Validation of Bioanalytical Methods for BE Studies

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Bioanalytical Methods
for BE Studies

Helmut Schütz
BEBAC
Moscow, 25 May 2012 1 • 74
Validation of Bioanalytical Methods for BE Studies

Main Topics

Validation = Suitability for Use?
 Method development – which Analyte?
 Matrix Effects in LC/MS-MS
 Ligand Binding Assays

Method Validation (Arlington Conferences I-III)

 Validation Plan
 Pre-Study Validation
 Validation Report
 Analytical Protocol / In-Study Validation

Plausibility
Review

Open (?) Issues
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Validation of Bioanalytical Methods for BE Studies

Validating Methods

Methods used for quantitative measurement of
analytes in any given biological matrix
must be
reliable and reproducible for the intended use…
 Accuracy  Cmax(ULOQ)
 Precision  AUCt/AUC∞ ≥80% (LLOQ)
 Selectivity
 Carry-over (LLOQ ≤5% Cmax)
 Sensitivity
 15–20% Bias / Precision
 Reproducibility
 Stability

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Validation of Bioanalytical Methods for BE Studies

Validating Methods

Level of Regulations
 Non-clinical studies: GLP
 Clinical studies:
 FDA: non-GLP
 EMA: The bioanalytical part of bioequivalence trials
should be conducted according to the applic-
able principles of Good Laboratory Practice
(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 compli-
ance programme.

Moscow, 25 May 2012 4 • 74

Validation of Bioanalytical Methods for BE Studies

Validating Methods

Reference standard
 FDA
 If possible, identical to the analyte.
 If not, an established chemical form (free base or acid, salt
or ester) of known purity can be used.
 Types
 Certified reference standards (e.g., USP compendial
standards)
 Commercially supplied reference standards obtained
from a reputable commercial source
 Other materials of documented purity custom-
synthesized by an analytical laboratory or other non-
commercial establishment.

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Validation of Bioanalytical Methods for BE Studies

Validating Methods

Reference standard
 FDA
 The source and lot number, expiration date, certificates
of analyses when available, and/or internally or externally
generated evidence of identity and purity should be
furnished for each reference standard.

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Validation of Bioanalytical Methods for BE Studies

Validating Methods

Reference standard
 EMA (GL 2011)
 Authentic and traceable source.
 Suitable reference standards, include certified standards
such as compendial standards (EPCRS, USP, WHO),
commercial available standards, or sufficiently
characterised standards prepared in-house or by an
external non-commercial organisation.
 CoA required (purity, storage conditions, expiration date,
batch number).
 Certified internal standard not required if suitability
demonstrated.
 Stable isotope-labelled IS for MS-methods recommended;
highest possible isotope purity, no isotope exchange.
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Validation of Bioanalytical Methods for BE Studies

Validation Plan

Written Document describing which steps will
be performed in the Validation.
 Purpose of Validation (e.g., ‘Validation of bio-
analytical method X for the determination of Y in
matrix Z’).
 Reference to established method (working instruc-
tion, SOP).
 If another document exists (already describing the
usal steps in validation) – cross-reference is
enough – otherwise detailed description is
necessary.

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Validation of Bioanalytical Methods for BE Studies

Pre-Study Validation

Terminology
 Specificity vs. Selectivity (IUPAC)
 Specific is considered to be the ultimate of selective,
meaning that no interferences are supposed to occur.
 selective (in analysis)
A term which expresses qualitatively the extent
to which other substances interfere with the determination
of a substance according to a given procedure.
 Specificity is a rather theoretical state; in the real world
we should assess selectivity only – which depends on the
analyte, metabolites, degradents, co-administered
compounds, matrix components,…
S Bansal and A DeStefano
Key Elements of Bioanalytical Method Validation for Small Molecules
The AAPS Journal 9(1), E109-E114 (2007)
http://www.aapsj.org/articles/aapsj0901/aapsj0901011/aapsj0901011.pdf

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Validation of Bioanalytical Methods for BE Studies

Pre-Study Validation

Full Validation
 Selectivity (FDA: mixed up with specificity)
Ability of an analytical method to differentiate and quantify the
analyte in the presence of other components in the sample.
 ≥6 sources of blank samples of the appropriate biological
matrix (ANVISA: +1 hemolytic, +1 lipemic) should be
tested for interference, and selectivity should be ensured
at the lower limit of quantification (LLOQ).
 Potential interfering substances: endogenous matrix
components, metabolites, decomposition products, and
in the actual study, concomitant medication and other
exogenous xenobiotics.
 Acceptable limit: ≤20% of response at LLOQ.

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Validation of Bioanalytical Methods for BE Studies

Pre-Study Validation

Full Validation
 Selectivity (cont’d)
Matrix Effects in MS-based Assays
 Matrix Factor
peak response in presence of matrix ions
MF=
peak response in mobile phase

MF=1: no matrix effects

MF<1: ion suppression
MF>1: ion enhancement or analyte loss
in the presence of matrix during analysis.

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Validation of Bioanalytical Methods for BE Studies

Pre-Study Validation

Full Validation
 Selectivity (cont’d)
Matrix Effects in MS-based Assays
 Suitability of internal standards (IS) in MS
 Stable isotope – labelled IS:
2H, 15N, 180 at 3 – 6 positions: different m/z, but

similar extraction and chromatography.

Should be used whenever possible!
 Structural analog IS
 Neutral radical (e.g., -CH3, -C2H5) preferred
 Radicals of different polarity/pK less suitable (e.g., -
OH, NH2), because extraction and/or chromato-
graphy will be influenced.
 Last resort: any other compound of similar polarity…

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Validation of Bioanalytical Methods for BE Studies

Pre-Study Validation

Full Validation
 Selectivity (cont’d)
A MF of ∼1 not necessary for a reliable bioanalytical assay.
However, a highly variable MF in individual subjects would
be a cause for the lack of reproducibility of analysis.
 If no stable isotope – labelled IS is used,
 to predict the variability of matrix effects in samples
from individual subjects, MF should be determined
in six individual lots of matrix.
 Variability in matrix factors (measured by CV)
should be less than 15%.
 If the matrix is rare and hard to obtain, the requirement
for assessing variability of MFs in six lots can
be waived.

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Validation of Bioanalytical Methods for BE Studies

Pre-Study Validation

Full Validation
 Selectivity (cont’d)
Do not forget separation of analyte and metabolite(s) in
LC/MS-MS!
 If using poor extraction and/or short run times:
 in-source dissociation of:
 acyl-glucuronides,
 esters,
 N-oxides,
 lactone-rings.
 Insuch a case interference is not the metabolite itself,
but the resulting parent-compound itself!
 Should be evaluated according to EMA’s GL.

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Validation of Bioanalytical Methods for BE Studies

Pre-Study Validation
low accuracy high accuracy
inaccurate accurate

low precison
high inprecison
inprecise

high precison
low inprecison
precise

bias, inaccuracy
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Validation of Bioanalytical Methods for BE Studies

Pre-Study Validation

Full Validation (cont’d)
 Precision
Replicate (≥5) analysis of known concentrations measured
at ≥4 levels (LLOQ, low=≤3×LLOQ, intermediate, high).
 Imprecision (CV%):
≤15% at each concentration (except at LLOQ, where
≤20% is acceptable.
 Inaccuracy (absolute mean bias – RE%):
≤15% at each concentration (except at LLOQ, where
≤20% is acceptable.
 Both parameters
 intra-batch (within analytical run).
 inter-batch (between analytical runs; aka repeatability).

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Validation of Bioanalytical Methods for BE Studies

Pre-Study Validation

Full Validation (cont’d)
 Precision
EMA: To enable evaluation of any trends over time within one
run, it is recommended to demonstrate accuracy of QC
samples over at least one of the runs with a size equivalent to
a prospective analytical run of study samples.
 Not required in FDA’s guidance.
Partial revalidation of ‘old’ methods for EMA!

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Validation of Bioanalytical Methods for BE Studies

Pre-Study Validation

Full Validation (cont’d)
 Precision (cont’d)
In 2006 problems were evident if trying to work according to
FDA’s bioanalytical guideline (2001)…
Survey on Ligand-Binding Assays (Arlington III)

32%
30%
23% 22% 23%

20%

10%

0%
15/20 20/25 30/30 other

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Validation of Bioanalytical Methods for BE Studies

Pre-Study Validation

Full Validation (cont’d)
 Precision (cont’d)
Ligand-binding assays according to
Arlington III White-Paper:
 Replicate (≥6) analysis of known concentrations
measured at ≥5 levels in duplicate.
 Anticipated LLOQ
 ∼3× LLOQ
 Midrange (geometric mean of LLOQ and ULOQ)
 High (∼75% of ULOQ)
 Anticipated ULOQ

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Validation of Bioanalytical Methods for BE Studies

Pre-Study Validation

Full Validation (cont’d)
 Precision (cont’d)
Ligand-binding assays according to Arlington III WP:
 Inter-batch imprecision (CV%) and inaccuracy (absolute
mean bias (RE%):
 ≤20% at each concentration (except at LLOQ and
ULOQ, where ≤25% is acceptable).
 Target total error (sum of the absolute value of the
RE% [accuracy] and inprecision [%CV%] should be
less than ≤±30% [≤±40% at the LLOQ and ULOQ]).
The additional constraint of total error allows for
consistency between the criteria for pre-study method
validation and in-study batch acceptance.

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Validation of Bioanalytical Methods for BE Studies

Pre-Study Validation

Full Validation (cont’d)
 Recovery
 The detector response obtained from an amount of the
analyte added to and extracted from the biological matrix,
compared to the detector response obtained for the true
concentration of the pure authentic standard.
 Recovery of the analyte does not need to be 100%, but
the extent of recovery of an analyte and of the internal
standard should be consistent, precise, and reproducible.
 Measured at low/intermediate/high level.
 Not required according to EMA’s GL!

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Validation of Bioanalytical Methods for BE Studies

Pre-Study Validation

Full Validation (cont’d)
 Carry-over
 Only required according to EMA’s GL!
 Assessment by injecting blank samples after a high
concentration sample or calibration standard during
method development.
 If unavoidable:
 Specific measures should be considered.
 Tested during the validation.
 Applied during the analysis of the study samples.
 Injection of blank samples after samples with an expected
high concentration, before the analysis of the next study
sample.
 Randomisation of samples should be avoided.

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Validation of Bioanalytical Methods for BE Studies

Pre-Study Validation

Full Validation (cont’d)
 Calibration/Standard Curve
Same matrix as the samples in the intended study spiked
with known concentrations (on basis of the concentration
range expected).
Number of standards: function of the anticipated range of
analytical values, nature of the analyte/response relationship.
 Blank sample (matrix sample processed without internal
standard),
 Zero sample (matrix sample processed with internal
standard),
 6 – 8 non-zero samples covering the expected range,
including LLOQ.

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Validation of Bioanalytical Methods for BE Studies

Pre-Study Validation

Full Validation (cont’d)
 Calibration/Standard Curve (cont’d)
 Simplest model that adequately describes the
concentration-response relationship should be used
(e.g., Almeida et al. 2002).
 Selection of weighting and use of a complex regression
equation should be justified (analysis of residuals; F-test,
Minimum AIC).
 Response at LLOQ: ≥5 times response of blank.
 Response at LLOQ: imprecision ≤20%, accuracy ±20%
from nominal concentration.
 Response at other levels: accuracy ±15% from nominal
concentration.

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Validation of Bioanalytical Methods for BE Studies

Pre-Study Validation

Full Validation (cont’d)
 Calibration/Standard Curve (cont’d)
 At least four out of six non-zero standards should
meet the above criteria, including the LLOQ and the
calibration standard at the highest concentration.
 Excluding individual standard points must not change
the model used.

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Validation of Bioanalytical Methods for BE Studies

LBA Calibration

Recommendations for 4-PL model
 Optimal Assay Design for Calibration
 ≥5 calibration concentrations (according to
Arlington III WP ≥6) and not more than 8.
 Calibrators should be prepared and analyzed in
duplicate or triplicate.
 Concentration progression should be logarithmic,
typically of the power of 2 or 3.
 Midpoint concentration of calibrators should be somewhat
greater than IC50.
 Anchor concentrations outside the expected validated
range should be considered for inclusion to optimize
the fit.
 Suboptimal plate layouts should be avoided.

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Validation of Bioanalytical Methods for BE Studies

LBA Layout
At left is a commonly used layout for an
assay in which the calibrators are pre-
pared in duplicate. In this plate confi-
guration calibrators are always located
in the same wells on the upper right of
the plate. This layout helps to ensure
proper identification of calibrators, but
it is a scheme that is susceptible to
positional effects on the plate.
The layout on the right is a much better
choice. In this scheme the calibrators
(as well as quality control [QC] samples
and study samples) are distributed more widely on the plate, with one of the replicat-
es positioned on the left side and the other on the right. The dilution direction is also
reversed, with increasing dilution going down the plate on the left side and up the
plate on the right.

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Validation of Bioanalytical Methods for BE Studies

Pre-Study Validation

Full Validation (cont’d)
 Stability
Stability of the analytes during sample collection and handling.
 Three Freeze-Thaw Cycles
≥3 aliquots at low and high levels stored for 24 hours
and thawed unassisted (?!) at room temperature.
When completely thawed, refrozen for 12 to 24 hours.
This cycle two more times repeated, then analyzed after
the third cycle.
If instable: samples should be frozen at -70 °C dur ing
another FT-cycle.

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Validation of Bioanalytical Methods for BE Studies

Pre-Study Validation

Full Validation (cont’d)
 Stability (cont’d)
 Short-Term Storage (bench top, room temperature)
Three aliquots of each of the low and high concentrations
should be thawed at room temperature and kept at this
temperature from 4 to 24 hours (based on the expected
duration that samples will be maintained at room
temperature in the intended study) and analyzed.

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Validation of Bioanalytical Methods for BE Studies

Pre-Study Validation

Full Validation (cont’d)
 Stability (cont’d)
 Long-Term Storage (frozen at the intended storage
temperature) should exceed the time between the date
of first sample collection and the date of last sample
analysis.
Determined by storing ≥3 aliquots of low/high levels under
the same conditions as the study samples.
Volume should be sufficient for analysis on three
occasions. Concentrations of all samples should be
compared to the mean of back-calculated values for the
standards at the appropriate concentrations from the first
day of long-term stability testing.

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Validation of Bioanalytical Methods for BE Studies

Pre-Study Validation

Full Validation (cont’d)
 Stability (cont’d)
 Long-Term Storage
Often not finished when clinical phase already starts
(Validation report contains a phrase like: ‘long-term
stability in progress’). Not recommended, see
http://www.emea.europa.eu/pdfs/human/chmptemplates/D80_AR_Generics_Non-
Clinical_Clinical_Guidance.pdf,
http://www.emea.europa.eu/Inspections/docs/gcp/INS-GCP-3a7.pdf
Brief description of analytical methods used, with emphasis on the
performance characteristics of assay validation and quality control.
Provide information regarding where the bioanalysis was performed.
In addition, it is essential to include the date of the start and finish of
the bio-analytical phase to see if the long-term stability data of the pre-
study validation is enough. Storage conditions of the samples should be
stated.

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Validation of Bioanalytical Methods for BE Studies

Pre-Study Validation

Full Validation (cont’d)
 Stability (cont’d)
 Stock Solution Stability of drug and the internal standard
should be evaluated at room temperature for ≥6 hours.
If the stock solutions are refrigerated or frozen for the
relevant period, the stability should be documented.
After completion of the desired storage time, the stability
should be tested by comparing the instrument response
with that of freshly prepared solutions.
Arlington III WP: If the reference standard is within its
expiration date when the stock solution is prepared, there
is no need to prepare a new stock solution when the
reference standard expires.

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Validation of Bioanalytical Methods for BE Studies

Pre-Study Validation

Full Validation (cont’d)
 Stability (cont’d)
 Post-Preparative Stability
Stability of processed samples, including the resident
time in the autosampler, should be determined.
The stability of the drug and the internal standard should
be assessed over the anticipated run time for the batch
size in validation samples by determining concentrations
on the basis of original calibration standards.

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Validation of Bioanalytical Methods for BE Studies

Pre-Study Validation

Full Validation (cont’d)
 Sample dilutions
of concentrations above the ULOQ.
 E.g., ∼150% of ULOQ diluted 1:1.
 Blank matrix should be used in dilution.
 Replicate (≥5) analysis.
 Imprecision (CV%): ≤15%
 Inaccuracy (absolute mean bias – RE%): ≤15%

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Validation of Bioanalytical Methods for BE Studies

Pre-Study Validation

Partial Validation (cont’d)
 Method transfers between laboratories
(or analysts…).
 Change in analytical methodology
(e.g., change in detection systems).
 Change in anticoagulant in harvesting
biological fluid.
 Change in matrix within species
(e.g., human plasma to human urine).
 Change in sample processing procedures.

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Validation of Bioanalytical Methods for BE Studies

Pre-Study Validation

Partial Validation (cont’d)
 Change in species within matrix (e.g., rat plasma to
mouse plasma).
 Change in relevant concentration range.
 Changes in instruments and/or software
platforms.
 Limited sample volume (e.g., paediatric study).
 Rare matrices.
 Selectivity – demonstration of an analyte in the
presence of concomitant medications and/or
specific metabolites.

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Validation of Bioanalytical Methods for BE Studies

Performing the Validation

Conducting the Validation strictly according
to the Validation Plan!
 Results must comply with limits set in the
Validation Plan.
 If not: The method is validated, but not valid!
Improve the method and start the cycle again.

Report of Results:
 Method Validation Report;
 will be referred in the Analytical Protocol of
PK/BA/BE-studies.

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Validation of Bioanalytical Methods for BE Studies

In-Study Validation

Application of Validated Method to Routine
Analysis
 System Suitability (SS)
 FDA (2001): Based on the analyte and technique, a specific SOP
(or sample) should be identified to ensure optimum operation of
the system used.
 Arlington III (2007): As part of qualifying instruments, performance
of SS ensures that the system is operating properly at the time of
analysis.
 SS checks are more appropriately used for chromatographic methods
to ensure that the system is sufficiently sensitive, specific, and
reproducible for the current analytical run.
 However, the SS tests do not replace the required run acceptance
criteria with calibration standards and QC samples.
 SS tests, when appropriate, are recommended to ensure success, but
are not required, nor do they replace the usual run acceptance criteria.
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Validation of Bioanalytical Methods for BE Studies

In-Study Validation

Study Samples should be analyzed according
to the Analytical Protocol.
 Minimum number of QCs (in multiples of three) should be
at least 5% of the number of unknown samples or six total
QCs, whichever is greater.
 Low / intermediate / high concentration levels
At least duplicates at each level.
Low within ≥LLOQ and 3×LLOQ
Intermediate near the center of the calibration range
(‘center’ according to Arlington III WP:
geometric mean of LLOQ and ULOQ)
High near the ULOQ (≥75% ULOQ)

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Validation of Bioanalytical Methods for BE Studies

In-Study Validation

Study Analyses (cont’d)
 Standards and QC samples can be prepared from the same
spiking stock solution, provided the solution stability and
accuracy have been verified (FDA 2001).
 Some kind of a vicious circle:
 One can use the same stock solution for calibration
and QC samples.
 One has to demonstrate that the stock solution was
prepared correctly and that its concentration is
accurate.
 How can one do that without comparing it to another,
– independently – prepared stock solution?
 Recommendation: Use two independent stock solutions
for standards and QCs to avoid trouble.

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Validation of Bioanalytical Methods for BE Studies

In-Study Validation

Study Analyses (cont’d)
Typical
Batch
System Suitability

unknown samples (period I / II staggered)

Don’t inject
unknown
samples in

Calibrators (Set II)

Calibrators (Set I)

random
order (carry-
QCs (Set I)

QCs (Set II)

over)!
Blank + IS
Blank

Solvent injections to prevent carry-over

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Validation of Bioanalytical Methods for BE Studies

In-Study Validation

Study Analyses (cont’d)
 Quality Control Samples (QCs) should be analyzed together
with Calibrators and study samples.
 Acceptance Criteria for an analytical run
QCs
85% – 115% accuracy for single determinations of QCs;
not more than 33% (two different out of six) per run should
be out of range.
Standard Curve
85% – 115% accuracy for 75% of standard points, except
at LLOQ (80% – 120%).
Values outside this ranges can be discarded, provided
they do not change the model established in validation.
No rejection of calibrators based on results of QCs!

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Validation of Bioanalytical Methods for BE Studies

In-Study Validation

Study Analyses (cont’d)
 Do not try to fool inspectors!

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Validation of Bioanalytical Methods for BE Studies

In-Study Validation

Study Analyses (cont’d)
 Samples can be analyzed with a single determination […]
if the assay method has acceptable variability as defined
by validation data.
 For a difficult procedure with a labile analyte*) where high
precision and accuracy specifications may be difficult to
achieve, duplicate or even triplicate analyses can be per-
formed for a better estimate of analyte. *) removed in Arlington III

replication CV [%]
Imprecision improves 0 (single) 20.0% 25.0% 30.0% 40.0% 50.0%
by ~25% going from
singlets to triplicates! 1 (duplicate) 16.8% 21.0% 25.2% 33.6% 42.4%
2 (triplicate) 15.2% 19.0% 22.8% 30.4% 38.0%
3 (quadruplicate) 14.1% 17.7% 21.1% 28.3% 35.4%

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Validation of Bioanalytical Methods for BE Studies

In-Study Validation

Reanalyses
 Acc. to Arlington III WP:
 Mandatory SOPs (additional to the ‘common’ ones…):
 Reintegration (incl. audit trail),
 Reassay criteria.
 EMA GL:
 Number of samples (and % of total number of samples)
should be discussed in the study report. Samples should
be identified and the initial value, the reason for reanalysis,
the values obtained in the reanalyses, the finally accepted
value and a justification for the acceptance should be
provided.

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Validation of Bioanalytical Methods for BE Studies

In-Study Validation

Study Reanalyses (cont’d)
 EMA GL (cont’d):
 Normally reanalysis of study samples because of a
pharmacokinetic reason is not acceptable. This is
especially important for bioequivalence studies, as this
may affect and bias the outcome of such a study. […]
reanalysis might be considered as part of laboratory
investigations, to identify possible reasons for results
considered as abnormal and to prevent the recurrence of
similar problems in the future.
 EMA BE GL (2010)
 First two sentences above stated in
Section 4.1.7 Bioanalytical methodology

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Validation of Bioanalytical Methods for BE Studies

In-Study Validation

Repeated samples
 SOP or guideline including acceptance criteria must be
established explaining the reasons for repeating sample
analysis.
Reasons for repeat analyses could include:
 repeat analysis of clinical or preclinical samples for
regulatory purposes
 inconsistent replicate analysis
 samples outside of the assay range
 sample processing errors
 equipment failure
 poor chromatography
 inconsistent pharmacokinetic data (EMA: not acceptable!)

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Validation of Bioanalytical Methods for BE Studies

In-Study Validation

Repeated samples (cont’d)
 Reassays should be done in triplicate if sample volume
allows.
The rationale for the repeat analysis and the reporting of the
repeat analysis should be clearly documented.
 Currently no specific guidelines, but all repeated samples
must be reported (original value, repeated value(s), used
value, justification):
 EU (Day 80 Critical Assessment Report, Generic medicinal
product, 2006):
http://www.emea.europa.eu/pdfs/human/chmptemplates/D80_AR_Generics_Non-
Clinical_Clinical_Guidance.pdf
Reasons for any reanalysis of samples and if the final value has been
decided correctly according to the relevant SOP.

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Validation of Bioanalytical Methods for BE Studies

In-Study Validation

Repeated samples (cont’d)
 FAD/CDER/OGD (Jan 2007):
http://www.fda.gov/downloads/Drugs/DevelopmentApprovalProcess/HowDrugsareDevelo
pedandApproved/ApprovalApplications/AbbreviatedNewDrugApplicationANDAGenerics/
UCM120957.pdf
Table 9 Reanalysis of Study Samples
Study No.
Additional information in Volume(s), Page(s)
Number of recalculated values used
Number of samples reanalyzed
Reason why assay was after reanalysis
repeated Actual number % of total assays Actual number % of total assays
T R T R T R T R
Pharmacokinetic1
Reason A (e.g. below
LOQ)
Reason B
Reason C
Etc.
Total
1 - If no repeats were performed for pharmacokinetic reasons, insert “0.0.”

Please provide a separate table for each analyte measured for each in-vivo study.

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Validation of Bioanalytical Methods for BE Studies

Plausibility Review

Plausibility Review of analytical data
 If ever possible, plan a blinded (!) Plausibility Review of
analytical data by an independent Pharmacokineticist as early
as possible.
 QC-cleared data only; start of review earliest if analyses of
∼50% of subjects are completed.
 Consistency within subjects!
 Pre-dose concentrations?
 Rising values in the terminal phase?
 Fluctuating values at Cmax?
 Re-analysis (‘pharmacokinetic repeats’):
values confirmed/rejected?
 Reanalysis of study samples because of a PK reason is
not acceptable (EMA BE GL 2010, bioanalytical GL 2011).
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Validation of Bioanalytical Methods for BE Studies

Case Study

concentration [ng/ml]
10 Plausibility Review: Subject 002 period 1
period 2

8
concentration [ng/ml]

10 Plausibility Review: Subject 001 period 1

period 2 6

8
4

6
2

4
0
0 4 8 12 16 20 24
2
time [h]

concentration [ng/ml]
10 Plausibility Review: Period 1 subject 001
subject 002
0
0 4 8 12 16 20 24 8
time [h]
6

subject time analyte γ-GT albumine

[h] [ng/ml] [U/l] [g/dl] 4

001 4.0 2.572 13 3.8

2
001 4.5 6.330 9 3.5
001 5.0 2.615 14 3.9
0
002 4.0 6.956 9 3.4 0 4 8 12 16 20 24
002 4.5 2.561 14 4.0 time [h]

002 5.0 9.262 8 3.4

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Validation of Bioanalytical Methods for BE Studies

Cross-Validation

Comparison of validation parameters when ≥2
analytical methods are used to generate data within
the same study or across different studies. Example:
an original validated bioanalytical method serves as
the reference and a revised bioanalytical method is the
comparator.

Cross-validation should also be considered when data
generated using different analytical techniques (e.g.,
LC/MS-MS vs. ELISA) in different studies are included
in a regulatory submission.

No specific recommendations in Arlington III WP.

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Validation of Bioanalytical Methods for BE Studies

Case Study
clindamycin 600 mg p.o.
100000
HPLC/UV (n=24)
t½: 5.61 h; r=0.981
concentration [ng/ml]

10000
GC/MS (n=20)
t½: 2.22 h; r=0.999
1000
LOQ: 20 ng/ml

100

10
0 4 8 12 16 20 24
time [h]

Identical LLOQ, but

bias in lower concen-
tration range?

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Validation of Bioanalytical Methods for BE Studies

Case Study

y=25.947+0.957*x+eps
4000
HLPC/UV [ng/ml]

3000

2000

1000 y=11.964+0.908*x+eps
150

HLPC/UV [ng/ml]
0
0 1000 2000 3000 4000
LC-MS/MS [ng/ml] 100

50

Bias in lower
0
concentration range! 0 50 100 150
LC-MS/MS [ng/ml]

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Validation of Bioanalytical Methods for BE Studies

Reporting Results

Avoid discrepancies between electronic data
and paper reports.
 Problems arise if electronic data in full precision
are transferred to the statistical database.
 Generally (paper-)reports contain only modified
results (rounded to decimal places or significant
figures, or – even worse – truncated values).
 If PK-metrics have to be re-calculated from the
paper-version or a PDF-file (i.e., during an
inspection), results may differ from reported ones…

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Validation of Bioanalytical Methods for BE Studies

Reporting Results

Reasons for rounding of analytical data:
 Pragmatic: Avoid discrepancies between paper
and electronic data which may raise unnecessary
questions.
 Scientific: Use of full precision data implies a
degree of accuracy/precision which is illusionary.
Raw data 3 decimal places 3 significant figures
31.41592653589793 31.416 31.4
3.141592653589793 3.142 3.14
0.3141592653589793 0.314 0.314

Rounding to three decimal places is suggest-

ing ability to distinguish between 31.4154
and 31.4165 – a difference of 0.0035% Better, but implies
still ~0.2% precision!
from the reported value!

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Validation of Bioanalytical Methods for BE Studies

Reporting Results

Personal opinion:
 Most analysts have digested Arlington I-II and are
familiar with 15% accuracy / precison (20% at
LLOQ), but routinely come up with results like
3.141 592653 589793.*)
 Subconsciously they believe, that such a result
is more correct than 3.14.
 If suggesting next time they should come up with

3.141 592653 589793238462 643383 279502 88,

they tell me, that I am a funny person…
*) at 15% CV: 95% Confidence Interval [2.21 – 4.07]
at 5% CV: 95% Confidence Interval [2.83 – 3.45]

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Validation of Bioanalytical Methods for BE Studies

Open (?) Issue

I have no opinion about ‘incurred samples’ –
an expression which has no easily
understandable meaning for me in the
English language. Nick Holford (2007)

http://www.pharmpk.com/PK07/PK2007028.html

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Validation of Bioanalytical Methods for BE Studies

Open (?) Issue

Incurred Sample Re-Analysis (Arlington III)
 Situations, where standards and QCs may not adequately
mimic that of study samples form dosed subjects.
 Metabolites converting to parent compound,
 Proteinbinding differences in patient samples,
 Recovery issues,
 Sample inhomogeneity,
 Mass spectrometric ionization matrix effects.
 It is generally accepted that the chance of incurred sample
variability is greater in humans than in animals, so the
following discussion pertains primarily to clinical studies.
 Final decision as to the extent and nature of the incurred sample
testing is left to the analytical investigator, and should be based on
an in-depth understanding of the method, the behavior of the drug,
metabolites, and any concomitant medications in the matrices of
interest.
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Validation of Bioanalytical Methods for BE Studies

Open (?) Issue

Incurred Sample Re-Analysis (cont’d)
 Considerations in selecting samples to be reassayed:
 concentration,
 patient population, and
 special populations (e.g., renally impaired),
 depending on what is known
 about the drug,
 its metabolism,
 and its clearance.
 Examples of studies that should be considered for incurred-
sample concentration verification are
 First-in-human,
 Proof-of-concept in patients,
 Special population, and
 Bioequivalence (!) studies.

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Validation of Bioanalytical Methods for BE Studies

Open (?) Issue

Incurred Sample Re-Analysis (cont’d)
 Re-assay of 15% of samples was required in Canada since
1992, but was removed in Sep 2003.
 Health Canada on 09 Jan 2008 published a ‘Notice: Replica-
tion of Incurred Samples in Bioavailability/Bioequivalence
Studies’:
 ‘[…] a voluntary submission of data collected on replicate samples since
2000. […] This information will be used for research purposes only and
will in no way affect past regulatory decisions. [...] Release of the
information will be limited to summary statistics, with no linkage between
the sponsor and the data.’
 HPB hopes ‘… to be able to present our findings at the next Canadian
Workshop on Recent Issues in GLP Bioanalysis on April 17-18, 2008 in
Montreal.’
http://www.hc-sc.gc.ca/dhp-mps/alt_formats/hpfb-
dgpsa/pdf/prodpharma/notice_bioan_avis_anbio_e.pdf

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Validation of Bioanalytical Methods for BE Studies

Open (?) Issue

Incurred Sample Re-Analysis (cont’d)
 European Initiative started by the ‘European Bioanalysis
Forum’:
http://www.aapspharmaceutica.com/meetings/files/112/PhilipTimmermanebfperspective.pdf
Until now only open to the industry, but collaboration planned
with other scientific and interprofessional groups on BA related
topics (academia, vendors, CROs, or regulatory bodies)…
 AAPS Workshop on Current Topics in GLP Bioanalysis:
Assay Reproducibility for Incurred Samples Samples –
Implications of Crystal City Recommendations (Feb 2008)
http://www.aapspharmaceutica.org/meetings/meeting.asp?id=112
http://www.aapspharmaceutica.org/GLP/
 EBF/EUFEPS Workshop (Apr 2010)
http://www.eufeps.org/spring10041516.html

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Validation of Bioanalytical Methods for BE Studies

Open (?) Issue

Incurred Sample Re-Analysis
 Expected to be mandatory for the FDA (sometimes already
requested).
 Will not be necessary in Canada.
 EMA: ‘For BE studies analysis of incurred samples should
always be carried out.’ (see below)

EMA
 GL on Validation of Bioanalytical Methods (Jul 2011)
http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC5
00109686.pdf
 Draft GL on the Validation of Bioanalytical Methods (Nov 2009)
http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/12/WC5
00018062.pdf
EMA received 436 pages [!] of comments from 55 sources
http://www.ema.europa.eu/docs/en_GB/document_library/Other/2011/08/WC500109687.pdf

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Validation of Bioanalytical Methods for BE Studies

ICSR / EMA

EMA 2011, Sections 6 and 7.3.3
 Recommended to evaluate accuracy of incurred
samples by reanalysis of study samples in separate
runs at different days. Extent of testing depends on
the analyte and the study samples, and should be
based upon in-depth understanding of the analytical
method and analyte. Should provide sufficient
confidence that the concentration being reported is
accurate.
 For BE studies ICSR should always be carried out.
 From several subjects around Cmax and in the
elimination phase.
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Validation of Bioanalytical Methods for BE Studies

ICSR / EMA

EMA 2011, Sections 6 and 7.3.3
 Samples should not be pooled.
 Difference between two results should be ≤20% of
the mean (≤30% for LBAs) for ≥⅔ of repeats.
 In case incurred sample analysis showed deviating
results, this should be investigated, and adequate
steps should be taken to minimize inaccuracy (and
imprecision).
 Remark: Not expected to reject the first study with
the method – but subsequent ones without improving
of the method!

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Validation of Bioanalytical Methods for BE Studies

EMA Reflection Paper

Reflection paper for laboratories that perform
the analysis or evaluation of clinical trial
samples (EMA, Feb 2012)
 Clarification
on GCP / GLP compliance
 Organisational details…

http://www.ema.europa.eu/docs/en_GB/document_library/Regulatory_and_procedural_guideline
/2012/03/WC500124406.pdf

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Validation of Bioanalytical Methods for BE Studies

Thank You!
Ensuring bioanalytical
compliance of BA/BE studies
Open Questions?

Helmut Schütz
BEBAC
Consultancy Services for
Bioequivalence and Bioavailability Studies
1070 Vienna, Austria
helmut.schuetz@bebac.at

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Validation of Bioanalytical Methods for BE Studies

References

Collection of links to global documents
US-FDA
http://bebac.at/Guidelines.htm  Center for Drug Evaluation and Research (CDER)

ICH Reviewer Guidance: Validation of Chromatographic
 Q2A: Validation of Analytical Methods: Methods (Nov 1994)
Definitions and Terminology (1994)  Center for Drug Evaluation and Research (CDER),
 Q2B: Validation of Analytical Methods: Center for Veterinary Medicine (CVM)
Methodology (1996) Guidance for Industry. Bioanalytical Method Validation
(May 2001)

OECD
 OECD Environmental Health and Safety
Brazilian Sanitary Surveillance Agency (ANVISA)
Publications, Series on Principles of Good  Manual for Good Bioavailability and Bioequivalence
Laboratory Practice and Compliance Monitoring Studies.
(1995-2002) Volume 1, Module 2: Analytical Step (2000)

WHO
European Directorate for the Quality of Medicines &
 Handbook for GLP (2001) HealthCare (EDQM)
 Validation of Analytical Procedures (Jun 2005)
 Fortieth Report - TRS No. 937 (2006)
 Uncertainty of Measurements (Dec 2007)
 Annex 7: Multisource (generic)
Part I – compliance testing
pharmaceutical products: guidelines on Part II – other than compliance testing
registration requirements to establish  Qualification of Equipment (core document, Jul 2007 )
interchangeability Annex 1: Qualification of HPLC Equipment (Feb 2007)
 Annex 9: Additional guidance for Annex 2: Qualification of GC Equipment (Oct 2006)
organizations performing in vivo
EMA
bioequivalence studies  Guideline on bioanalytical method validation (2011)

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Validation of Bioanalytical Methods for BE Studies

References
EMEA GCP Inspector’s Group DeSilva B et al.
Annex VII to Procedure for Conducting GCP Inspec- Recommendations for the Bioanalytical Method Validation of
tions requested by the EMEA: Bioanalytical Part, Phar- Ligand-binding Assays to Support Pharmacokinetic
macokinetic and Statistical Analyses of Bioequivalence Assessments of Macromolecules
Trials (2008) Pharm Res 20, 1885–90 (2003)
Reflection paper for laboratories that perform the Shah VP et al.
analysis or evaluation of clinical trial samples (2012) Analytical methods validation: Bioavailability, bioequivalence
and pharmacokinetic studies
Pachla LA, Wright DS, and DL Reynolds Int J Pharm 82, 1–7 (1992)
Bioanalytical Considerations for Pharmacokinetic and → ‘Arlington I’ (Dec 3-5, 1990)
Biopharmaceutic Studies Shah VP, et al.
J Clin Pharmacol 21, 332–5 (1981) Bioanalytical Method Validation—A Revisit with a Decade of
Cartwright AC et al. Progress
International harmonization and consensus DIA Pharm Res 17, 1551–7 (2000)
meeting on bioavailability and bioequivalence testing → ‘Arlington II’ (Jan 12-14, 2000)
requirements and standards Viswanathan CT, et al.
Drug Information Journal 25, 471 (1991) Workshop/Conference Report—Quantitative Bioanalytical
Methods Validation and Implementation: Best Practices for
Karnes ST, Shiu G, and VP Shah Chromatographic and Ligand Binding Assays
Validation of Bioanalytical Methods The AAPS Journal 9(1) Article 4, E30–41 (2007)
Pharm Res 8, 421–6 (1991) http://www.aapsj.org/articles/aapsj0901/aapsj0901004/aapsj0901004.pdf
Almeida AM, Castel-Branco MM, and AC Falcão → ‘Arlington III’ (May 1-3, 2006)
Linear regression for calibration lines revisited: JWA Findlay and R Dillard
weighting schemes for bioanalytical methods Appropriate Calibration Curve Fitting in Ligand Binding Assays
The AAPS Journal 9(2) Article 29, E260–7 (2007)
Journal of Chromatography B 774, 215–22 (2002) http://www.aapsj.org/articles/aapsj0902/aapsj0902029/aapsj0902029.pdf

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Validation of Bioanalytical Methods for BE Studies

Routine Drug Analysis Process and

Run Acceptance Criteria (Arlington III WP)
Process or Criteria Chromatographic Assays Ligand-Binding Assays
Preparation of Standards and QC samples can be prepared from the same spiking stock solution,
standards and QC provided the solution stability and accuracy have been verified. A single source of
samples matrix may also be used, provided selectivity has been verified.
Placement of samples Standard curve samples, blanks, QCs, and study samples can be arranged as
considered appropriate within the run, and support detection of assay drift over the
run.
Number of calibration Include with each analytical batch: Include with each analytical batch or
standards in a run  Blank matrix (sample without IS) microtiter plate:
 Zero standard (matrix sample with IS)  Blank matrix
 Non-zero calibration standards:  Non-zero calibration standards:
≥6 standard points ≥6 standard points. Can include
anchor points (below LLOQ or
above ULOQ in the asymptotic
low- and high-concentration end
of the standard curve).

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Validation of Bioanalytical Methods for BE Studies

Routine Drug Analysis Process and

Run Acceptance Criteria (Arlington III WP)
Process or Criteria Chromatographic Assays Ligand-Binding Assays
Acceptance criteria for Residuals (absolute difference between the Residuals for each calibration
calibration standards back calculated and nominal concentration) standard should meet the following
for each calibration standard should meet limits:
the following limits:  LLOQ and ULOQ standards
 LLOQ standard <20% <25%
 All other standards <15%  All other standards <20%
 Any anchor points if used, are
not to be included in the above
acceptance criteria.
A minimum of 75% standards (at least 6 nonzero points) should be within the
above limits for the analytical run to qualify. Values falling outside these limits can
be discarded, provided they do not change the established model.

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Validation of Bioanalytical Methods for BE Studies

Routine Drug Analysis Process and

Run Acceptance Criteria (Arlington III WP)
Process or Criteria Chromatographic Assays Ligand-Binding Assays
Number of QC samples Include QC samples at the following 3 con- QC samples at the following 3 con-
in a batch centrations (within the calibration range) in centrations (within the calibration
duplicate with each analytical batch: range) in duplicate should be added
 Low: near the LLOQ (up to 3× LLOQ) to each microtiter plate:
 Medium: midrange of calibration curve  Low: above the second non-
 High: near the high end of range anchor standard, ~3× LLOQ
 Medium: midrange of calibration
curve
 High: below the second non-
anchor point high standard at
~75% of ULOQ
Each analytical batch should contain 6 or a minimum of 5% of the total number of
unknown samples. Add QCs in multiples of three concentrations (low, medium,
high) when needed.

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Validation of Bioanalytical Methods for BE Studies

Routine Drug Analysis Process and

Run Acceptance Criteria (Arlington III WP)
Process or Criteria Chromatographic Assays Ligand-Binding Assays
Acceptance criteria for Allowed % deviation from nominal values: Allowed % deviation from nominal
QC samples  QCs prepared at all concentrations values:
greater than LLOQ <15%  QCs prepared at all concen-
 Low QC (if prepared at LLOQ) <20% trations other than LLOQ and
ULOQ <20%
 Low and high QC (if prepared at
LLOQ or ULOQ) <25%
 In certain situations wider
acceptance criteria may be
justified, e.g., when total error
during assay validation
approaches 30%.
At least 67% (4 of 6) of the QC samples should be within the above limits; 33% of
the QC samples (not all replicates at the same concentration) can be outside the
limits. If there are more than 2 QC samples at a concentration, then 50% of QC
samples at each concentration should pass the above limits of deviation.

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Validation of Bioanalytical Methods for BE Studies

Routine Drug Analysis Process and

Run Acceptance Criteria (Arlington III WP)
Process or Criteria Chromatographic Assays Ligand-Binding Assays
Replicate analysis In general, samples can be analyzed with Accuracy can generally be improved
a single determination without replicate by replicate analysis. Therefore,
analysis if the assay method has accept- duplicate analysis is recommended.
able variability as defined by the validation If replicate analysis is performed, the
data. Duplicate or replicate analysis can be same procedure should be used for
performed for a difficult procedure where samples and standards.
high precision and accuracy may be
difficult to obtain.
Multiple analytes in a Samples involving multiple analytes in a run should not be rejected based on the
run data from one analyte failing the acceptance criteria.
Rejected runs The data from rejected runs need not be documented, but the fact that a run was
rejected and the reason for failure should be reported.

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