SOP for Handling of Out of Specification Results in Microbiological Testing

1.0 PURPOSE

To lay down the procedure for handling of out of specification results in microbiological testing.

2.0 SCOPE

It is applicable to handling and investigation of Out Of Specification results in Sterility Test, Microbiological Testing of Non-Sterile Products and Bacterial Endotoxin Tests in Quality Control Microbiology Laboratory.

3.0 RESPONSIBILITY

Quality Control Microbiology
Quality Control Microbiology Officer is responsible for reporting any Out of Specification Results.
Head - Microbiology or his designee is responsible to notify QA and initiate necessary investigations.
To implement necessary corrective actions
Quality Assurance Department
To participate in investigations
To review and approve investigation reports
To review action taken reports
Production Department
To participate in investigations
To implement necessary corrective actions

4.0 ACCOUNTABILITY

Head of Department

5.0 PROCEDURE

5.1 Notification and allotment of number for Out Of Specification Result

5.1.1 On observation of an out of specification (OOS) result in microbiological testing, the microbiologist shall notify the Microbiology In –Charge or his designee and they shall notify QA.
5.1.2 On receipt of a microbiological testing OOS, the details shall be entered in the log book and a number shall be allotted as OOS/M/YY/ZZZZ, where
OOS: Out of Specification
M: Microbiology
YY: Represents last two digits of the current year
ZZZZ: Represents serial number starting with 0001
Example: First OOS reported in the year 2013: OOS/M/13/0001

5.2 Handling and Investigation of Sterility Test Failures

5.2.1 On observation of growth in sterility test media, record the results on the sterility test report and notify the Microbiology In - Charge or his designee. The Microbiology In - Charge or his designee shall notify QA.
5.2.2 Forward the media container showing growth for identification.
5.2.3 If the growth is observed in only one of the media container, continue the incubation of other media container.

5.2.4 Identification of Contaminants

5.2.5.1 Subculture from the container showing growth on Soybean Casein Digest Agar plate, incubate the plate at 30 – 35°C.
5.2.5.2 If growth is observed in Fluid Thioglycollate Medium perform sub-culturing in duplicate and incubate one set in aerobic conditions and one set in anaerobic conditions.
5.2.5.3 Perform the identification of the isolates as per SOP.
5.2.5.4 Store the media container at 2 – 8°C till isolated colonies are obtained and investigation is completed.
5.2.5.5 If more than one type of colony is observed, identify all the types of colonies recovered.
5.2.5.6 Make the subculture of all the isolates and store at 2 – 8°C till the investigation is completed.
5.2.5.7 If the contaminant is slow growing or does not grow on subculturing to solid media, then use other appropriate methods to culture.
5.2.5.8 Similarly identify the colonies from sterility testing area environmental and personnel monitoring plates sampled on the day of test if available or of subsequent days to determine the similarities.
5.2.5.9 For final confirmation of similarities, send the isolates for Genetic Identification to the external agency

5.2.5 Laboratory Investigation

5.2.5.1 Review the microbiological monitoring records of sterility testing area to determine any faults on the day of the test. Also, review the reports of a week prior to and after the day of the test.
5.2.5.2 Review the microbiological monitoring records of laboratory personnel involved in the test to determine any faults on the day of the test. Also, review the reports of a week prior to and after the day of the test.
5.2.5.3 Review the trends for at least last 2 months for an upward trend in microbial load in the sterility testing area.
5.2.5.4 Review the data of non-viable particulate monitoring for any anomalies.
5.2.5.5 Verify the results for negative product control test of the particular test session.
5.2.5.6 Review the sterility test report and verify whether all media and sterile materials used were within their expiration dates.
5.2.5.7 Review the preparation records of all media, rinsing fluid and diluents used in the test. Verify the media containers for sterility check if they are still under incubation or if any containers of the particular lot are available for use.
5.2.5.8 Review the sterilization records of all the materials prepared and sterilized in the laboratory.
5.2.5.9 Review the differential pressure, temperature and relative humidity records (as applicable) for any aberrations or unusual results.
5.2.5.10 Review the cleaning records of the sterility testing area and verify whether correct disinfects and concentration is used and cleaning is performed as per procedure.
5.2.5.11 Review the instrument usage and maintenance logs and verify if any activities were performed that may have an impact on the results. If appropriate verify the current operational status of critical equipment like cRABS, LAF and Steritest Equinox systems.
5.2.5.12 Contact Engineering / Maintenance Department and review the HVAC system logs of sterility testing area for their operation status on the day of sterility test and any maintenance performed or other aberrations.
5.2.5.13 Interview the microbiologist who performed the sterility test and involved in the preparation of materials or any activity associated with the test and verify that:
All the materials are properly disinfected and transferred to the testing area as per procedure or any anomalies observed during the process.
• The condition of the packs of sterilized material was integral or any damages were observed during the transfer and before use.
• The test was performed as per procedure or if any incidents occurred during testing.
If any other anomalies were observed which might impact results.
5.2.5.14 Review the identification results of the isolates from the sterility failure and determine the presence of the identified isolates in the laboratory or production environment. Also compare with identification results from microbiological monitoring of sterility test area, microbiological monitoring of personnel, negative controls or other sources if any.
5.2.5.15 Record all the information in Sterility Failure Investigation Report.
5.2.5.16 Evaluate the information gathered and determine whether the test can be considered invalid as per SOP and pharmacopeias. Only if conclusive and documented evidence shows that the contamination has occurred as part of testing, and then only the test should be considered invalid.
5.2.5.17 If the test is considered invalid, the schedule for a repeat of the test with the same number of units as in the original test. If no evidence of microbial growth is found in the repeat test, the product examined complies with the test.
5.2.5.18 When available evidence does not indicate laboratory error or inconclusive or repeat test shows growth conclude the investigations, as sterility failure and the batch shall be rejected.
5.2.5.19 Proceed to an investigation at the manufacturing facility.
5.2.5.20 If sterility test is considered invalid, take necessary corrective and preventive actions for causes identified.

5.2.6 Manufacturing Facility Investigation

5.2.6.1 Review the microbiological monitoring records of the production facility on the day and a week prior to and after a day of subject batch manufacturing and filling.
5.2.6.2 Review the microbiological monitoring records of production personnel involved on the day and a week prior to and after the day of subject batch manufacturing and filling.
5.2.6.3 Review the trends for at least last 2 months for an upward trend in microbial load in the production facility.
5.2.6.4 Review the non-viable particulate monitoring in the cRABS and background area for any anomalies.
5.2.6.5 Review the cleaning records of the production facility and verify whether correct disinfects and concentration is used and cleaning is performed as per procedure.
5.2.6.6 Review the critical equipment usage and maintenance logs and verify if any activities were performed that may have an impact on the results. If appropriate verify the current operational status of critical equipment like cRABS, LAFs, Depyrogenation tunnel, Filling Machine, Autoclaves, Mobile LAFs, etc.,
5.2.6.7 Contact Engineering / Maintenance Department and review the HVAC system logs of manufacturing facility area for their operation status on the day of batch and any maintenance performed or other anomalies.
5.2.6.8 Review the batch record for following:
• Batch (bulk) manufacturing records and pre-filtration bulk bio-burden data.
• Product and Process air and gas sterilizing grade filter integrity test results.
• Preparation and sterilization records of all components, machine parts and garments.
• Preparation and depyrogenation records of glass vials.
• Batch filling and sealing records (Lyophilization records if applicable).
• Environmental conditions (Differential pressure, Temperature and Relative Humidity) during critical operations.
• Hold times of pre-filtration and filtered bulk against the validated hold times.
• Hold times of sterilized materials against the validated hold times.
• Any deviations or events that could have impacted the critical zones.
5.2.6.9 Review the number of personnel involved/present during critical operations and their training to perform the operations.
5.2.6.10 Review the records batches manufactured previously and after the subject batch for any deviations or events that could have an adverse impact.
5.2.6.11 Review the previous deviations, changes or problems (e.g., process, components, equipment) that may have an impact on sterility assurance of product.
5.2.6.12 Record all the information in Sterility Failure Investigation Report.
5.2.6.13 Evaluate the information gathered and if assignable root cause for sterility failure can be determined. Also, evaluate the impact on other batches manufactured before and after the subject batch.
5.2.6.14 Based on the investigation report, recommend the necessary corrective and preventive actions.

5.3 Handling and Investigation of Out of Specification Results in Microbiological Testing of Non-Sterile Products

5.3.1 Out of specification (OOS) results in the bacteriological testing may be due to high total bacterial count and total fungal count or due to the presence of any specified organism in the sample.

5.3.2 Laboratory Investigation

5.3.2.1 Verify the results for negative control test performed along with the sample analysis. If the negative control results show growth then the test can be considered invalid.
5.3.2.2 Review the preparation records of all media, rinsing fluid and diluents used in the test. Verify the media containers for sterility check if they are still under incubation or if any containers of the particular lot are available for use.
5.3.2.3 Review the sterilization records of all the materials prepared and sterilized in the laboratory.
5.3.2.4 Review the differential pressure, temperature and relative humidity records (as applicable) for any aberrations or unusual results.
5.3.2.5 Review the cleaning records of the laboratory and verify whether correct disinfects and concentration is used and cleaning is performed as per procedure.
5.3.2.6 Interview the microbiologist who performed the test and verify that:
• The sample preparation and testing were performed as per procedure.
• If the sample was received in appropriate sterile container and condition of sample container was proper before testing.
• If any anomalies observed during any stage of testing and incubation
5.3.2.7 Interview the person who performed the sampling and verify that:
• If the product was stored at recommended conditions and containers were intact.
• Sampling is performed as per procedure using sterile sampling tools.
• If any anomalies observed during sampling.
5.3.2.8 Review other activities performed along with subject sample and evaluate if those activities have any impact on test results.
5.3.2.9 If the negative control shows growth or if the out of specification can be conclusively attributed to laboratory error, then the test can be considered invalid.
5.3.2.10 If the test is considered then perform resample and retest.
5.3.2.11 If the retest results comply with acceptance criteria then the batch complies with test requirements.
5.3.2.12 If there is no conclusive laboratory error or if the retest results do not comply then proceed to manufacture facility investigation for finished products. In case of raw materials, communicate the results to the manufacturer.

5.3.3 Manufacturing Facility Investigation

5.3.3.1 Review the results and trends of the applicable water system (purified water and potable water) for an upward trend.
5.3.3.2 Review the concerned batch manufacturing record for following:
• Microbiological results of water used at different stages of batch processing
• Hold times of product at following stages of processing as applicable
o After dispensing of raw materials
o After sifting (API an Excipients)
o After granulation
o After milling and sifting
o After blending
o After compression of tablets before coating
o After coating of tablets
• Hold times of binder solution and coating solutions
• Hold times of cleaned equipment and accessories
• Environmental conditions (Temperature and Relative Humidity) during critical operations.
• Any deviations or events that may have an impact on the microbiological parameter of the product.
5.3.3.3 Review the analytical reports of all the raw materials used in the batch for microbiological results and verify if any of the material showed upward results.
5.3.3.4 Review the records of batches manufactured previously and after the subject batch for any deviations or events that could have an adverse impact on microbiological quality of the product.
5.3.3.5 Review the previous deviations, changes or problems (e.g., process, components, equipment) that may have an impact on microbiological quality of the product.
5.3.3.6 Record all the information in Microbiological Testing of Non-Sterile Products Investigation Report.
5.3.3.7 Evaluate the information gathered and if assignable root cause for can be determined. Also, evaluate the impact on other batches manufactured before and after the subject batch.
5.3.3.8 Based on the investigation report, recommend the necessary corrective and preventive actions.

5.4 Handling and Investigation of Out of Specification Results in Bacterial Endotoxin Test

5.4.1 On obtaining an out of specification result in bacterial endotoxin, notify the Microbiology In-Charge. Do not discard the sample or the sample preparations; they should be saved for an investigational purpose and retesting during laboratory investigation.

5.4.2 Laboratory Investigation

5.4.2.1 Review the analytical report and interview the analyst for following:
• Calculation errors
• Sample preparation details (dilution and concentration)
• LAL reagents preparation details and their validity at the time of use
• Test dilution/concentration used against valid dilution/concentration
• The pyrogen free glassware and accessories used in the test and their status
• Negative water control results
• Any other analytical errors
5.4.2.2 Verify the sample container for any physical damages and its storage. Similarly verify the sampling details for sampling methodology, sampling tools used and their status.
5.4.2.3 If out of specification results are found to be due to a calculation error, then correct the calculations and stop further investigations.
5.4.2.4 If the investigation reveals that out of specification results are due to analytical error, for example, incorrect sample dilutions, use of unsuitable reagents, glassware and accessories or negative water results showing positive, then perform repeat analysis using the same sample after rectification of identified error.
5.4.2.5 If the investigation reveals that out of specification results are due to sampling error, for example, incorrect sampling methodology, incorrect or non-depyrogenated sampling tools used, then perform re-sampling using the correct methodology and depyrogenated sampling tools. Perform testing using re-sample.
5.4.2.6 If the repeat analysis results are within specifications, then the batch shall be released.
5.4.2.7 If the investigation does not reveal any laboratory error, proceed to the investigation at the manufacturing facility.

5.4.3 Manufacturing Facility Investigation

5.4.3.1 Review the results and trends of Water for Injection for any upward endotoxin recoveries.
5.4.3.2 Review the concerned batch manufacturing record for following:
• Batch (bulk) manufacturing records and pre-filtration bulk bacterial endotoxin test results and bio-burden data.
• Preparation and sterilization records of all components, machine parts and garments.
• Preparation and depyrogenation records of glass vials.
• Batch filling and sealing records (Lyophilization records if applicable).
• Environmental conditions (Differential pressure, Temperature and Relative Humidity) during critical operations.
• Hold times of pre-filtration and filtered bulk against the validated hold times.
• Hold times of sterilized materials against the validated hold times.
• Any deviations or events that could have an impact on bacterial endotoxin results.
5.4.3.3 Review the analytical reports of all raw materials used for endotoxin test results and microbiological test results specifically for Gram-negative specified organisms.
5.4.3.4 Review the records batches manufactured previously and after the subject batch for any deviations or events that could have an adverse impact.
5.4.3.5 Review the previous deviations, changes or problems (e.g., process, components, equipment) that may have an impact on quality of the product.
5.4.3.6 Record all the information in Bacterial Endotoxin Test Investigation Report.
5.4.3.7 Evaluate the information gathered and if assignable root cause for bacterial endotoxin out of specification results can be determined.
5.4.3.8 Based on the investigation if any assignable cause can attribute for out of specification results in bacterial endotoxin test, then the subject batch shall be rejected. Also, evaluate the impact on other batches manufactured before and after the subject batch.
5.4.3.9 If the investigation cannot attribute the cause for out of specification results, then proceed for re-sampling and re-testing as follows:

5.4.4 Retesting

5.4.5.1 The re-testing shall be performed by a different analyst other than one performed the initial tests and it shall be performed in triplicate.
5.4.5.2 If the original sample is available, properly stored and suitable, then the retesting can be performed with the original sample. If original sample is not sufficient or suitable, then re-sampling shall be performed for re-testing.
5.4.5.3 If all the results are conforming to the specifications, then the subject batch can be released.
5.4.5.4 If the result does not comply with the specifications, the subject batch shall be rejected. A further investigation shall be conducted to determine the root cause and possible impact on other batches.

5.5 Investigation Flowchart, Investigation Closure and Reporting

5.5.1 An overview of the investigation and decision flow are provided as a flow chart for each investigation. This flowchart provides the logical set of procedural steps to perform an investigation.
5.5.2 The investigation shall be closed within 30 days. If not completed within 30 working days this shall be justified.
5.5.3 The investigation shall be reviewed and approved by Head QA.
5.5.4 If any probable causes for out of specification results are determined at manufacturing, the impact on other batches manufactured should be evaluated.

6.0 ABBREVIATIONS

6.1 SOP - Standard Operating Procedure
6.2 OOS - Out of Specification

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