Out of Specification

MHRA
MEDICINE AND HEALTHCARE
PRODUCTS REGULATORY AGENCY

M.KOUPPARIS 1
M.KOUPPARIS 2
 Laboratory Analysis (1)
• Investigations of "Out of Specification (OOS) /
Out of Trend (OOT)/ Atypical -results" have to
be done in cases of:
– Batch release testing and testing of starting
materials.
– In-Process Control testing: if data is used for
batch calculations/decisions and if in a
dossier and on Certificates of Analysis.
– Stability studies on marketed batches of
finished products and or active pharmaceutical
ingredients, on-going / follow up stability (no
M.KOUPPARIS 3
stress tests)
 Laboratory Analysis (2)
– Previous released batch used as
reference sample in an OOS investigation
showing OOS or suspect results.
– Batches for clinical trials.

M.KOUPPARIS 4
 Laboratory Analysis (3)
• All solutions and reagents must be retained until
all data has been second person verified as
being within the defined acceptance criteria.
• Pharmacopoeia have specific criteria for
additional analyses of specific tests (i.e.
dissolution level specification for S1, S2 & S3
testing; Uniformity of dosage units specification
for testing of 20 additional units; Sterility
Testing).

M.KOUPPARIS 5
 Laboratory Analysis (4)
• However if the sample test criteria is
usually the first level of testing and a
sample has to be tested to the next
level this should be investigated as it is
not following the normal trend.

M.KOUPPARIS 6
 Laboratory Analysis (5)
• The OOS process is not applicable for In-
process testing while trying to achieve a
manufacturing process end-point, i.e.:
- adjustment of the manufacturing process.
(e.g. pH, viscosity),
- for studies conducted at variable
parameters to check the impact of drift
(e.g.process validation at variable
parameters).
M.KOUPPARIS 7
 OOS / OOT Result (1)
• Out-of-Specification (OOS) Result
– Test result that does not comply with the
predetermined acceptance criteria, for
example:
- filed applications, drug master files,
approved marketing submissions,
- official compendia
- internal acceptance criteria

M.KOUPPARIS 8
 OOS / OOT Result (2)
• Out-of-Specification (OOS) Result
- Test results that fall outside of
established acceptance criteria which
have been established in official
compendia and/or by company
documentation:
- Raw Material Specifications
- In-Process/Final Product Testing,
- etc.
M.KOUPPARIS 9
 OOS / OOT Result (3)
• Out of Trend (OOT) Result
– Is generally a stability result that does
not follow the expected trend,
- either in comparison with other
stability batches
- or with respect to previous results
collected during a stability study.

M.KOUPPARIS 10
 OOS / OOT Result (4)
• Out of Trend (OOT) Result
However the trends of starting materials and in-
process samples may also yield out of trend
data.
The result is not necessarily OOS but does not
look like a typical data point.
Should be considered for environmental trend
analysis, such as for viable and non viable data
(action limit or warning limit trends)

M.KOUPPARIS 11
 OOS / OOT Result (5)
• Atypical / Aberrant / Anomalous Result
– Results that are still within specification
but are unexpected, questionable,
irregular, deviant or abnormal.
- Examples would be chromatograms that
show unexpected peaks,unexpected
results for stability test point, etc.

M.KOUPPARIS 12
M.KOUPPARIS 13
 Phase Ia Investigation – Obvious
Error (1)
Examples
• Calculation error
- analyst and supervisor to review, both initial and date
correction.
• Power outage
- analyst and supervisor document the event, annotate
“power failure; analysis to be repeated” on all associated
analytical documentation.
• Equipment failure
- analyst and supervisor document the event, annotate
“equipment failure; analysis to be repeated” cross
reference the maintenance record.
M.KOUPPARIS 14
 Phase Ia Investigation – Obvious
Error (2)
• Testing errors
- for example, spilling of the sample solution, incomplete
transfer of a sample; the analyst must document
immediately.
- for microbiology it could be growth on a plate not in the
test sample area, negative or positive controls failing.

• 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.
M.KOUPPARIS 15
Phase Ia Investigation – Obvious
Error (3)
If no error was noted, and none of the
above conditions were met Phase Ib
investigation must take place.

M.KOUPPARIS 16
Phase Ib Investigation –Definitions
(1)
• Specification
- A specification is defined as a list of
tests, references to analytical procedures,
and appropriate acceptance criteria which
are numerical limits, ranges, or other
criteria for the tests described.
- It establishes the set of criteria 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. M.KOUPPARIS 17
Phase Ib Investigation –Definitions
(2)
• Specification
- “Conformance to specification” means
that the drug substance and drug product,
when tested according to the listed
analytical procedures, will meet the
acceptance criteria.
- Specifications are critical quality
standards that are proposed and justified
by the manufacturer and approved by
regulatory authorities as conditions of
approval.
M.KOUPPARIS 18
Phase Ib Investigation –Definitions
(3)
• Regulatory Approved Specification
- Specifications for release testing. If no release
specifications have been established then the
internal specification becomes the release
specification.

• Acceptance Criteria
- Numerical limits, ranges, or other suitable
measures for acceptance of the results of
analytical procedures which the drug substance
or drug product or materials at other stages of
their manufacture should meet.
M.KOUPPARIS 19
Phase Ib Investigation –Definitions
(4)
• Internal Specification
- Are also action limits within regulatory
specifications.
• Assignable Cause
- An identified reason for obtaining an
OOS or aberrant/anomalous result.
• No Assignable Cause
- When no reason could be identified.

M.KOUPPARIS 20
Phase Ib Investigation –Definitions
(5)
• Invalidated test
- A test is considered invalid when the
investigation has determined the assignable
cause.
• Reportable result
- 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.

M.KOUPPARIS 21
Phase Ib Investigation –Definitions
(6)
• Warning Level or Trend excursions
- 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 the results as action level
excursions.

M.KOUPPARIS 22
Phase Ib Investigation –Definitions
(7)
• Hypothesis/Investigative Testing
- Is 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.

M.KOUPPARIS 23
 Phase Ib
Investigation by Analyst and Supervisor (1)

• Phase Ib Investigation
– Initial Investigation conducted by
the analyst and supervisor using the
Laboratory Investigation Checklist

• Contact Production/Contract
Giver/QP/MAH as appropriate
M.KOUPPARIS 24
 Phase Ib
Investigation by Analyst and Supervisor (2)
• For microbiological analysis where possible
once a suspect result has been identified ensure
all items related to the test failure are retained
such as other environmental plates, dilutions,
ampoules/vials of product, temperature data,
autopipettes, reagents – growth media.
No implicated test environmental plates should
be destroyed until the investigation has been
completed.

M.KOUPPARIS 25
 Phase Ib
Investigation by Analyst and Supervisor (3)
• The Analyst and Supervisor investigation should
be restricted to data / equipment /analysis
review only
• On completion of the Analyst and Supervisor
investigation re-measurement can start once
the hypothesis plan is documented and is
only to support the investigation testing.
• This initial hypothesis testing can include the
original working stock solutions but should not
include another preparation from the original
sample (see: re-testing)
M.KOUPPARIS 26
 Phase Ib
Investigation by Analyst and Supervisor (4)
• The checklist 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 followed e.g.. 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 or
problem).
- How were sample containers stored prior to use.
- Correct sampling procedure followed e.g. version
number.
M.KOUPPARIS 27
 Phase Ib
Investigation by Analyst and Supervisor (5)
- Assessment of the possibility that the sample
contamination has occurred during the testing/ re-testing
procedure (e.g. sample left open to air or unattended).
- All equipment used in the testing is within calibration
date.
- Review equipment log books.
- Appropriate standards used in the analysis.
- Standard(s) and/or control(s) 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.
M.KOUPPARIS 28
 Phase Ib
Investigation by Analyst and Supervisor (6)
-Media/Reagents prepared according to
procedure.
- Items were within expiry date
- A visual examination (solid and solution)
reveals normal or abnormal
appearance
- Data acceptance criteria met
- The analyst is trained on the method.
- Interview analyst to assess knowledge of the
correct procedure.

M.KOUPPARIS 29
 Phase Ib
Investigation by Analyst and Supervisor (7)
- 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.
- 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.
M.KOUPPARIS 30
 Phase Ib
Investigation by Analyst and Supervisor (8)
• Additional considerations for microbiological
analysis:
- Are the isolates located as expected
– on glove dab marks, SAS ‘dimples’, 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.

M.KOUPPARIS 31
 Phase Ib
Investigation by Analyst and Supervisor (9)
- 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.
- Was the media/reagent stored correctly before use
- Were the incubation conditions satisfactory.
- Take photographs to document the samples at time of
reading (include plates, gram stains and any thing else
that may be relevant).

M.KOUPPARIS 32
M.KOUPPARIS 33
 Phase II Investigation (1)
• Conducted when the phase I investigations did not reveal
an assignable laboratory error.
- Phase II investigations are driven by written and
approved instructions against hypothesis.
- Prior to further testing a manufacturing
investigation should be started to determine whether
there was a possible manufacturing root cause.
• If not already notified the contract giver/MAH/QP (in
accordance with the responsibilities in the TA) should be
notified along with production and QA if a manufacturing
site.

M.KOUPPARIS 34
 Phase II Investigation (2)
• It is important when considering performing
additional testing that it is performed using a
predefined retesting plan to include retests
performed by an analyst other than the one who
performed the original test.
- A second analyst performing a retest
should be at least as experienced and
qualified in the method as the original
analyst.
• If the investigation determines analyst error all
analysis using the same technique performed by
the concerned analyst should be reviewed.
M.KOUPPARIS 35
 Phase II Investigation (3)
• Hypothesis/Investigative Testing
- Is 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.
M.KOUPPARIS 36
 Phase II Investigation (4)
• Re-Test
- Performing the test over again using material from the
original sample composite, if it has not been
compromised and/or is still available.
- If not, a new sample will be used.
• Re-sample
- A new sample from the original container where
possible, required in the event of insufficient material
remaining from original sample composite or proven
issue with original sample integrity.
• Most probable cause
- Scientifically justified determination that the result
appears to be laboratory error.

M.KOUPPARIS 37
M.KOUPPARIS 38
 Phase II
Conduct Laboratory Failure Investigation (1)
• Investigations of "Out of Specification (OOS) / Out of
Trend (OOT)/ Atypical -results" have to be done in cases
of:
– Batch release testing and testing of starting materials.
– In-Process Control testing: if data is used for batch
calculations/decisions and if in a dossier and on
Certificates of Analysis.
– Stability studies on marketed batches of finished
products and or active pharmaceutical ingredients, on-
going / follow up stability (no stress tests)
– Previous released batch used as reference sample in
an OOS investigation showing OOS or suspect results.
– Batches for clinical trials.

M.KOUPPARIS 39
 Phase II
Conduct Laboratory Failure Investigation (2)
• All solutions and reagents must be retained until all data
has been second person verified as being within the
defined acceptance criteria.
• Pharmacopoeia have specific criteria for additional
analyses of specific tests (i.e. dissolution level
specification for S1, S2 & S3 testing; Uniformity of
dosage units specification for testing of 20 additional
units; Sterility Testing).
• However if the sample test criteria is usually the first
level of testing and a sample has to be tested to the
next level this should be investigated as it is not
following the normal trend.

M.KOUPPARIS 40
 Phase II
Conduct Laboratory Failure Investigation (3)
• The OOS process is not applicable for In-
process testing while trying to achieve a
manufacturing process end-point i.e.:
- adjustment of the manufacturing
process. (e.g. pH, viscosity),
- and for studies conducted at variable
parameters to check the impact of drift
(e.g. process validation at variable
parameters).

M.KOUPPARIS 41
M.KOUPPARIS 42
 Phase II Investigation
Unknown Cause / No Assignable Cause (1)
Hypothesis Testing (Applicable to Phase Ia and
Phase II):
• Should be started as part of Phase Ia and
continue into Phase II if no assignable
cause found.
• Description of the testing should be written, and
then approved by QA/Contract Giver/QA
equivalent prior to initiating investigational
testing.
The requirements of investigational testing
listed below:

M.KOUPPARIS 43
 Phase II Investigation
Unknown Cause / No Assignable Cause (2)
• The description must fully document
– 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
• This Hypothesis testing may continue from the re-
measurement of the original preparations.
• Investigational testing may not be used to replace an
original suspect analytical results.
- It may only be used to confirm or discount a probable
cause.

M.KOUPPARIS 44
 Phase II Investigation
Unknown Cause / No Assignable Cause (3)
• 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.

M.KOUPPARIS 45
 Phase II Investigation
Unknown Cause / No Assignable Cause (4)
• Retesting:
– Performed on the original sample not a different
sample.
– Can be a 2nd aliquot from the same sample that was
the source of the original failure.
– If insufficient quantity of the original sample remains to
perform all further testing then the procedure for
obtaining a resample must be discussed and agreed by
QA/Contract Giver/QA equivalent.
- The process of obtaining the resample should be
recorded within the laboratory investigation.

M.KOUPPARIS 46
 Phase II Investigation
Unknown Cause / No Assignable Cause (5)
• Retesting:
– The decision to retest should be based on
sound scientific judgement. The test plan
must be approved before re testing occurs.
– The minimum number of retests should be
documented within the procedure and be based
upon scientifically sound principles.
- Any statistical review with regards to
%RSD and repeatability should relate to the
values obtained during method validation
(accuracy, precision, and intermediate
precision).
M.KOUPPARIS 47
 Phase II Investigation
Unknown Cause / No Assignable Cause (6)
• Retesting:
- The number of retests should be
statistically valid; papers have suggested
5, 7, or 9.
– The retests should be performed by a
different analyst where possible. The
second analyst should be at least as
experienced and qualified in the method
as the original analyst.
M.KOUPPARIS 48
 Phase II Investigation
Unknown Cause / No Assignable Cause (7)
Re-sampling:
– Should rarely occur!
– If insufficient quantity of the original sample
remains to perform all further testing then the
procedure for obtaining a resample must be
discussed and agreed by QA/Contract Giver/QA
equivalent.
- The process of obtaining the resample
should be recorded within the laboratory
investigation.

M.KOUPPARIS 49
 Phase II Investigation
Unknown Cause / No Assignable Cause (8)
Re-sampling:.
– Re-sampling should be performed by the
same qualified methods that were used for
the initial sample.
- However, if the investigation
determines that the initial sampling
method was in error, a new accurate
sampling method shall be developed,
qualified and documented.
M.KOUPPARIS 50
 Phase II Investigation
Unknown Cause / No Assignable Cause (9)
Re-sampling:.
– It involves the collecting a new sample from the
batch.
– Will occur when the original sample was not truly
representative of the batch or there was a
documented/traceable lab error in its
preparation.
– Evidence indicates that the sample is
compromised or invalid.
– Sound scientific justification must be employed if
re-sampling is to occur.

M.KOUPPARIS 51
 Phase II Investigation
Unknown Cause / No Assignable Cause (10)
• Averaging:
– The validity of averaging depends upon the sample and
its purpose.
- Using averages can provide more accurate results.
- For example, in the case of microbiological assays,
the use of averages because of the innate variability
of the microbiological test system.
-The kinetic scan of individual wells, or endotoxin data
from a number of consecutive measurements,
- or with HPLC consecutive replicate injections from the
same preparation (the determination is considered one
test and one result), however, unexpected variation in
replicate determinations should trigger investigation and
documentation requirements.
M.KOUPPARIS 52
 Phase II Investigation
Unknown Cause / No Assignable Cause (11)
• Averaging:

- Averaging cannot be used in cases when

testing is intended to measure variability
within the product, such as powder
blend/mixture uniformity or dosage form
content uniformity.

M.KOUPPARIS 53
 Phase II Investigation
Unknown Cause / No Assignable Cause (12)
• Averaging:
- Reliance on averaging has the disadvantage of hiding
variability among individual test results.
- For this reason, all individual test results should
normally be reported as separate values.
- Where averaging of separate tests is appropriately
specified by the test method, a single averaged result
can be reported as the final test result.
- In some cases, a statistical treatment of the variability
of results is reported.
- For example, in a test for dosage form content
uniformity, the standard deviation (or relative standard
deviation) is reported with the individual unit dose test
results.
M.KOUPPARIS 54
 Phase II Investigation
Unknown Cause / No Assignable Cause (13)
• Averaging:
– In the context of additional testing performed during an
OOS investigation, averaging the result (s) of the original
test that prompted the investigation and additional retest
or resample results obtained during the OOS
investigation is not appropriate because it hides
variability among the individual results.
- Relying on averages of such data can be particularly
misleading when some of the results are OOS and
others are within specifications.
- It is critical that the laboratory provide all individual
results for evaluation and consideration by Quality
Assurance (Contract Giver/QP).

M.KOUPPARIS 55
 Phase II Investigation
Unknown Cause / No Assignable Cause (14)
• Averaging:
- All test results should conform to
specifications
(Note: a batch must be formulated with the
intent to provide not less than 100 percent of
the labelled or established amount of the
active ingredient.
– Averaging must be specified by the test
method.
– Consideration of the 95% Confidence Limits (CL
95% ) of the mean would show the variability
when averaging is used.
M.KOUPPARIS 56
 Phase II Investigation
Unknown Cause / No Assignable Cause (15)
• Averaging:
– Consideration of using 95% Confidence
Limits (CL 95% ) of the mean would show
the variability when averaging is used.

• The confidence interval is calculated from

the formula:

M.KOUPPARIS 57
M.KOUPPARIS 58
 Phase II Investigation
Unknown Cause / No Assignable Cause (16)
Outlier test:
– An outlier may result from a deviation from
prescribed test methods, or it may be the
result of variability in the sample.
- It should never be assumed that the
reason for an outlier is error in the testing
procedure, rather than inherent variability
in the sample being tested.

M.KOUPPARIS 59
 Phase II Investigation
Unknown Cause / No Assignable Cause (17)
Outlier test:
– Statistical analysis for Outlier test results can be as part
of the investigation and analysis.
- However for validated chemical tests with relatively
small variance and that the sample was considered
homogeneous it cannot be used to justify the
rejection of data.
– While OOS guidance is not directly intended for bioassay
analysis, it can be used as a starting point for the
investigation.
- Compendia such as the BP; PhEur and USP, provide
guidance on outliers for these types of analysis.

M.KOUPPARIS 60
 Phase II Investigation
Unknown Cause / No Assignable Cause (18)
Stability – OOS/OOT:
• Stability OOS/OOT situations should be escalated as soon as
the suspect result is found.
– Follow the investigation as above for Phase I and Phase II.
– For OOS Situations Regulatory agencies will require
notification within a short time point of discovery due to
recall potential.
• 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.
• The stability OOS should link to the Product Recall procedures.

M.KOUPPARIS 61
 Phase II Investigation
Unknown Cause / No Assignable Cause (19)

Stability – OOS/OOT:
• OOT
• To facilitate the prompt identification of
potential issues, and to ensure data
quality, it is advantageous to use objective
(often statistical) methods that detect
potential out-of-trend (OOT) stability data
quickly.
M.KOUPPARIS 62
 Phase II Investigation
Unknown Cause / No Assignable Cause (20)
Stability – OOS/OOT:
• OOT
• OOT alerts can be classified into three categories to help
identify the appropriate depth for an investigation.
- OOT stability alerts can be referred to as:
- 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.

M.KOUPPARIS 63
 Phase II Investigation
Unknown Cause / No Assignable Cause (21)
Stability:
– A compliance alert defines a case in which an OOT result
suggests the potential or likelihood for OOS results to
occur before the expiration date within the same
stabilitystudy (or for other studies) on the same product.
– The stability OOS should link to the Product Recall
procedures.
– Historical data are needed to identify OOT alerts.
– An analytical alert is observed when a single result is
aberrant but within specification limits (i.e., outside
normal analytical or sampling variation and normal
change over time).
M.KOUPPARIS 64
 Phase II Investigation
Unknown Cause / No Assignable Cause (22)
Microbiological investigations:
- These are difficult to perform as the result can be
1 to 2 weeks after the analysis was performed
and may be weeks after the batch was
manufactured.
- It is important to evaluate the test conditions
carefully and determine what the boundary of
samples/products/manufacturing area is.
- It you do not determine the boundary of the suspect
results it is difficult to determine if it one or more
batches impacted.
M.KOUPPARIS 65
 Phase II Investigation
Unknown Cause / No Assignable Cause (23)
Microbiological investigations:
- The laboratory and manufacturing investigations need
to be in depth.
- The investigations should clearly state the hypothesis
and who will be responsible for the identified tasks.
- Are the organisms of an expected type, determine likely
source – would it be likely to be found where it was?
- Review the media – prepared in house or bought in
pre-prepared, supplier history, sterilisation history
- Equipment/utilities used – validation, maintenance and
cleaning status.
- Evaluate area/environmental trends for test area and
support areas.

M.KOUPPARIS 66
 Phase II Investigation
Unknown Cause / No Assignable Cause (24)
Microbiological investigations:
- Cleaning and maintenance of the test environment
- Disinfectant used
- Use appropriate root cause analysis to help brain storm
all possibilities
- It is likely that there may be more than one root cause
- Review decisions and actions taken in light of any new
information.
- 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

M.KOUPPARIS 67
 Phase II Investigation
Unknown Cause / No Assignable Cause (25)
Microbiological investigations:
-Unusual events should be included to understand
potential impacts.
- What is the justification to perform a repeat analysis (is
sample left); re-test or resample
- Any identifications may need to be at DNA/RNA level
(bioburden failures)
- All potential sources of contamination need to be
considered – process flow the issue from sample storage
to the test environment.
- Use scientific decisions/justifications and risk based
analysis.
M.KOUPPARIS 68
 Phase II Investigation
Unknown Cause / No Assignable Cause (26)
Microbiological investigations:
- The investigation may include working closely with the
manufacturing team
- During the investigation it is an advantage to go and look
at where the contamination occurred.
- Ask how relevant plant is cleaned, tested for integrity,
checked for wear, checked for material suitability and
maintained at the occurrence site may reveal possible
causes.
- Where possible talk directly to the staff involved as some
information may be missed if not looked at from the
chemist/ microbiologist point of view.

M.KOUPPARIS 69
 Phase II Investigation
Unknown Cause / No Assignable Cause (27)
Microbiological investigations:
- Look for other documentation such as deviations and
engineering notifications around the area of concern (this
is applicable to the laboratory as well as manufacturing).
- Trending can have species drift which may also be worthy
of an action limit style investigation.
- Statistical analysis for microbiology can include lots of
zero results so recovery rates or similar may have to be
used.
- If a sample is invalidated the remaining level of assurance
needs to be carefully considered, is their sufficient
residual information?
- Corrective actions may be appropriate for more than one
root cause.
M.KOUPPARIS 70
 Phase III Investigation (1)
• 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 preventative action.

M.KOUPPARIS 71
 Phase III Investigation (1)
• 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 preventative action.

M.KOUPPARIS 72
 Phase III Investigation (2)
• 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.
• To conclude the investigation all of the results
must be evaluated.
• The investigation report should contain a
summary of the investigations performed; and a
detailed conclusion.
M.KOUPPARIS 73
 Phase III Investigation (3)
• 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.
• Once a batch has been rejected there is no limit to
further testing to determine the cause of failure, so
that corrective action can be taken.
• The decision to reject cannot be reversed as a result
of further testing.

M.KOUPPARIS 74
 Phase III Investigation (4)
• 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.

M.KOUPPARIS 75
 Batch Disposition (1)
• Conclusion:
– 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 favour
of passing retest results.
- All test results, both passing and suspect,
should be reported (in all QC documents and
any Certificates of Analysis) and all data has to
be considered in batch release decisions.

M.KOUPPARIS 76
 Batch Disposition (2)
• Conclusion:
– If the investigation determines that the
initial sampling method was inherently
inadequate, a new accurate 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 sampled by the same method.
M.KOUPPARIS 77
 Batch Disposition (3)
• Conclusion:
• An initial OOS result does not necessarily 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.

M.KOUPPARIS 78
 Batch Disposition (4)
• Conclusion:
– 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 or lot.
- A confirmed OOS result indicates that the
batch 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.
M.KOUPPARIS 79
 Batch Disposition (5)
• Conclusion:
- For inconclusive investigations — in cases where an
investigation:
(1) does not reveal a cause for the OOS test result
and
(2) does not confirm the OOS result
– the OOS result should be given full consideration
(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.

M.KOUPPARIS 80
 Batch Disposition (6)
• Conclusion:
- 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.
- In making such a decision, Quality
Assurance/QP should always err on the side of
caution.

M.KOUPPARIS 81
M.KOUPPARIS 82
M.KOUPPARIS 83
M.KOUPPARIS 84
 Capability of an analytical
Incapable method
method
Capable method
0,250 0,900

lower spec. lower spec.

upper spec. 0,800
limit limit upper spec.
0,200 limit limit
0,700

0,600
0,150
probability

probability
0,500

0,400
0,100
0,300

0,050 0,200

0,100

0,000 0,000
90 95 100 105 110 90 95 100 105 110
Concentration Concentration

M.KOUPPARIS 85
 How to measure capability?
Use measures from statistical process control
0,250

USL  LSL
lower spec.
upper spec.
cp 
limit
0,200 limit

C.I .
0,150

probability
e.g., specification between
97 mg/l and 103 mg/l, width 0,100
of confidence interval of
12mg/l: 0,050
Conf.
Interval
103  97
cp 
12 0,000
90 95 100 105 110
 0.5 Concentration

M.KOUPPARIS 86
 One-sided specifications
USL  x 0,250

c p ,u  lower spec. expected

 1   C .I . limit value

  0,200

 2
0,150
x  LSL

probability
c p ,l 
 1   C .I .
 
0,100 Half Conf.

 2
Interval

0,050

Where x is the expected 0,000

average value of the 90 95 100 105 110
strength
parameter.

M.KOUPPARIS 87
 Interpreting cp
Batch failure rate purely due to variability in analytical
method.
50
Bx failure rate due to

40
analysis / %

30

20

10

0
0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8 2

cp

M.KOUPPARIS 88
 Example 1

• Specification 0,60 LSL

>97.0% 0,50

• OOS result 96.5% OOS

Probability of
0,40
Re-test
with confidence 0,30

interval +/- 2.1%. 0,20

• Re-test 97.7% with 0,10

confidence interval 0,00

+/- 2.1%. 90 92 94 96 98 100

Strength
• No evidence that the
OOS and re-test are M.KOUPPARIS 89
 Example 2
LSL
• Specification 0,90
1,00

>97.0% 0,80
OOS
0,70
• OOS 96.0% with

Probability
Re-test
0,60
confidence interval 0,50
0,40
+/- 0.9%. 0,30

• Re-test 98.0% with

0,20
0,10
confidence interval 0,00
90 92 94 96 98 100
+/- 0.9%.
Strength
• No evidence that the
OOS and re-test are M.KOUPPARIS 90
M.KOUPPARIS 91