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

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

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

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 m 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

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

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

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)

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

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

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

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

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 M

Filters to remove microorganisms: 0.22 m, 0,45 m

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

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

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

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

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

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

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)

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

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

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

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

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

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

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

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.

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)
Chromogenic
Endpoint

Chromogenic
Kinetic

Turbidimetric

Semiquantitative

Quantitative

Quantitative

Quantitative

Simple Least
expensive,
Requires 37
degree bath

Requires
spectrophotomet
er
or plate reader

Requires
Requires
incubating plate incubating plate
or tube reader
or tube reader

Manually read
and recorded

Can be
automated,
allows electronic
data storage

Can be
automated,
allows electronic
data storage

Can be
automated,
allows electronic
data storage

Sensitive down
to 0.03 EU/ml

Sensitive down
to 0.1 EU/ml

Sensitive down
to .005 EU/ml

Sensitive down
to .001 EU/ml *

Gel Clot

* (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
They

are the fundamental basis of all life.

determine the properties of all living forms.

Genes

are dened 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

Bacterial chromosome
and plasmid

The flow of genetic mat

Bacteriophage

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 rst 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

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 ofmicroorganisms to exclude contaminating
microorganisms.
Examples include the production ofantibiotics, 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

Recent developments in
Biotechnology Examples
Category
Medicine- 1

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

4Environment

-Improvement

5- Food

-Single

6- Industry

- Use of enzymes in detergent industry, textile and energy

production

acids (citric, gluconic), mineral extraction.

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 puried.

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 Proces

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

Mix and connect

A chain

B chain
Insulin

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:
-Interferon is produced by leukocytes.
-interferon is produced by broblasts.
-interferon is produced by sensitized T cells.

Interferon can be produced (commercially) by

two methods:
1- Cultures of human diploid broblasts attached too a solid
support.
2- Bacteria in which the interferon gene is cloned and expressed ,
the interferon is then puried.
Used

for treatment of Hepatitis B and C and many other Cancer

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 dwarsm and other conditions related to
growth hormone deciency.

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 obtainunlimited quantities of HBs antigen particles.

This was the rst 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 identied 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 modication of the
molecule to produce the desired change such as substrate specicity
and temperature stability.
Substitution of certain amino acid at a specic position can be
achieved by site-directed mutation in the cloned gene.

fication of macroscopic animals- 6

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 sh.

Production of foreign proteins in transgenic farm animals nd a

more signicant progress.

For example 1-antitrypsin, a protein used as replacement

therapy for genetically-decient 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 eld, 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 specic 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 benets. 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

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