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Objectives
Understand controlled environment for
manufacturing process of sterile product
Source & incidence of microbial contamination.
Process of sterilization to revise
Control on sterile product
Sterility test and other relevant tests
Evaluation of Sterile
Pharmaceutical
Sterility test and other relevant tests
1. The Needs of Controlled Environment for
Manufacturing Process of sterile product
Hygienic condition is required for the preparation
of pharmaceuticals.
Thermolabile nature increase in aeptic
manufacturing
Hospital setting TPN, CDR
1. The Needs of Controlled Environment for
Manufacturing Process of sterile product
Sterile products need to be:
Free of m/o
There is not a level of survivors which is so small
as to be negligible and acceptable
Sterilised
2. Source and incidence of microbial
contamination
Major source of contamination are water and raw
material used.
Crude drugs of nature origin present a particular
hazard
Contamination by people
Skin cells
Fragment of hair
Exhaled liquid droplets
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3. Process of sterilisation
Sterilisation is a complex & expensive process
BP processes of sterilisation include:
Steam sterilisation (autoclave heating)
Dry heat sterilisation (hot air oven)
Ionising radiation sterilisation
Gas sterilisation (ethylene oxide)
Filtration
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4. Control on Sterile Product
Evaluation of Parenteral Products
There are 2 types of control on sterile products to
ensure the absence of m/o from the products.
Control on the sterilisation process
Sterility test on the product
Sterility Testing
Pyrogen Testing
Clarility Testing
Leaker Testing
Sterility Testing
Sterility Testing
Intended for detecting the presence of aerobic
& anaerobic viable forms of bacteria, fungi
and yeasts in pharmaceutical preparations.
Is NOT meant to prove that a product is sterile
but to estimate the probable sterility of a
batch of articles.
Sterility test attempts to reveal the presence or
absence of viable m/o in a sample number of
containers taken from batch of product. Based on
results obtained from testing the sample a
decision is made as to the sterility of the batch.
Sterility Testing
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Sterility Testing
Is made after the product exposition to the one of
the possible sterilisation procedures
Can only provide partial answers to the state of
sterility of the product batch under test.
Is inadequate as an assurance of sterility for a
terminally sterilised product.
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These are fallibly, i.e. contaminants might not be
detected & missed.
A sample of material to be tested is added to
culture medium which is incubated & then
examined for signs of microbial growth.
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Sterility Testing
Pyrogen Test
If growth occurs the assumption is made that the
contamination arose from the sample, which
consequently fails the test.
Sample:
First entry points for fallible
Assume we have a batch of 100 iv vial.
the effect of pyrogens in human
Pyrogen can cause a rise in body temperature, malaise, and
headache
the detection methods of pyrogens
1. Involves injecting the test solution and measuring the rise in
body temperature of healthy mature rabbit, or
2. Bacterial endotoxin test- Limulus Amoebocyte Lysat test (LAL):
The test is based on the primitive blood-clotting mechanism of
the American horseshoe crab (Limulus Polypphemus). Several
Enzyme located with the crabs amoebocyte blood cells are
triggered by endotoxin to initiate an enzymatic coagulation
cascade that concludes with the production of proteinaceous
gel
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Pyrogen Test
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Pyrogen Testing
If pyrogens are detected in a sterile product, that means
that bacteria have proliferated somewhere along during
the formulation process.
Employs rabbit as the test animal
Because it is highly sensitive to pyrogens.
Samples to be tested are injected into the ear
vein of 3 rabbits.
The body temperatures was measured by
inserting thermometer into rabbits anus
employing automatic temperature recording
device.
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Pyrogen Testing
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Pyrogen Testing
The sample passes the test if at no time during
a 3 hour period following injection, the
temperature of any rabbit rises by more than
0.6C or the sum of the rise for the 3 rabbits
exceeds 1.4C.
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Limulus Amebocyhte Lysate (LAL) test
The test solution is combined with a cell lysate
from the amoebocytes (blood cells) of the
horseshoe crab.
Any endotoxin that might be present will be
coagulated with the fraction of the amoebocytes
and result in the formation of a gel.
This test is considered to be simple, rapid and of
greater sensitivity than the rabbit test.
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Clarity Testing
Are performed to prevent the distribution and
use of parenteral product which contain
particulate matter.
All injectable solutions including solutions
constituted from sterile solids must be
essentially free from particles of
approximately 50 m or more that can be
observed by inspection with the unaided eye.
Clarity Test
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The permitted limits of particulate matter are
given below:
Particle Size in m
(equal to or larger
than)
10
Maximum No. of
Particles per ml
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50
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Clarity Testing
Particulate matter is defined as :
extraneous,
mobile,
undissolved substances,
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Other than gas bubbles, unintentionally
present in injections.
nil
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Clarity Testing
Clarity Testing
1. Visual inspection
3. Other devices are based on light absorption,
light scattering or change in electrical
resistance
4. Coulter counter
Human visual inspection with the aid of good direct
lighting on the containers with the product against a
black and white background.
Thus transparent particles would be visible against black
background and the white background would reveal
coloured particles.
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2. Filtration
Detected by passing the solution through a filter and
examining the filter microspically.
Automatic image analysis devices
Focus the image of particles on a TV screen and record the image
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Leaker Testing
Method I
is performed by dipping the sealed container
in colored solution and producing ve
pressure.
Leaker Test
A 1% methylene blue solution is most commonly
employed.
Release of vacuum is accompanied by the
entry of the colored solution into the
imperfectly sealed ampoule.
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Leaker Testing
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Leaker Testing
A leaking ampoule is called a leaker and all
leakers are to be discarded.
Vials and bottles are NOT subjected to a leaker
test.
Method II
The ampoules are autoclaved in a dye bath
Method III
The hot ampoules are removed from the
autoclave and quickly dipped into a cool bath
of dye solution.
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The End
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