genetic engineering.
Microbiological control of medicines in
pharmaceutical manufacturing and
pharmaceutical companies.
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.
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
Microbiological Samplings
Methods
Air Sampling:
 Active
 Passive
 Surface Sampling:
 Contact Plates
 Swabs
 Rinse Sampling
Manufacturing facility
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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
Clean rooms
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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 :
- 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


Europe :
5 μm particle dia
Grade A :
0
B:
0
C : 2,000
D : 20,000
USA :
class 100 (grade A/B),
class 10,000(grade C),
class 100,000 (grade D)
viable microorganisms
<1
5
100
500
Factors affecting the clean room condition
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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
- 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
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Generalized clean room design:
- Separated entries and exits for personnel, raw materials,
and products
-
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
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High standard of personnel hygiene
-
Only the minimum number of personnel required should be
present in the clean area at any given time
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
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CDS of process-scale chromatography systems :
challenging
ex) Processing of product derived from microbial sources :
contamination with lipid, endotoxins, nucleic acids, proteins
Water for pharmaceutical processing
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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
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Water for injection (WFI)
- Highest purity
- Extensive use in biopharmaceutical manufacturing
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 MΩ
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Filters to remove microorganisms: 0.22 µm, 0,45 µm
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Reverse osmosis (RO) membrane : Semi-permeable
membrane (permeable to the solvent, water, but
impermeable to solute, i.e., contaminants)
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)
Sterility Testing
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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
Sterility Testing
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Facilities
 Sterility
testing should be carried out under
the same conditions as aseptic manufacture
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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
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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
Sterility Testing
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Methods are defined in Pharmacopoeia
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membrane filtration is the preferred method if product is filterable
direction innoculation is alternative
Media types
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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
Sterility Testing
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Media
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should be tested for growth promoting qualities prior to use (low
number of organisms)
should have batch number and shelf life assigned
Incubation Period
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At least 14 days incubation
20-25°C for SCD/TSB, 30-35°C 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
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
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facilitate interpretation of test results
negative control contamination rate should be calculated
and recorded
Sterility Testing
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Positive Test Controls
 bactiostasis/fungistasis
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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)
Sterility Testing
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Positive Controls
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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
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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
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
Sterility Testing
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Interpretation and Repeat Tests
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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
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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
Sterility Testing
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Interpretation and Repeat Testing
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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
Other Microbiological Laboratory Issues
Testing of Biological Indicators
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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
Other Microbiological Laboratory Issues
Endotoxin Testing
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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
Other Microbiological Laboratory Issues
Endotoxin Testing (2)
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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
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Equipment used in test must be endotoxin free
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Validation of accuracy and reliability of the method
for each product is essential
Other Microbiological Laboratory Issues
Endotoxin Testing (3)
Gel Clot Method
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Original method
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The official “referee test”
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The specimen is incubated with LAL of a known
senstivity. Formation of a gel clot is positive for
endotoxin.
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.
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
Other Microbiological Laboratory Issues
Endotoxin Testing (6)
Gel Clot
Semiquantitative
Simple Least
expensive,
Requires 37
degree bath
Manually read
and recorded
Sensitive
down to 0.03
EU/ml
Chromogenic
Endpoint
Chromogenic
Kinetic
Turbidimetric
Quantitative
Quantitative
Quantitative
Requires
spectrophotome
ter
or plate reader
Can be
automated,
allows
electronic
data storage
Sensitive down
to 0.1 EU/ml
Requires
incubating plate
or tube reader
Can be
automated,
allows
electronic
data storage
Sensitive down
to .005 EU/ml
Requires
incubating plate
or tube reader
Can be
automated,
allows
electronic
data storage
Sensitive down
to .001 EU/ml *
* (Sensitivities vary by reagent manufacturer, instrumentation and
testing conditions)
Fundamentals of
biotechnology and genetic
engineering.
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.
The Biotechnology Tree
Biotechnology and Genetic Engineering
•Genes are the fundamental basis of all life.
•They determine the properties of all living forms.
•Genes are defined 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.
RNA and DNA
The flow of genetic material
Bacterial chromosome
and plasmid
Bacteriophage
Historical Development of Biotechnology
•Sumarians and Babylonians were drinking beer by 6000 BC, they were the first
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.
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.
Recent developments in Biotechnology
Category
Examples
1- Medicine
- Production of antibiotics, steroids, monoclonal antibodies,
vaccines, gene therapy, recombinant DNA technology
drugs and
improving diagnosis by enzymes and enzyme sensors.
2- Agriculture Plant tissue culture, protoplast fusion, introduction of
foreign genes into plants and nitrogen fixation.
3- Chemicals
-Organic
acids (citric, gluconic), mineral extraction.
4Environment
-Improvement
5- Food
-Single
6- Industry
- Use of enzymes in detergent industry, textile and energy
production
of waste treatment, replacement of chemical
insecticides by biological ones and biodegradation of
xenobiotics.
cell protein (SCP), use of enzymes in food
processing and food preservation.
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.
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).
Overview of a Biotechnological Process
Applications in Genetic Engineering
1- Therapeutic proteins and peptides
A. A- Insulin production
Insulin = protein = 2 polypeptide chains
A chain = 30 amino acids
B chain = 21 amino acids
Synthesize A-chain gene and
insert into a plasmid
Synthesize B-chain gene and
insert into a plasmid
Cloned plasmids are inserted
separately into E. coli
A chain
B chain
Lyse cells and purify the proteins
A chain
Mix and
connect
Insulin
B chain
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:
•α-Interferon is produced by leukocytes.
• -interferon is produced by fibroblasts.
• γ-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.
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.
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.
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.
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.
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 sitedirected mutation in the cloned gene.
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 α1-antitrypsin, a protein used as replacement therapy for geneticallydeficient individuals at risk from emphysema, have been produced in transgenic
sheep. The compound is excreted in their milk.
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.
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)
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 specific genetic message.
Within the cells, the DNA molecules may provide new genetic instructions to
correct the host phenotype.
Ex Vivo gene therapy:
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.
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