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BM Rao, Ph.D.21st July, 2011

Analytical method validation approaches

from Development to Launch

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Analytical Method Validation ( What, Why, When and How much)

Drug Development Phases

Validation Requirement (Innovator Vs Generics & regulatory Perspective)

Validation Prerequisites

Validation Activity Flow

Validation Parameters

System Suitability

Out of Acceptance – case studies

Recent FDA 483’s & Warning Letters

References

Summary recommendations & Conclusions

Q & A session

Topics

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RT : Reporting Threshold DL : Detection Limit QL : Quantitation Limit SL : Specification Limit API : Active Pharmaceutical Ingredient DS : Drug Substance DP : Drug Product IND : Investigational New Drug CTA : Clinical Trial Application NDA : New Drug Application MAA : Marketing Authorization Application  Sample Matrix: Other possible ingredient of drug product except the API

Glossary

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What is Method Validation

The FDA defines the term as :

“Established documented evidence which provides a high degree of assurance that a

specific process will consistently produce a product meeting its pre-determined

specifications and quality attributes.” - General Principles of Validation (1987)

ICH guideline :

“A documented program that provides a high degree of assurance that a specific

process, method, or system will consistently produce a result meeting pre-determined

acceptance criteria.” - Q7A-GMP for active pharmaceutical ingredients (2000)

EU-guideline :

Action of proving, in accordance with GMP-principles that any procedure, process,

equipment, material, activity or system actually leads to the expected results.

Method validation is the “process used to confirm that the analytical procedure employed for a specific test is suitable for its intended use”

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Why Method Validation• To obtain reliable analytical results & comply with international regulations

• Essential component of the measure that laboratories should employ to ensure that

they produce accurate and reliable results • Universally recognized comprehensive system of quality assurance • Identification of sources and quantitation of potential errors

• Determination if method is acceptable for Intended Use

• Establish proof that a method can be used for decision making

• Satisfy FDA requirements

• To meet accreditation requirement

• Ensure that the test method give “correct” results

• Customers want to be “assured of the correctness of result”

• Objective evidence for defense against challenges

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When Method Validation

Method validations are required when

• New method is developed

• Existing method is significantly modified (optimized)

• Existing validated method is applied to a different sample matrix

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Method Validation – how much is adequate

Depends on

• Phase of drug for which method is to be used

• The critically of the measurement

• The scope of the method

“Validation is always a balance between costs, risks and technical possibilities”

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Method Validation – how much contd..Test method description Validation or Verification requirements

Standard methods with performance data (e.g. compendia method USP/EP etc.)

Verification of performance, but validation may be required if any changes made

in-house developed methods Full validation

Published in the literature without any performance data Full validation

Published in the literature with performance data Verification of performance but more likely full validation required

Changes in implementation of previously validated method - i.e. changes to equipment, reagents, lab environment or staff.

Verification

Existing validated method applied to different matrices, different concentration ranges

Validation - extent will vary - e.g. having similar properties to those of representative matrices

Existing validated method applied to additional analytes Full Validation

Commercial Test Kits - collaboratively tested, third party evaluation (e.g. AOAC)

Verification

Commercial Test Kits - no performance data available, incomplete or not applicable

Full Validation

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Different Phases During New Drug Development

Pre-phase-I Phase-I Phase-IIIFiling /

Approvals NDA

Phase-II

Phase-II A Phase-II B

Early phase

Late phase

Pre-phase-I : Pharmacology and toxicology studies

Phase-I : Testing the simplest formulation of drug in healthy volunteers

Phase-IIA : Evaluation of drug for the clinical effectiveness in the target patient population (for fixing the proper and safe dosage range)

Phase-II B and Phase-III : Testing in thousands of patients with proposed marketed formulation after the establishment of safe and clinical

effectiveness (Late Phase Development)

Filing / Approvals : Regulatory submissions

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Drug Product Development and GMP

Full characterization

Good Manufacturing Practices

Full GMP 21 CFR 210, 211

Product Characterization

Phase III

Phase I

Phase II A

Pre-clinical

Validated MethodStandard screening methods

Phase II B

Clinical Monitoring ProgramEarly Phase Late Phase

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• Pre- Phase1 to Phase I – Limited validation, focussing on key method attributes eg. specificity, limits of quantitation and linearity

• Up to Phase IIA – Starting to include accuracy and precision data to support specifications

• From Phase IIB to Phase III – Full validation according to ICH guidelines will be completed for all analytical methods prior to submission of marketing applications

PHASED APPROACH TO ANALYTICAL VALIDATION

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Innovator Vs GenericsInnovatorInnovator GenericsGenerics

R & D on APIR & D on API --

Preclinical trialsPreclinical trials --

Clinical trials pre -phase I to IIa Clinical trials pre -phase I to IIa

(Early Development)(Early Development)Method validation Method validation

summarysummary--

Clinical trials phase IIb to IIIClinical trials phase IIb to III

(Late Development)(Late Development)

Full Method Full Method validationvalidation

--

Post marketing phase IVPost marketing phase IV Validated methodsValidated methods --

Entering of Generics; Entering of Generics; Pharmaceutical Pharmaceutical development, Comparability development, Comparability with Innovatorwith Innovator

Validated methodsValidated methods Validated Validated methods: GMP methods: GMP

and GLPand GLP

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Validation Parameters defined in ICH & USP

USP ( USP 33, NF 28) ICH (Q2 (R1))

Specificity Specificity

Linearity & Range Linearity

Accuracy Range

Precision Accuracy

Limit of DetectionPrecision

(Repeatability, Intermediate Precision, Reproducibility)

Limit of Quantitation Detection Limit

Ruggedness Quantitation Limit

Robustness Robustness

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Assessment of method validation requirements

•What analytes should be detected?

• What are the expected concentration levels?

• What are the sample matrices?

• Are there interfering substances expected, and, if so, should they be detected and quantified?

• Are there any specific legislative or regulatory requirements?

• Should information be qualitative or quantitative?

• What are the required detection and quantitation limits?

• What is the expected concentration range?

•What precision and accuracy is expected?

•How robust should the method be?

•Which type of equipment should be used? Is the method for one specific instrument, or should it

be used by all instruments of the same type?

•Will the method be used in one specific laboratory or should it be applicable in all laboratories at

one side or around the globe?

•What skills do the anticipated users of the method have?

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Prerequisites for analytical method validation– Six “M”s

Quality of theQuality of theanalytical methodanalytical method

MManan MMachineachine

qualifiedqualified

calibratedcalibrated

robustrobust

qualifiedqualified

MMethodsethods

suitablesuitable

characterisedcharacterised

documenteddocumented

MMilieuilieuMMaterialaterial MManagementanagement

QualityQuality

ReferenceReferencestandardsstandards

TemperatureTemperature

Analysts´Analysts´supportsupport

skilledskilled

HumidityHumidity

VibrationsVibrations TimeTime

SuppliesSupplies

IrradiationsIrradiations

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Validation Activity Flow

Not meeting Acceptance Criteria

Initiate the event to Identify the root cause

Define Corrective action

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Required Validation Parameters IdentificationIdentification ImpuritiesImpurities AssayAssay

quantitativequantitative limitlimit

Accuracy Accuracy -- ++ -- ++

Precision Precision

•Repeatability (System Repeatability & Analysis Repeatability)

•Intermediate precision

•Reproducibility

--

++ --

++

SpecificitySpecificity ++ ++ ++ ++

Detection LimitDetection Limit -- -- ++ --

Quantitation LimitQuantitation Limit -- ++ -- --

LinearityLinearity -- ++ -- ++

RangeRange -- ++ -- ++

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SPECIFICITY• Specificity is the ability to assess unequivocally the analyte in the presence of

components which may be expected to be present. Typically these might include impurities, degradants, matrix, etc.

• Specificity is demonstrated by representative chromatograms of appropriate solutions which may include but not limited to , reference, selectivity batch, stressed sample, placebo and stressed placebo solutions that contains all compounds for which specificity has to be proven.

Most common techniques are used to determine specificity:• Photo-diode array detector• LC-MS

The chromatographic signal does not

indicate any impurity in either peak.

Spectral evaluation, however, identifies

the peak on the left as impure.

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ACCURACY & PRECISIONAccuracyThe closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found.

Precision

The closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions.

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ACCURACY• Should be established across specified range of analytical procedure.

• Should be assessed using a minimum of 3 concentration levels covering the specified

range in presence of sample matrix, each in triplicate (total of 9 determinations).

• Should be evaluated as Percent recovery of known amount added.

Test Type Concentration in % w.r.t. nominal sample concentration

Assay 70-80-100-120-130

Related Substances a

QL-SL- atleast 1.2 times SL

Content Uniformity 70-100-130

Dissolution 20b-100-120

Residual Solvent QL-SL- atleast 1.2 times SL

Concentration 70% 100% 120%

% Recovery 1 100.6 99.7 99.7

% Recovery 2 100.2 99.9 99.4

% Recovery 3 99.0 100.2 99.2

Mean Recovery 99.9 99.9 99.4

a: using impurity for specified impurity and using active for unspecified degradants b: it should be below the value at 1st timepoint of profile. Ex. extended release it may be below 20%.

Concentration 70% 100% 120%

% Recovery 1 100.6 99.7 99.7

% Recovery 2 100.2 99.9 99.4

% Recovery 3 99.0 100.2 99.2

Mean Recovery 99.9 99.9 99.4

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PRECISION• The precision of an analytical procedure expresses the closeness of agreement

(degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision and reproducibility.

• Precision should be investigated using homogeneous, authentic samples. However, if it is not possible to obtain a homogeneous sample it may be investigated using artificially prepared samples or a sample solution.

• Repeatability (System Repeatability & Analysis Repeatability)

Repeatability expresses the precision under the same operating conditions over a short interval of time. Repeatability is also termed intra-assay precision .

• Intermediate precision

Intermediate precision expresses within-laboratories variations: different days, different analysts, different equipment, etc.

• Reproducibility

Reproducibility expresses the precision between laboratories (collaborative studies, usually applied to standardization of methodology).

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Variations affecting method reproducibility

  PrecisionIntermediate

PrecisionReproducibility

Instrument same different different

Batches of accessories e.g. chrom. columns

same different different

Operators same different different

Sample matrices different different different

Concentration different different different

Batches of material, e.g., reagents same different different

Environmental conditions, e.g., temperature

same different different

Laboratory same same different

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PRECISION - System Repeatability• It will be determined by injecting at least 5 consecutive injections of the same solution

. RSD of the response will be evaluated.Test Type Concentration in % w.r.t. nominal sample

concentration

Assay 100

Related Substances SL - individual Impurity & API (for unknown)

Content Uniformity 100

Dissolution 100

Residual Solvent SL

Determination 100% API SL (0.5%) of API

1 19770367 99353

2 19748915 99342

3 19726133 99749

4 19776942 99407

5 19847909 99584

% RSD 0.2 0.2

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PRECISION - Analysis Repeatability• Should be assessed using a minimum of 3 concentration levels covering the specified

range in presence of sample matrix, each in triplicate (total of 9 determinations).

or• It will be determined by analyzing at least 6 sample preparations by one person on

one instrument in same sample set . • Sample should contain all impurities of interest. If single sample does not contain all,

multiple samples can be used or spiking can be preferred. • RSD of the assay will be evaluated (for active as well as impurities).

Determination % API % Imp - A % Imp - B

1 98.9 0.08 0.25

2 99.4 0.09 0.28

3 100.2 0.10 0.26

4 101.1 0.10 0.29

5 99.2 0.11 0.25

6 98.7 0.10 0.25

% RSD 0.9 10.7 6.7

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PRECISION - Intermediate Precision• It will be determined by analyzing at least 6 sample preparations each by two

different person, instrument on different day. • Sample should contain all impurities of interest. If single sample does not contain all,

multiple samples can be used or spiking can be preferred. • RSD/Pooled RSD of the assay for all samples will be evaluated (for active as well as

impurities) or Absolute/% relative difference between two analyst can be evaluated.• Intermediate precision may not be needed if reproducibility is performed.

Active Impurity

Determination Analyst 1 Analyst 2 Analyst 1 Analyst 2

1 98.9 100.8 0.25 0.23

2 99.4 100.3 0.28 0.26

3 100.2 101.1 0.26 0.25

4 101.1 99.8 0.29 0.24

5 99.2 100.5 0.25 0.22

6 98.7 100.1 0.25 0.24

% RSD 0.8 7.7

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PRECISION - Reproducibility• It will be determined by analyzing at least 6 sample preparations each by two

different laboratories. • Sample should contain all impurities of interest. If single sample does not contain all,

multiple samples can be used or spiking can be preferred. • RSD/Pooled RSD of the assay for all samples will be evaluated (for active as well as

impurities) or Absolute/% relative difference between two laboratories can be evaluated.

Active Impurity

Determination laboratory 1 laboratory 2 laboratory 1 laboratory 2

1 98.9 100.8 0.25 0.23

2 99.4 100.3 0.28 0.26

3 100.2 101.1 0.26 0.25

4 101.1 99.8 0.29 0.24

5 99.2 100.5 0.25 0.22

6 98.7 100.1 0.25 0.24

% RSD 0.8 7.7

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DETECTION LIMIT & QUANTITATION LIMIT DETECTION LIMIT : The detection limit of an individual analytical procedure is the

lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value.

QUANTITATION LIMIT : The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy.

Based on Visual Evaluation Based on Signal-to-Noise Based on the Standard Deviation of the Response and the Slope

Limit of detection and limit of quantitation via signal to noise

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Quantitation Limit When impurity is not available When impurity is available

3 separate solutions are prepared containing the API at a reporting threshold concentration. These three solutions can be prepared with 100% placebo.

3 separate solutions are prepared containing the Impurity at a reporting threshold concentration with 100% API and 100% placebo.

Note: •Three RT solutions must be prepared from three different stock solutions.•These solutions are analysed and the recovery & repeatability is evaluated. •First Blank injection is considered for S/N ratio calculation.

Quantitation limit: Acceptance Criteria

Method type Concentration Range ≤ Mean % recovery ≤ % RSD ≤(n=3)

Early Phase (IND/CTA) Target QL ≤ RT 50.0 - 150.0 25.0

Late Phase (NDA/MAA) Target QL ≤ RT 70.0 - 130.0 15.0

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LINEARITY The linearity of an analytical procedure is its ability (within a given range) to obtain

test results which are directly proportional to the concentration (amount) of analyte in the sample.

A linear relationship should be evaluated across the range of the analytical procedure at minimum 5 levels.

Linearity should be evaluated by visual inspection of a plot of signals as a function of analyte concentration or content.

Test results should be evaluated by appropriate statistical methods, for example, by calculation of a regression line by the method of least squares.

Correlation coefficient, % RSD of the response factor can be evaluated. Y-intercept, slope of the regression line and residual sum of squares should be

submitted .

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LINEARITY

Signal height or peak area as a function of analyte concentration

Divide signal data by their respective concentrations, yielding the relative responses. A graph is plotted with the relative responses on the y-axis and the corresponding concentrations on the x-axis, on a log scale

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RANGE The range of an analytical procedure is the interval between the upper and lower

concentration (amounts) of analyte in the sample (including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity.

The range is normally derived from linearity studies and depends on the intended application of the procedure. It is established by confirming that the analytical procedure provides an acceptable degree of linearity, accuracy and precision.

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ROBUSTNESS• The robustness of an analytical procedure is a measure of its capacity to remain

unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage.

• The evaluation of robustness should be considered during the development phase and depends on the type of procedure under study.

• If measurements are susceptible to variations in analytical conditions, the analytical conditions should be suitably controlled or a precautionary statement should be included in the method.

• Examples of typical variations– Influence of variations of pH in a mobile phase– Influence of variations in mobile phase composition– Different columns (different lots and/or suppliers)– Temperature– Flow rate

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OTHER ADDITIONAL PARAMATERS• Stability of the Solutions

• Filtration Study

• Relative Response factor

• Automation

– Method Equivalency

– Carry over

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SYSTEM SUITABILITY• System suitability tests are an integral part of gas and liquid chromatographic

methods. They are used to verify that the resolution and reproducibility of the chromatographic system are adequate for the analysis to be done. The tests are based upon the concept that the equipment, electronics, analytical operations, and samples to be analyze d constitute an integral system that can be evaluated as such.

• System Suitability Test characteristics and limits are recommended as a component of any analytical method and are established to ensure the validity of the analytical method whenever used.

Parameters Recommendations

K’ In general k’ ≥ 2.0

RR > 2, between the peak of interest and the closest potential interferent

(degradant, internal STD, impurity, excipients, etc…..)

T T ≤ 2

N In general N > 2000

Repeatability RSD ≤ 2.0% (n ≥ 5)

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Revalidation & Bracketing ValidationRevalidation is performed under following situations

• Change in the synthetic route• Changes in sample preparation procedure where recovery or sample matrix effect

may change• Changes in Analyte detection method e.g. Change in UV wavelength, UV to

Fluorescence detector etc.• Changes in Chromatographic Operational parameters ( Column packing, Separation

technique, sample load, etc.)• Elucidation of new Impurities or Degradation products.

Only above conditions are not limited. Case to case evaluation is needed !!

Bracketing approach for validation

• Dose Proportional Formulations : Validation can be shown for Lowest strength & additionally intermediate precision or reproducibility shall be done for Highest strength.

• Non-dose proportional formulations : worst case placebo shall be used.

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Out of Acceptance case studies

Observation Root cause Corrective action

Accuracy passing at 70 & 100% but failing at 130%.

The peak height of sample and standard solution is higher than max linearity range of UV detector.

Revise the test method to reduce the concentration.

Accuracy is not meeting the acceptance criteria for specified impurity

Impurity was not completely soluble in dilution solvent of the method.

Identify the correct dilution solvent in which impurity is soluble.

Accuracy is failing for impurity at quantitation level (0.05%)

One of the excipient is trapping the API. Acidic sample diluent improved the recovery

Issue with original diluent. Modified the method prior to revalidation

Accuracy is not passing for drug Substances at 130%.

Solubility issue at higher concentration

Method validated in 80-120%.

Linearity test is failing for RSD of Response factor but correlation coefficient is passing.

Lower concentration solutions (0.05%) injected after 100% level.

Blank injection before lower concentration solution.

% Dissolution is variable during reproducibility test.

Improper homogenization of dissolution media.

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FDA- Form 483• There was inadequate method validation specificity data to demonstrate that each

method was capable of distinguishing the active ingredient from its impurities and

degradation products.

• Specificity studies did not include the minimum stress conditions of acid and base

hydrolysis, oxidation, thermal degradation and photolysis, degradation schematic for

the active ingredient that identifies the major degradation products was not included

for each product.

• Stress studies conducted as part of method validation do not target a minimum

amount of degradation. … a standard period of two hours as commonly used for

stress studies with no justification…

• Spreadsheets used to calculate linearity, percent recovery, and final assay results for

the cleaning validation of …were not validated and the data transcribed from

chromatographs to the spreadsheets were not checked for accuracy.

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FDA- Warning Letters• On addition to the example of modifying both compendia methods and customer

supplied methods, we also observed the use of un-validated in-house methods as well as invalidated modifications to in-house methods.

• Change control procedures in the laboratory failed to document test method changes to

assure accurate, reliable, and reproducible results. The test method did not state

whether a helix was to be used during dissolution testing. A … was reportedly used

during method development, validation and daily method runs, but there is no

documentation of a … being used in any of the documents.

• There is no assurance that qualification or maintenance of the laboratory equipment can

consistently produce valid and accurate analytical results in that numerous examples of

test data were invalidated due to instrument malfunction.

• Attempts to corroborate data in the validation report with supporting raw data in the

laboratory were difficult and frustrating for the FDA personnel conducting the inspection.

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FDA- Warning Letters• OOS accuracy results reported by analyst 3 were never submitted in the final report.

Repeat analysis performed in a different system passed specifications and these

results were submitted in the report.

• Raw data and calculations were not checked by a second responsible individuals

required by your procedures. Inaccurate calculations were noted in the report.

• The process validation samples were assays using an HPLC method that had not

been validated. The method validation used for both products … did not include a

protocol that included specification and acceptance criteria. … The method validation

was not reviewed and approved until during the current inspection. Lots of both

products were released for distribution prior to completion of the method validation.

• Method validation for the product Sennosides is inadequate in that the data does not

assess all variables, such as different mobile phase concentrations and analytes, to

demonstrate that the method can sustain variance.

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References

• ICH Q2 (R1) Validation of Analytical Procedures: Text and Methodology, International Conference on Harmonization.

• ICH Q3A (R2): Impurities in New Drug Substances, International Conference on Harmonization.

• ICH Q3B (R2): Impurities in New Drug Products, International Conference on Harmonization.

• ICH Q6A: Specifications: Test Procedures and Acceptance Criteria for New Drug Substances and New Drug Products: Chemical Substances, International Conference on Harmonization.

• ICH Q7: Good Manufacturing Practice Guidance for Active Pharmaceutical Ingredients, International Conference on Harmonization.

• FDA Draft Guidance for Industry on Analytical Procedures and Methods Validation: Chemistry, Manufacturing, and Controls Documentation. August 2000

• Center for Drug Evaluation and Research (CDER) Guidance: Guideline for Submitting Samples and Analytical Data for Methods Validation. February 1987

• Center for Drug Evaluation and Research (CDER) Reviewer Guidance: Validation of Chromatographic methods. November 1994

• US Pharmacopoeia General chapters: General tests and assays

• US Pharmacopoeia chapter <1225> Validation of Compendial Procedures

• US Pharmacopoeia chapter <1226> Verification of Compendial Procedures

• US Pharmacopoeia chapter <1092> The Dissolution Procedure: Development and Validation

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Summary recommendations

• Develop a validation master plan or an operating procedure for method validation

• For individual method validation projects, develop a validation project plan

• Define intended use of the method and performance criteria• Check all equipment and material for performance and quality• Perform validation experiments• Summarize the Validation outcome (include the critical method validation

observations in the respective methods)• Develop an operating procedure for method transfer between

laboratories

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Conclusions Analytical Method Validation is not jus a “routine” activity.

Need to be done in a high level GMP environment

Results generated throughout the validation activity needs to be “reviewed carefully”

Successful validation provides – “Successful Method Transfers & Satisfactory performance

of the Analytical method throughout the Lifecycle”

Quality issues if not addressed during method validations may have severe impact during

drug development (loss of time, costs, regulatory queries etc.)

Method development

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QUESTIONS ???

Contact details:BM Rao, Ph.D.Director – Analytical DevelopmentPharmaceutical Development & Manufacturing SciencesJanssen India – pharmaceutical companies of Johnson & Johnson Ltd.Email : drbmrao@hotmail.com