Global Regulatory Issues: one BA method, one validation, one report ...

Global Regulatory Issues:
one BA method, one validation, one report …
Peter van Amsterdam (Abbott/EBF/GBC)
18 July 2013
14th Annual Land O’ Lakes Bioanalytical Conference

http://www.europeanbioanalysisforum.eu
Contents
1. Some history lessons
2. Guidance
3. Method validation
4. Sample analysis
5. Quality systems
6. Further reading
18-Jul-2013 2

The evolution of Regulated Bioanalysis
18-Jul-2013 http://www.europeanbioanalysisforum.eu 3

The early years of regulations
 1965: EEC 65/65 (reaction to Thalidomide)
o No real focus on bioanalysis
 1978: 21 CFR 58
 1982: OECD 1
o Both are General GLP guidelines (preclinical safety)
o Quality system ensure the uniformity, consistency, reliability, reproducibility,
quality, and integrity pre-clinical safety tests.
 Eighties (flowing over in the Nineties)
o Increased focus on Bioequivalence studies (including paragraphs on
bioanalytical methodology to be applied
o EU, FDA, Australia, Canada to lead
 BMV workshop – (Crystal City-I)
o < 1990 = lack of uniformity in industry wrt validation bioanalytical methods
o Crystal City-I was first international conference with focus on Bioanalytical
method validation and sample analysis
o Resulted in Shah paper (Pharm Res. 1992;9:588-592).

Crystal City I
Shah
2001 FDA
Guidance
CC-I CC-II CC-III
Crystal City
Conferences
Conference
papers
Regulatory
Guidance
Additional white
papers
CC-IV
(ISR)
CC-IV
Fast
1990 2000 2010
CC III
Viswanathan
CC II
Shah (chrom.)
Miller (LBA)
DeSilva
Bioanalysis regulations >1990 (simplified)

The broader and global context
18-Jul-2013
20201960 1970 1980
1965:
65/65/EEC
1979:
US 58cfr21
1982:
OECD GLP
A B C
Anvisa RDC 899
HC removes ISR
1988:
Australian draft
CO6: 7581c
EMA draft
Anvisa update
Open letter to
FDA & EMA
GBC
formed
More countries or
regions likely to issue
Guidelines
D
E
1990 2000 2010
Thalidomide
A. scientist adopting home designed quality systems
B. scientist shopping for inspiration in other areas –
peers, DIN, EPA,..
C. scientist regrouped around Shah paper
D. multiple countries issuing regulations of BA included in BE
guidelines
E. Industry increase meeting frequency (e.g. APA, EBF, CVG)
recommendation papers after (broad) internal discussions
EMA final
MHLW draft
http://www.europeanbioanalysisforum.eu 6

The broader and global context: more
detail
18-Jul-2013
20201960 1970 1980
1965:
65/65/EEC
1979:
US 58cfr21
1982:
OECD GLP
A B C
Anvisa RDC 899
HC removes ISR
1988:
Australian draft
CO6: 7581c
EMA draft
Anvisa update
Open letter to
FDA & EMA
GBC
formed
More countries or
regions likely to issue
Guidelines
D
E
1990 2000 2010
Thalidomide
CFR 21 part 11, ICH
S3A, ICH-E6, ICH
M3 (R2), MHRA
GcLP, Etc…
A bucket full of other
adjacent regulations:
EMA final
MHLW draft
http://www.europeanbioanalysisforum.eu 7

Technology developments
8
20201960 1970 1980 1990 2000 2010
TLC, GC
(LC-UV)
immunoassays
Sub μg/mL
GC2, GC-MS
GC-NPD/ECD
HPLC-
UV/FL/EC
immunoassays
ng/mL
TLC
Immunoassays
bioassays
μg/mL
GC2, GC-MS,
GC-NPD/ECD
HPLC-
UV/FL/EC, LC-
MS/MS,
Old school
Immunoassays
Sub ng/mL
LC-MS/MS,
New generation
Binding assays
AMS
ICP-MS
pg/mL
New generation
LC and MS(/MS)
and Binding
assays
Sub pg/mL?
18-Jul-2013 http://www.europeanbioanalysisforum.eu
Sensitivity doubles every 2 years (Moore’s law of bioanalysis)

Manual low throughput
μg limits of quantification
Chromatography: Multiple assay formats
LBA: Limited assay formats
Paper raw data
PK of unchanged drug
 automated high throughput
 sub-pg limits of quantification
 1 single assay format (LC-MS/MS)
 multiple (and novel) assay formats
 electronic raw data
 PK/PD, TK, metabolites, biomarkers,..
20201960 1970 1980 1990 2000 2010
a lot
happened
9
Highlights from technology

Evolutions in the Pharma landscape
around the turn of the century
and how it (may have) impacted regulated bioanalysis across industry:
 Portfolio changes in industry: new targets, new disease models
o Increased development time for small molecule scaffold  less NCE
o Increased emphasis on peptides and proteins  more NBE
• Enabling also faster development from Discovery to market
• Creating a boost in (new and innovative) LBA developments
 Patent expirations (of multi-billion dollar selling drugs):
o R&D optimise life cycle management
• More Bioequivalence (BEq) studies filed from R&D Pharma
o Generic Pharma boosting
• More BEq studies (with bioanalysis often outsourced) filed from generic Pharma
o Economic pressure on R&D Pharma calling for re-organisations
• More (bioanalytical) outsourcing
o CROs growing their business exponentially (also outside EU/US)
• More people involved = more difference in how quality is achieved and documented
• More regions involved

Guidance

What to look for
 ‘Global’
– OECD
– ICH
– WHO
 ‘Regional’
– FDA
– EMA
– ASEAN
 National
– HPFB
– MHLW
– CFDA
– etc.
http://www.europeanbioanalysisforum.eu 1318-Jul-2013
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

‘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)
Draft Guideline on Bioanalytical Method Validation in
Pharmaceutical Development

‘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
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

‘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)
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

… let us try to simplify the matter

… and concentrate on EMA BMV (2011)
http://www.europeanbioanalysisforum.eu18-Jul-2013 18
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
 Final & Current
– FDA is from 2001*)
– ANVISA is more directed to BE studies
– MHLW is a draft
*) not taking ‘updates’ of CC-III & CC-IV into account

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

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

Method Validation

Full Validation
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.
FDA: Less defined and seems to be more lenient
ANVISA: Less defined, but follows same principles. Requires chromatographic
method for ‘chromatographable analytes’
MHLW: Quite similar to EMA

Reference standards
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.
FDA: Similar to EMA, but requires same salt, base or free acid form. SILIS not
mentioned
ANVISA: Prefers pharmacopeia reference standards, recommends SILIS and has
detailed requirements on CoA information
MHLW: Similar to EMA, but less explicit

Selectivity
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).
FDA: Similar, but a bit less explicit
ANVISA: Similar, but no co-med or metabolites mentioned. Requires hemolyzed and
lipemic
MHLW: Similar, but no co-med or metabolites mentioned

Carry-Over
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
FDA: Not addressed
ANVISA: Similar, but also specifies how to conduct the experiment
MHLW: Similar

Lower limit of quantification
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
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

Calibration curve (1)
EMA:
The calibration standards should be prepared in the same matrix as the intended study
samples. There should be one calibration curve for each analyte studied in the method
validation and for each analytical run.
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 pharmacokinetics of the
analyte of interest.
A minimum of six calibration concentration levels should be used, in addition to the blank
sample (processed matrix without analyte and IS) and a zero sample (processed matrix
with IS). Each calibration standard can be analysed in replicate.
A relationship which can simply and adequately describe the response of the instrument
with regard to the concentration of analyte should be applied. The blank and zero samples
should not be taken into consideration to calculate the calibration curve parameters.
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 calibration standards, with a minimum of six calibration standard levels, must fulfil this
criterion.

Calibration curve (2)
EMA:
In case replicates are used, the criteria (within ±15% or ±20% for LLOQ) should also be
fulfilled for at least 50% of the calibration standards tested per concentration level.
In case all replicates of the LLOQ or the ULOQ calibration standard are rejected then the
batch should be rejected from the validation, the possible source of the failure be
determined and the method revised (if necessary). If the next validation batch also fails,
then the method should be revised before restarting validation.
Although the calibration curve should preferably be prepared using freshly spiked
samples, it is allowed to use previously prepared and stored calibration samples, if
supported by appropriate stability data.
FDA: Similar, but (far) less detail. Goodness of fit
ANVISA: Quite similar plus requirement for weighting and minimal 8 calibration
standards for non-linear models
MHLW: Similar

Accuracy
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
FDA: Similar, however minimum of 3 concentrations
ANVISA: Similar, but 5 concentrations required: LLOQ, Lo, Me, Hi & ULOQ
MHLW: Similar. Me QC at midpoint cal. curve

Precision
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’
FDA: Similar, however minimum of 3 concentrations
ANVISA: Similar, but 5 concentrations required: LLOQ, Lo, Me, Hi & Dilution QC
MHLW: Similar

Dilution integrity
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
FDA: As part of sample analysis, no criteria
ANVISA: As part accuracy & precision
MHLW: Similar

Matrix effect
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)
FDA: Indicated
ANVISA: Similar, but: 8 samples (4 normal, 2 lipemic, 2 hemolyzed) and ‘the degree
of hemolysis’
MHLW: Similar, but CV MF < 15%

Stability (1)
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.

Stability (2)
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.
FDA: Similar, but some aspects in greater detail and some in less detail. Triplicate
analysis, but no criteria
ANVISA: Quite similar, but triplicate analysis
MHLW: Similar, but triplicate analysis

Recovery
EMA: Not addressed
FDA: Recovery of the analyte need not be 100%, but the extent of recovery of an analyte
and of the internal standard should be consistent, precise, and reproducible. Recovery
experiments should be performed by comparing the analytical results for extracted
samples at three concentrations (low, medium, and high) with unextracted standards that
represent 100% recovery.
ANVISA: Not addressed
MHLW: Similar to FDA

Endogenous analytes
EMA: Not addressed
FDA: Not addressed
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

Partial validation
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)
FDA: Similar, but OK with partial for matrix change within species & species change
within matrix
ANVISA: Mentioned
MHLW: Similar

Cross validation
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 no criteria given
MHLW: Similar, but 20% for QCs is acceptable

Reporting
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
FDA: Similar, lot of detail
MHLW: Similar

One BA 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: 5th = 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

One BA method, one validation, one report
(2)
Recovery FDA, MHLW
Endogenous analyte ANVISA
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
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
 Note: ANVISA requires dilution QC in all A & P validation batches

Sample Analysis

Analytical run
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
FDA: Similar
ANVISA: Not specifically addressed
MHLW: Not specifically addressed

Acceptance criteria of an analytical run
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..
FDA: Similar
ANVISA: Simiar
MHLW: Similar

Calibration range
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.
FDA: Not addressed
MHLW: Similar

Reanalysis of study samples
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
FDA: Only the formal part is addressed
ANVISA: Similar except re: safety
MHLW: Similar except re: safety

Integration
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.
FDA: Similar
MHLW: Similar

Incurred samples reanalysis
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.
FDA: Not addressed
MHLW: Similar

System suitability
EMA: Not addressed
FDA: SOP is needed
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

Reporting
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
FDA: Similar, lot of detail
MHLW: Similar

One study, one method, one report
Analytical run EMA, FDA
Acceptance criteria for
an analytical run
Any
Calibration range EMA
Reanalysis of study
samples
EMA, MHLW,
ANVISA
Note: EMA requirement with
respect to patient safety
Reintegration EMA, FDA, MHLW
Incurred sample
reanalysis
EMA, MHLW
System suitability FDA, MHLW
Reporting EMA, FDA
Conclusions
 System suitability: check FDA & MHLW
 Note: EMA accepts/advocates reanalysis if an outlying result may indicate
issues with patient safety

Quality systems

GxP
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
FDA: Pre-clinical adhere to GLP (21 CFR 58), Clinical adhere to 21 CFR 320.29
MHLW: Not clearly addressed

One study, one method, one quality system ..
Method validation EMA Note: discrepancy between EMA
BMV (2011) & EMA BE (2010)
Sample analysis EMA, FDA
Conclusions
 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

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