Explaining the conditions when Sterility test may be considered invalid?

 Hi Friends,

Today, I am going to explain you very important point of sterility testing. I ask this question in interview but a very few candidates can give clear answer. So, I think I have to explain this here on the blog so that it can be helpful for everyone. 

We know that when we have growth in any of SCDM and FTM media in sterility analysis, we consider that product is fail. But there are certain condition in which we can consider that the test is invalid.

Here important thing is understanding the meaning of invalid. In any case, if test is fail we can not repeat the tests but if sterility test is considered as invalid it means we can repeat the sterility test with same number of units as used in original test. On the basis of below mentioned conditions we can invalid the test.

1. The data of microbiological monitoring of the sterility testing facility show a fault.

Explanation: If environment monitoring results of sterility testing area on the day of sterility testing show a fault means results found fail then sterility testing can be considered as invalid.

2. A review of testing procedure used during the test in question reveals a fault.

Explanation: It means after getting sterility failure results we have to initiate the investigation. During investigation we need to review the procedure. During review of sterility testing procedure if we found any fault in sterility test then sterility test can be considered as invalid.

3. Microbial growth is found in the negative control.

Explanation: During sterility testing, we use media negative control (NC) and negative diluting fluid control (NDFC). Media negative control is used to check any contamination in the media after sterilization and media negative control incubated for 14 days along with test sample. Negative diluting fluid control is used to verify the testing conditions in which we only filter the diluent used during the sterility testing. NDFC also incubated for 14 days along with test sample. If growth is found in any of negative control or negative diluting fluid control, the test may be considered as invalid because there may be problem with the media used during sterility testing or problem with the testing conditions.

4. After determination of the identity of the microorganisms isolated from the test, the growth of the species may be ascribed unequivocally to faults with respect to the material or technique used in conducting sterility test procedure.

Explanation: After identification of the contaminating microorganism up to species level, if identified microorganisms species is same as identified from the material used in sterility testing or from the environment of sterility testing area or personnel monitoring then sterility test considered as invalid.

With proper investigation and based on scientific justification, we can repeat the sterility test by using same number of units as we used in the original test. If no evidence of microbial growth is found in repeat test then sample complies for the sterility test. If growth is found in the repeat test then sample does not comply for test of sterility.

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How sterility test method validation performed and what is the purpose of sterility test method validation?

In sterility testing, we test different types products like general products and antibiotics etc. Every product is having different type of nature with respect to its mode of action. In case of antimicrobial product they can kill the contamination if present in the product and we are not able to detect the contamination inside the product. We may get false negative results and fail product can be released into the market. It may become threat to patient safety.

To avoid this situation we first need to perform the sterility test method validation. In sterility test method validation, we perform the sterility of the product, positive product control (PPC) and positive control (PC). In sterility testing of product, product is reconstituted by rinsing fluid A,D or K as per product requirement and filter through 0.45 micron filter. Rinsing shall be given with the diluent. Basically 100 mL of pre-wetting and 3x100 mL final rinsing shall be given. After that cut the filter paper into two equal halves and one half shall be inoculated in SCDM media and another half shall be inoculated in FTM media. Sample shall be incubated for up to 14 days. In case of PPC, sample shall be tested as in case of product by giving 2x100 mL rinsing of fluid A,D or K depending upon the product and in last 1x100 ml of rinsing not more than 100 cfu per 0.1 mL microorganisms specified in the pharmacopoeia shall be inoculated. Membrane filter shall be cut into two equal halves and incubated in respective media as per the used microorganisms. PPC sample shall be incubated for not more than 5 days. In case of PC, product is not reconstituted and filtered. In PC, 2x100 mL rinsing of fluid A,D or K depending upon the product shall be given and in last 1x100 ml of rinsing not more than 100 cfu per 0.1 mL microorganisms specified in the pharmacopoeia shall be inoculated and filtered.  PC also incubated for not more than 5 days. No growth shall be observed in product up to 14 days of period. Comparable growth of PPC with PC shall be observed. If comparable growth observed it means either product does not contain any antimicrobial property or antimicrobial property of the product has been successfully removed. 

In sterility method validation we need to validate the number of rinsing to remove any antimicrobial property of the product so that it does not inhibit the growth of microorganisms which may be transferred into the product through the process. 

If antimicrobial property of the product can not be removed by rinsing only then neutralizing agents by proving its efficacy and toxicity or different rinsing fluid can be used.

Thanks