Out of Specification Result in Microbiology – Guideline

Standard Operating Procedure (SOP) for Handling of Out of Specification (OOS) Test Result in Microbiological Analysis. OOS results include all test results that fall outside of the specification or acceptance criteria established in drug applications, drug master files, official compendia, or by the manufacturer.

Handling of (Microbiology) Out-of-Specification (OOS) Results

1.0   Purpose

    • The objective of SOP is to define and describe the requirements for handling of Microbiology Laboratory Out of Specification (OOS) test result.
    • This involves the execution of a detailed sequential investigation process that includes documenting results, performing additional testing needed to evaluate Out of Specification result(s), completing product impact and risk assessments, regulatory notification (if applicable), making a final conclusion, completing material disposition, and Corrective and Preventive Action (CAPA).

2.0   Scope

    • This document reflects the current regulatory policies regarding the investigation and evaluation of microbiological Out of Specification (OOS) test results.
    • The scope includes Out of Specification (OOS) results for in-house testing of incoming material, manufactured raw materials and finished products, and laboratory work performed by the contract laboratory.
    • This SOP is applicable to test results determined to be out of specification during sample testing, including:
      • Microbiology Laboratory testing associated with regulated drug products/drug substances for distribution (inclusive of Exhibit Batches).
      • This includes testing performed on Raw and Packaging Materials, In- Process Materials, Semi-Finished/Intermediates, and Final Products for drug substances and drug products.
    • This SOP is applicable to microbiological Out of Specification results of tests, including, but not limited to,

      • Bio-Assay,
      • Sterility,
      • Antimicrobial Effectiveness Bacterial Endotoxin,
      • Aseptic Process Simulation (media fill),
      • Total Microbial Aerobic Count,
      • Total Combined Yeast and Mold Counts, and
      • Specified Microorganisms in Finished Products (DP and API), Raw Materials, Water (DI, PW, WFI, and Raw Water/Potable Water).
    • Wherever gram-negative bacteria handling is involved during Out of Specification management of Microbiological Results, applicable general procedures shall be followed.
    • Exclusions:

    • Analytical laboratory Out of Specification results of tests. These are addressed in a separate SOP for Handling of Out-Of-Specification (OOS) Results.
    • Product complaint samples sent by Complainant but evaluated by QA to be not fit for testing and/or having an “unknown” or “unacceptable” handling/storage.
    • Testing performed as part of the Product Development.
    • Field/Market Samples for which the storage conditions and transportation along with authenticity are not traceable.
    • Microbial environmental monitoring (Active Air Sampling, Passive Air Sampling, and Personnel Monitoring).
    • Microbial monitoring of Factory Garments, Compressed Air, and Clean Room Drain.

3.0   Procedure for Handling of Out of Specification Result

  • Investigation of Out of Specification (Microbiology) Result

  • After obtaining an Out of Specification (OOS) result, the Microbiologist /Analyst/Initiator who performed the initial test shall immediately inform the Functional Supervisor that an OOS result occurred.
  • The Functional Supervisor and the Microbiologist /Analyst/Initiator shall immediately check whether the Out of Specification result is due to an “Obvious Error” that will negate the requirement for further investigations Some examples of “Obvious Errors” are as follows:
    • Calculation/Transcription Error:

    • The Microbiologist /Analyst and Functional Supervisor shall review for calculation/transcription. If an error is found, corrections shall be made as per the SOP- Good Documentation Practices (GDP).
    • Power Outage:

    • The Microbiologist /Analyst and Supervisor shall document the event, annotate as “power failure. analysis to be repeated”, where applicable on all associated analytical documentation.
    • Equipment Failure:

    • The Microbiologist /Analyst and Supervisor shall document the event, annotate it as “equipment failure; analysis to be repeated”.
    • The maintenance record shall be cross-referenced with this The equipment shall be tagged as being “out of service” until it is returned to a validated state.
    • Incorrect Instrument Parameters:

    • For example, incorrect setting of the dry bath temperature. In such a case, the Microbiologist/Analyst and Functional Supervisor shall document the event, annotate it as “incorrect instrument parameter, analysis to be repeated” on all associated analytical documentation and it shall be logged as Unplanned Deviation/Incident (refer to the current version of SOP – Deviations/Incidents).
    • If the cause of the Out of Specification is determined to be an “obvious error”, the concerned Microbiologist/Analyst/Initiator shall take necessary steps, as specified above, and also make corrections in the Analytical Raw Data Sheet/Notebook by following the SOP for Good Documentation Practices.
    • If the root cause is determined to be an “obvious error”, no further Out of Specification investigation is necessary, since the “OOS” result in such a case can be considered as “Invalid”.
    • However, it shall be logged as an “Unplanned Deviation/Incident” and processes as per the Unplanned Deviation / Incident workflow (refer to the current SOP – Deviations/Incident) version.
  • Initiation of Out of Specification (OOS) Record. 
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    • If “obvious error” is not the root cause of the OOS, as verified based on the parameters specified in the earlier section, the Microbiologist/Analyst/Initiator shall initiate an “OOS Record” (either electronically or manually, as per the tracking system available).
    • The following information shall be documented during initiation of the OOS Record and
    • Each OOS Record shall be assigned a unique identification number:

      • Short description of the Out of Specification
      • Name of the reporting Microbiologist/ Analyst
      • Due date for Out of Specification closure (30-calendar days from the discovery date of OOS)
      • Department, Area, etc.
      • Standard Test Procedure (STP) Number
      • Specification Number
      • Stage
      • Test
      • Details of affected samples (batch numbers, test specification, status of the batch, sample type, stability station, reference raw datasheet, and market)
      • Date of analysis
      • Discovery date of Out of Specification
    • The Microbiologist / Analyst/Initiator shall log these details and submit the “OOS Record” to the Functional supervisor.
    • If an Out of Specification result is obtained for a stability sample/control sample of a batch(s) distributed in the market, a written/electronic notification shall be sent to Regulatory Affairs (RA), Manufacturing Group, Site QA Group, and Head-QA/QC.
    • This notification shall occur within 24 hours of the discovery of the initial Out of Specification result even if the Phase I Preliminary Laboratory Investigation for laboratory error has not been completed.
    • Out of Specification Reporting to Regulatory Agencies:

      • OOS reporting to Regulatory Agencies shall be performed, as applicable, based on regional and/or site-specific procedures and the guidance documents of the applicable regulatory authorities.
    • After the creation of an “OOS Record”, a sequential detailed Phase I Investigation process shall be conducted to determine the root cause.

      • Each step of the investigation process shall be clearly defined, including the number of replicates and the outcome of each investigational step shall be evaluated to determine whether the root cause has been identified and the investigation can be concluded or if additional investigation is necessary, and the appropriate course of action to be taken in the next investigational step.
      • If the investigation cannot be concluded after the completion of an investigational step. the next sequential step shall be performed in order to determine the root cause.
      • As part of the continuance to the next investigational step. the outcome of the previous step shall be summarized and, if applicable, a detailed analysis plan inclusive of the purpose of action shall be chosen based on the outcome of the previous investigative
      • Management oversight during every step of the investigation process is essential to ensure that the purpose of each investigational step is scientifically substantiated and approved prior to its execution in order for a timely, thorough root cause investigation to be performed.

PHASE I – Preliminary Laboratory Investigation

    • Phase I Laboratory Investigation shall be conducted by the Functional Supervisor with the concerned Microbiologist / Analyst/Initiator on a paper form or validated electronic versions thereof (refer to Attachment I)
    • Example Phase I Laboratory Investigation Report.) The purpose of this investigation is to determine whether the Out of Specification result is caused by a laboratory error.
    • The Phase I Investigation shall be completed within three working/business days from the date of discovery of Out of Specification result (except sterility failures).
    • In case there is a need to extend the investigation, the Functional Supervisor shall prepare an interim report with reason and justification for This shall be approved by the QA/QC Designee.
    • During the Phase I Laboratory Investigation, the following shall be evaluated, but not limited to:

    • Discussion of the test method with the Microbiologist / Analyst to confirm the Microbiologist / Analyst’s knowledge/competency.
    • Evaluate Microbiologist/ Analyst training/qualification.
    • Examination of raw data obtained in the analysis, printouts, temperature charts, etc., to identify anomalously or suspect information.
    • Confirm the performance of any instrument(s) used in the test.
    • Determine that appropriate working/reference standards, media, reagents, and other solutions were used and that they meet relevant acceptance criteria.
    • Compare the test method performed against approved procedures.
    • Evaluate system suitability data, where applicable.
    • Evaluate measurements, calculations, conversion factors, and formulas associated with testing and reagent/media preparation.
    • Examine glassware used in the preparation of samples and reagents/media.
    • Evaluation of other tests performed on the batch in question.
    • Identification and evaluation of any unusual events, malfunctions, or unexpected circumstances associated with the test environment.
    • Evaluation of test results obtained from the same sample type and from the same instrument run.
    • Inspection of the work area to determine if any environmental or facility conditions could have adversely impacted the testing.
    • Additionally, during a Phase I Microbiology Investigation, the following test-specific requirements shall also be evaluated:
    • Bacterial Endotoxin Test (Out of Specification Results):

      • Evaluation of validity of reagents.
      • Depyrogenation of glassware used in the preparation of samples and reagents/media.
      • Evaluation of product positive control, product negative control, etc.
      • Calculations in case of Kinetic Turbidimetric LAL reagent (KTA) analysis.
      • Confirm if Dry Heat Sterilizer was calibrated/qualified, and its temperature checked on a routine basis.
      • Confirm if the heating block was calibrated and displays the correct temperature.
      • Ensure/Confirm if (Limulus Amebocyte Lysate) (LAL)/Control Standard Endotoxin/LAL Reagent water was stored at the correct temperature.
      • Confirm if reconstituted lysate/Control Standard Endotoxin/LAL Reagent water were within the shelf life.
      • Confirm if the test for confirmation of labeled lysate sensitivity of the LAL reagent was within the acceptance criteria.
    • Sterility Test (Out of Specification Results):

      • Examination of sterilization of components used in the preparation of samples and reagents/media.
      • Confirm if glassware/media/garments were sterilized, as per the validated sterilization cycle.
      • Evaluate/Confirm if the manometer reading of the Laminar Air Flow hood (LAF) is within the acceptance criteria.
      • Confirm if the sample containers were dried after sterilization.
      • Evaluation of product positive controls, negative control, etc.
      • Evaluation of test results obtained from other material of the same sample type and from the same instruments, review of other samples tested on the same day, and preceding and succeeding day results by the same Microbiologist/Analyst.
      • Environmental monitoring of sterility testing facility (including personnel monitoring data) during the test period.
      • Confirm if the Growth Promotion Test (GPT) results of the media used in the test and negative control were as per acceptance criteria.
    • Bio-Assay (Out of Specification Results):

      • Check for any error in the preparation, handling, and storage of samples.
      • Confirm if the Zone Reader/Vernier Caliper was calibrated.
      • Confirm if culture identity, performance, and passage number meet the requirement.
    • Microbiological Examination of Non-Sterile Products:

    • Evaluate if the differential pressure of the dynamic pass box was within the acceptance criteria
    • Evaluate if the differential pressure/manometer reading of the LAF was within acceptance criteria
    • Determine if the microbial environment monitoring of LAF count and Laboratory area count observation was as per acceptance criteria
    • Confirm if the media was sterilized, as per validated sterilization cycle.
    • Confirm if the samples were incubated at correct temperature, as stated in the procedure.
    • Check if the culture media pH observation was as per acceptance criteria.
    • Confirm if culture identity, performance, and passage number meet the Pharmacopeial requirements.
    • Comparison with previous trend, whether any excursions beyond limits were received in the recent past.
    • Result of Growth Promotion Tests (GPT) on Media used, evaluation of positive and negative control, incubation conditions,, etc.
    • Confirm if sampling tools used for sampling were sterilized.

    • After completing the Phase I Laboratory Investigation, the Functional Supervisor shall determine if a laboratory error occurred or not, and subsequently forward the OOS Record to the Head Microbiology /Head Quality Control (HQC) for review.
    • If the root cause of the OOS remains undetermined, the Functional Supervisor shall document in the OOS Record, e., that the cause of OOS result is “unknown at this stage”.
    • The HQC/ Head Microbiology shall review the OOS Records and, if needed, request additional details from the Functional Supervisor.
    • The HQC/ Head Microbiology shall review the reason for OOS as documented by the Functional Supervisor, and edit/update/comment, It may also attach additional documents, if needed.
    • If the root cause for OOS was not clearly identified previously by the Functional Supervisor, i.e., whether it is due to a laboratory error or not, the HQC/Head Microbiology shall make a final judgment on the same and document the decision in the record.
    • Post review, the HQC / Head Microbiology shall forward the OOS Record to the QA Designee for review.

    • The QA Designee shall examine all the related records of Preliminary Laboratory Investigation along with conclusions/recommendation made by the Functional Supervisor / HQC / Head If needed, the QA Designee may request further details from HQC / Head Microbiology.
    • Based on the Phase I Laboratory Investigation records, the QA designee shall make the final determination whether the root cause of OOS was caused by a Laboratory Error.
    • If the result of Phase I Laboratory Investigation of the OOS is: “No Laboratory Error Found”/”No Assignable Root Cause Found”, then the investigation shall continue to a Full Scale Investigation, as in section 6.
    • If the result of Phase I Laboratory Investigation of the OOS Result is: “Laboratory Error Found”/”Assignable Root Cause Found”, follow the steps explained in section 5.
  • CAUSE OF OOS – “LABORATORY ERROR” / “ASSIGNABLE ROOT CAUSE” FOUND:

    • If “Laboratory Error” or “Assignable Root Cause” is established during the Phase I Laboratory Investigation, the QA Designee shall perform a Root-Cause Analysis in order to identify the root cause so that CAPA may be initiated accordingly.
    • Additionally, a Retest shall be conducted to confirm the investigation findings, where applicable.
  • CAPA Plan for Phase I:

    • A CAPA of similar nature is already in place – in such a case, a cross-reference to the existing CAPA shall be given.
    • CAPA is not needed based on justifiable parameters – in such a case, a “No CAPA Required” justification shall be provided.
    • Re-Test Plan and Reporting of Results: Re-test shall be initiated by QA and the Re-test plan shall be communicated by the QA designee to the Responsible Person selected to perform the Re-test to confirm the “Laboratory Error”. Supporting evidence in combination with other evidences that may be used to invalidate original results may include, but not limited to:
    • Re-test results that do not confirm with the original result such as, when inadequate extraction/dilution of the sample is observed.
    • Product history and results of other tests from the same sample that do not confirm the original result.
    • The determination that the sample is not representative of the product or material. A specimen template for Re-Test Report is given in Attachment The test specific Re-test requirements are described below:
  • Microbiological Examination of Non-Sterile Products:

    • Re-testing shall be performed by two Microbiologists/Analysts, each in duplicate on fresh aliquot of products/material (typically prepared and analyzed in the same manner that generated the original OOS result).
    • The Microbiologist/ Analyst who had performed the original testing shall be preferably selected as one of the Microbiologist/Analysts. If the results of Re-Test meet the specification limit (individually), then the average of the Re-test result shall be reported as final value for the material release.
    • The original OOS shall be invalidated.
    • In case of sample constraint for Re-Test, special instructions shall be part of the task record, created by QA.
    • Bio-Assay:

    • Re-testing shall be performed by two Microbiologists/ Analysts, each in duplicate, on a fresh aliquot of products/material (typically prepared and analyzed in the same manner that generated the original OOS result).
    • The Microbiologist/Analyst who had performed the original testing shall be preferably selected as one of the Microbiologist / Analysts.
    • If the results of Re-Test meet the specification limits (individually), then the re-test results shall be averaged and reported as a final value for the material release. The original OOS shall be invalidated.
    • Sterility Testing:

    • A test may be repeated only when it can be demonstrated that the test was invalid for causes unrelated to the product being examined, restricted to one of the criteria as follows:
      • Data of   the   microbiological monitoring of the sterility testing facility (which includes personnel monitoring data) shows a fault.
      • Review of the testing procedure used during the test in question reveals a fault.
      • Microbial growth is found in the negative controls.
    • After determination of the identity of the micro-organism isolated from the test, the growth of this species (or these species) may be ascribed unequivocally to faults with respect to the Material and/or the technique used in conducting the sterility test procedure.
  • Conclusion and Batch Disposition:

    • Based on investigation, if it is evident that the root cause of “OOS” result from original testing is due to “Laboratory Error”, and if the Re-Test results by the Microbiologist / Analyst(s), both individual and average, meet the specifications the original OOS results shall be substituted with the average of the Re-test results as explained in each test. all data, individuals and average must be reported in the testing.
    • Additionally, a notation shall be included in the Certificate of Analysis (COA) documenting that an OOS result occurred for the specific test referencing the unique investigation The average value may be assigned to the batch on the COA, but the individual values shall be listed as part of the OOS note.

 

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