Sterility validation

Rahul Kashyap
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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.



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