Bioanalytical method validation - Global regulatory chalenges

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

Evaluating EMA and FDA guidance on bioanalytical method validation

Contents

1. Guidance

2. Method validation

3. Sample analysis

4. Quality systems

---------------------

Further reading

14 May 2014 2

Guidance

14 May 2014 Evaluating EMA and FDA guidance on bioanalytical method validation 3

What to look for

‘Global’ – OECD

– ICH

– WHO

‘Regional’ – FDA

– EMA

– ASEAN

National – HPFB

– MHLW

– CFDA

– etc.

Evaluating EMA and FDA guidance on bioanalytical method validation 4 14 May 2014

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) Guideline on Bioanalytical Method Validation in Pharmaceutical Development

USA: FDA (2013) DRAFT Guidance for Industry: Bioanalytical Method Validation


http://www.ich.org/LOB/media/MEDIA417.pdf





http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM070107.pdf

http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC500109686.pdf

http://bvsms.saude.gov.br/bvs/saudelegis/anvisa/2012/rdc0027_17_05_2012.pdf









http://wwwhourei.mhlw.go.jp/hourei/doc/tsuchi/T130717I0010.pdf







‘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


http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm070124.pdf





http://www.cde.org.cn/attachmentout.do?mothed=list&id=167



http://cdsco.nic.in/html/BE Guidelines Draft Ver10 March 16, 05.pdf

http://www.ema.europa.eu/pdfs/human/qwp/140198enrev1fin.pdf

http://www.sda.gov.cn/WS01/CL0844/67395.html

http://www.sda.gov.cn/WS01/CL0844/67395.html

http://www.hc-sc.gc.ca/dhp-mps/alt_formats/pdf/prodpharma/applic-demande/guide-ld/bio/gd_cbs_ebc_ld-eng.pdf



‘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


http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?CFRPart=58





http://www.oecd.org/document/63/0,3343,en_2649_34381_2346175_1_1_1_1,00.html

http://apps.who.int/tdr/publications/tdr-research-publications/gclp-web/pdf/gclp-web.pdf

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

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

http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/cfrsearch.cfm?cfrpart=11




http://ec.europa.eu/health/files/eudralex/vol-4/annex11_01-2011_en.pdf














14 May 2014 Evaluating EMA and FDA guidance on bioanalytical method validation 8

… let us try to simplify the matter


… and concentrate on EMA BMV (2011)

Evaluating EMA and FDA guidance on bioanalytical method validation 14 May 2014 10

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








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


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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.

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

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.

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

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).

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

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

Draft FDA: Described, but leaving judgment to scientists

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

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

Calibration curve


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

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

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

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’, 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

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


ANVISA: As part accuracy & precision

MHLW: Similar to EMA

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)


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.

Draft FDA: Similar, but also IS stock stability and reinjection reproducibility

ANVISA: Quite similar, but triplicate analysis

MHLW: Similar, but triplicate analysis

Recovery


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

Endogenous analytes


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

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)

Draft 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 leaving judgment to scientists


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

Draft FDA: Similar, but with more detail and recommendation for a summary table


MHLW: Similar, but a bit less detail

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


One method, one validation, one report (2)


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


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

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

Draft FDA: Similar, lot of detail

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..

Draft FDA: Similar

ANVISA: Similar

MHLW: Similar, but multi analyte case not addressed

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.

Draft FDA: Similar, but less attention to inadequate calibration range


MHLW: Similar, but less attention to inadequate calibration range

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

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

(Re)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.

Draft FDA: Similar, but requires reporting of both original and re-integration data


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.

NOTE: Since 2012, EMA does not accept BE studies anymore without ISR

Draft FDA: Similar, but recommended ISR size is 7%


MHLW: Similar

ISR Considerations


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

System suitability


EMA: Not addressed

FDA: SOP is needed. Do not use CALs, QCs or samples


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

Draft FDA: Similar, lot of detail


MHLW: Similar

One study, one method, one report


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


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

Draft 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

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.


Bioanalytical method validation - Global regulatory chalenges

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Transcript of Bioanalytical method validation - Global regulatory chalenges