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SOP for Sterility Testing


Standard operating procedure to ensure the sterility of the Pharmaceutical products.

1.0  OBJECTIVE

       To ensure the that batch of product is sterile or has been sterilized.

2.0  SCOPE

       This procedure is applicable to all parenteral pharmaceutical dosage forms and any specific material for which this test is mentioned in specification.

3.0  RESPONSIBILITY

3.1  Doing       : Technical Assistant (Microbiologist)/Executive
3.2  Checking  : Executive /Manager   

4.0  ACCOUNTABILITY

       Head of the department.

5.0  PROCEDURE

5.1  MEDIA PREPARATION FOR STERILITY TESTING

5.1.1  Fluid Thioglycollate medium as per SOP for Media Preparation.
5.1.2  Soybean casein digest medium as per SOP for Media Preparation.

5.2  GROWTH PROMOTION TEST OF MEDIUM

5.2.1  Perform a growth promotion test as per method.

5.3  STERILITY TESTING PROCEDURE FOR MATERIALS & PRODUCT OTHER THAN INSULIN

5.3.1  Sterilize all required accessories for sterility test in autoclave at 121°C for 30 minutes as per SOP and glassware and forcep/cutter by DHS at 180°C for 2 hrs. as per SOP for Cleaning and Sterilization of Glassware.
5.3.2  Enter in sterility testing room and follow gowning procedure as per SOP
5.3.3  Switch “OFF” the UV light of sterility room and Switch “ON” the tube light.
5.3.4  Operate the LAF as per SOP
5.3.5  Mop the LAF bench with 70% filtered IPA.
5.3.6  Disinfect the outer surface of container of sample with 70% filtered IPA prior to testing and allow it to dry.
5.3.7  Expose the media plates and monitor the area as per SOP for Enverionmental Monitoring.
5.3.8  SAMPLE QUANTITY FOR STERILITY TEST
          For finish product : 20 containers of every batch or every lot.
          For Bulk sample   : 2X50 ml sample in sterile container.
5.3.9  Arrange the filtration assembly in LAF and connect it with vacuum line / vacuum pump.
5.3.10  Place sterile 0.45µ membrane on filter holder with the help of sterile forcep and  clamped it properly.
5.3.11  In another LAF, collect the sample solution from ampoules / vials / bottles in a sterile flask with help of sterile syringe. For dry powder or liophilized container, add the sterile water / 0.1% peptone  dissolve and then collect it in sterile flask.
5.3.12  Filter the collected solution aseptically through 0.45µ membrane filter with help of vacuum.
5.3.13  If the test sample have any anti microbial properties / preservatives, filter with 3X100 ml of freshly prepared  sterile 0.1% peptone as rinsing solution.
5.3.14  Simultaneously perform the sterility test of 0.1% peptone water or any other diluent used for rinsing the filter.
5.3.15  After completion of filtration, open the assembly and cut the membrane filter in two equal parts with sterile cutter.
5.3.16  Inoculate aseptically one half membrane filter with help of sterile forcep in fluid Thioglycollate medium and other half membrane filter in Soybean casein digest medium. Keep one tube of each medium without inoculated  as a –ve control.
5.3.17  Transfer the medium tubes to walk in incubator through hatch.
5.3.18  Incubation time and temperature  is as follow.
            A)  Fluid Thioglycollate medium    : Incubate at 30 – 35°C for not less than 14 days as per required pharmacopoeia.
            B)  Soyabean casein digest medium : incubate at 20 - 25°C for not less than 14 days as per required pharmacopoeia.
5.3.19  The products terminally sterilized by a validated moist heat process, incubate the test specimen for not less than 7 days.
5.3.20  Daily observe the incubated media for presence (turbidity) or absence (clear) of microbial growth and note down the observation as per annexure – I.
5.3.21  Check the growth promoting properties of preservative containing product, bacteriostatic and fungistatic products after completion of incubation period of sterility test as per 5.5 and note down the observation.

5.4  STERILITY TESTING PROCEDURE FOR INSULIN

5.4.1  All steps to be followed as per 5.3.1 to 5.3.17.
5.4.2  In case of suspension, dissolve the insulin crystals in sterile 20% ascorbic acid (Approx  50ml).
5.4.3  Incubation time & temperature is follow.
          A)  Fluid Thioglycollate medium      : 30 – 35°C for NLT 14 Days
          B)  Soybean casein digest medium  : 20 – 25°C for NLT 14 Days
5.4.4  Daily observe the incubated media for presence (turbidity) or absence (clear) of microbial growth and note down the observation as per annexure – I.
Note: For Insulin Finished product, Record the results in Insulin Finished product Data Sheet
5.4.5  Check the growth promoting properties of medium after completion of incubation period 14 days, as per 5.5
5.5  Checking of Growth Promoting Properties Of Medium After Completion Of Incubation Period For Sterility Test.
5.5.1  After completion of incubation period, distribute the each medium in three tubes. Inoculate the test organisms (approx  100 cfu/ml) in respective medium as follow.
1)  Fluid Thioglycollate medium : (For aerobic & anaerobic bacteria)
P.aeruginosa - ATCC – 9027
S.aureus         - ATCC – 6538
Cl.sporogenes - ATCC – 1437
Incubated at 30 – 35°C for 2 – 3 Days.
2)  Soyabean casein digest medium : (For bacteria & fungus)
S.aureus      - ATCC –  6538
C.albicans    -  ATCC – 10231
A.niger         -  ATCC – 16404
Incubated at 20 – 25°C for 5 Days
5.5.2  Acceptance criteria : Medium should be respond to growth within its incubation period.
5.5.3  Observed the incubated media tubes for growth promotion and note down the observation.
Note: For Insulin Finished product, Record the results in Insulin Finished product Data Sheet

5.6  BLIND TEST (CONTROL TEST)

5.6.1  Perform a sterility test with using sterile water for injection.
5.6.2  Follow the steps 5.3.1 to 5.5.4.
5.6.3  Record the observations.
5.6.4  Frequency : Conduct this test once in a week and cross check by other person every six month.
5.6.5  Acceptance Criteria : The test sample should  complies in sterility test
5.6.6  Interpretation of results for bind test.
         1)  If the sample passes in sterility test, the analyst is qualified to perform a sterility test.
         2)  If the sample fails in sterility test, the analyst require retraining for sterility testing.

5.7  Observation and interpretation for Sterility test

5.7.1  If no evidence of growth is found, the preparation being examined passes the test for sterility.
5.7.2  If evidence of Microbial growth is found, reserve the container showing this, and unless and it is demonstrated by any other means that their presence is due to causes unrelated to the preparation being examined, then the test for sterility is invalid and perform a retest on the same number of the sample.
5.7.3  If no evidence of microbial growth is found on retesting, the preparation being examined passes the test for sterility.
5.7.4  If the evidence of microbial growth is found on retesting, the preparation being examined fails the test for sterility.
5.7.5  As per IP If no evidence of microbial growth is found the preparation being examined complies with the test for sterility.
5.7.6  If evidence of microbial growth is found the preparation being examined does not complies with tests for sterility. Do not repeat the test unless it can be clearly shown the test was invalid for causes unrelated to the preparation being examined.
5.7.7  The test may consider invalid only when one or more of the following conditions are fulfilled.
5.7.7.1  If the microbial growth found in negative control.
5.7.7.2  Data on microbial monitoring of the sterility testing facility show a fault.
5.7.7.3  A review of the testing procedure used for the test in question reveals a fault.
5.7.7.4  After identifying the micro organisms isolated from the containers showing microbial growth, the growth may be ascribed without any doubt to faults with respect to the material and / or the techniques used in conducting the test procedure.
5.7.8  If the test is declared to be invalid repeat with the same numbers of unit as in original test. It no evidence of microbial growth is found in the repeat test the preparation being examined complies with the test for sterility. It microbial growth is found in the repeat test and confirmed microscopically, the preparation being examined does not comply with the tests for sterility.

6.0  ABBREVIATIONS

6.1  ATCC = American Type culture collection
6.2  IP = Indian Pharmacopoeia 
6.3  LAF = Laminar Air Flow
6.4  UV = Ultra Violet
6.5  IPA = Isopropyl alcohol


ANNEXURE – I

Quality Control Department

Microbiology  Laboratory
RECORD OF STERILITY TEST BY MEMBRANE FILTRATION.
 S.O.P No.:- ___________
  
Product Name.:-
Batch. No.:-
Mfg.Date.:-
Exp.Date.:-
Batch. Size.:-
Sample.:- Bulk / Finished
Sample Qty.:-
S.T.R.No.:-
Preservative:-
Date of Testing.:-
LAF-I ID.No.:-
LAF-II ID.No.:-

Medium Name.
Temp
pH
Lot. No.
Medium – A   Fluid Thioglycollate (100 ml) for aerobes & Facultative Anaerobes
30°C- 35°C


Medium – B   Soybean Casein Digest Broth (100 ml) for Aerobes & Fungi
20°C – 25°C



Day
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Remarks
Date.:















Medium A
Test Lot















Medium B
Test Lot















Medium Control















Peptone
Control















Ascorbic Acid control















Signature















Positive control _________

+ Ve Growth Observed
- Ve Growth Not Observed.

Remarks :- The Product Complies / Does not Comply the sterility test as per IP/ BP / EP / USP





Analyst
(Date & Sign)
Date of Report
(Date & Sign)
Checked By
(Date & Sign)






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