Drug Master File Some Significant Results

Overview of the Validation Elements

Accuracy is the closeness of the test results obtained by the RMM to the value obtained by the compendia or reference method. Accuracy is demonstrated across the practical range of the test, and is usually expressed as the percentage of recovery of microorganisms by the method.

Precision is the degree of agreement among individual test results when the procedure is applied repeatedly to multiple samplings of suspensions of microorganisms across the range of the test. Precision is usually expressed as the variance, standard deviation or coefficient of variation of a series of measurements.

Specificity is the ability to detect a range of microorganisms demonstrating that the method is adequate for its intended purpose, and may also include an evaluation of whether the method will be susceptible to false positives or interference from components in the sample, other microorganisms, diluents or media (e.g., detecting dead microorganisms, or detecting certain species of microorganisms when the same species should not be detected [inclusivity and exclusivity]). When conducting tests for sample interference, background noise should be nonexistent or at an acceptable low level, and the sample components should not interfere with the detection of a known microorganism challenge.

 

Limit of Detection is the lowest number of microorganisms in a sample that can be detected under the stated experimental conditions. The test may also determine the presence or absence of microorganisms in the original sample.

 

Ruggedness is the degree of precision of test results obtained by analysis of the same samples under a variety of normal test conditions, such as different analysts, instruments, lots of reagents, laboratories, etc. It can also be considered as the intrinsic resistance to the influences exerted by operational and environmental variables on the results of the method.

Robustness is a measure of a method’s capacity to remain unaffected by small but deliberate variations in method parameters.  Parameters such as sample volume, instrument temperature and medium volume in the vial were tested.

 

Comparison to USP <61> and <62>

Total Aerobic Count: (i) naturally contaminated samples:  Two hundred and one product-samples were analyzed for total aerobic count at three specification levels.  Fifty samples were inoculated.  All un-inoculated samples were below the specified level by both methods. There was 100% agreement between the two methods. This indicates the ability of the BioLumix system to yield equivalent results to the plate count method over the range of 10 cfu/g to 1,000 cfu/g.

(ii) Inoculated products: Fifty-nine tests samples were inoculated with all the organisms sited in USP <61at various levels in the range of 10-100,000 cfu/ml. Ten inoculated samples that had counts below the specified level and all were correctly classified by the BioLumix system.  There was 100% agreement between the two methods in determining that the number of organisms found in each sample were either above or below the specification level of <10-100,00 cfu/ml. The data shows the ability of the BioLumix system to determine if a sample is above or below the specified level at the range of 10-100,000 cfu/ml.

Yeast and Molds:  (i) naturally contaminated samples: One hundred forty-seven product samples were analyzed for Yeast and Mold Count at two specification levels.  Forty-four samples were inoculated.  All un-inoculated samples were below the specified level by both methods. There was 100% agreement between the two methods. This indicates the ability of the BioLumix system to yield equivalent results to the plate count method over the range of 100 cfu/g to 10 cfu/g.

(ii) Inoculated products:  Thirty-three samples were inoculated with Aspergillus niger, Candida albicans, or Saccharomyces cerevisiae at various levels in the range of 10-1,000 cfu/ml.  E. coli was tested as a challenge organism that should not grow in this medium.  Fifteen inoculated samples that had count below the specified level and all were correctly classified by the BioLumix system.  The E. coli did not grow in the medium and did not detect by the instrument.  There was 100% agreement between the two methods in determining that the number of organisms found in each sample were either above or below the specification level of <10-1,00 cfu/ml. The data shows the ability of the BioLumix system to determine if a sample is above or below the specified level at the range of 10-1,000 cfu/ml.

Gram Negative Bile Tolerant (Enterobacterial counts): (i) naturally contaminated samples: One hundred forty-seven product samples were analyzed for Enterobacterial Count at two specification levels.  Forty-four samples were inoculated.  All un-inoculated samples were below the specified level by both methods. There was 100% agreement between the two methods. This indicates the ability of the BioLumix system to yield equivalent results to the plate count method over the range of 100 cfu/g to 10 cfu/g.

(ii) Inoculated products: Thirty-six samples were inoculated with Citrobacter freundii, Escherichia coli, or Salmonella enteritidis at various levels in the range of 10-1,000 cfu/ml.  Staphylococcus aureus was tested as a challenge organism that should not grow in this medium.  The samples were tested for Enterobacterial Count.  The S. aureus did not grow in the medium and did not detect by the instrument.  There was 100% agreement between the two methods in determining that the number of organisms found in each sample were either above or below the specification level of <10-1,00 cfu/ml. The data shows the ability of the BioLumix system to determine if a sample is above or below the specified level at the range of 10-1,000 cfu/ml.

Specificity

Total Aerobic Count: one hundred and twenty five well diverse bacteria were inoculated into the TAC vial. All organisms tested were detected in the TAC vials and DT data were provided by the BioLumix instrument and software.

Yeast and Molds:  Forty two yeast and molds were tested in the YM vial.  All detected in the vial.  Twenty-five strains of various bacteria were also tested in the YM vial and none of them detected in the vial.  Therefore, inclusivity and exclusivity has been demonstrated for the YM vial.

Gram Negative Bile Tolerant (Enterobacterial counts): Fifty-two bacteria belonging to the Enterobacteriaceae family were tested.  All of the organisms were detected in the ENT vial.  Twenty-three gram negative strains and 28 gram positive strains of bacteria strains that do not belong to the family Enterobacteriaceae were also tested in the Enterobacteriaceae vial and none of them were detected in the vial.  Therefore, inclusivity and exclusivity have been demonstrated for the ENT vial.

Similar results were obtained for all other BioLumix vials.

Detection Limit

Total count:  The data showed that the limit of detection for the BioLumix system is nearly equivalent to or slightly more sensitive in detecting low numbers of microorganisms as directly compared to the plate count method.  However, the difference between the two methods is not statistically significant by theChi Square test. The lower limit of detection is approximately 1-2 organisms per sample vial.

Yeast and Mold Count:  The data showed that the limit of detection for the BioLumix system is equivalent to the plate method in detecting low numbers of yeast or mold as directly compared to the plate count method.

Gram Negative Bile Tolerant: The data showed that the limit of detection for the BioLumix system is equivalent to the plate method in detecting low numbers of Enterobacteriaceae as directly compared to the plate count method.

Precision or Repeatability of Data

Precision or Repeatability of DataTotal count:  Seventy-one quadruplicates sets were tested in the BioLumix system, with seven different types of bacteria.  The average Standard Deviation (SD) for the total count medium was 0.27 hours, the average Coefficient of Variation was 7.07%.

Yeast and Mold Count:   Seven yeast strains and eight mold stains were tested in quadruplicates, some of them multiple times.  The detection times found for individual microorganisms tested using quadruplicate vials were found to be similar within the YM test.  The average Standard Deviation (SD) for the yeast and mold was 0.39 hours.  It is similar to that found for bacteria.  Molds had longer detection times and an average SD of 0.66 hours and the average Coefficient of Variation was 4.72%.

Gram Negative Bile Tolerant: Twenty-three Enterobacteriaceae strains were tested in quadruplicates.  The detection times found for individual microorganisms tested using vials were found to be similar within this test.  The average Standard Deviation (SD) for the Bile tolerant Gram negative (ENT) vial was 0.17hours, the average Coefficient of Variation was 2.94%.

Similar results were obtained for other BioLumix vials such as E. coli, S. aureus, P. aeruginosa, and Salmonella.

 

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