Summary of the Draft Guideline on Bioanalytical Method ...bioanalysisforum.jp/images/2014_5thJBFS/041_2_5thJBF...Japan Bioanalysis Forum 5th JBF Symposium, Draft BMV Guideline for

Japan Bioanalysis Forum

5th JBF Symposium, Draft BMV Guideline for LBA 1

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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

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

Takahiro Nakamura, PhD, DJSOT (Shin Nippon Biomedical Laboratories, Ltd. , SNBL)



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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.

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.

Contents (2/2)



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Guideline/Guidance Comparison on BMV for LBA



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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.

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 low-molecular-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).

Scope in MHLW

Analyte is the same as for endogenous compounds

(endogenous compounds)



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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.

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.

Reference Standard



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EMA Guideline:

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.

Reference Standard



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FDA Draft Guidance:

A. Key Reagents

Key reagents, such as reference standards, antibodies, tracers, and matrices should be characterized appropriately and stored under defined conditions.

Reference Standard



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Reference Standard

MHLW Draft Guideline (intended): Well-established characteristics of reference standard:

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.

Structure, physico-chemical characterization, amount, purity, potency, immunological characterization, or etc. are well-established.



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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.


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.

Full Validation - Kits

An analytical method validation should be performed at every relevant facility.



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EMA Guideline:

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.




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FDA Draft Guidance:

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.

……..




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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


a full validation.


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.

Full Validation – MRD



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Re

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10％ Serum（10-fold dilution） 20％ Serum（5-fold dilution) 33％ Serum（3-fold dilution） 50％ Serum（2-fold dilution）

Different dilution fold ↓

Standard curve changes ↓

Analysis with the same dilution fold

Determination of MRD

Analysis with the same dilution fold for calibration standards, QCs, and samples

Concentration (log)

MRD



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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


a full validation.


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.




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EMA Guideline:

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.

FDA Draft Guidance: No statement




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5. 特異性を評価する Specificity should be evaluated.

MHLW Draft Guideline: 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.

Full Validation - Specificity

Analyte: anti-X human IgG Sensitivity: ～1 ng/mL in matrix Matrix: ～10 mg/mL IgG in human serum



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EMA Guideline:

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.

……..




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FDA Draft Guidance: No statement in LBA section

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.

To see selectivity section




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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.

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 near-LLOQ QC samples should be within ±20% of the theoretical concentration (or ±25% at the LLOQ).

Full Validation - Selectivity



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0

1

2

1 2 3 4 5 6 7 8 9 10

レスポンス

(濃度）

LBAブランク試料の80%以上が定量下限（1.0）未満

LLOQ添加試料の80%以上の真度が25%以内

定量下限

±25%ブランク添加試料

Res

po

nse

（

Co

nc.）

0

1

2

1 2 3 4 5 6

レスポンス

低分子・LCブランク試料が定量下限（1.0）の20％未満

定量下限

Res

po

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LBA At least 80% of the blank samples should be below the LLOQ At least 80% of the LLOQ QC samples should be within ±25%

±25% LLOQ

LLOQ

Response of blank sample is less than 20%


LC-MS

Blank sample

LLOQ QC



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EMA Guideline:

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.

……….



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EMA Guideline:

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.



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FDA Draft Guidance:

1. Selectivity (1/2)

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. ……




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FDA Draft Guidance:

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.




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Full Validation - Calibration Curve

MHLW Draft Guideline: 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.



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Concentration (log)

Res

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●

●

●

●

●: Calibration standards

ULOQ

LLOQ



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Concentration (log)

Res

po

nse

●

●

●

●

●

●

●

●


◎: Anchor points

ULOQ

LLOQ

◎

◎

4-parameter logistic model



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Concentration (log)

Res

po

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●

●

●

●

●

●

●

●


◎: Anchor points

ULOQ

LLOQ

◎

◎

Accuracy*: within ±25％



*: Back-calculated concentrations

MHLW, EMA, FDA: At least 75% of calibration standards meet the criteria

MHLW and EMA: →FDA: within ±20%




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Concentration (log)

Res

po

nse

●

●

●

●

●

●

●

●


◎: Anchor points

ULOQ

LLOQ

◎

◎




*: Back-calculated concentrations

FDA: Total error: not exceed 30% (MHLW, EMA: No statement in calibration curve section)

MHLW, EMA, FDA: At least 75% of calibration standards meet the criteria

MHLW and EMA: →FDA: within ±20%

Total error: a sum of absolute accuracy (-100%)**

and precision

**: relative error




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Total Error

Modified from B. DeSilva et al., Pharm. Res., 20: 1885-1900 (2003)

CV

RE

Total error = |RE| + CV

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . .

. .

. . . . .

.

. . .

. . . . . . . . .

. .

. . . . .

. . .



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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.

……..

Full Validation – Accuracy & Precision



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Full Validation - QCs

Concentration (log)

Res

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●

●

●

●

●

●

●： Calibration standards ◎

◎

◎： Anchor points

ULOQ

LLOQ



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Concentration (log)

Res

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●

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●

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◎


ULOQ

LLOQ ●

●

●

HQC

MQC

LQC

●

●

ULOQ

LLOQ



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Concentration (log)

Res

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◎


ULOQ

LLOQ

Accuracy: within ±20％ Precision: not exceed 20%

MHLW and EMA: FDA: → Accuracy: within ±20% Precision: not exceed 20%



●

●

●

HQC

MQC

LQC

●

●

ULOQ

LLOQ



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Concentration (log)

Res

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◎


ULOQ

LLOQ


MHLW, EMA: Total error: not exceed 40%

MHLW and EMA: FDA: → Accuracy: within ±20% Precision: not exceed 20%





(FDA: No statement in QC section)

●

●

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HQC

MQC

LQC

●

●

ULOQ

LLOQ



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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.


……..

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.




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Concentration (log)

Res

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◎


3x

x1/3

3x

3x ●

●

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HQC

MQC

LQC

●

●

ULOQ

LLOQ



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EMA Guideline:

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. ……..




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FDA Draft Guidance:

2. Accuracy, Precision and Recovery

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%. ……




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9. 希釈直線性の評価が必要

Dilutional linearity should be evaluated.

MHLW Draft Guideline: 4.1.5. Dilutional linearity

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.

Full Validation – Dilutional Linearity



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Modified from B. DeSilva et al., Pharm. Res., 20: 1885-1900 (2003)

OD

45

0

Drug (ng/ml)

Data 1

0.1 1 10 100 10000.0

0.5

1.0

1.5

Hook effectProzone



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Nominal conc.

(ng/mL)Dilution

Measured conc.

(ng/mL)

Final conc.

(ng/mL)

Accuracy

(%)

100000 1 >ULOQ >ULOQ -

1000 1 >ULOQ >ULOQ -

100 1 >ULOQ >ULOQ -

40.0 5000 42.54 212700 106.4

20.0 10000 18.51 185100 92.6

10.0 20000 9.76 195200 97.6

5.00 40000 5.34 213600 106.8

2.50 80000 2.48 198400 99.2

1.25 160000 1.23 196800 98.4

Mean 200300

SD 10987

%CV 5.5

Ref. standard：200000 ng/mL ULOQ: 50 ng/mL



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EMA Guideline:

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 back-calculated 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%.



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FDA Draft Guidance:

2. Accuracy, Precision and Recovery

…….

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.

……



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6. 注意事項 6.5. 重要試薬

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.

…..

Critical reagent



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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.

Critical reagent



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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.

……..

Partial Validation



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Partial Validation

EMA Guideline: No statement about MRD

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.



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Partial Validation

FDA Draft Guidance:

No statement about critical reagent or MRD

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: ……



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Analysis of Study Samples



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Incurred sample reanalysis (ISR)

MHLW Draft Guideline: The assay variability should be

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.

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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Incurred sample reanalysis (ISR)

MHLW Draft Guideline: 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: The total number of ISR samples should be 7% of the study sample size.



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Parallelism

MHLW Draft Guideline: No statement

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 Parallelism of diluted study samples should be evaluated with diluted standards to detect matrix effects.



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Parallelism

100256.251.56

Log (Dilution)

Lo

g (O

D)

Standard

Serum

Sample

v

Parallel Case

100256.251.56

Log (Dilution)

Lo

g (O

D)

Standard

Serum

Samplev

Non-Parallel Case

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)

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

Acknowledgements

Documents

Summary of the Draft Guideline on Bioanalytical Method ...bioanalysisforum.jp/images/2014_5thJBFS/041_2_5thJBF...Japan Bioanalysis Forum 5th JBF Symposium, Draft BMV Guideline for