1. No category

Japan Bioanalysis Forum
中村隆広
（株式会社新日本科学）
Summary of the Draft Guideline on Bioanalytical
Method (Ligand Binding Assay) Validation in
Pharmaceutical Development
http://bioanalysisforum.jp/
医薬品開発における生体試料中薬物濃度分析法
（リガンド結合法）のバリデーションに関する
カイドライン案の概要
Takahiro Nakamura, PhD, DJSOT
(Shin Nippon Biomedical Laboratories, Ltd. , SNBL)
5th JBF Symposium, Draft BMV Guideline for LBA
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Japan Bioanalysis Forum
5th JBF Symposium, Draft BMV Guideline for LBA
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1. Scope: リガンド結合法を用いて分析する低分子化合物も対象
The guideline also applies to low-molecular-weight drugs
that are analyzed by LBAs.
2. Reference standard: 標準物質はその特性の精査が必要
It is necessary to show well-established characteristics of
reference standard.
3. Full validation: 市販キットもフルバリデーションが必要
A full validation is required for a commercialized kit.
4. MRD: フルバリデーションの実施前にMRDを設定する.
Determination of minimum required dilution (MRD) before
a full validation.
5. Specificity: 特異性を評価する. Specificity should be evaluated.
http://bioanalysisforum.jp/
Contents (1/2)
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6. Selectivity: 選択性の評価方法が低分子ガイドラインとは異なる.
Evaluation method for selectivity differs from that of LMW.
7. Accuracy and precision: 真度及び精度は、定量上限のQC試料に
ついても評価が必要であり、トータルエラーについても評価する.
Accuracy and precision should be evaluated at ULOQ, and
total error should be evaluated.
8. QCs: 高濃度のQC試料の濃度を定量上限の1/3以上とする.
Concentration of high-level QC is at least 1/3 of ULOQ.
9. Dilutional linearity: 希釈直線性の評価が必要
Dilutional linearity should be evaluated.
10. Partial validation: 重要試薬のロット変更やMRDの変更時にパー
シャルバリデーションが必要である. Partial validation should be
performed after changing a critical reagent lot or MRD.
http://bioanalysisforum.jp/
Contents (2/2)
5th JBF Symposium, Draft BMV Guideline for LBA
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Guideline/Guidance Comparison on BMV for LBA
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Scope in MHLW
This guideline is applicable to the validation of analytical methods
based on ligand binding assays (LBAs) to measure concentrations
of drugs in biological samples obtained in toxicokinetic studies
and clinical trials, as well as to the analyses of study samples using
such methods. The information in this guideline generally applies
to the quantification of peptides and proteins as well as lowmolecular-weight drugs that are analyzed by LBAs. A typical
example of LBA is immunological assay based on antigen-antibody
reaction, such as enzyme immunoassay (EIA).
http://bioanalysisforum.jp/
1. リガンド結合法を用いて分析する低分子化合物も対象
The guideline also applies to low-molecular-weight drugs
that are analyzed by LBAs. (endogenous compounds)
Analyte is the same as for endogenous compounds
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Reference Standard
MHLW Draft Guideline:
A reference standard serves as the scale in quantifying an
analyte, ……... A certificate of analysis or an alternative
statement that provides information on lot number,
content (amount, purity, or potency), storage conditions,
and expiration date or re-test date should accompany the
standard. As a reference standard, it is necessary to show
its authenticated source and its characteristics should be
well-established.
5th JBF Symposium, Draft BMV Guideline for LBA
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2. 標準物質はその特性の精査が必要
It is necessary to show well-established characteristics of
reference standard.
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Reference Standard
Macromolecules are heterogeneous and their potency and
immunoreactivity may vary. The reference material should be
well characterised and documented (e.g. certificate of analysis
and origin).The purest reference standard available at the time
should be procured. It is strongly recommended that the
batch of the reference standard used for the preparation of
calibration standards and QC samples is the same as used for
dosing in the non clinical and clinical studies. In case of change
of batch, an analytical characterisation and bioanalytical
evaluation should be carried out prior to use to ensure that
the performance characteristics of the method are not altered.
5th JBF Symposium, Draft BMV Guideline for LBA
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EMA Guideline:
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Japan Bioanalysis Forum
Reference Standard
A. Key Reagents
Key reagents, such as reference standards, antibodies, tracers,
and matrices should be characterized appropriately and
stored under defined conditions.
5th JBF Symposium, Draft BMV Guideline for LBA
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FDA Draft Guidance:
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Reference Standard
MHLW Draft Guideline (intended):
Structure, physico-chemical characterization, amount,
purity, potency, immunological characterization, or etc.
are well-established.
It is not recommended that the batch of the reference
standard used for calibration standards and QC samples is
the same as used for dosing studies, or that an analytical
characterisation and bioanalytical evaluation are carried in
the case of change of batch of reference standard.
5th JBF Symposium, Draft BMV Guideline for LBA
http://bioanalysisforum.jp/
Well-established characteristics of reference standard:
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Full Validation - Kits
MHLW Draft Guideline:
A full validation should be performed when establishing a
new bioanalytical method for quantification of an
analyte/analytes. A full validation is also required when
implementing an analytical method that is disclosed in
literature or commercialized as a kit product.
http://bioanalysisforum.jp/
3. 市販キットもフルバリデーションが必要
A full validation is required for a commercialized kit.
An analytical method validation should be performed
at every relevant facility.
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Full Validation - Kits
7.1.1.13. Commercial kits
Commercial kits may have been developed for purposes
other than to support pharmacokinetics. Therefore,
commercial kits need to be revalidated to ensure that the
LLOQ and the QC samples in the actual concentration range
to be used for sample analysis perform accurately and
precisely. The principles of validation listed above apply.
5th JBF Symposium, Draft BMV Guideline for LBA
http://bioanalysisforum.jp/
EMA Guideline:
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Full Validation - Kits
C. Diagnostic Kits
……..
Diagnostic kit validation data provided by the manufacturer
may not ensure reliability of the kit method for drug
development purposes. The performance of diagnostic kits
should be assessed in the facility conducting the sample
analysis.
……..
5th JBF Symposium, Draft BMV Guideline for LBA
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FDA Draft Guidance:
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Japan Bioanalysis Forum
Full Validation – MRD
MHLW Draft Guideline:
In an LBA validation, full validation should be conducted
using samples diluted at a factor of minimum required
dilution (MRD), which has been determined in the course of
method development.
5th JBF Symposium, Draft BMV Guideline for LBA
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4. フルバリデーションの実施前にMRDを設定する
Determination of minimum required dilution (MRD) before
a full validation.
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MRD
Different dilution fold
↓
Standard curve changes
↓
Analysis with the same dilution fold
Determination of MRD
http://bioanalysisforum.jp/
Response
10％ Serum（10-fold dilution）
20％ Serum（5-fold dilution)
33％ Serum（3-fold dilution）
50％ Serum（2-fold dilution）
Analysis with the same dilution fold for
calibration standards, QCs, and samples
Concentration (log)
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Full Validation – MRD
MHLW Draft Guideline:
Glossary:
Minimum required dilution (MRD):
A dilution factor of samples (including calibration
standards and QC samples) with buffer to analyze samples
appropriately. MRD should be identical for all samples.
It may not necessarily be the exact minimum dilution
where samples can be analyzed.
5th JBF Symposium, Draft BMV Guideline for LBA
http://bioanalysisforum.jp/
4. フルバリデーションの実施前にMRDを設定する
Determination of minimum required dilution (MRD) before
a full validation.
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Full Validation – MRD
7.1.1.6. Minimum required dilution
Because matrices may exhibit a high background signal, it
may be necessary to determine the minimum required
dilution. The minimum required dilution is the smallest
dilution to which a sample must be diluted in buffer to
optimize accuracy and precision in an assay run by reducing
the signal to noise ratio. Spiked samples should be prepared
in the same matrix as the study samples for determination
of the minimum required dilution.
http://bioanalysisforum.jp/
EMA Guideline:
FDA Draft Guidance: No statement
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Full Validation - Specificity
5. 特異性を評価する Specificity should be evaluated.
Specificity is the ability of an analytical method to detect and
differentiate the analyte from other substances such as its related
substances. For LBA, it is important that binding reagent specifically
binds to the target analyte but does not cross-react with coexisting
substances that are structurally similar to the analyte. If similar
substances are expected to be present in biological samples of interest,
the extent of the impact of such substances on the analysis of analyte
should be evaluated. It is acceptable that specificity is evaluated in the
course of method development or after completing a method validation.
http://bioanalysisforum.jp/
MHLW Draft Guideline:
Analyte: anti-X human IgG
Sensitivity: ～1 ng/mL in matrix
Matrix: ～10 mg/mL IgG in human serum
5th JBF Symposium, Draft BMV Guideline for LBA
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Full Validation - Specificity
7.1.1.2. Specificity
Specificity of the binding reagent(s) refer(s) to its (their) ability to bind
solely to the analyte of interest. Specificity is related to the concept of
cross-reactivity. Ideally the binding reagent should be specific such
that no cross-reactivity occurs with structurally “related compounds”
(e.g. endogenous compounds, isoforms, variants forms of the analyte,
or physico-chemically similar compounds) or with anticipated
concomitant medication. During method development and validation,
frequently these “related molecules” are not available. Evaluation of
specificity may be conducted after the original validation is completed
as more data on the behaviour of the analyte become available.
……..
5th JBF Symposium, Draft BMV Guideline for LBA
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EMA Guideline:
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Full Validation - Specificity
VIII. GLOSSARY
Selectivity/Specificity: The ability of the bioanalytical method to
measure and differentiate the analytes in the presence of
components that may be expected to be present. These could
include metabolites, impurities, degradants, or matrix
components.
http://bioanalysisforum.jp/
FDA Draft Guidance: No statement in LBA section
To see selectivity section
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Full Validation - Selectivity
MHLW Draft Guideline:
Selectivity is the ability of an analytical method to measure and
differentiate the analyte in the presence of other components in samples.
Selectivity is evaluated using blank samples obtained from at least 10
individual sources and near-LLOQ QC samples prepared from individual
blank samples. In case of a matrix with limited availability, it is acceptable
to use matrix samples obtained from less than 10 sources.
Assay results for at least 80% of the blank samples should be below the
LLOQ, and accuracy of the measurements of at least 80% of the nearLLOQ QC samples should be within ±20% of the theoretical
concentration (or ±25% at the LLOQ).
5th JBF Symposium, Draft BMV Guideline for LBA
http://bioanalysisforum.jp/
6. 選択性の評価方法が低分子ガイドラインとは異なる
Evaluation method for selectivity differs from that of LMW.
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Full Validation - Selectivity
2
LLOQ
定量下限
1
0
1
2
3
Response
（Conc.）
5
±25%
±25%
Blank
ブランク 添加試料
LLOQ QC
sample
レスポ
ンス
1
(濃度）
4
6
ブランク試料が定量下限（1.0）の20％未満
Response
of blank sample is less than 20%
LC-MS
低分子・LC
2
http://bioanalysisforum.jp/
Response
レ
ス
ポ
ン
ス
LLOQ
定量下限
0
1
LBA
LBA
2
3
4
5
6
7
8
9
10
At least
80% of the blank samples should be below the LLOQ
ブランク試料の80%以上が定量下限（1.0）未満
At least
80% of the LLOQ QC samples should be within ±25%
LLOQ添加試料の80%以上の真度が25%以内
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Full Validation - Selectivity
7.1.1.3. Selectivity (1/2)
Selectivity of a ligand-binding assay is the ability to measure the
analyte of interest in the presence of unrelated compounds in the
matrix. Generally there is no extraction due to the inherent
characteristics of macromolecules. Then, unrelated compounds
present in matrix e.g. degrading enzymes, heterophilic antibodies or
rheumatoid factor, may interfere with the analyte of interest in the
ligand binding assay. Selectivity is tested by spiking at least 10 sources
of sample matrix at or near the LLOQ. These sources should include
lipemic and haemolysed samples.
……….
5th JBF Symposium, Draft BMV Guideline for LBA
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EMA Guideline:
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Full Validation - Selectivity
7.1.1.3. Selectivity (2/2)
…….. It is also strongly recommended that sources from relevant
disease population be included. Selectivity should be evaluated at the
low end of an assay where problems occur in most cases. It may be
prudent also to evaluate selectivity at higher analyte concentrations.
In cases where interference is concentration dependent, it is essential
to determine the minimum concentration where interference occurs.
It may be necessary to adjust the lower level of quantification
accordingly, before assay validation. The accuracy should be within
20% (25% at the LLOQ) of the nominal spiked concentration in at least
80% of the matrices evaluated.
5th JBF Symposium, Draft BMV Guideline for LBA
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EMA Guideline:
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Full Validation - Selectivity
FDA Draft Guidance:
As with chromatographic methods (described in Section III), LBAs
should be shown to be selective for the analyte. The following
recommendations for dealing with two selectivity issues should
be considered:
a. Interference from Substances Physiochemically Similar to the
Analyte
Cross-reactivity of metabolites, concomitant medications,
and their significant metabolites, or endogenous compounds
should be evaluated individually and in combination with the
analyte of interest.
……
5th JBF Symposium, Draft BMV Guideline for LBA
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1. Selectivity (1/2)
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Full Validation - Selectivity
1. Selectivity (2/2)
……
b. Matrix Effects
Matrix effects should be evaluated. For example:
The calibration curve in biological fluids should be
compared with calibrators in buffer to detect matrix
effects using at least ten sources of blank matrix.
5th JBF Symposium, Draft BMV Guideline for LBA
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FDA Draft Guidance:
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Full Validation - Calibration Curve
4.1.3. Calibration Curve
A calibration curve demonstrates the relationship between a theoretical
concentration and a response variable for an analyte.
A calibration curve should be prepared using the same matrix as the
intended study samples, whenever possible, by spiking the blank matrix
with known concentrations of the analyte. A calibration curve should be
generated with at least 6 concentration levels of calibration standards,
including LLOQ and ULOQ samples, and a blank sample. Anchor point
samples at concentrations below LLOQ and above ULOQ may also be
used to improve curve fitting. A 4- or 5-parameter logistic model is
generally used for the regression equation of calibration curve. The
validation report should include the regression equation and weighting
conditions used.
5th JBF Symposium, Draft BMV Guideline for LBA
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MHLW Draft Guideline:
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http://bioanalysisforum.jp/
Full Validation - Calibration Curve
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Full Validation - Calibration Curve
●: Calibration standards
●
ULOQ
Response
●
●
●
●
LLOQ
http://bioanalysisforum.jp/
●
●
●
Concentration (log)
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Full Validation - Calibration Curve
●
◎: Anchor points
●
◎
ULOQ
Response
●
●
●
4-parameter logistic model
●
LLOQ
http://bioanalysisforum.jp/
●: Calibration standards
●
●
◎
Concentration (log)
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Full Validation - Calibration Curve
MHLW and EMA:
Accuracy*: within ±25％ →FDA: within ±20%
●
◎: Anchor points
●
*: Back-calculated concentrations
◎
ULOQ
Response
●
MHLW, EMA, FDA:
At least 75% of calibration
standards meet the criteria
●
●
4-parameter logistic model
Accuracy*: within ±20％
●
LLOQ
●
●
◎
http://bioanalysisforum.jp/
●: Calibration standards
Accuracy*: within ±25％
Concentration (log)
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Full Validation - Calibration Curve
MHLW and EMA:
Accuracy*: within ±25％ →FDA: within ±20%
●
◎: Anchor points
●
*: Back-calculated concentrations
Response
**: relative error
4-parameter logistic model
Accuracy*: within ±20％
FDA:
Total error: not exceed 30%
(MHLW, EMA: No statement
in calibration curve section)
●
●
●
◎
Total error:
a sum of absolute
accuracy (-100%)**
and precision
●
●
LLOQ
ULOQ
●
MHLW, EMA, FDA:
At least 75% of calibration
standards meet the criteria
◎
http://bioanalysisforum.jp/
●: Calibration standards
Accuracy*: within ±25％
Concentration (log)
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Total Error
.........
..
RE
CV
..........
..
......... .
. .... . .
..
..... ....
..... . .
. .
http://bioanalysisforum.jp/
Total error = |RE| + CV
Modified from B. DeSilva et al., Pharm. Res., 20: 1885-1900 (2003)
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7. 真度及び精度は、定量上限のQC試料についても評価が必要
であり、トータルエラーについても評価する
Accuracy and precision should be evaluated in ULOQ, and
total error should be evaluated.
MHLW Draft Guideline:
Accuracy of an analytical method describes the degree of closeness
between analyte concentration determined by the method and its
theoretical concentration. Precision of an analytical method
describes variation between individual concentrations determined
in repeated measurements.
……..
5th JBF Symposium, Draft BMV Guideline for LBA
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Full Validation – Accuracy & Precision
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http://bioanalysisforum.jp/
Full Validation – Accuracy & Precision
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Full Validation - QCs
●
◎： Anchor points
●
◎
ULOQ
Response
●
●
●
●
LLOQ
http://bioanalysisforum.jp/
●： Calibration standards
●
●
◎
Concentration (log)
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Full Validation - QCs
●： Calibration standards
●
◎： Anchor points
Response
●
●
ULOQ
HQC
●
●
●
MQC
●
http://bioanalysisforum.jp/
●
◎
●
LLOQ
●
LQC
●
◎
Concentration (log)
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Full Validation - QCs
◎： Anchor points
●
Response
●
●
ULOQ
HQC
●
●
●
MQC
Accuracy: within ±20％
Precision: not exceed 20%
●
●
LLOQ
●
●
◎
http://bioanalysisforum.jp/
●： Calibration standards
FDA:
MHLW and EMA:
→ Accuracy: within ±20%
Accuracy: within ±25％
Precision: not exceed 20%
Precision: not exceed 25%
◎
●
LQC
Accuracy: within ±25％
Precision: not exceed 25%
Concentration (log)
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Full Validation - QCs
◎： Anchor points
MHLW, EMA:
Total error: not exceed 40%
●
Response
●
●
ULOQ
HQC
●
●
●
MQC
Accuracy: within ±20％
Precision: not exceed 20%
MHLW, EMA:
Total error: not exceed 40% ●
●
LLOQ
●
●
◎
LQC
MHLW, EMA:
Total error: not exceed 30%
(FDA: No statement in QC section)
http://bioanalysisforum.jp/
●： Calibration standards
FDA:
MHLW and EMA:
→ Accuracy: within ±20%
Accuracy: within ±25％
Precision: not exceed 20%
Precision: not exceed 25%
◎
●
Accuracy: within ±25％
Precision: not exceed 25%
Concentration (log)
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Full Validation – Accuracy & Precision
MHLW Draft Guideline:
……..
The low-level should be within 3 times the LLOQ, the mid-level is
around the midpoint on the calibration curve, and the high-level
should be at least one-third of the ULOQ of the calibration curve.
Within-run and between-run accuracy and precision should be
evaluated by repeating the analysis run at least 6 times.
5th JBF Symposium, Draft BMV Guideline for LBA
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8. 高濃度のQC試料の濃度を定量上限の1/3以上とする
Concentration of high-level QC is at least 1/3 of ULOQ.
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Full Validation - QCs
●
◎： Anchor points
HQC
●
Response
●
●
◎
ULOQ
x1/3
●
MQC
●
3x
●
●
http://bioanalysisforum.jp/
●： Calibration standards
●
LLOQ
●
LQC
●
◎
3x
Concentration (log)
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Full Validation – Accuracy & Precision
7.1.1.8. Precision and accuracy
For the estimation of precision and accuracy QC samples should not be
freshly prepared, but should be frozen and treated the same way as for
the analysis of study samples. At least 5 QC samples (anticipated LLOQ,
less than 3 times the LLOQ, mid, high and anticipated ULOQ) should be
used to assess accuracy, precision and the total error of the method.
……..
5th JBF Symposium, Draft BMV Guideline for LBA
http://bioanalysisforum.jp/
EMA Guideline:
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Full Validation – Accuracy & Precision
FDA Draft Guidance:
Accuracy is determined by replicate analysis of samples containing
known amounts of the analyte (QCs). Accuracy should be
measured using a minimum of five determinations per
concentration. A minimum of three concentrations in the range of
expected study sample concentrations is recommended. The
mean value should be within 20% of the actual value except at
LLOQ, where it should not deviate by more than 25%.
……
5th JBF Symposium, Draft BMV Guideline for LBA
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2. Accuracy, Precision and Recovery
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Full Validation – Dilutional Linearity
9. 希釈直線性の評価が必要
Dilutional linearity should be evaluated.
4.1.5. Dilutional linearity
http://bioanalysisforum.jp/
MHLW Draft Guideline:
Dilutional linearity is assessed to confirm that the method can appropriately
analyze samples at concentrations exceeding the ULOQ without influence of a
hook effect or prozone effect and that these measurements are not affected
by dilution within the calibration range. Dilutional linearity is evaluated by
analyzing a QC sample exceeding the ULOQ and its serially-diluted samples at
multiple concentrations. The absence or presence of response reduction (hook
effect or prozone effect) is checked in the analyzed samples, and measures
should be taken to eliminate response reduction in study sample analysis, when
applicable. Accuracy and precision in the measurements corrected for the
dilution factor should be within ±20% deviation of the theoretical
concentration and not more than 20%, respectively.
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Full ValidationData
– Dilutional
Linearity
1
Hook effect
Prozone
OD450
1.0
0.5
0.0
0.1
1
10
100
http://bioanalysisforum.jp/
1.5
1000
Drug (ng/ml)
Modified from B. DeSilva et al., Pharm. Res., 20: 1885-1900 (2003)
5th JBF Symposium, Draft BMV Guideline for LBA
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Nominal conc.
Measured conc.
Dilution
(ng/mL)
(ng/mL)
100000
1
>ULOQ
1000
1
>ULOQ
100
1
>ULOQ
40.0
5000
42.54
20.0
10000
18.51
10.0
20000
9.76
5.00
40000
5.34
2.50
80000
2.48
1.25
160000
1.23
Mean
Ref. standard：200000 ng/mL
SD
ULOQ: 50 ng/mL
%CV
Final conc.
(ng/mL)
>ULOQ
>ULOQ
>ULOQ
212700
185100
195200
213600
198400
196800
200300
10987
5.5
5th JBF Symposium, Draft BMV Guideline for LBA
Accuracy
(%)
106.4
92.6
97.6
106.8
99.2
98.4
http://bioanalysisforum.jp/
Full Validation – Dilutional Linearity
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Japan Bioanalysis Forum
Full Validation – Dilutional Linearity
7.1.1.9. Dilutional linearity
Because the narrow range of the calibration standard curve, it is
necessary to demonstrate with QC samples that the analyte of interest,
when present in concentrations exceeding the range of quantification
(above ULOQ), can be accurately measured by the assay after dilution
in blank matrix to bring the analyte concentrations into the validated
range for analysis. An additional reason for conducting dilutional
experiments is to detect a possible prozone or “hook effect” i.e. a
signal suppression caused by high concentrations of analyte. The backcalculated concentration for each dilution should be within 20% of the
nominal concentration after correction for dilution and the precision of
the final concentrations across all the dilutions should not exceed 20%.
5th JBF Symposium, Draft BMV Guideline for LBA
http://bioanalysisforum.jp/
EMA Guideline:
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Full Validation – Dilutional Linearity
FDA Draft Guidance:
…….
Samples with concentrations over the ULOQ should be diluted
with the same matrix as used for the study samples, and accuracy
and precision should be demonstrated.
……
5th JBF Symposium, Draft BMV Guideline for LBA
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2. Accuracy, Precision and Recovery
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Critical reagent
MHLW Draft Guideline:
重要試薬とは，リガンド結合法による生体試料中薬物濃度分析におい
て分析結果に直接影響する試薬を指し，主に結合試薬（抗体及びその
標識体等）が該当する．
…..
6. Points to note 6.5. Critical reagent
Critical reagents are usually binding reagents (labeled or
unlabeled antibodies) that have a direct impact on the results of
ligand-binding-based bioanalytical methods.
…..
5th JBF Symposium, Draft BMV Guideline for LBA
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6. 注意事項 6.5. 重要試薬
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Critical reagent
6. 注意事項 6.5. 重要試薬
….. 重要試薬のロット変更の際には原則としてパーシャルバリデー
ションが必要である．
6. Points to note 6.5. Critical reagent
….. Partial validation is required when the critical reagent lot is
changed in principle.
5th JBF Symposium, Draft BMV Guideline for LBA
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MHLW Draft Guideline:
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10. 重要試薬のロット変更やMRDの変更時にパーシャルバリデーション
が必要である
Partial validation should be performed after changing a critical
reagent lot or MRD. (in principle)
MHLW Draft Guideline:
……..
Typical bioanalytical method changes subject to a partial validation are as
follows: analytical method transfers between laboratories, changes in
analytical instruments, changes of the critical reagent lot, changes in
calibration range, changes in MRD, changes in anticoagulant, changes in
analytical conditions, changes in sample storage conditions, confirmation
of impact by concomitant drugs, and use of rare matrices.
……..
5th JBF Symposium, Draft BMV Guideline for LBA
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Partial Validation
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Partial Validation
7.1.1.12. Reagents
Critical reagents, including binding reagents (e.g. binding proteins,
aptamers, antibodies or conjugated antibodies) and those containing
enzymatic moieties have direct impact on the results of the assay and
therefore their quality must be assured. Accordingly, when changing
reagent batches during validation or sample analysis the analytical
performance of the method must be verified to ensure that it is not
altered compared with the original or previous batch.
5th JBF Symposium, Draft BMV Guideline for LBA
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EMA Guideline: No statement about MRD
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Partial Validation
FDA Draft Guidance:
II. BACKGROUND Partial Validation
Partial validations evaluate modifications of already validated
bioanalytical methods. Partial validation can range from as little as
one intra-assay accuracy and precision determination to a nearly
full validation. Typical bioanalytical method modifications or
changes that fall into this category include but are not limited to:
……
5th JBF Symposium, Draft BMV Guideline for LBA
http://bioanalysisforum.jp/
No statement about critical reagent or MRD
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http://bioanalysisforum.jp/
Analysis of Study Samples
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Analysis of Study Samples
Incurred sample reanalysis (ISR)
within ±30% for at least two-thirds of the samples
analyzed in ISR.
EMA Guideline: The concentration obtained for the
initial analysis and the concentration obtained by reanalysis
should be within 30% of their mean for at least 67% of the
repeats.
http://bioanalysisforum.jp/
MHLW Draft Guideline: The assay variability should be
FDA Draft Guidance: Two-thirds (67%) of the repeated
sample results should be within 20% for small molecules
and 30% for large molecules.
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Analysis of Study Samples
Incurred sample reanalysis (ISR)
Less than 1000 samples, approximately 10% samples
Exceeding 1000 samples, approximately 5% samples
EMA Guideline:
Approximately 10% samples, less than 1000 samples
Approximately 5% samples, exceeding 1000 samples
FDA Draft Guidance:
http://bioanalysisforum.jp/
MHLW Draft Guideline:
The total number of ISR samples should be 7% of the study
sample size.
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Parallelism
EMA Guideline: 7.1.1.10. Parallelism
If study samples are available, parallelism between the
calibration standard curve and serially diluted study
samples should be assessed to detect possible matrix effect
or differing affinities for metabolites. ……..
FDA Draft Guidance: 1. Selectivity b. Matrix Effects
http://bioanalysisforum.jp/
MHLW Draft Guideline: No statement
Parallelism of diluted study samples should be evaluated
with diluted standards to detect matrix effects.
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Parallelism
Standard
v
Serum
Sample
1.56
100
25
6.25
Log (Dilution)
Log (OD)
Log (OD)
Standard
Serum
Sample
v
1.56
http://bioanalysisforum.jp/
Non-Parallel Case
Parallel Case
100
25
6.25
Log (Dilution)
Modified from Plikaytis et al. J. Clin. Microbiol. 32: 2441-2447 (1994)
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1. The guideline also applies to low-molecular-weight (LMW)
drugs that are analyzed by LBAs. (endogenous compounds)
2. It is necessary to show well-established characteristics of reference
standard.
3. A full validation is required for a commercialized kit.
4. Determination of minimum required dilution (MRD) before a full
validation.
5. Specificity should be evaluated.
6. Evaluation method for selectivity differs from that of LMW.
7. Accuracy and precision should be evaluated in ULOQ, and
total error should be evaluated.
8. Concentration of high-level QC is at least 1/3 of ULOQ.
9. Dilutional linearity should be evaluated.
10. Partial validation should be performed after changing a critical
reagent lot or MRD. (in principle)
5th JBF Symposium, Draft BMV Guideline for LBA
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Summary
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Ministry of Health, Labour and Welfare <MHLW>:
Toshinari Mitsuoka
National Institute of Health Sciences <NIHS>:
Akiko Ishii, Noriko Katori, Haruhiro Okuda,
Nana Kawasaki, Shingo Niimi
Japan Pharmaceutical Manufacturers Association <JPMA>:
Masataka Katashima (Astellas Pharma)
Kotaro Maekawa (Hisamitsu Pharmaceutical)
Japan Bioanalysis Forum <JBF>:
All Steering Committee members,
and LBA Task Force members
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Acknowledgements
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