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SOP for Investigation of Sterility Failure to Find Out the Root Cause

Standard operating procedure to investigate the root cause of sterility failure when the sterile sample fails in sterility test.


To lay down a Procedure is to provide guidelines for investigation on sterility failure, which is required to find out the root cause, and to take the appropriate actions for future analysis.


This procedure is applicable for investigating the sterility test failure.




Head QA / QC


5.1 If evidence of microbial growth is observed during observation of incubated tube of the batch, the batch tested does not meet the requirement of sterility test. Do not repeat the test unless it can be clearly shown that the test was invalid for causes unrelated to the preparation under examination.
(a) Microbial growth is found in the negative controls.
(b) Data on microbial monitoring of the sterility testing facility show a fault.
(c) A review of the testing procedure used for the test in the question reveals a fault.
(d) After identifying the microorganisms isolated from the containers Showing microbial growth may be ascribed without any doubt to faults with respect to the materials and/ or technique used in conducting the test procedure.
5.2 Reason for positive result must be investigated as per the following.
5.2.1 Preserve the positive tube along with positive and negative control till to complete investigation.
5.2.2 Immediately streak the loop full suspension from sterility positive tubes on the pre-incubated SCDA plate in a duplicate.
5.2.3 If the suspension is streaked from FTM, incubate the plates at 30 to 35°C for 48 hrs.
5.2.4 If the suspension is streaked from SCDM, incubate the plates at 20 to 25°C for 72 hrs.
5.2.5 After Incubation, observe the cfu and find out the colony by staining techniques or other biochemical techniques.
5.2.6 Simultaneously check the results of microbiological monitoring taken while performing sterility test and during manufacturing and filling of the suspected product.
5.2.7 Identify the colony found during the plate exposure on the testing date and also the microorganism found in a preserved positive tube.
5.2.8 Also check the area cleaning and sanitization record for sterility testing room and manufacturing area.
5.2.9 Check the sterilization cycle details of the media, filtration assembly and utensils which is used for sterility testing including sterilization temperature, pressure, holding time, chemical sterilization indicator.
5.2.10 Carefully review the manufacturing steps; special care has to be taken for sterilization of bung & machine parts and depyrogenation of vials.
5.2.11 Simultaneously review the sterility testing procedure and training of sterility test operator.
5.2.12 Compile all the above data and find out the root cause.
5.2.13 If colony observed during Sterility testing is the same characteristics as found in the positive tube, there are chances of contamination during testing. The test may be considered invalid and repeat the test using the same quantity of sample.
5.2.14 If no evidence of microbial growth is found in the repeat test, the preparation under examination complies with the test of sterility. If microbial growth is found in the repeat test and confirmed microscopically, the preparation under examination does not comply with the test for sterility.

5.3 Precaution

5.3.1 Each step must be review carefully and record the investigation in details.
5.4 Limits/ Acceptance Criteria
5.4.1 If the investigation report shows fault in sterility test operator or any environmental or personnel cross-contamination during the sterility test, repeat the test using the same number of containers which is used for the first test.
5.4.2 If the investigation report shows fault during manufacturing or filling or during sterilization cycle of a suspected product, do not repeat the test and reject the batch.


SOP: Standard Operating Procedure
QA: Quality Assurance
QC: Quality Control
NA: Not Applicable
FTM: Fluid Thioglycollate Medium
SCDM: Soybean casein digest medium
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