PROTOCOL APPROVAL
Signing
of this approval page of protocol indicates agreement with the Testing approach
described in this document. If any process, procedure changes protocol shall be
prepared and approved. This protocol cannot be executed until signed and
approved by following personnel
Quality Control--Microbiology Execute, Participate and provide necessary support for the validation activity. Preparation and review of the validation Report of the documents and its compliance to meet the acceptance criteria of the protocol.
Quality Assurance--Monitoring the validation activities, compilation, review and authorization of the method validation report and its compliance to meet the acceptance criteria of the protocol.
1.PURPOSE
To establish a protocol test for the Validation of Sterility testing by membrane filtration method will produce the consistent results analyzed as per the standard operating procedure.
2. SCOPE
The scope of the test extends to the Validation of Sterility testing by membrane filteration method
3. RESPONSIBILITY
The responsibility for executing the protocol shall be with the technical director under whose guidance the analysis shall be carried out. The following persons shall be involved in the execution of the protocol.
Head Quality Control:
Microbiologist 1 :
Microbiologist 2 :
4. DOCUMENT REFERENCE:
1. Sterility test validation USP
5.PROCEDURE FOR ANALYSIS:
Equipment:
* Analytical balance, able to determine 0.1 mg
* Conical flasks, Volumetric flasks, 100ml, 50 ml and 20 ml
* Magnetic stirrer or ultrasonic bath
* Micropipette
* Petriplates
* Test tubes
* LAF
* Autoclave
* Dry heat sterilizer
Reagents & Materials:
* Sodium chloride
* Soyabean casein digest broth
* Soyabean digest agar
* Sabouraud Dextrose Agar
* Fluid thioglycollate medium
Organisms:
Pseudomonas aeruginosa-ATCC 9027;
Staphylococcus aureus-ATCC 6538;
Escherichia coli-ATCC 8739
Candida albicans-ATCC 10231;
Aspergillus niger-ATCC 16404;
Pseudomonas aeruginosa-ATCC 9027;
Staphylococcus aureus-ATCC 6538;
Escherichia coli-ATCC 8739
Candida albicans-ATCC 10231;
Aspergillus niger-ATCC 16404;
Preparation of Inoculum: Prepare the Slants and incubate for 24 hours for pre incubation.
Take 24 hours active culture slant transfer all five cultures and incubate respective temperatures mentioned in the table.
|
Name of the organism |
Medium |
Incubation temperature |
Incubation period |
|
Escherichia coli |
Soyabean Casein Digest agar |
32.5˚±2.5˚C |
24 hours |
|
Pseudomonas aeruginosa |
Soyabean Casein Digest agar |
32.5˚±2.5˚C |
24 hours |
|
Styphylococcus aureus |
Soyabean Casein Digest agar |
32.5˚±2.5˚C |
24 hours |
|
Candida albicans |
Sabouraud Dextrose Agar |
22.5˚±2.5˚C |
3 days |
|
Aspergillus niger |
Sabouraud Dextrose Agar |
22.5˚±2.5˚C |
6 to 10days |
Name of the organism | Medium | Incubation temperature | Incubation period | Observation | |
Positive | negetive | ||||
Escherichia coli | Soyabean Casein Digest agar | 32.5˚±2.5˚C | 24 hours | ||
Pseudomonas aeruginosa | Soyabean Casein Digest agar | 32.5˚±2.5˚C | 24 hours | ||
Styphylococcus aureus | Soyabean Casein Digest agar | 32.5˚±2.5˚C | 24 hours | ||
Candida albicans | Sabouraud Dextrose Agar | 22.5˚±2.5˚C | 24 hours | ||
Aspergillus niger | Sabouraud Dextrose Agar | 22.5˚±2.5˚C | 24 hours | ||
Validation of Sterility Test by Membrane Filtration method is done by following procedures
A. Test for Residual Antimicrobial Activity
The Test for Residual Antimicrobial Activity is carried out the test procedure as described in general sterility test, up to the final wash procedure. To the final wash add an inoculum of viable cells of the specific bacteria and fungi. After the final wash with the added microorganisms has been passed through the filter, inoculate filter paper in FTM & incubate at 30 to 35ºC or in SCDM and incubate at 20 to 25ºC as per table 1.
Growth of each of the added microorganisms should be apparent within 48 hrs. If conspicuous growth does not occur within 3 days for bacteria and 5 days for fungi, the
Test procedure is not
valid and must be modified.
B. Test for Antimicrobial Activity
To demonstrate that the mixture does not manifest antimicrobial activity, carry out the test as described in sterility test procedure, up to the incubation step and add an inoculum of viable cells of the specific bacteria and fungi respectively to FTM and SCDM and incubate at 30 to 35ºC and 20 to 25ºC respectively.
Growth of each of each of the added microorganisms should be apparent within 48 hrs. If conspicuous growth does not occur within 3 days for bacteria and 5 days for fungi, the test procedure is not valid and must be modified
C. Stasis Test (Efficacy of the test media at the end of incubation period)
The Stasis Test is designed to demonstrate that the media (i.e. FTM and SCDM) inoculated with the test preparations will support growth for full incubation period. After incubation of the media has been completed in accordance with the instruction given in the sterility test for negative control, add to representative tube containing FTM that has been incubated at 30-35ºC, an inoculum of viable cells of specific bacteria. To the next tube containing SCDM that has been incubated at 20-25ºC, add an inoculum of viable cells of specific fungi. Return all the inoculated tubes to their previous temperature and incubation continued.
All the tubes should show growth of added microorganisms within 48 hours. If conspicuous growth does not occur within 3 days for bacteria and 5 days for fungi, the test is considered invalid.
A. TEST FOR RESIDUAL ANTIMICROBIAL ACTIVITY)
Objectives:
The test is performed to ensure that; any residual of Antimicrobial Activity is satisfactory eliminated by using the steps mentioned in this protocol.
Procedure:
Acceptance Criteria
1. If the conspicuous growth is observed within 3 days for bacteria and 5 days for fungi, and the growth of each challenge microorganisms in the Positive Product control containers are visually comparable to the growth in the positive control and there is no growth in Negative control & Negative Product control, the product possess no antimicrobial activity under the condition of the test or such a activity has been satisfactory eliminated. The test for sterility may be carried out routinely without further modifications.
2. If the conspicuous growth is not observed within 3 days for bacteria and 5 days for fungi, or growths of each test organism in the Positive Product Control containers are visually not comparable with positive control containers respectively, the product possesses antimicrobial activity that has not been satisfactory eliminated under the conditions of the test. Modify the conditions in order to eliminate the antimicrobial activity and repeat the validation test. (i.e. by using additional washes)
† Negative Product Control Test: -
The result of negative product control test facilitates the interpretation of sterility test results, particularly when used to declare a test invalid because of contamination in negative product control. The essential element of the negative control is to simulate the testing method.
B. TEST FOR ANTIMICROBIAL ACTIVITY
Objectives:
The test is performed to ensure that, the absence of Antimicrobial Activity under the experimental conditions.
Procedure:Acceptance Criteria:
1. Growth of each of the added microorganisms in Product Control should be apparent within 3 days for bacteria and 5 days for fungi & should be comparable to positive controls.
2. If growth does not occur within 3 days for bacteria and 5 days for fungi, the test procedure is not valid and must be modified (e.g. using additional washes) until growth does occur when tests as above are carried out.
3. Growth should be absent in Negative Controls.
C. STASIS TEST (Efficacy of the test Media at the end of Incubation Period)
Introduction:
The Stasis Test is designed to demonstrate that the media (i.e. FTM and SCDM) inoculated with the test preparations will support growth for full incubation period. It is also necessary to demonstrate that growth-promoting qualities of media are retained and stable for the full test period.
Objectives:
The test is performed to ensure that, the growth promoting qualities of fluid thioglycollate and Soyabean casein digest media are stable for the full test period.
Procedure:
1. Carry out the negative control for FTM (4 tubes) and SCDM (3 tubes) along with the Test for Residual of Antimicrobial Activity and Test for Antimicrobial activity without any inoculation up to the incubation Period.
2. After incubation, observe all the negative control of each medium i.e. FTM and SCDM for absence of growth.
3. After confirmation of absence of growth, add aseptically 10 to 100 viable cells of microorganisms in their respective tubes as described in Table – I.
4. Return the tubes to their previous temperature and continue the incubation for NMT 3 days for bacteria and NMT 5 days for fungi.
5. Observation: - daily observe the tubes for evidence of microbial growth by means of turbidity.
Acceptance Criteria:
1. The test is valid if the growth of each of the added microorganisms observed within 3 days for bacteria and 5 days for fungi.
2. If growth is not observed within 3 days for bacteria and 5 days for fungi the test is considered invalid.
10.0 METHOD FOR
PREPARATION OF CELL SUSPENSION
10.1 Prepare
soybean casein digest Agar medium & Sabouraud Dextrose agar medium in a
conical flask as per Media Preparation SOP.
10.2 Prepare 0.9 %
w/v solution of Sodium chloride with distilled water in a conical flask &
transfer to the test tubes in the following manner (for one microorganism): 9
ml Normal Saline containing 5 test tubes, 45 ml Normal Saline containing 1 test
tube & 90 ml Normal Saline containing 3 test tubes, plug the tubes with
cotton plug and sterilize in an autoclave at 121ºC temperature for 20 minutes.
10.3 Sterilized
Petri plates in hot air oven at 180ºC for 1hour.
10.4 Perform the
activity of serial dilution in the microbiological room.
10.5 Remove
culture slant from the refrigerator i.e. MC1/M1/W1/D1, and allow it to attain
ambient temperature.
10.6 Transfer 1 ml
of sterile normal saline to the slant of freshly grown culture, mix using
inoculation loop and suspend it in 9 ml sterile Normal Saline. Homogenize the
suspension by gentle shaking.
10.7 Prepare
serial dilutions (by diluting each time 1 ml of culture suspension with 9 ml of
Normal Saline) up to 10-4 dilution, from 10-4 dilution
take 5 ml and add it to 45 ml sterile Normal Saline(10-5) & from
10-5 dilution take 10 ml and add it to 90 ml sterile Normal
Saline (10-6) & repeat the procedure till 10-8 dilution.
10.8 Preserve the
original undiluted culture suspension & 10-5 to 10-8 dilutions
in a refrigerator between 2ºC to 8ºC.
10.9 Aseptically
pour 1 ml each of 10-3 to 10-8 dilution of each
organism in each of the sterilized dry petridishes.
10.10
Aseptically pour in each plate about 20 ml sterilized
Soyabean Casein Digest agar medium for bacterial culture and sterilized
Sabouraud Dextrose agar for fungal culture, previously liquified and cooled to
around 45ºC, mix and allow the medium to solidify. Incubate the Plates
at 32.5±2.5ºC for 24 to 48 hours for bacterial culture and 22.5±2.5ºC for
48 to 72 hours for fungal culture.
10.11 At the end
of incubation count the number of Colony Forming Units developed on each of the
petridishes.
10.12 Select the
culture suspension containing 10-100 cfu/ml and preserve in refrigerator
between 2ºC to 8ºC.
10.13 A volume of
individual suspensions so prepared equivalent to about 100 cells of each
organism per ml shall be used for Sterility Test Validation.
11.0 PRECAUTION
1. Validation
tasks are to be carried out by trained personnel using techniques and
equipment, which minimize the risk of accidental microbial contamination of the
test and of the testing environment.
2. Validation
tests should be conducted in Sterility Test Room and positive control tests in
Microbiological testing room under LAF.
3. Personnel
conducting sterility testing or associated aseptic manipulations should wear
sterilized garments.
4. All equipment,
vessels and materials, which are used for validation of sterility test, should
be sterilized by autoclaving or by dry heat sterilization.
12.0 DISCREPANCY AND CORRECTIVE ACTION REPORT
Document any
discrepancies observed during the validation in annexure -1. Include the
corrective actions of the same. When all the discrepancies are satisfactorily
resolved or an approved action plan is developed which ensures that the
discrepancy will be resolved.
13.0 COMPILATION, REVIEW AND SUMMARY REPORT
Compile and review that
all test procedures have been completed, reconciled and attached to this
protocol. Verify that the approvals for deviations have been taken and are
resolved appropriately to the satisfaction.
Testing method
validation shall be considered acceptable when all the conditions specified in
the test procedures have been met.
REPORT
: The tests are conducted with different organisms
and the concentration
CONCLUSION: The
conclusion is drawn as per the log reduction reports of the microbial growth of
different organisms in the product.
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