Open Access

Austin Pharmacology & Pharmaceutics

Review Article

Reference for Investigation of Out of Specification Results

in Pharmaceutical Industry
Mote NN*
Abstract
Assistant Manager in Pharmaceutical Industry, Master of
Science in Analytical Chemistry from Shivaji University, The main objective of selecting this topic because of many pharmaceutical
FDA Approved in Instrumentation and Chemical companies receives the 483 observations/ warning letters from the USFDA. The
Analysis, Kolhapur, Maharashtra, India reason of most of 483 observations/warning letters was inadequate investigation
*Corresponding author: Nivrutti Narayan Mote, of Laboratory Non-Conformances. It includes Out of specification/Out of
Assistant Manager in Pharmaceutical Industry, Master of trend investigation. The inadequate investigation because of unawareness of
Science in Analytical Chemistry from Shivaji University, regulatory guidance requirements while performing the investigation. If any
FDA Approved in Instrumentation and Chemical company receives 483 observations/warning letter it will impact on company
Analysis, Kolhapur, Maharashtra, India reputation in the market and also impact on company revenue. So as to avoid
this we should know the some basic concept while performing the out of
Received: May 10, 2021; Accepted: June 07, 2021; specification/Out of trend investigation.
Published: June 14, 2021
Keywords: Applicability; Definitions; Phase I (Initial Laboratory Investigation);
Phase II (Full Scale OOS investigation); Manufacturing investigation hypothesis
testing/experimentation; Re-sampling; Retesting; Inconclusive OOS; Key point
to be considered while performing OOS investigation; Conclusion; USFDA
Observation on OOS investigation

Introduction (no stress tests) 83 observation/warning letters.

As per current good manufacturing practice for finished • Previous released batch used as reference sample in an OOS
pharmaceuticals. Subpart J--Records and Reports (211.192 investigation showing OOS or suspect results.
Production record reviews). All drug product production and control
• Pharmacopoeias have specific criteria for additional
records, including those for packaging and labeling, shall be reviewed
analyses of specific tests (i.e. dissolution level specification for S1,
and approved by the quality control unit to determine compliance
S2 & S3 testing; Uniformity of dosage unit’s specification for testing
with all established, approved written procedures before a batch is
of 20 additional units; Sterility Testing). However if the sample test
released or distributed [1]. Any unexplained discrepancy (including a
criteria is usually the first level of testing and a sample has to be tested
percentage of theoretical yield exceeding the maximum or minimum
percentages established in master production and control records) to the next level this should be investigated as it is not following the
or the failure of a batch or any of its components to meet any of its normal trend.
specifications shall be thoroughly investigated, whether or not the • Batches for clinical trials.
batch has already been distributed [2]. The investigation shall extend
to other batches of the same drug product and other drug products OOS Investigation NOT Applicable To
that may have been associated with the specific failure or discrepancy • In-process tests that are performed for the purpose of
[3-5]. A written record of the investigation shall be made and shall monitoring and/or adjusting the process (e.g. pH, viscosity).
include the conclusions and follow-up. Even if a batch is rejected
based on an OOS result, the investigation is necessary to determine if • The studies conducted at variable parameters to check the
the result is associated with other batches of the same drug product or impact of drift (e.g. process validation at variable parameters).
other products. Batch rejection does not negate the need to perform • Stress tests conducted on sample ex. Temperature excursion
the investigation. The investigation should be thorough, timely, study, photo stability study.
unbiased, well-documented, and scientifically sound [6].
Important Definitions
Applicability: OOS/OOT/Atypical Results
Investigation Applicable To 1) Out-of-Specification (OOS) Result: Test results that fall
outside of established acceptance criteria which have been established
• Batch release testing and testing of starting materials.
in official compendia and/or by company documentation (i.e., Raw
• In-Process Control testing: if data is used for batch Material Specifications, In-Process/Final Product Testing, etc.).
calculations/decisions and if in a dossier and on Certificates of
2) Atypical/Aberrant/Anomalous Result: Results that are still
Analysis.
within specification but are unexpected, questionable, irregular,
• Stability studies on marketed batches of finished products deviant or abnormal. Examples would be chromatograms that show
and or active pharmaceutical ingredients, on-going/follow up stability unexpected peaks, unexpected results for stability test point, etc.

Austin Pharmacol Pharm - Volume 6 Issue 1 - 2021 Citation: Mote NN. Reference for Investigation of Out of Specification Results in Pharmaceutical Industry. Austin
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3) Obvious Error: Investigation is to determine whether there potential non-conformity or other undesirable potential situation.
has been a clear obvious error due to external circumstances such as
Note: Preventive action is taken to prevent occurrence whereas
power failure or those that the analyst has detected prior to generating
corrective action is taken to prevent recurrence.
data such as spilling sample.
Examples:
Phase I: Investigation (Initial Laboratory
Investigation)
Calculation error: Analyst and supervisor to review both initial
The phase I investigation involves the assessment of laboratory
and date correction.
data and Verifications of initial preparation. This activities performed
Power outage: Analyst and supervisor document the event, by analyst and supervisor. Whenever possible, this should be done
annotate “power failure; analysis to be repeated” on all associated before test preparations (including the composite or the homogenous
analytical documentation. source of the aliquot tested) are discarded. This way, hypotheses
regarding laboratory error or instrument malfunctions can be
Equipment failure: Analyst and supervisor document the event,
tested using the same test preparations. Examination of the retained
annotate “equipment failure; analysis to be repeated” cross reference
solutions should be performed as part of the laboratory investigation
the maintenance record.
[7,8].
Testing errors: for example, spilling of the sample solution,
incomplete transfer of a sample; the analyst must document
• Re-injection of same solution ---To rule out the error
related to instrument malfunctioning.
immediately. For microbiology it could be growth on a plate not in
the test sample area, negative or positive controls failing. • Re-dilution or Re-pipetting of same solution----To rule out
the error related to dilution or pipetting.
Incorrect instrument parameters: For example setting the
detector at the wrong wavelength, analyst and supervisor document It should not be assumed that OOS test results are attributable
the event, annotate “incorrect instrument parameter”; analysis to be to analytical error without performing and documenting an
repeated” on all associated analytical documentation. investigation.
4) Assignable cause: An identified reason for obtaining an OOS If cause of out of specification is identified then retesting or
or aberrant/anomalous result. recalculation to be performed. If results found within the limit, initial
data to be invalidated. Retesting or recalculated data to be consider
5) No assignable cause: When no reason could be identified.
for final reporting and release.
6) Invalidated test: A test is considered invalid when the
For better understanding of phase-I investigation refer flow
investigation has determined the assignable cause.
diagram-A (Figure 1).
7) Reportable result – The final analytical result. This result
When the initial assessment does not determine that laboratory
is appropriately defined in the written approved test method and
error caused the OOS result and testing results appear to be accurate,
derived from one full execution of that method, starting from the
a full-scale OOS investigation using a predefined procedure should
original sample.
be conducted [7].
8) Hypothesis/Investigative testing: Testing performed to
help confirm or discount a possible root cause i.e. what might have Phase II: Investigation (Full Scale OOS
happened that can be tested: - for example it may include further Investigation)
testing regarding sample filtration, sonication /extraction; and Phase II investigation involves:
potential equipment failures etc. Multiple hypothesis can be explored
• Production process review/ Manufacturing Investigation
9) Re-test: Performing the test over again using material from the
original sample composite, if it has not been compromised and/or is • Hypothesis testing/Experimentation
still available. If not, a new sample will be used. • Additional laboratory testing
10) Re-sample: A new sample from the original container where Production process review/manufacturing Investigation
possible, required in the event of insufficient material remaining When the initial laboratory investigation does not determine
from original sample composite or proven issue with original sample the cause of OOS results, full-scale OOS investigation using a
integrity. predefined procedure should be conducted. This investigation may
11) Most probable cause: Scientifically justified determination consist of a production process review and/or additional laboratory
that the result appears to be laboratory error. work. The objective of such an investigation should be to identify
the root cause of the OOS result and take appropriate corrective
12) Corrective action: Action to eliminate the cause of a detected and preventative action. A full-scale investigation should include a
non-conformity or other undesirable situation. Note: Corrective review of production and sampling procedures, and will often include
action is taken to prevent recurrence whereas preventive action is additional laboratory testing. A full-scale OOS investigation should
taken to prevent occurrence. consist of a timely, thorough, and well-documented review [8].
13) Preventative action: Action to eliminate the cause of a If this part of the OOS investigation confirms the OOS result

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Figure 1: Flow diagram- A (Phase I Investigation).

and is successful in identifying its root cause, the OOS investigation Re-injections can provide strong evidence that the problem should be
may be terminated and the product rejected. However, a failure attributed to the instrument, rather than the sample or its preparation.
investigation that extends to other batches or products that may have
• Re-dilution from original solution in case of multiple
been associated with the specific failure must be completed.
dilutions to find out dilution error.
Hypothesis testing/experimentation
• Re-extraction of a dosage unit, where possible, can be
Hypothesis/Investigative testing: Description of the testing performed to determine whether it was fully extracted during the
should be written, and then approved by QA prior to initiating original analysis.
investigational testing. The requirements of investigational testing
listed below: Investigational testing may not be used to replace original suspect
analytical results. It may only be used to confirm or discount a
The description must fully document; probable cause.
• The hypothesis to the test the root cause being investigated. Additional laboratory testing
• What samples will be tested. Phase II investigation may includes additional laboratory testing.
• The exact execution of the testing. These include:
• How the data will be evaluated • Re-sampling and
On completion of the Analyst and Supervisor investigation • Retesting a portion of the original sample.
hypothesis testing can be started. Hypothesis testing performed to
Criteria for Re-sampling: It involves the collecting a new sample
help confirm or discount a possible root cause i.e. what might have
from the batch.
happened that can be tested. Hypothesis testing applicable to Phase
I and Phase II. The initial hypothesis testing can include the original • If insufficient quantity of the original sample remains
working stock solutions but should not include another preparation to perform all further testing then the procedure for obtaining a
from the original sample. The initial hypothesis testing can involve resample must be discussed and agreed by QA/Contract Giver/QA
re-measurement of the original preparation or working solutions, equivalent. The process of obtaining the resample should be recorded
however retesting is when the original sample or composite sample is within the laboratory investigation.
used to perform analysis [9].
• Re-sampling should be performed by the same qualified,
Example: validated methods that were used for the initial sample.
It may include further testing regarding sample filtration, • Sound scientific justification must be employed if re-
Sonication /extraction; and potential equipment failures etc. sampling is to occur.
• Original Solutions can be re-injected as part of an • An investigation might conclude that the original sample
investigation where a transient equipment malfunction is suspected. was prepared improperly and was therefore not representative of the

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Figure 2: Flow diagram- B (Phase-II Investigation).

batch quality. judgment. The test plan must be approved before re testing occurs.
• If the investigation determines that the initial sampling • Retesting Performed on Original sample.
method was inherently inadequate, a new accurate sampling method
• Can be a 2nd aliquot from the same sample that was the
must be developed, documented, and reviewed and approved by the
source of the original failure.
QCU.
• The sample used for the retesting should be taken from the
• If the initial sample given to the laboratory for analysis has
same homogeneous material that was originally collected from the
become in some way adulterated (e.g., through breakage or exposure lot, tested, and yielded the OOS results. For a Liquid it may be from
to heat, light, or moisture) or was exhausted in the testing process, it the original unit liquid product or composite of the liquid product
may be acceptable to resample the batch under the same constraints and for a solid, it may be an additional weighing from the same
as described earlier. sample composite prepared for the original test.
• Control mechanisms for examination of additional • Retests performed by an analyst other than the one who
specimens should be in accordance with predetermined procedures performed the original test. (It’s a part of retesting Plan).
and sampling strategies (§ 211.165(c)).
• A second analyst performing a retest should be at least as
Criteria for retesting: experienced and qualified in the method as the original analyst.
• The decision to retest should be based on sound scientific • The minimum number of retests should be documented

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within the procedure and be based upon scientifically sound principles. original test. A second analyst performing a retest should be at least as
Any statistical review with regards to %RSD and repeatability should experienced and qualified in the method as the original analyst.
relate to the values obtained during method validation (accuracy,
• FDA inspections have revealed that some firms use a
precision, and intermediate precision). The number of retests should
strategy of repeated testing until a passing result is obtained, then
be statistically valid; papers have suggested 5, 7 or 9 (Figure 2).
disregarding the OOS results without scientific justification. This
Handling of Inconclusive OOS practice of “testing into compliance” is unscientific and objectionable
under CGMPs.
Inconclusive OOS, Product /Material can be released by verifying
following Key points (Figure 3): • The firm’s predetermined retesting procedures should
contain a point at which the additional testing ends and the batch is
• A comprehensive laboratory investigation (Phase 1) fails to
evaluated.
reveal any laboratory error.
• Failure investigation that extends to other batches or
• Review of events during production of the batch reveals no
products that may have been associated with the specific failure must
aberrations or indication of unusual process variation.
be completed.
• Review of the manufacturing process and product history
• Investigational testing/Hypothesis testing may not be used
demonstrates that the process is robust.
to replace an original suspect analytical result. It may only be used to
• The Six/seven passing retest results are all well within the confirm or discount a probable cause.
known limits of variability of the method used.
• This Hypothesis testing may continue from the re-
• Batch results from in-process monitoring, content measurement of the original preparations.
uniformity, dissolution, and other tests are consistent with the
• Once a batch has been rejected there is no limit to further
passing retest results.
testing to determine the cause of failure, so that corrective action can
After a comprehensive investigation, Quality control might be taken.
conclude that the initial OOS result did not reflect the true quality
• The decision to reject cannot be reversed as a result of
of the batch. Any decision to release a batch, in spite of an initial
further testing.
OOS result that has not been invalidated, should come only after a
full investigation has shown that the OOS result does not reflect the • If the investigation determines that the initial sampling
quality of the batch. In making such a decision, Quality Assurance/ method was inherently inadequate, a new accurate sampling method
QP should always err on the side of caution [10]. must be developed, documented, and reviewed and approved by the
Quality Assurance responsible for release. A consideration should be
Key Point to be Consider during OOS given to other lots sampled by the same method.
Investigation
• Any decision to release a batch, in spite of an initial OOS
• Initial hypothesis testing can include the original working result that has not been invalidated, should come only after a full
stock solutions but should not include another preparation from the investigation has shown that the OOS result does not reflect the
original sample. quality of the batch. In making such a decision, Quality Assurance/
• Even if a batch is rejected based on an OOS result, the QP should always err on the side of caution.
investigation is necessary to determine if the result is associated • Products that are the subject of approved full and
with other batches of the same drug product or other products and abbreviated new drug applications, regulations require submitting
identification and implementation of corrective and preventative within 3 working days a Field Alert Report (FAR) of information
action. Batch rejection does not negate the need to perform the concerning any failure of a distributed batch to meet any of the
investigation. specifications established in an application.
• It is important when considering performing additional Conclusion
testing that it is performed using a predefined retesting plan to include
retests performed by an analyst other than the one who performed the • 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.
• When clear evidence of laboratory error exists, laboratory
testing results should be invalidated. The firm should determine the
source of that error and take corrective action to prevent recurrence.
• When evidence of laboratory error remains unclear, a full-
Figure
3: Flow3: Flow chart of OOS.
Inconclusive OOS.
scale OOS investigation should be conducted by the manufacturing
Figure chart of Inconclusive

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Mote NN Austin Publishing Group

firm to determine what caused the unexpected results (FDA). the OOS results without any scientific justification and released these
lots into the U.S. market (21 CFR 211.192).
• An initial OOS result does not necessarily mean the subject
batch fails and must be rejected. The OOS result should be investigated, Observation 6: You firm failed to establish and follow adequate
and the findings of the investigation, including retest results, should written procedures for cleaning and maintenance of equipment (21
be interpreted to evaluate the batch and reach a decision regarding CFR 211.67(b)). The cleaning validation for your non-dedicated tank,
release or rejection which should be fully documented. used to manufacture your drug product was inadequate. Your High-
Performance Liquid Chromatography (HPLC) chromatograms for
• In those cases where the investigation indicates an OOS
residual disinfectant showed significant peaks for rinse samples with
result is caused by a factor affecting the batch quality (i.e., an OOS
a retention time similar to that of your cleaning agent. You failed to
result is confirmed), the result should be used in evaluating the quality
investigate these peaks. During the inspection, you integrated these
of the batch or lot. A confirmed OOS result indicates that the batch
peaks, which yielded OOS results for residual disinfectant. Your
does not meet established standards or specifications and should
cleaning validation failed multiple rinse samples tested for residual
result in the batch’s rejection, in accordance with § 211.165(f), and
disinfectant.
proper disposition. Other lots should be reviewed to assess impact.
Observation 7: Failed to thoroughly investigate any unexplained
• In case of confirmed OOS, the investigation changes from discrepancy or failure of a batch or any of its components to meet
an OOS investigation into a batch failure investigation, which must be any of its specifications, whether or not the batch has already been
extended to other batches or products that may have been associated distributed (21 CFR 211.192). Your original atomic absorption
with the specific failure (§ 211.192). analysis of sample was Out-Of Specification (OOS). A retest of the
• If the investigation determines that the initial sampling sample was also OOS. A third sample was retested and found within
method was inherently inadequate, a new accurate sampling method specifications. You invalidated the OOS results without justification
must be developed, documented, and reviewed and approved by the or documented investigation.
Quality Assurance responsible for release. A consideration should be Observation 8: Lacked in thorough investigations into root
given to other lots sampled by the same method. causes, and failed to implement prompt and effective Corrective
• Investigational/Hypothesis testing may not be used to Actions and Preventive Actions (CAPA).
replace an original suspect analytical result. It may only be used to Observation 9: Failed to establish adequate written
confirm or discount a probable cause. responsibilities and procedures applicable to the quality control unit
USFDA Observations on OOS Investigation and to follow such written procedures (21 CFR 211.22(d)). You lacked
adequate written procedures for various functions, including, but not
Observation 1: Invalidated Out-Of-Specification (OOS) results limited to customer complaints, recalls, annual product review, out-
without adequate investigation and scientific justification. Example: of-specification (OOS) or deviation investigations, change control,
Obtained OOS results for the impurity during stability testing of CGMP-related training, issuing batch records, documenting batch
injection batches. OOS investigation reports stated that the postulated record review, cleaning, storage conditions.
cause was “poor column efficiency”, although no chromatographic
abnormalities were noted and system suitability criteria were met. Observation 10: Your firm does not ensure that complete data
During the inspection, lab management indicated that retention from testing of your API are included in the official batch record and
times, theoretical plates, and tailing factor appeared appropriate and reviewed by your quality unit. For example, you reported passing
results for related substances. However, our investigator found
no specific root cause had been demonstrated. Repeated the analyses,
unreported analyses including out-of-specification (OOS) results for
obtained passing results, and invalidated the OOS results.
the same lot acquired earlier on the same date, and on the next day as
Observation 2: OOS investigation of the failure of to meet the the reported results.
specifications under accelerated stability conditions. While the
Observation 11: Lacked of adequate procedures for investigating,
investigation lacked a demonstrated assignable root cause in the
and scientific justification to invalidate, OOS results.
laboratory, obtained passing results during repeat analysis and
invalidated the OOS without a Phase II production investigation. Observation 12: OOS Results for Residual Solvent You initiated
investigation for an initial OOS. The investigation did not reveal
Observation 3: Investigations of Out-Of-Specification (OOS)
laboratory testing anomalies. You tested another sample preparation
results were inadequate. For example, in multiple instances, you
three times and obtained results very close to the specification
disregarded the original failing result based on a retest, but you
upper limit. You invalidated the initial failing result, stating that
lacked a Phase 1 laboratory investigation to support invalidation of
your statistical analysis showed a significant difference between the
the result. You also often lacked Phase 2 investigations to evaluate
original value and the retest results. Your investigation lacked further
your manufacturing operation for potential root causes.
assessment of the root cause of the failing result.
Observation 4: Investigated numerous OOS results as “incidents”
Observation 13: Out-Of-Specification (OOS) results observed for
and not as OOS results.
viscosity test. The next two retest values were also OOS. You failed to
Observation 5: Failed to thoroughly investigate Out-of- conduct laboratory and manufacturing investigations into these OOS
Specification (OOS) assay test. Retested the samples and invalidated results, which included identifying a root cause and implementing

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Mote NN Austin Publishing Group

Corrective Actions and Preventive Actions (CAPA). Instead, you continued to distribute other batches of the same product while your
rejected the batch without conducting an adequate investigation. OOS investigation remained open for more than five months.
Observation 14: Quality control laboratory disregarded multiple References
Out-Of-Specification (OOS) impurity results without justification. 1. Guidance for Industry Investigating Out-Of-Specification (OOS) Test Results
for Pharmaceutical Production.
Observation 15: Your firm failed to thoroughly investigate any
2. MHRA Guideline on Out of Specification. 2018.
unexplained discrepancy or failure of a batch or any of its components
to meet any of its specifications, whether or not the batch has already 3. Reference book on Handling Laboratory and Manufacturing Deviations
Robert B. Kirsch R. B. Kirsch Consulting, Arlington Heights, Illinois, USA.
been distributed (21 CFR 211 192). Your firm frequently invalidated
initial Out-Of-Specification (OOS) laboratory results without an 4. The GMP Questions & Answers Guide - GMP Advisor Version 02 of 2020.
adequate investigation that addressed potential manufacturing 5. Guide To Inspections of Pharmaceutical Quality Control Laboratories.
causes.
6. Current Good Manufacturing Practice For Finished Pharmaceuticals 21 CFR
Observation 16: Our investigators documented that your 211, 21 CFR 210.

investigations into Out-Of-Specification (OOS) test results were 7. Guidance for Industry Q7A Good Manufacturing Practice Guidance for Active
not thorough, timely, or based on scientific rationales. Your Pharmaceutical Ingredients.
investigations did not adequately determine root cause. Stability 8. Stability testing of active pharmaceutical ingredients and finished
Failure: Investigation of Two different batches failed stability testing. pharmaceutical products WHO Technical Report Series, No. 1010. 2018.
During the inspection, we reviewed your initial OOS investigation 9. USFDA issue Warning Latter to pharmaceutical industry.
in which you determined that the stability failures were caused by
10. WHO guidelines on quality risk management. WHO Technical Report Series
an excipient used to manufacturing. In the same investigation, you No. 981. 2013.
also concluded, without performing a science-based health hazard
evaluation, that such impurities do not pose health risks. You

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