validation protocol of sterility test

SR. NO.	PARTICULARS
1.0	APPROVAL SIGNATURES
2.0	OBJECTIVE
3.0	SCOPE
4.0	PRE-REQUISITES
5.0	RESPONSIBILITIES
6.0	REFERENCE DOCUMENTS
7.0	EXPERIMENTAL DETAILS
7.1	PRODUCT NAME
7.2	BATCH NO.
7.3	QUANTITY OF SAMPLE
7.4	FILTER TYPE
8.0	PROCEDURE
8.1	PRODUCT CONTROL
8.2	TEST CONTROL
8.3	POSITIVE CONTROL
9.0	ACCEPTANCE CRITERIA
10.0	ABBREVIATIONS
11.0	CHANGE HISTORY LOG FORMAT

1.0 APPROVAL SIGNATURES

Signing of this protocol indicates agreement with the validation study of sterility test of finished product IP/BP/USP.

Further if any changes in this protocol are required, protocol will be revised and duly approved.

Prepared By:

NAME

DESIGNATION

DEPARTMENT

SIGN.

DATE

Checked By:

Approved By:

NAME

DESIGNATION

DEPARTMENT

SIGN.

DATE

2.0 OBJECTIVE :
To carry out validation of sterility test of the finished products IP/BP/USP

3.0 SCOPE:
This protocol is applicable for conducting validation of sterility test of finished products IP/BP/USP

4.0 PRE-REQUISITES
4.1 Materials: Bacterial & Fungal cultures as per IP/BP/USP, Fluid Thioglycollate medium (pH 7.1+ 0.1 ), Soybean Casein Digest Medium (pH 7.3+ 0.2),
Normal saline, Peptone water, 0. 45μ filter paper.
4.2 Equipments: Membrane Filtration Assembly, Filtration cups, Vacuum flask, Vacuum pump, Forceps, Scissors, Surgical blades,

Bunsen burners, Validated LAF and Validated incubators.

5.0 RESPONSIBILITIES

Sr. No.	Responsibilities	Department
1.0	Preparation of protocol	Q.C. Officer (Micro)
2.0	Provision of qualified personnel to assist in the protocol preparation and execution	Q.C. Executive
3.0	Verification of Protocol	Head Q.C.
4.0	Approval of Protocol	Head Q.A.
5.0	Final determination of system Acceptability	Head Q.C. and Head Q.A.
6.0	Review and assembling of data into a final report	Q.C. Executive

6.0 REFERENCE DOCUMENTS
IP/BP/USP

7.0 EXPERIMENTAL DETAILS

7.1 PRODUCT NAME :

7.2 BATCH NO. :

7.3 QUANTITY OF SAMPLE :

7.4 FILTER TYPE :

8.0 PROCEDURE

8.1 PRODUCT CONTROL :
8.1.1 Aseptically filter the product from sample container through
Membrane filtration unit.
8.1.2 Give washing with suitable volume of peptone water (Volume to be mentioned in the validation report)
8.1.3 Cut the membrane filter paper into two halves.
8.1.4 Transfer one half into 100 ml of Soya bean Casein Digest Medium & second half into Fluid Thioglycollate Medium.
8.1.5 Incubate the tubes for 14 days.
8.1.6 Examine the media tubes daily for any evidence of growth.

8.2 TEST CONTROL :
8.2.1 Test for Bacillus subtilis ATCC 6633 (MTCC 441)
8.2.1.1 Aseptically filter the product from sample container through Membrane filtration unit.
8.2.1.2 Give washing with suitable volume of 0.1% peptone water (volume to be mentioned in the validation report.
8.2.1.3 Inoculate the final rinse with 10-100 cfu/ml of B. subtilis ATCC 6633 (MTCC 441).
8.2.1.4 Transfer the membrane filter in 100 ml of Fluid Thioglycollate Medium tube.
8.2.1.5 Incubate the tube at 32.5 ˚C+ 2.5 ˚C for not more than 3 days.
8.2.1.6 Examine the media daily for evidence of growth.

8.2.2 Test for Clostridium sporogenes ATCC 19404 (NCIM 5125)
8.2.2.1 Aseptically filter the product from sample container through Membrane filtration unit.
8.2.2.2 Give washing with suitable volume of 0.1% peptone water (volume to be mentioned in the validation report).
8.2.2.3 Inoculate the final rinse with 10-100 cfu/ml of Clostridium sporogenes ATCC 19404 (NCIM 5125)
8.2.2.4 Transfer the membrane filter in 100 ml of Fluid Thioglycollate Medium tube.
8.2.2.5 Incubate the tube at 32.5 ˚C+ 2.5 ˚C for not more than 3 days.
8.2.2.6 Examine the media daily for evidence of growth.

8.2.3 Test for Candida albicans ATCC 10231 (MTCC 227)
8.2.3.1 Aseptically filter the product from sample container through Membrane filtration unit.
8.2.3.2 Give washing with suitable volume of 0.1% peptone water (volume to be mentioned in the validation report).
8.2.3.3 Inoculate the final rinse with 10-100 cfu/ml of Candida albicans ATCC 10231 (MTCC 227).
8.2.3.4 Transfer the membrane filter in 100 ml of Soyabean Casein Digest Medium tube.
8.2.3.5 Incubate the tube at 22.5 ˚C+ 2.5 ˚C for not more than 5 days.
8.2.3.6 Examine the media daily for evidence of growth.

8.2.4 Test for Environmental isolate
8.2.4.1 .
8.2.4.2 Give washing with suitable volume of 0.1% peptone water (volume to be mentioned in the validation report)
8.2.4.3 Inoculate the final rinse with 10-100 cfu/ml of Environmental isolates.
8.2.4.4 Transfer the membrane filter in 100 ml of Soybean casein digest medium tube.
8.2.4.5 Repeat the above procedure with Fluid Thioglycollate medium.
8.2.4.6 Incubate Fluid Thioglycollate Medium tube at 32.5 ˚C+ 2.5 ˚C for not more than 3 days.
8.2.4.7 Incubate Soyabean casein digest medium tube 22.5 ˚C+ 2.5 ˚C for not more than 5 days.
8.2.4.8 Examine the media daily for the evidence of growth.

8.3 POSITIVE CONTROL :
8.3.1 Transfer 10 – 100 cfu/ml suspension of each test culture and environmental isolate individually in 100ml Fluid Thioglycollate medium or Soyabean Casein Digest medium as per requirement.
8.3.2 Incubate Fluid Thioglycollate Medium tube at 32.5 ˚C+ 2.5 ˚C for not more than 3 days.
8.3.3 Incubate Soyabean casein digest medium tube 22.5 ˚C+ 2.5˚C for not more than 5 days.
8.3.4 Examine the media daily for the evidence of growth.

9.0 ACCEPTANCE
Growth of each test organism in the test container is visually comparable to the growth
in the positive control. If the growth of the test organisms in the test container is not
visually comparable to that in the positive control, then test is repeated by modifying
the method as given below.
9.1 Repeat test using larger number of rinse.
9.2 Change in the type of membrane filter paper used.

10.0 ABBREVATION

MIC. Microbiology
PRO Protocol
SVAL Sterility test Validation
QC Quality Control
IP Indian Pharmacopoeia
BP British Pharmacopoeia
USP United States pharmacopoeia
ATCC American Type Culture Collection
MTCC Microbial Type Culture Collection
NCIM National Collection of Industrial Microorganisms
Q.A. Quality Assurance
No. Number

11.0 CHANGE HISTORY LOG FORMAT

Rev. No.

Details of changes

Reason for change

Effective Date

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