Sterility validation
Procedure:
Validation has been divided into two
parts i.e. Part I & Part II. Since the product contains substances which
are likely to interfere with the test results i.e. some of the active
ingredients or preservatives in the formulation are likely to inhibit growth of
microorganism.
Part I of the protocol establishes the quantity of water or solvent
that is required to remove to the
maximum possible extent all traces of active ingredients & preservatives retained on the membrane. Protocol
for testing the traces of preservatives and active ingredients
for individual product shall be separately prepared and record shall
be generated.
Part II of the protocol will help in establishing that after
the membrane is treated for inactivation of active ingredients and preservatives
in Part I of the protocol, it is still capable of supporting growth in the
media defined in the test.
Procedure
Part
I: [Inactivation of active ingredients
and preservatives shall done by washing with sterilized 0.1 % Peptone Water.]
Aseptically
half of the contents from each of 20 containers of the products
Shall
be transferred and filtered through
sterile 0.45 m, 47 mm diameter. Membrane
filter and the filtrate shall be discarded.
The
membrane filter shall be washed with 100 ml of sterilised 0.1% peptone water
and the filtrate shall be collected and labeled as first washing.
The
membrane filter shall be washed with further two quantities of 100 ml sterilized
0.1% peptone water and the filtrates shall be preserved and labeled
as second and third washing respectively.
The filtrate obtained from the
first, second and third washings shall be tested for traces of active ingredients and
preservatives using the validated colorimetric method or UV Spectrophotometric method.
Acceptable limits: Concentration of
active ingredients and preservatives in filtrate obtained from final wash
should not be more than 0.5 ppm
Part II
:
[ Testing the ability of the membrane to support growth of
microorganisms after inactivation of the active ingredients and preservatives.]
Sterility testing procedure shall be
strictly followed as per STP No. QA/STP-019.
Aseptically more than half contents
from each of 20 containers of the
product shall be filtered through seven 0.45 m ,
47 mm diameter membrane filters separately.
The membrane filters shall be washed
using the procedure established in part I of the protocol.
In final washing of each of 100 ml
0.1 % peptone solution inoculate 10 -100 cfu of respective microorganisms. For
detail refer following table.
Sr. No.
|
Name
|
Catalog No.
|
Incubation
|
|
Temperature
|
Period
|
|||
1.
|
Staphylococcus
aureus
|
ATCC – 6538
|
32.5 ± 2.5 °C
|
NMT 3 days
|
2.
|
Pseudomonas aeruginosa
|
ATCC –
9027
|
32.5 ± 2.5 °C
|
NMT 3 days
|
3.
|
Bacillus
subtilis
|
ATCC – 6633
|
22.5 & 32.5 ± 2.5 °C
|
NMT 3 days
|
4.
|
Clostridium
sporogenes
|
ATCC – 19404
|
32.5 ± 2.5 °C
|
NMT 3 days
|
5.
|
Bacteroides
vulgatus
|
ATCC – 8482
|
32.5 ± 2.5 °C
|
NMT 3 days
|
6.
|
Candida albicans
|
ATCC –
10231
|
22.5 & 32.5 ± 2.5 °C
|
NMT 5 days
|
7.
|
Aspergillus
niger
|
ATCC – 16404
|
22.5 ± 2.5 °C
|
NMT 5 days
|
For Bacillus subtilis and Candida albicans :
Cut the filter paper into two halves with the
help of sterile scissor.
Put one half into sterile Fluid Thioglycolate
Medium and another half into the Soyabean Casein Digest Medium tubes.
Incubate Fluid Thioglycolate Medium tube at 32.5 ± 2.5 °C and Soyabean Casein Digest Medium
tubes at 22.5 ± 2.5 °C.
For Staphylococcus
aureus, Pseudomonas aeruginosa, Clostridium sporogenes
and Bacteroides vulgatus :
Cut the filter paper into two halves
with the help of sterile scissor.
Put one half into sterile Fluid
Thioglycolate Medium and destroy the another half.
Incubate Fluid Thioglycolate Medium tube at 32.5 ± 2.5 °C .
For Aspergillus niger :
Cut the filter paper into two halves
with the help of sterile scissor.
Put one half into sterile Soyabean
Casein Digest Medium and destroy the
another half.
Incubate Soyabean Casein Digest Medium tube at 22.5 ± 2.5 °C.
For Positive Control:
For every selected microorganism
Inoculate 10 – 100 cfu of respective
microorganisms into 0.1 % of 100 ml
sterile peptone water and filter through 0.45
m,
47 mm diameter membrane filters
separately.
Cut the filter paper into two halve
with the help of sterile scissor.
Put one half into the respective
medium tube and destroy the another half.
For Negative Control:
incubate one tube of Fluid
Thioglycolate Medium at 32.5 ± 2.5 °C and
Soyabean Casein Digest Medium tube
at 22.5 ± 2.5 °C.
Results:
All the inoculated membranes should show growth comparable
to the growth observed in the positive controls within 3 days in case of
Bacteria and 5 days in case of fungi.
Inference:
If growth is observed as expected it
positively confirms that the inactivation procedure is complete and it does not
affect the sensitivity of the Test for Sterility described in the respective
Pharmacopoeia.
Frequency:
Validation of sterility test shall
be done on all new products or whenever there is a change in formulation /
immediate (primary) pack of the product or change in the manufacturing facility
and testing of the product.
Responsibility:
Q. A. in- charge shall be
responsible for effective implementation of this work instruction.