The document outlines procedures and considerations for pharmaceutical sterility testing, including sources of contamination, investigation phases for out-of-specification (OOS) results, and the roles of analysts and supervisors in identifying root causes. It emphasizes the importance of thorough laboratory and manufacturing investigations, particularly in microbiological contexts, and provides guidelines for batch disposition decisions based on OOS findings. The investigation process includes phases for identifying errors, retesting, and implementing corrective and preventive actions, ensuring compliance with regulatory standards.

1. Hardik Mistry
Date : 02/05/2014

2. Sterility

3. http://medicaldesign.com/site-files/medicaldesign.com/files/archive/medicaldesign.com/Whitepapers/SterilityTestin_00000021071.pdf
Pharmaceutical Sterility Testing Essential things to know, Steven Richter February 25, 2008

4. Source of Contamination in the cleanroom
External Internal
Introduction of contaminated outside
air or circulating air *
Staff
Staff Process
Impure process media or raw
materials
Production equipment, machines,
tools etc.
Inadequately cleaned materials, tools
etc.
Unsuitable building materials, work
materials
Mechanical abrasion in the cleanroom
Poor filter quality, not airtight filter seal surfaces, leakage in the ducting
system, abrasion in air recirculation equipment and in the ducting system

5. Cont….
Percentage Distribution of the Sources of Contamination
Process
20 - 30 %
People
30-40 %
Equipment
20 - 30 %
Process Media
5 - 10 %
Air
5 - 10 %

6. Investigation Area
M
M
M
M
M

7. INVESTIGATING STERILITY TEST FAILURES
(OOS)

8. Out Of Specification
• Test results that falls outside or does not comply with the pre-
determined acceptance criteria which have been established in
official compendia and/or company documentation
• (i.e. Filed application, Drug Master Files, Approved marketing
submissions, or Official compendia or Internal acceptance
criteria, Raw material specifications, In-process final product
testing etc.).

9. Laboratory Analysis
OOS (OOT) Result
Phase Ia Investigation
Phase Ib Investigation
Phase II Investigation
Phase III Investigation
Batch
Disposition
Satisfactory
Product Impact
Assessment
Manufacturing
Investigation

10. Phase Ia :
Preliminary Laboratory
investigation
No error
found
Initiate Phase Ib
Laboratory
Investigation
Obvious Error
No further
Investigation Required
Document and
Correct invalid Result

11. Definition
• Phase Ia investigation whether there has been a clear obvious errors
due to external circumstances as power failure or those that the analyst
has detected prior to generating data such as spilling sample that will
negate the requirement of Phase Ib investigation.
• For microbiological analysis this may be after analysis has been
completed and received during reading of the samples.
• It is expected that these issues are trended even if a laboratory
investigation Ib or II was not raised.
Phase Ia Investigation - Obvious Error

12. Obvious Errors
o Calculation error – Analyst and supervisor to review, both initial
and date correction
o Power outage – Analyst and supervisor document the event, “power
failure; analysis to be repeated on all associated analytical
documentation
o Equipment failure – Analyst and supervisor document the event,
annotate “Equipment failure’ analysis to be repeated“ Cross reference
the maintenance record.

13. Cont…
o Testing errors – For example, spilling of the sample, incomplete
transfer of sample, the analyst must document immediately.
For microbiology it could be growth on plate not in the test sample
area, negative or positive control failing
o Incorrect instrument parameters –
For example setting the detector at the wrong wavelength analyst and
supervisor document the event, annotate “incorrect instrument
parameter” analysis to be repeated on all associated analytical
documentation.

14. Phase Ib Investigation
Investigation by Analyst and
Supervisor
No assignable Cause
or
Evidence of Error Remains
Unclear
Contact –
Production/QA/Contract
Giver/QP
Phase II Investigation
Assignable Cause
(Root Cause Identified)
Test Data
Invalidated
Repeat
Analysis
Close Investigation
Generate CAPA
Record Results

15. Phase Ib Investigation - Definitions
Specification :
 Specifications are the critical quality standards and set of acceptance
criteria proposed and justified by the manufacturer and approved by the
regulatory authorities to which a drug substance, drug product or
materials at other stages of its manufacture should conform to be
considered acceptable for its intended use.
 Specification can be list of tests, references to analytical procedures and
appropriate acceptance criteria which are numerical limits, ranges or
other for the tests described.
Specifications are the condition of approval.

16. • Assignable cause – An identified reason for obtaining an OOS or
aberrant/anormalous result.
• No assignable cause – When no reason could be identified.
• Invalidated test – A test is considered invalid when the investigation
has determined the assignable cause.
• Reportable result – It is the final analytical result. This result is
appropriately defined in the written approved test method and derived
from one full execution of that method, starting from the original
sample.
• Warning level or trend execution : If two or more consecutive
samples exceed warning (alert) or if an increasing level of counts, or
same organisms identified, over a short period was identified
consideration should be given to treat results as action excursions.
Cont…

17. Hypothesis/Investigative Testing
It is the testing performed to help, confirm or discount a possible root
cause i.e what might have happened that can be tested.
for example it may include further testing regarding sample
filtration, sonication /extraction; and potential equipment failures etc.
Multiple hypothesis can be explored.

18. Investigation by Analyst and Supervisor
• Conducted using the laboratory investigation checklist.
• Contact production/contract giver/QP/MAH as appropriate.
• For microbiological OOS ensure all items related to the test failure are
retained such as other environmental plates, dilutions, ampoules/vials
of product, temperature data, auto pipettes, reagent- growth media.
• No implicated test environmental plates should be destroyed until the
investigation has been completed.
• The analyst and supervisor investigation should be restricted to
data/equipment/analysis review only.

19. • It may not be all-inclusive, but should be a good guideline to cover the
pertinent areas that need to be covered in any laboratory investigation.
• Correct test methodology and Correct sampling procedure followed eg.
Version number
• Correct sample(s) taken/tested (Check labels was it taken from correct
place)
• Sample integrity maintained, correct container and chain of custody
(was there an unusual event of problem)
• How were sample containers stored prior to use
Checklist

20. • Assessment of the possibility that the sample contamination has
occurred during the testing/re-testing procedure (eg. Sample left open
to air or unattended)
• Review equipment log books
• Appropriate standards and Controls used in the analysis which
performed as expected.
• System suitability conditions met (those before analysis and during
analysis)
• Correct and clean glassware used
• Correct pipette/ volumetric flasks volumes used
• Correct specification applied
Cont…

21. • Media/Reagents prepared according to procedure.
 Items were within expiry date
 A visual examination (solid and solution) reveals normal or
abnormal appearance
• The analyst is trained on the method.
• Interview analyst to assess knowledge of the correct procedure.
• Examination of the raw data, including chromatograms and spectra;
any anomalous or suspect peaks or data.
• Any previous issues with this assay
• Other potentially interfering testing/activities occurring at the time of
the test.
Cont…

22. • Any issues with environmental temperature/humidity within the area
whilst the test was conducted.
• Review of other data for other batches performed within the same
analysis set.
• Consideration of any other OOS results obtained on the batch of
material under test.
• Assessment of method validation.
Cont…

23. Additional consideration for microbiological analysis
• Are the isolates located as expected – on gloves, Finger dab marks, Contact
plates, filter membrane etc
• Was the sample media integral – i.e no cracks in plates
• Was there contamination present in other tests (or related tests) performed at the
same time, including environmental controls
• Were negative and positive controls satisfactory
• Were the correct media/reagents used
• Were the samples integral (Not leaking )
• Were the samples stored correctly (refrigerated)
• Were the samples held for the correct time before being tested.
• Were the media/reagents stored correctly before use
• Were the incubation conditions satisfactory
• Take photographs to document the sample at time of reading (include
plates, gram stains and any thing else that may be relevant)

24. 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 on the testing procedure used for the test in the question
reveals a fault.
(d) After identifying the micro-organisms 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.

25. Phase II
Conducting Failure Investigation
Follow Approved Protocol
Execute Investigation (Hypothesis Testing)
No Assignable Cause
OOS results Obtained
Confirm OOS
Assignable Cause
No further Retest
Report all Analysis
Batch
Disposition
Phase III Investigation
Report
Retest Results
Impact Assessment
Disposition Batch
Invalidate
original result
General
CAPA

26. Unknown Cause
No Assignable Cause
Hypothesis Testing
Re-Test
Re Sampling
Stability
Microbiology
Averaging
Outlier test

27. Phase II Investigation
• Conducted when phase I investigation did not reveal an assignable
laboratory error.
• Phase II are driven by written and approved instruction. Then started
manufacturing investigation to check whether there was a possible
manufacturing root cause..
• If not already notified the contract giver/MAH/QP should be notified
along with production and QA if a manufacturing site.
• Additional testing including the retest performed by the analyst other
than one performed the original test using the predefined retesting plan
and who should be experienced and qualified in the method.
• If the investigation determined analyst error all analysis using the same
techniques performed by the concerned analyst should be reviewed.

28. • Description of the testing should be written, and then approved by QA/Contract
giver/QA equivalent prior to initiating investigational testing and this testing
does not continue with the re measurement of the original preparation and may
not be used to replace the original result.
• Such description should include
 The hypothesis to the test the root cause being investigated
 What samples will be tested
 The exact execution of the testing
 How the data will be evaluated.
• If no assignable cause that could explain the results can be identified during the
manufacturing investigation or the assay failure investigation retesting may be
considered. Part of the investigation may involve retesting a portion of the
original sample.
Phase II Investigation – Unknown Cause/No Assignable Cause

29. • Performed on the original sample not a different sample which can be a 2nd
aliquot from the same sample that was the source of the original failure.
• In case of the insufficient quantity of the original sample remains to perform, for
further testing resample must be done with the approval of the QA/Contract
giver/ QA equivalent and whose records included in laboratory investigation.
• The decision to retest should be based on sound scientific judgement and the
test plan must be approved before re testing occurs which must include the
minimum number of the retests..
• Any statistical review with regards to % RSD and repeatability should relate the
values obtained during method validation (Accuracy, precision, and
intermediate precision) The number of retest should be statistically valid.
• Summarizes : Different sample – Approval of QA – Minimum Retest
Retesting

30. Microbiological Investigation
• The laboratory and manufacturing investigation need to be in depth
because the microbiological investigations are difficult to perform as the
result can be 1 to 2 weeks after the analysis was performed or may be
weeks after the batch was manufactured.
• It you do not determine the boundary of the suspect results it is difficult
to determine if it one or more batches impacted.
• The investigations should clearly state the hypothesis and who will be
responsible for the identified tasks.

31. • Source of the expected organism
• Review of the media
• Review of the Equipments/Utilities
• Environmental Monitoring trend
• Cleaning procedure review
• Disinfectant review
• All the possible root causes
• Review of decisions and actions taken in light of any new information
Microbiological Investigation check list
Due to the variability of microbiological results don’t limit the investigation to the
specific batch it should be broader to review historical results and trends

32. • Use scientific decisions/justifications and risk based analysis.
• Unusual events should be included to understand potential impacts.
• All potential sources of contamination need to be considered – process
flow the issue from sample storage to the test environment.
• Any identification may need to be at DNA/RAN level (bioburden
failures).
• Justification to perform repeat analysis (is sample left) retest or
resample if any.
Cont…

33. Stability – OOS
• Follow the investigation as above for phase I and Phase II.
• For Stability OOS situations regulatory agencies will require notification
within a short time point of discovery because the stability OOS should
link to the products Recall procedures.
• If abnormal results are found at any stability interval which predicts that
the test results may be OOS before the next testing interval, schedule
additional testing before the next scheduled testing interval. This will
help better determine appropriate actions to be taken.
• A case in which an OOT result suggests the potential or likelihood for
OOS results to occur before the expiration date within the same stability
study (or for other studies) on the same product is compliance alert.

34. OOT -OOS
• OOT is when a result is aberrant but within specification limits and
Historical data are needed to identify the OOT alerts.
• To facilitate the prompt identification of potential issues & to ensure
data quality, it is advantageous to use objective (often statistical)
methods that detect potential out-of-trend (OOT) stability data quickly.
• OOT alerts can be classified into three categorized to help identify the
appropriate depth for an investigation. It can be
− Analytical
− Process control and
− Compliance alerts
• As the alert level increases from analytical to process control to
compliance alert, the depth of investigation should increase.

35. Phase III Investigation
• If the batch is rejected, there still needs to be an investigation
 To Determine if other batches or products are affected
 Identification and implementation of corrective and preventive action
• The phase 3 investigation should review the completed manufacturing
investigation and combined laboratory investigation into the suspect
analytical results, and/or method validation for possible causes into the
results obtained.
• In the conclusion all of the results must be evaluated and investigation
report should contain a summary of the investigations performed and a
detailed conclusion.

36. • For microbiological investigations where appropriate use risk analysis
tools to support the decisions taken and conclusions drawn.
• It may not have been possible to determine the actual root cause
therefore a robust most probable root cause may have to be given.
• The batch quality must be determined and disposition decision taken.
• The decision to reject cannot be reversed as a result of further testing.
• The impact of OOS result on other batches, on going stability
studies, validated processes and testing procedures should be
determined by Quality control and Quality Assurance and be
documented in the conclusion along with appropriate corrective and
preventive actions.
Cont…

37. Batch Disposition
A. No Phase I or II obvious error
• If no laboratory or calculation errors are identified in the Phase I and
Phase II, there is no scientific basis for invalidating initial OOS results in
favor of passing retest results.
• All test results, both passing and suspect, should be reported (in all QC
documents and any Certificate of Analysis) and all data has to be
considered in batch release decisions.

38. B. Initial Sampling method inadequate
• If the investigation reveals that the initial sampling method was
inherently inadequate, a new sampling method must be developed
documented and reviewed and approved by the Quality Assurance
responsible for release.
• A consideration should be given to other lots samples by the same
method.
• An initial OOS result does not necessary mean the subject batch fails
and must be rejected.
• The OOS result should be investigated and the findings of the
investigation, including retest results, should be interpreted to evaluate
the batch and reach a decision regarding release or rejection which
should be fully documented.

39. Cont…
C. Where OOS result affect the batch quality
• In those cases where the investigation indicates an OOS result is caused
by a factor affecting the batch quality (i.e an OOS result is confirmed) the
result should be used in evaluating the quality of the batch.
• A confirmed OOS result indicates that the batches does not meet
established standards or specifications and should result in the batch’s
rejection and proper disposition.
• Other lots should be reviewed to assess impact.

40. D. For inconclusive investigations –
in case where an investigation does not reveal a cause for the OOS test
result and
does not confirm the OOS result
the OOS result should be given full consideration with the most
probable cause determined, in the batch or lot disposition decision by
the certifying QP and the potential for a batch specific variation also
needs considering.
• Any decision to release a batch, in spite of an initial OOS result that has
not been invalidated, should come only after a full investigation has
shown that the OOS result does not reflect the quality of the batch by
Quality Assurance/QP.

41. Sterility Failure Investigation

48. THANK YOU
Failure to analyze the failure is real failure.