Your firm failed to thoroughly investigate aberrant potency values obtained for drug product batches according to regulatory requirements. Considerable time and effort should be spent investigating the root cause of aberrant potency results through a documented, multistep process including ruling out laboratory errors, reviewing manufacturing changes, modifying analytical methods, and confirming method robustness. Thorough investigations are necessary to ensure the quality, safety and efficacy of drug products.

1. Investigating Aberrant Potency Values
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2. - Introduction
- Roles & Responsibilities
- Reasons for aberrant values
- Investigating Aberrant Potency Values
- Preventing aberrant values
- Warning letter citations
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Contents

3. Introduction
• Accurate potency and purity data are critical in the development of drug products. These
results are used to make decisions regarding formulation development/selection,
formulation stability, and process robustness, and are used to release clinical supplies
and set clinical use periods.
• Sometimes aberrant potency values (e.g., assay values, content uniformity results,
stratified core test results) are obtained and significant efforts are spent investigating
these issues to identify the root cause, which may be manufacturing or method related.
• Sample preparation and extraction is a critical component of the method used for
potency and purity testing of drug products. All of the active ingredient and impurities
must be recovered from the dosage form to achieve accurate quantitative results.
• The sample preparation method should also be robust enough to serve its intended
purpose (e.g., use by only the project lab or by multiple labs, low or high sample
throughput).

4. Introduction
• Although the analytical measurement is at
the heart of the analytical process, it is not
the only source of error (systematic or
random) which affects the overall trueness
of the end result.
• It is apparent that one analytical
measurement does usually not constitute
a route to a reportable value.
• Additionally, there are variance
contributions which arise from other parts
of the process, particularly in sample
preparation and sub-sampling.

5. Introduction
• After a sample preparation/extraction method has been developed, validated and is in
use, issues, such as obtaining low, high, out of trend, out of specification, or variable
potency values (e.g., assay values, content uniformity results, stratified core test
results), may arise.
• These aberrant values raise questions about the analytical method (e.g., was there
insufficient extraction of the API from the dosage form, is the method non-robust) and
about the manufacturing process (e.g., was API lost during the process, is the process
non-robust).
• Considerable time and effort can be spent troubleshooting these aberrant potency
results to find the root cause.

6. Roles and Responsibilities
The role and responsibilities of the analyst and the supervisor are critical to the
performance of within-laboratory failure investigations. The analyst’s role and
responsibilities are as follows:
1. The first responsibility for achieving accurate laboratory testing results lies with the analyst
who is performing the test.
2. The analyst should be aware of potential problems that could occur during the testing process
and should watch for problems that could create aberrant results.
3. The analyst should ensure that only those instruments meeting established specifications are
used and that all instruments are properly calibrated.

7. Roles and Responsibilities
4. Analytical methods that have system suitability requirements which, if not met, should not be
used or continued. Analysts should not knowingly continue an analysis they expect to
invalidate at a later time for an assignable cause (i.e., analyses should not be completed for
the sole purpose of seeing what results can be obtained when obvious errors are known).
5. Before discarding test preparations or standard preparations, analysts should check the data
for compliance with specifications.
6. When unexpected results are obtained and no obvious explanation exists, test preparations
should be retained and the analyst should inform the supervisor.

8. Roles and Responsibilities
The analyst’s direct line manager or supervisor must be informed of an aberrant result
occurrence as soon as possible. The supervisor is then involved in a formal and
documented evaluation. Their role and responsibilities are as follows:
1. To conduct an objective and timely investigation and document it.
2. To discuss the test method and confirm the analyst’s knowledge of the procedure.
3. To examine the raw data obtained in the analysis, including chromatograms and spectra, and
identify anomalous or suspect information.
4. To confirm the performance of the instruments.
5. To determine that appropriate reference standards, solvents, reagents and other solutions
were used and that they met quality control specifications.

9. Roles and Responsibilities
6. To evaluate the performance of the testing method to ensure that it is performing according to
the standard expected based on method validation data.
7. To document and preserve evidence of this assessment.
8. To review the calculation.
9. To ascertain, not only the reliability of the individual value obtained, but also the significance
of these aberrant results in the overall quality assurance program. Laboratory error should be
relatively rare. Frequent errors suggest a problem that might be due to inadequate training of
analysts, poorly maintained or improperly calibrated equipment or careless work.
10. When clear evidence of laboratory error exists, the laboratory testing results should be
invalidated.

10. Reasons for aberrant values – (Questions raised)
• Sometimes these aberrant potency values are method related (e.g., is the
method robust/rugged) and may arise when the method has been transferred to
a new testing group or when there has been a change in the manufacturing site
or process.
• Sometimes the aberrant potency values are accurate and are the result of a
sample, a sampling, or a manufacturing related issue (e.g., non-homogeneity
issues for a given batch or non-robust manufacturing process leading to batch-
to-batch variability).

11. Reasons for aberrant values for fixed combination tablets
Potential causes related to the analytical assay
• The tablet disintegration step is insufficient in the tablet assay preparation
– Shaker to shaker variability is the cause (shaker rpm or stroke length discrepancy)
– The shake time is insufficient
– Shaker rpm is insufficient
• A residue in the volumetric flasks (e.g., soap or methanol used for cleaning) is interfering with
the tablet disintegration step
• The tablet extraction solvent is not strong enough or optimal to extract the actives
• The pH of the disintegration step is in a non-robust area for disintegration or extraction
• Tablets stick to the bottom of the flasks prior to disintegration by some mechanism not
allowing complete disintegration

12. Reasons for aberrant values for fixed combination tablets
Tablet related causes and tablet assay causes
• Excipient segregation is generating true low assay tablets
• Hard tablets are not easily disintegrated or extracted by the analytical method
• Tablets with a visible shine are not easily disintegrated or extracted by the analytical method.
It was noted that tablets manufactured by Site X have a distinct shine as compared to lab
prototypes
• Over lubricated tablets or granules are not easily disintegrated or extracted by the analytical
methods

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Investigating Aberrant
Potency Values

14. Step 1: Rule Out Laboratory Error
• All measurements and responses are subject to error. These errors may be
random or systematic, or a combination of both.
• The first step of the investigation should be to conduct a general laboratory
investigation to rule out possible laboratory error.
• The basic question to be answered is, “Was the method followed as written?” Items
to check include:
• use of the correct size volumetric flask(s) and volumetric pipette(s),
• use of the correct agitation mechanism and time,
• use of correct filters, assignment of correct API potency factor, etc.

15. Step 1: Rule Out Laboratory Error
• It is important to not dispose of any sample solutions (e.g., stock solutions, final sample
solutions) as these can prove valuable if additional testing is needed to try to determine the
cause of the aberrant results.
• The analyst can talk through the method with the analyst who performed the test to see if
there were any issues with interpretation of steps written in the method. If there were any
unusual or atypical observations, these should be discussed.
• Although every step of the sample preparation method is important, a step worth particular
attention is ensuring adequate mixing of the sample solution. Samples are often prepared
in volumetric flasks.
• The sample (e.g., tablet) is placed in the volumetric flask along with an approximate
volume of dilute and then is shaken or stirred for a specified length of time. The method
then typically specifies to dilute to volume and “mix thoroughly.”

16. Step 1: Rule Out Laboratory Error
• If adequate mixing is not performed, the solution will not be homogeneous and low or
variable potency values may result when an aliquot is taken from the flask for analysis or
to make a sub-dilution for analysis.
• This is especially true for viscous solutions or solutions with insoluble excipients.
• In addition, when preparing a large number (e.g., >50–100) of samples, if manual mixing is
performed, the consistency and/or vigor of mixing may vary from sample to sample and
there may be insufficient mixing for some samples.

17. Step 1: Rule Out
Laboratory Error
• Possible variables to study to assess
sample preparation/extraction
method robustness and ruggedness

18. Step 2: Review Available Information, Discuss,
Brainstorm, and Plan Next Steps
• After laboratory error has been ruled out, the analytical team should meet to review the
available information and discuss the next steps of the investigation.
• This discussion should involve all relevant personnel. This includes the analyst who
performed the testing and individuals from the laboratory that developed the method or
who have extensive experience with the method.
• It is also useful, if possible, to have individuals involved who have previous experience
investigating aberrant results.
• The team should review and discuss the issue and any relevant information, observations,
and data.

19. Step 2: Review Available Information, Discuss,
Brainstorm, and Plan Next Steps
This should include information to answer the following types of questions:
• When were the aberrant values observed?
• Were the aberrant values obtained on the first batches (prototypes or lab scale batches) of the
formulation? (e.g., there is limited experience with the method.)
• Were the aberrant values obtained on the first batches at a new manufacturing site or post scale-
up of the process? (e.g., did something change?)
• Were the aberrant values obtained on batches that had a change in the API (e.g., change in
particle size) or drug product manufacturing process (e.g., change in tablet hardness)? (e.g., is
there a change that may impact the method’s ability to extract the sample?)
• Were the aberrant values obtained by a new testing laboratory or new analyst?
• What type of testing or method related issues have been encountered in the past with this
method or with this type of dosage form?
Analysis of the above information may provide clues as to the cause of the aberrant values. If
the incidence of aberrant values correlates with a change in the manufacturing process,
this process change and its potential impact on the method should be evaluated.

20. Step 2: Review Available Information, Discuss,
Brainstorm, and Plan Next Steps
Some manufacturing changes and their potential impact on the sample preparation method
are listed in the Table

21. Step 2: Review Available Information, Discuss,
Brainstorm, and Plan Next Steps
• When high variability in potency values are obtained (e.g., higher %RSD than typical for
content uniformity), questions are raised regarding the analytical method (e.g., is the
method robust/rugged) and the manufacturing process (e.g., nonhomogeneity issues for a
given batch or non-robust manufacturing process leading to batch to batch variability).
• One should ensure that the method is being followed properly (e.g., analyst is adequately
mixing, etc). A second area that should be evaluated is method robustness.

22. Step 3: Try to Confirm Aberrant Results with
Current Method
• One should try to confirm the aberrant results (e.g., low potency values) using the current
method.
• This often involves reanalysis/reinjection of the sample solutions (if not previously done in
Step 1), analyzing additional samples along with a control sample or having another
analyst perform additional testing.
• At this stage, the analyst who developed the method or who has extensive experience with
the method may perform testing or may watch the testing being performed.
• If higher potency values are obtained at this stage, then it is likely that the original low
potency results are due to either laboratory error, which was not identified during Step 1, or
are due to a possible method robustness issue.

23. Step 4: See if Modifications to the Current Method
Will Improve Aberrant Potency Values
One should try modifications to the current method to see if increased potency values can be
obtained on the sample(s) in question.
Compare methods for similar formulations to get ideas. Method modifications can include,
but are not limited to:
• Letting sample solutions sit/stir overnight and then analyzing them
• Increasing agitation time (e.g., shake time)
• Increasing extraction solvent volume
• Changing method of agitation
• Changing extraction solvent(s) (e.g., change pH, change organic content)
• Using increased temperature (e.g., using a stirring hot plate or accelerated solvent extraction
[ASE] or microwave assisted extraction [MAE])

24. Step 5: Try to Improve Results Using Alternative
Methods
• Alternative analysis methods and alternative sample preparation methods should be used
to try to improve results (e.g., if original results were low potency then try to increase
recovery of samples) or to try to confirm the original aberrant values obtained for the
samples.
• Testing should be performed on the samples in question along with a control sample.
• The control sample is one with a known potency value, typically close to 100% label claim.
• Alternative analysis methods that can be used include dissolution and near infrared (NIR).
For solid oral dosage forms, there is typically a dissolution or drug release method that is
available.
• The dissolution or drug release method can be used to see the maximum amount of drug
dissolved or released at the end of the test or after an infinity point. NIR can be used as a
qualitative method to compare the response of the sample in question vs. a control
sample. NIR is a good technique to consider since no sample preparation is required and
hence sample extraction is not a factor.

25. Step 6: Confirm Current Method Able to Extract
API from Dosage Form
• A good way to confirm that the current method is able to completely extract API from the
dosage form is to analyze tablets that have been individually made with a known quantity
of API.
• If low recoveries are obtained for these samples, then this suggests that the current
method is not extracting all API.
• Analysis of control samples or previously analyzed samples can also be performed to
demonstrate that the method is capable of obtaining reproducible results.
• If drug–excipient interactions are suspected, such as adsorption of drug to an insoluble
excipient, then one can prepare drug–excipient blends for analysis or wet spike API
solution onto dry excipient blend and prepare the sample per the method.
• If there is drug–excipient interaction, a low recovery will be obtained and the extraction/
dissolving solvent or sample preparation method will need to be changed to address this
issue.

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Preventing aberrant Values

27. Preventing aberrant Values
• Follow the Validated procedure for analysis
• Follow the steps for sample extraction as mentioned in the procedure (Stirring, Centrifuge,
Sonication, Filtration etc.)
• Use blanks, controls or checks wherever required.
• Use qualified & Calibrated instruments.
• Analyst qualification, training and adequate knowledge about the method, technique &
product behavior (hygroscopic / light or temperature sensitive etc.) is required.
• Proper storage & handling of sample is important.
• Maintain the instrument condition properly.
• Use Valid standards and Reagents of prescribed grade.
• Verify the system suitability criteria before injecting samples.

28. Preventing aberrant Values
• Follow basic operating procedures
• Follow Good laboratory practices.
• Maintain the room temperature & humidity.
• Use trend charts to monitor the product / instrument behavior.
• Document all the steps during and/or immediately after performing the activity.
• Follow internal specifications or limits (weights. pH adjustments).
• Monitor the solubility of the product during the extraction.
• Investigate for root cause of aberrant values and document.
• Implement effective CAPA to avoid re-occurence.

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Warning letter citations

30. Warning letter Citations
• Your firm failed to thoroughly investigate any 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 been distributed (21 CFR 211.192).
PART 211 -- CURRENT GOOD MANUFACTURING PRACTICE FOR FINISHED PHARMACEUTICALS
Subpart J--Records and Reports
Sec. 211.192 Production record review.
All drug product production and control records, including those for packaging and labeling, shall be
reviewed and approved by the quality control unit to determine compliance with all established, approved
written procedures before a batch is released or distributed.
Any unexplained discrepancy (including a percentage of theoretical yield exceeding the maximum or
minimum percentages established in master production and control records) or the failure of a batch or
any of its components to meet any of its specifications shall be thoroughly investigated, whether or not
the batch has already been distributed. The investigation shall extend to other batches of the same drug
product and other drug products that may have been associated with the specific failure or discrepancy.
A written record of the investigation shall be made and shall include the conclusions and follow-up.

31. Warning letter Citations
• Your firm failed to investigate and document out-of-specification results according to a
procedure.
• Your laboratory’s written procedure failed to establish proper retesting practices for out-of-
specification results.
• Recognizing that an Out-of-Specification Test Result was obtained
in [REDACTION] specification of [REDACTION] as per requirement of your Quality
Control/Quality Assurance Standard Operating Procedure for OOS Investigation, SOP
Number 2802, Reporting, Investigation and Disposition of Out of Specification (OOS)
Laboratory Results, effective 02/05/09 and previous revisions, it appears that you avoided
to perform an Investigation for the assay test result of [REDACTION].
• The Quality Unit is not initiating investigations and documentation of those investigations in
a timely manner.

32. Warning letter Citations
• The investigation [REDACTION] (115576) into the misplaced
report [REDACTION] (114632) concluded that human error was the root cause, however,
the investigation failed to examine other possible causes such as the lack of an oversight
system to alert the validation group that equipment qualifications were past due.
• Specifically, your firm failed to perform adequate investigations with scientifically justifiable
conclusions to incidents of out-of-specification results or production deviations and/or
failed to implement appropriate corrective actions for the root cause determination.
• The firm failed to investigate out of specification (OOS) laboratory results as per SOP QCP
05.002, “Laboratory Investigations Procedure”. Additionally, laboratory investigations have
lacked scientific justification to support the dismissal of OOS/OOT results and conclusions
of the investigations.

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