Detailed comparison of the bioanalytcial method validation guidelines of Europe, Japan, Brazil and the United States

1. Evaluating EMA and FDA guidance on
bioanalytical method validation
Peter van Amsterdam (Abbott/EBF/GBC)
10th Bioavailability / Bioequivalence, Dissolution and Biowaivers
14 May 2014, Budapest

2. Evaluating EMA and FDA guidance on bioanalytical method validation
Contents
1. Guidance
2. Method validation
3. Sample analysis
4. Quality systems
---------------------
Further reading
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3. Guidance
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4. What to look for
 ‘Global’
– OECD
– ICH
– WHO
 ‘Regional’
– FDA
– EMA
– ASEAN
 National
– HPFB
– MHLW
– CFDA
– etc.
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Where to look …..
 Bioanalytical method
validation guidelines
 BA/BE specific
guidelines
 PK / phase I specific
guidelines
 TK guidelines
 Analytical chemistry
guidance documents
 GLP
 GC(L)P
 G-other-P

5. ‘BMVs’
 International: ICH (2005)
Q2(R1): Validation of Analytical Procedures: Text and Methodology
 USA: FDA (2001)
Guidance for Industry: Bioanalytical Method Validation
 Europe: EMA (2011)
Guideline on Bioanalytical Method Validation
 Brazil: ANVISA (2012)
RESOLUÇÃO - RDC Nº 27, DE 17 DE MAIO DE 2012
Dispõe sobre os requisitos mínimos para a validação de métodos bioanalíticos
empregados em estudos com fins de registro e pós-registro de medicamentos.
 Japan: MHLW (2013)
Guideline on Bioanalytical Method Validation in Pharmaceutical
Development
 USA: FDA (2013)
DRAFT Guidance for Industry: Bioanalytical Method Validation
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6. ‘BABEs’
 USA: FDA (2003)
Bioavailability and Bioequivalence Studies for Orally Administered
Drug Products - General Considerations
 China: CFDA (2005)
Technical guideline for human bioavailability and bioequivalence
studies on chemical drug products
 India (2005)
Guidelines for Bioavailability & Bioequivalence Studies
 Europe: EMA (2010)
Guideline on the Investigation of Bioequivalence
 China: CFDA (2011)
Guidance on Management of Laboratory for Drug Clinical Trial
Biological Sample Analysis (interim)
 Canada: HPFB (2012)
Conduct and Analysis of Comparative BA Studies
… and many more
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7. ‘GLPs’
 USA: FDA (1978)
21CFR Part 58 Good Laboratory Practice for Nonclinical Laboratory
Studies
 International: OECD (current)
Principles of Good Laboratory Practice and Compliance Monitoring
 International: WHO (2009)
Good Clinical Laboratory Practice (GCLP)
 Europe: EMA (2012)
Reflection paper for laboratories that perform the analysis or
evaluation of clinical trial samples
 USA: FDA (1997)
21CFR Part 11 Electronic Records; Electronic Signatures
 Europe: Eudralex (2010)
Good Manufacturing Practice - Medicinal Products for Human and
Veterinary Use - Annex 11: Computerised Systems
… and many more
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8. 14 May 2014 Evaluating EMA and FDA guidance on bioanalytical method validation 8

9. … let us try to simplify the matter
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10. … and concentrate on EMA BMV (2011)
Evaluating EMA and FDA guidance on bioanalytical method validation
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Guideline on the validation of bioanalytical methods
http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC500109686.pdf
 Well written with a clear structure
 Clear distinction between method validation and
sample analysis
 First BMV guideline addressing ‘all’ the specifics for
LBA/macromolecules
 Defines applicable quality systems: GLP (pre-clinical)
and GCP (clinical)
 Good match with current thinking in BA community
 Good fit with EMA Bioequivalence guideline
 Fits with developing concepts on GCP for
bioanalytical laboratories
 Well accepted within the global BA community
 Final & Current
– FDA is from 2001 + a challenged 2013 draft
(not taking ‘updates’ of CC-III & CC-IV into account)
– ANVISA is specific for Brazil
– MHLW is quite similar to EMA

11. Table of contents EMA BMV
 1. Introduction
 2. Scope
 3. Legal basis
 4. Method validation
– 4.1. Full validation of an analytical method
o 4.1.1. Selectivity
o 4.1.2. Carry-over
o 4.1.3. Lower limit of quantification
o 4.1.4. Calibration curve
o 4.1.5. Accuracy
o 4.1.6. Precision
o 4.1.7. Dilution integrity
o 4.1.8. Matrix effect
o 4.1.9. Stability
– 4.2. Partial validation
– 4.3. Cross validation
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12. Table of contents (continued)
 5. Analysis of study samples
– 5.1. Analytical run
– 5.2. Acceptance criteria of an analytical run
– 5.3. Calibration range
– 5.4. Reanalysis of study samples
– 5.5. Integration
 6. Incurred samples reanalysis
 7. Ligand binding assays
– 7.1. Method validation
o 7.1.1. Full validation (multiple subchapters)
– 7.2. Partial validation and cross-validation
– 7.3. Analysis of study samples
o 7.3.1. Analytical run
o 7.3.2. Acceptance criteria for study sample analysis
o 7.3.3. Incurred samples reanalysis
 8. Reports
– 8.1. Validation report
– 8.2. Analytical report
 Definitions
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13. Method Validation
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14. Full Validation
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EMA:
A full method validation should be performed for any analytical method whether new or
based upon literature.
Generally a full validation should be performed for each species and matrix concerned
Validation should be performed using the same anticoagulant as for the study samples
If problematic for validation purposes to obtain an identical matrix compared to the matrix
of the study samples, a suitable alternative matrix may be used.
The main characteristics of a bioanalytical method that are essential to ensure the
acceptability of the performance and the reliability of analytical results are: selectivity,
LLOQ, the response function and calibration curve performance, accuracy, precision,
matrix effects, stability of the analyte(s) in the biological matrix and stability of the
analyte(s) and of the IS in the stock and working solutions and in extracts under the entire
period of storage and processing conditions.
The principles of validation and analysis apply to all analytes of interest.
Draft FDA: Less defined and seems to be more lenient, leaving judgment to
scientists
ANVISA: Less defined, but follows same principles. Requires chromatographic
method for ‘chromatographable analytes’
MHLW: Quite similar to EMA

15. Reference standards
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EMA:
Suitable reference standards, include certified standards such as compendial standards
(EPCRS, USP, WHO), commercially available standards, or sufficiently characterised
standards prepared in-house or by an external non-commercial organisation. A certificate
of analysis is required to ensure purity and provide information on storage conditions,
expiration date and batch number.
The use of certified standards is not needed for IS, as long as the suitability for use is
demonstrated, e.g. lack of analytical interference is shown for the substance itself or any
impurities thereof. A certificate of analysis is not required.
Recommended to use stable isotope labeled IS for MS based assays
However, it is essential that the labeled standard is of the highest isotope purity and that
no isotope exchange reaction occurs. The presence of any unlabeled analyte should be
checked and if relative amounts of unlabeled analyte are detected the potential influence
has to be evaluated during method validation.
Draft FDA: Similar to EMA, but requires expiration date and purity for IS. Stock
solutions should not be used after ‘powder’ expires. SILIS not mentioned
ANVISA: Prefers pharmacopeia reference standards, recommends SILIS and has
detailed requirements on CoA information
MHLW: Similar to EMA, but less explicit

16. Selectivity
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EMA:
Selectivity should be proved using at least 6 individual sources of the appropriate blank
matrix
Normally, absence of interfering components is accepted where the response is less than
20% of the LLOQ for the analyte and 5% for the IS.
It may also be necessary to investigate the extent of any interference caused by
metabolites of the drug(s), degradation products and possible co-administered
medications.
Co-medications normally used in the subject population studied which may potentially
interfere should be taken into account at the stage of method validation, or on a study
specific and compound specific base.
The possibility of back-conversion of a metabolite into parent analyte during the
successive steps of the analysis should also be evaluated, when relevant (i.e. potentially
unstable metabolites).
Draft FDA: Similar, but a bit less explicit & no acceptance criteria given
ANVISA: Similar, but no co-med or metabolites mentioned. Requires hemolyzed and
lipemic
MHLW: Similar, but no co-med or metabolites mentioned

17. Carry-Over
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EMA:
Carry-over should be addressed and minimised during method development
During validation carry-over should be assessed by injecting blank samples after a high
concentration sample or calibration standard at the ULOQ.
Carry over in the blank sample following the high concentration standard should not be
greater than 20% of the LLOQ and 5% for the internal standard.
If it appears that carry-over is unavoidable, study samples should not be randomised.
Specific measures should be considered, tested during the validation and applied during
the analysis of the study samples, so that it does not affect accuracy and precision
Draft FDA: Described, but leaving judgment to scientists
ANVISA: Similar, but also specifies how to conduct the experiment
MHLW: Similar

18. Lower limit of quantification
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EMA:
The LLOQ is the lowest concentration of analyte in a sample which can be quantified
reliably, with an acceptable accuracy and precision...
The LLOQ is considered being the lowest calibration standard.
The analyte signal of the LLOQ sample should be at least 5 times the signal of a blank
sample
The LLOQ should be adapted to expected concentrations and to the aim of the study, e.g.
for bioequivalence studies the LLOQ should be not higher than 5% of the Cmax
Draft FDA: Similar plus 20% CV & 20% bias requirement, no words on adapting
ANVISA: No separate section on LLOQ, but similar wording is in calibration section
MHLW: Similar plus 20% CV & 20% bias requirement, no words on adapting

19. Calibration curve
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EMA:
The calibration standards should be prepared in the same matrix as the intended study
samples. A minimum of six calibration concentration levels should be used, in addition to
the blank sample and a zero sample (processed matrix with IS).
Ideally it should be known what concentration range is expected. This range should be
covered by the calibration curve range, defined by the LLOQ and the ULOQ. The range
should be established to allow adequate description of the PK of the analyte of interest.
A relationship which can simply and adequately describe the response of the instrument
with regard to the concentration of analyte should be applied.
All the available (or acceptable) curves obtained during validation, with a minimum of 3
should be reported. The back calculated concentrations of the calibration standards
should be within ±15% of the nominal value, except for the LLOQ for which it should be
within ±20%. At least 75% of the standards, with a minimum of six calibration standard
levels, must fulfill this criterion.
The calibration curve should preferably be prepared using freshly spiked samples.
Draft FDA: Similar. Instructions on exclusion of calibrators
ANVISA: Quite similar plus requirement for weighting and a minimum of 8
calibration standards for non-linear models
MHLW: Similar

20. Accuracy
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EMA:
To enable evaluation of any trends over time within one run, it is recommended to
demonstrate accuracy and precision of QC samples over at least one of the runs in a size
equivalent to a prospective analytical run of study samples.
Within-run accuracy should be determined by analysing in a single run a minimum of 5
samples per level at a minimum of 4 concentrations which are covering the calibration
curve range: the LLOQ, within three times the LLOQ (low QC), around 50% of the
calibration curve range (medium QC), and at least at 75% of the upper calibration curve
range (high QC). The mean concentration should be within 15% of the nominal values for
the QC samples, except for the LLOQ which should be within 20% of the nominal value.
For the validation of the between-run accuracy, LLOQ, low, medium and high QC samples
from at least three runs analysed on at least two different days should be evaluated. The
mean concentration should be within 15% of the nominal values for the QC samples,
except for the LLOQ which should be within 20% of the nominal value.
Note: between-run = ‘total’, interpret Me QC at 50% as on a geometric scale
Draft FDA: Similar, however include outliers in calculation of accuracy and QC
concentrations are to be related to sample concentrations rather than cal range.
ANVISA: Similar, but 5 concentrations required: LLOQ, Lo, Me, Hi & ULOQ
MHLW: Similar. Me QC at midpoint cal. curve

21. Precision
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EMA:
Precision is expressed as the coefficient of variation (CV). Precision should be
demonstrated for the LLOQ, low, medium and high QC samples, within a single run and
between different runs, i.e. using the same runs and data as for the demonstration of
accuracy.
For the validation of the within-run precision, there should be a minimum of five samples
per concentration level at LLOQ, low, medium and high QC samples in a single run. The
within-run CV value should not exceed 15% for the QC samples, except for the LLOQ
which should not exceed 20%.
For the validation of the between-run precision, LLOQ, low, medium and high QC samples
from at least three runs analysed on at least two different days should be evaluated. The
between-run CV value should not exceed 15% for the QC samples, except for the LLOQ
which should not exceed 20%
Note: between-run = ‘total’, interpret Me QC at 50% as on a geometric scale
Draft FDA: Similar, however include outliers in calculation of accuracy and QC
concentrations are to be related to sample concentrations rather than cal range.
ANVISA: Similar, but 5 concentrations required: LLOQ, Lo, Me, Hi & Dilution QC
MHLW: Similar

22. Dilution integrity
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EMA:
Dilution of samples should not affect the accuracy and precision.
If applicable, dilution integrity should be demonstrated by spiking the matrix with an
analyte concentration above the ULOQ and diluting this sample with blank matrix (at least
five determinations per dilution factor). Accuracy and precision should be within the set
criteria, i.e. within ±15%. Dilution integrity should cover the dilution applied to the study
samples.
Use of another matrix may be acceptable, as long as it has been demonstrated that this
does not affect precision and accuracy
Draft FDA: Described, but leaving judgment to scientists
ANVISA: As part accuracy & precision
MHLW: Similar to EMA

23. Matrix effect
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EMA:
Matrix effects should be investigated when using mass spectrometric methods, using at
least 6 lots of blank matrix from individual donors. Pooled matrix should not be used.
For each analyte and the IS, the matrix factor (MF) and the IS normalised MF should be
calculated for each lot of matrix. The CV of the IS-normalised MF should not be greater
than 15 %. This determination should be done at a low and at a high level of concentration
If the matrix is difficult to obtain, less than 6 different lots of matrix may be used, but this
should be justified. However, matrix effects should still be investigated.
If a formulation for injection to be administered to the subjects or animals contains
excipients known to be responsible for matrix effects, matrix effects should be studied with
matrix containing these excipients, in addition to blank matrix.
In addition to the normal matrix it is recommended to investigate matrix effects on other
samples e.g. haemolysed and hyperlipidaemic plasma samples. If applicable also
samples from special populations (such as renally or hepatically impaired populations)
Draft FDA: Described, but leaving judgment to scientists
ANVISA: Similar, but: 8 samples (4 normal, 2 lipemic, 2 hemolyzed) and ‘the degree
of hemolysis’
MHLW: Similar, but CV MF < 15%

24. Stability (1)
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EMA:
Evaluation of stability should be carried out to ensure that every step taken … sample
preparation, analysis and storage … do not affect the concentration of the analyte.
Stability tests should be done similar to the conditions and materials used for the actual
study samples.
Reference to data published in the literature is not considered sufficient.
Stability of the analyte in the studied matrix is evaluated using low and high QC samples.
The QC samples are analysed against a fresh calibration curve and the mean
concentration at each level should be within ±15% of the nominal concentration.
The following stability tests should be evaluated:
• stock solution and working solutions of the analyte and IS. SILIS not needed
• freeze and thaw stability of the analyte in the matrix
• short term stability of the analyte in matrix at room temperature or sample processing
temperature
• long term stability of the analyte in matrix stored in the freezer
• stability of the processed sample at room temperature or under the storage conditions
to be used during the study (dry extract or in the injection phase),
• on-instrument/autosampler stability of the processed sample at injector or autosampler
temperature.

25. Stability (2)
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EMA:
WRT LTS: For small molecules it is considered acceptable to apply a bracketing
approach, i.e. in case stability has been proved for instance at -70°C and -20°C, it is not
necessary to investigate the stability at temperatures in between.
Study samples may be used in addition to QC samples, but the exclusive use of study
samples is not considered sufficient.
The results of long term stability should be available before the study report is issued.
In case of a multi-analyte study and specific for bioequivalence studies, attention should
be paid to stability of the analytes in the matrix containing all the analytes.
Sufficient attention should be paid to the stability of the analyte in the sampled matrix
directly after blood sampling of subjects and further preparation before storage, to ensure
that the obtained concentrations by the analytical method reflect the concentrations of the
analyte in the subject at the moment of sampling. A demonstration of this stability may be
needed on a case-by-case basis, depending on the structure of the analyte.
Draft FDA: Similar, but also IS stock stability and reinjection reproducibility
ANVISA: Quite similar, but triplicate analysis
MHLW: Similar, but triplicate analysis

26. Recovery
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EMA: Not addressed
MHLW: Recovery is a measure of the efficiency at which an analytical method recovers
the analyte through the sample-processing step. In order to elucidate the nature of
analytical method, it is advisable to evaluate the recovery.
The recovery is determined by comparing the analyte response in a biological sample that
is spiked with the analyte and processed, with the response in a biological blank sample
that is processed and then spiked with the analyte. It is important to demonstrate the
reproducibility, rather than to show a higher recovery rate.
ANVISA: Not addressed
Draft FDA: Similar to MHLW, but 3 concentrations required

27. Endogenous analytes
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EMA: Not addressed
Draft FDA: The biological matrix used to prepare calibration standards should be the same
as the study samples and free of the endogenous analyte. To address the suitability of an
analyte-free biological matrix, the matrix should be demonstrated to have (1) no
measurable endogenous analyte and (2) no matrix effect or interference when compared
to the biological matrix. The use of alternate matrices (e.g., buffers, dialyzed serum) for
the preparation of calibration standards is generally not recommended …
The QCs should be prepared by spiking known quantities of analyte(s) in the same
biological matrix as the study samples. The endogenous concentrations of the analyte in
the biological matrix should be evaluated prior to QC preparation (e.g., by replicate
analysis).
ANVISA: For endogenous compounds, the selectivity may be tested by a comparison of
the gradients of, as a minimum, 6 standard addition curves in 6 samples from different
sources of the biological matrix and the standard curve in solution or substitute matrix.
The method is considered selective if the slopes of the curves are not significantly
different.
The validation tests of the calibration curve, accuracy, and carry-over may be carried out
using calibration standards and QCs in solution or substitute matrix
The validation tests of precision and stability tests must be carried out in the same
biological matrix as the samples in the trial.
MHLW: Not addressed

28. Partial validation
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EMA:
In situations where minor changes are made to an analytical method that has already
been validated, a full validation may not be necessary, depending on the nature of the
applied changes. Changes for which a partial validation may be needed include transfer of
the bioanalytical method to another laboratory, change in equipment, calibration
concentration range, limited sample volume, another matrix or species, change in
anticoagulant, sample processing procedure, storage conditions etc. All modifications
should be reported and the scope of revalidation or partial validation justified.
Note: Generally a full validation should be performed for each species (4.1 Full
validation)
Draft FDA: Similar, but OK with partial for matrix change within species & species
change within matrix
ANVISA: Mentioned
MHLW: Similar

29. Cross validation
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EMA:
Where data are obtained from different methods within and across studies or when data
are obtained within a study from different laboratories, applying the same method,
comparison of those data is needed and a cross validation of the applied analytical
methods should be carried out.
For the cross validation, the same set of QC samples or study samples should be
analysed by both analytical methods
For QC samples, the obtained mean accuracy by the different methods should be within
15% and may be wider, if justified.
For study samples, the difference between the two values obtained should be within 20%
of the mean for at least 67% of the repeats.
FDA: Similar, but leaving judgment to scientists
ANVISA: Not addressed
MHLW: Similar, but 20% for QCs is acceptable

30. Reporting
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EMA:
The validation report should include at least the following information:
• summary of the validation performances,
• details of the applied analytical method
• details of the assay procedure (analyte, IS, sample pre-treatment and analysis),
• reference standards (origin, batch number, CoA, stability and storage conditions),
• calibration standards and QC samples (matrix, anticoagulant, preparation, preparation
dates, and storage conditions),
• run acceptance criteria,
• analysis:
• table of all runs with dates, passed or failed and the reason for the failure
• table of calibration results of all accepted analytical runs,
• table of QC results of all accepted analytical runs (precision and accuracy);
• stability data of stock solution, working solution, QC
• data on selectivity, LLOQ, carry-over, matrix effect, dilution integrity;
• unexpected results obtained during validation with full justification of the action taken,
• deviations from method and/or SOPs
Draft FDA: Similar, but with more detail and recommendation for a summary table
ANVISA: Not addressed
MHLW: Similar, but a bit less detail

31. One method, one validation, one report (1)
Aspect Follow … and
Full validation EMA, MHLW
Reference standards EMA ANVISA: CoA information
Selectivity EMA ANVISA: Lipemic & hemolyzed
Carry-over EMA, MHLW ANVISA: experimental
LLOQ Any Note: BE requirement EMA
Calibration curve EMA ANVISA: weighting and non-linear
models
Accuracy & Precision EMA, MHLW ANVISA: add 5th QC = dilution QC
Dilution integrity EMA, MHLW ANVISA: dilution is part of A & P
Matrix effect EMA ANVISA: 2x lipemic & hemolyzed
MHLW: CV_MF < 15%
Stability EMA ANVISA, FDA, MHLW: triplicate
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32. One method, one validation, one report (2)
Aspect Follow … and
Recovery FDA, MHLW
Endogenous analyte ANVISA, FDA
Partial validation EMA, MHLW Note: species change within a matrix
or matrix change within a species
may require a full validation
Cross validation EMA
Reporting EMA, FDA
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Conclusions
 You can use EMA BMV as the basis
 CoA & selectivity: check ANVISA
 Matrix effect: check ANVISA & MHLW
 Stability: check ANVISA, FDA or MHLW
 Recovery: use FDA or MHLW
 Endogenous analyte: check ANVISA and/or FDA
 Note: ANVISA requires dilution QC in all A & P validation batches

33. Sample Analysis
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34. Analytical run
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EMA:
An analytical run consists of the blank sample (processed matrix sample without analyte
and without IS) and a zero sample (processed matrix with IS), calibration standards at a
minimum of 6 concentration levels, at least 3 levels of QC samples (low, medium and
high) in duplicate (or at least 5 % of the number of study samples, whichever is higher),
and study samples to be analysed.
All samples (calibration standards, QCs, and study samples) should be processed and
extracted as one single batch of samples in the order in which they intend to be submitted
or analysed. A single batch is comprised of samples which are handled at the same time,
i.e. subsequently processed without interruption in time and by the same analyst with the
same reagents under homogeneous conditions
Draft FDA: Similar, lot of detail
ANVISA: Not specifically addressed
MHLW: Not specifically addressed

35. Acceptance criteria of an analytical run
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EMA:
The back calculated concentrations of the calibration standards should be within ±15% of
the nominal value, except for the LLOQ for which it should be within ±20%. At least 75% of
the calibration standards, with a minimum of six, must fulfil this criterion. If one of the
calibration standards does not meet these criteria, this calibration standard should be
rejected and the calibration curve without this calibration standard should be re-evaluated,
and regression analysis performed.
If the rejected calibration standard is the LLOQ, the LLOQ for this analytical run is the next
lowest acceptable standard of the calibration curve. If the highest calibration standard is
rejected, the ULOQ for this analytical run is the next acceptable lower standard of the
calibration curve. The revised calibration range must cover all QC samples (low, medium
and high).
The accuracy values of the QC samples should be within ±15% of the nominal values. At
least 67% of the QC samples and at least 50% at each concentration level should comply
with this criterion..
Draft FDA: Similar
ANVISA: Similar
MHLW: Similar, but multi analyte case not addressed

36. Calibration range
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EMA:
If a narrow range of analyte concentrations of the study samples is known or anticipated
before the start of study sample analysis, it is recommended to either narrow the
calibration curve range, adapt the concentrations of the QC samples, or add new QC
samples to adequately reflect the concentrations of the study samples.
If a narrow range of analysis values is unanticipated, but observed after the start of
sample analysis, it is recommended that the analysis is stopped and either the calibration
range narrowed, QC concentrations revised, or QC samples at additional concentrations
are added. It is not necessary to reanalyse samples analysed before optimising the
standard curve range or QC concentrations. The same applies if a large number of the
study samples appear to be above the ULOQ. The calibration curve range should be
extended, if possible, and QC samples added or their concentrations modified.
At least 2 QC sample levels should fall within the range of concentrations measured in
study samples. If the calibration curve range is changed, the bioanalytical method should
be revalidated (partial validation) to verify the response function and to ensure accuracy
and precision.
Draft FDA: Similar, but less attention to inadequate calibration range
ANVISA: Not addressed
MHLW: Similar, but less attention to inadequate calibration range

37. Reanalysis of study samples
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EMA:
Possible reasons for reanalysis of study samples and criteria to select the value to be
reported should be predefined in the protocol, study plan or SOP
The following are examples of reasons for study sample reanalysis:
• rejection of an analytical run
• IS response significantly different from calibration standard and QC samples,
• improper sample injection or malfunction of equipment,
• the obtained concentration is above the ULOQ or below the run’s LLOQ (truncated)
• identification of quantifiable analyte levels in pre-dose samples or placebo sample,
• poor chromatography
For bioequivalence studies, normally reanalysis of study samples because of a
pharmacokinetic reason is not acceptable,
The safety of trial subjects should take precedence over any other aspect of the trial.
Consequently, there may be circumstances when it is necessary to reanalyse specific study
samples, e.g. where an unexpected result is identified that may impact patient safety
Draft FDA: Similar, but specifies number of replicates and no special description about
safety concerns
ANVISA: Similar, but no special description about safety concerns
MHLW: Similar, but no special description about safety concerns

38. (Re)Integration
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EMA:
Chromatogram integration and re-integration should be described in a SOP. Any deviation
from this SOP should be discussed in the analytical report. Chromatogram integration
parameters and in case of re-integration, initial and the final integration data should be
documented at the laboratory and should be available upon request.
Draft FDA: Similar, but requires reporting of both original and re-integration data
ANVISA: Not addressed
MHLW: Similar

39. Incurred samples reanalysis
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EMA:
As a guide, 10% of the samples should be reanalysed in case the number of samples is
less than 1000 samples and 5% of the number of samples exceeding 1000 samples.
Furthermore, it is advised to obtain samples around Cmax and in the elimination phase.
The concentration obtained for the initial analysis and the concentration obtained by
reanalysis should be within 20% of their mean for at least 67% of the repeats. Large
differences between results may indicate analytical issues and should be investigated.
Incurred sample reanalysis should be done at least in the following situations:
• toxicokinetic studies once per species
• all pivotal bioequivalence trials
• first clinical trial in subjects
• first patient trial
• first trial in patients with impaired hepatic and/or renal function
Samples should not be pooled, as pooling may limit anomalous findings.
NOTE: Since 2012, EMA does not accept BE studies anymore without ISR
Draft FDA: Similar, but recommended ISR size is 7%
ANVISA: Not addressed
MHLW: Similar

40. ISR Considerations
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 At least 67% within ± 20% of the their mean value <
A few in every batch or all in one or more dedicated batches
Overall result is OK, but a random X% shows extreme values
Overall result is OK, but a subject/time/… specific X% shows extreme values
Overall result is OK, but indicative of systematic difference between 1st and 2nd result
Instability of the drug (analyte)
Instability of metabolites

41. System suitability
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EMA: Not addressed
FDA: SOP is needed. Do not use CALs, QCs or samples
ANVISA: Not addressed
MHLW: Analytical instruments used in bioanalysis should be well maintained and properly
serviced. In order to ensure optimum performance of the instrument used for bioanalysis,
it is advisable to confirm the system suitability prior to each run, in addition to periodical
check. However, confirmation of the system suitability is not mandatory in bioanalysis,
because the validity of analysis is routinely checked in each analytical run

42. Reporting
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EMA:
The analytical report should include at least the following information:
• reference standards (origin, batch, certificate of analysis, stability, storage conditions)
• calibration standards and QC samples (storage conditions)
• run acceptance criteria (short description, reference to specific protocol or SOP)
• assay procedure (short description)
• sample tracking (dates, sample conditions, storage location and conditions)
• study sample analysis:
• content of the analytical run,
• table identifying all analytical runs and study samples, with run dates and results,
• table of calibration results of all (passed) analytical runs,
• table of QC results of all (passed) analytical runs;
• failed analytical runs (identity, assay date, reason for failure),
• deviations from method and/or SOPs
• reassay, excluding reassay due to analytical reasons, such as failed run
For bioequivalence studies, all chromatograms from the runs which include 20% of the
subjects, including the corresponding QC samples and calibration standards. For other
studies representative chromatograms should be appended to the report
Draft FDA: Similar, lot of detail
ANVISA: Not addressed
MHLW: Similar

43. One study, one method, one report
Aspect Follow … and
Analytical run EMA, FDA
Acceptance criteria Any
Calibration range EMA
Reanalysis of study
samples
EMA, MHLW,
ANVISA
Note: EMA requirement with
respect to patient safety
Reintegration EMA, MHLW FDA: original & reintegration
data
Incurred sample reanalysis EMA, MHLW
System suitability FDA, MHLW
Reporting EMA, FDA
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Conclusions
 You can use EMA BMV as the basis
 System suitability: check FDA & MHLW
 Note: EMA accepts/advocates reanalysis if an outlying result may indicate
issues with patient safety

44. Quality systems
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45. GxP
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EMA:
The validation of bioanalytical methods and the analysis of study samples for clinical trials
in humans should be performed following the principles of GCP. Further can be found in
the “Reflection Paper for Laboratories That Perform The Analysis Or Evaluation Of Clinical
Trial Samples.” (EMA/INS/GCP/532137/2010).
The validation of bioanalytical methods used in non-clinical pharmaco toxicological studies
that are carried out in conformity with the provisions related to Good Laboratory Practice
should be performed following the Principles of GLP.
EMA BE: The bioanalytical part of bioequivalence trials should be performed in
accordance with the principles of GLP. However, as human bioanalytical studies fall
outside the scope of GLP, the sites conducting the studies are not required to be
monitored as part of a national GLP compliance programme.
Note: OECD GLP
Draft FDA: Pre-clinical adhere to GLP (21 CFR 58), Clinical adhere to 21 CFR 320.29
ANVISA: Not addressed
MHLW: Not clearly addressed

46. One study, one method, one quality system ..
Aspect Follow … and
Method validation EMA Note: discrepancy between EMA
BMV (2011) & EMA BE (2010)
Sample analysis EMA, FDA
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Conclusions
 You can use EMA BMV as the basis
 For method validation for pre-clinical and clinical use the GLP system as the
standard quality system for the laboratory.
 For sample analysis from GLP studies (pre-clinical): use GLP as the quality
standard and claim compliance to GLP.
 For clinical sample analysis: use the GLP system as the standard quality
system for the laboratory
 For clinical studies in Europe: adhere to the EMA ‘GCLP’ reflection paper

47. Acknowledgements
 EBF members and Steering Committee for the learning
experiences.
 The GBC Founding & Steering Committee members and
Harmonization Team leads for the learning experiences
 Informa Life Sciences for giving me the opportunity to present
at their meeting.
 The regulators and inspectors for stimulating us to
continuously improve our work
 You, for your attention
and prof. Ernő Rubik for the inventing the cube.
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48. Further reading
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49. Papers
 Analytical methods validation: bioavailability, bioequivalence and pharmacokinetic studies.
Shah VP , Midha KK , Dighe SV , et.al.
Pharm Res. 9, 588 - 592 (1992)
 Bioanalytical method validation - a revisit with a decade of progress.
Vinod P. Shah, Kamal K. Midha, John W. A. Findlay, et.al.
Pharmaceutical Research 17(12), 1551-1557 (2000)
 Validation of immunoassays for bioanalysis: a pharmaceutical industry perspective
J.W.A. Findlay, W.C. Smith, J.W. Lee, et.al.
J. Pharmaceutical and Biomedical Analysis 21, 1249-1273 (200)
 Workshop on bioanalytical methods validation for macromolecules: summary report
Krys J. Miller, Ronald R. Bowsher, Abbie Celniker et.al.
Pharmaceutical Research 18(9), 1373-1383 (2001)
 Recommendations for the bioanalytical method validation of ligand-binding assays to support
pharmacokinetic assessments of macrmolecules
Binodh DeSilva, Wendell Smith, Russell Weiner, et.al.
Pharmaceutical Research 20(11), 1885-1900 (2003)
 Workshop/Conference report - Quantitative Bioanalytical Methods Validation and
Implementation: Best Practices for Chromatographic and Ligand Binding Assays
C. T. Viswanathan, Surendra Bansal, Brian Booth, et.al.
Pharmaceutical Research 24(10), 1962-1973 (2007)

European Bioanalysis Forum and the way forward towards harmonized regulations
Berthold Lausecker, Peter van Amsterdam, Margarete Brudny-Kloeppel, et.al.
Bioanalysis 1(5), 873-875 (2009)
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50.  Workshop Report and Follow-Up-AAPS Workshop on Current Topics in GLP Bioanalysis:
Assay Reproducibility for Incurred Samples-Implications of Crystal City Recommendations
Douglas Fast, Marian Kelley, C. Viswanathan, et. al.
The AAPS Journal 11(2), 238-241 (2009)
 Incurred sample reproducibility: views and recommendations by the European Bioanalysis
Forum
Philip Timmerman, Silke Luedtke, Peter van Amsterdam, et.al.
Bioanalysis 1(6), 1049-1056 (2009)
 Request for global harmonization of the guidance for bioanalytical method validation and
sample analysis
Philip Timmerman, Steve Lowes, Douglas Fast et.al.
Bioanalysis 2(4), 683 (2010)
 International harmonization of bioanalytical guidance
Surendra Bansal, Mark Arnold, Fabio Garofolo
Bioanalysis 2(4), 685-687 (2010)
 Towards harmonized regulations for bioanalysis: moving forward!
Peter van Amsterdam, Berthold Lausecker, Silke Luedtke, et.al.
Bioanalysis 2(4), 689-691 (2010)
 Bioanalytical method validation: notable points in the 2009 draft EMA Guideline and differences
with the 2001 FDA Guidance
Greame Smith
Bioanalysis 2(5), 929–935 (2010)
 Regulated bioanalysis and the desire for harmonized regulations in China
Daniel Tang, Dafang Zhong
Bioanalysis 2(12), 1913-1919 (2010)
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51.  SQA opinion paper on global harmonization of the bioanalytical method validation guidances
Christopher Tudan, Stephen Rogenthien, Anthony Jones
Bioanalysis 2(12), 1921-1925 (2010)
 Building the Global Bioanalysis Consortium – working towards a functional globally acceptable
and harmonized guideline on bioanalytical method validation
Peter van Amsterdam, Mark Arnold, Surendra Bansal, et.al.
Bioanalysis 2(11), 1801-1803 (2010)
 Workshop/Conference Report on EMA Draft Guideline on Validation of Bioanalytical Methods
Henning Blume, Erich Brendel, Margarete Brudny-Klöppel, et.al.
European Journal of Pharmaceutical Sciences 42, 300–305 (2011)
 Implication of differences in bioanalytical regulations between Canada, USA and south America
Mark Arnold
Bioanalysis 3(3), 253-258
 Bioanalytical procedures and regulation: towards global harmonization
Howard Hill
Bioanalysis 3(4), 365-367 (2011)
 Bioanalysis in Latin America: where are we and where are we going?
Rafael Eliseo Barrientos-Astigarraga
Bioanalysis 3(10), 1043-1045 (2011)
 US FDA/EMA harmonization of their bioanalytical guidance/guideline and activities of the
Global Bioanalytical Consortium
Fabio Garofolo, Josée Michon, Virginie Leclaire, et.al.
Bioanalysis (4)3, 231-236 (2012)
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52.  2012 white paper on recent issues in bioanalysis and alignment of multiple guidelines
Binodh DeSilva, Fabio Garofolo, Mario Rocci et.al.
Bioanalysis 4(18), 2213-2226 (2012)
 Managing scientific, technical and regulatory innovation in regulated bioanalysis: a discussion
paper from the European Bioanalysis Forum
Philip Timmerman, Neil Henderson, John Smeraglia et.al.
Bioanalysis 5(2), 139-145 (2013)
 The European Bioanalysis Forum community’s evaluation, interpretation and implementation
of the European Medicines Agency guideline on Bioanalytical Method Validation
Peter van Amsterdam, Arjen Companjen, Margarete Brudny-Kloeppel et.al.
Bioanalysis 5(6), 645-659 (2013)
 Regulated bioanalysis in Japan: where do we come from and where are we going?
Noriko Katori
Bioanalysis 5(11), 1321-1323 (2013)
 Current regulations for bioanalytical method validations
Mark E. Arnold, Rafael E. Barrientos-Astigarraga, Fabio Garofolo, et.al. in:
Wenkui Li, Jie Zhang, Francis L.S. Tse (ed)
Handbook of LC-MS Bioanalysis: Best practices, experimental protocols and regulations
John Wiley & Sons, Inc. (2013)
 A comparison of FDA, EMA, ANVISA and others on bioanalysis in support of
bioequivalence/bioavailability studies
Bradley Nash in: Wenkui Li, Jie Zhang, Francis L.S. Tse (ed)
Handbook of LC-MS Bioanalysis: Best practices, experimental protocols and regulations
John Wiley & Sons, Inc. (2013)
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53. Evaluating EMA and FDA guidance on bioanalytical method validation
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Guidance
 ANVISA: Resolution- RDC Nº 27, Requirements for the validation of bioanalytical methods.
http://bvsms.saude.gov.br/bvs/saudelegis/anvisa/2012/rdc0027_17_05_2012.pdf
 CDSCO: Guidelines for bioavailability and bioequivalence studies
http://cdsco.nic.in/html/BE%20Guidelines%20Draft%20Ver10%20March%2016,%2005.pdf
 CFDA: Technical guideline for human bioavailability an bioequivalence studies on chemical drug
products
http://www.cde.org.cn/attachmentout.do?mothed=list&id=167
 EMA: Guideline on the investigation of Bioequivalence
http://www.emea.europa.eu/docs/en_GB/document_library/Scientific_guideline/2010/01/WC500070039.pdf
 EMA: Guideline on the validation of bioanalytical methods
http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC500109686.pdf
 EMA: Reflection Paper for Laboratories that perform the analysis or evaluation of clinical trial samples
http://www.ema.europa.eu/docs/en_GB/document_library/Regulatory_and_procedural_guideline/2010/09/WC50
0096987.pdf
 FDA: Guidance for Industry: Bioanalytical Method Validation
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM070107.pdf
 FDA: Guidance for Industry - Safety Testing of Drug Metabolites
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm079266.pdf

54.  HPFB: Conduct and Analysis of Comparative BA Studies
http://www.hc-sc.gc.ca/dhp-mps/alt_formats/pdf/prodpharma/applic-demande/guide-
ld/bio/gd_cbs_ebc_ld-eng.pdf
 ICH: E6(R!) Guideline for Good Clinical Practice
http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Efficacy/E6_R1/Step4/E6_R1_
_Guideline.pdf
 ICH: Q2(R1): Validation of Analytical Procedures: Text and Methodology
http://www.ich.org/LOB/media/MEDIA417.pdf
 ICH: M3(R2): Guidance on Non-Clinical Safety Studies for the Conduct of Human Clinical Trials
and Marketing Authorization for Pharmaceuticals
http://www.ich.org/MediaServer.jser?@_ID=5544&@_MODE=GLB
 MHLW:Draft Guideline on Bioanalytical Method Validation in Pharmaceutical Development
http://www.nihs.go.jp/drug/BMV/BMV_draft_130415_E.pdf
 MHLW: Guideline on Bioanalytical Method Validation in Pharmaceutical Development
http://bioanalysisforum.jp/images/T130918I0020.pdf
 OECD Series on Principles of Good Laboratory Practice (GLP) and Compliance Monitoring
http://www.oecd.org/chemicalsafety/testing/oecdseriesonprinciplesofgoodlaboratorypracticeglpandcom
pliancemonitoring.htm
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55. … and may our dreams come true
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