Analytical Method ValidationICH Q2A and USP • the ability to assess unequivocally the analyte in...

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RaziVaccine & SerumResearch Institute

Analytical MethodValidation

A. Es-haghiPh.D.

Dept. of Physico chemistryRazi vaccine and serum research institute

[email protected]

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Introduction

Test procedures for assessment of the quality levels of pharmaceutical products are subject to various requirementsThe CGMP regulations [21 CFR 211.194(a)] require that test methods, which are used for assessing compliance of pharmaceutical products with established specifications, must meet proper standards of accuracy and reliabilityaccording to these regulations [21 CFR 211.194(a)(2)], users of analytical methods described in the USP and the NF are not required to validate accuracy and reliability of these methods, but merely verify their suitability under actual conditions of useRecognizing the legal status of USP and NF standards, it is

essential, therefore, that proposals for adoption of new or revised compendial analytical methods be supported by sufficient laboratory data to document their validity

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What is Validation?

USP 30-NF 25, General Chapter <1225> Validation of Compendial Methods

• “Validation of an analytical method is the process by which it isestablished, by laboratory studies, that the performance characteristics of the method meet the requirements for the intended analytical applications.”

ICH Guideline Q2A – Text on Validation of Analytical Procedures

• “The objective of validation of an analytical procedure is to demonstrate that it is suitable for its intended purpose.”

FDA draft guidance – Analytical Procedures and Method Validation

• “Methods validation is the process of demonstrating that analytical procedures are suitable for their intended use.”

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Analytical Method Validation

In actual practice, analytical methods validation involves

• Established analytical method• Data from controlled samples• Statistical evaluation• Assessment of performance characteristics and applicability

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USP Performance Characteristics

Performance Characteristics are discrete attributes, derived by actual execution of an analytical method using controlled samples, which demonstrate the suitability and proper use of that method for a specific application

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USP Performance Characteristics

SPECIFICITYLINEARITYRANGEDETECTION LIMITQUANTITATION LIMITACCURACYPRECISIONRUGEDNESSROBUSTNESSSYSYEM SUITABILITY

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Specificity / Selectivity

ICH Q2A and USP• the ability to assess unequivocally the analyte in the presence of

components that may be expected to be present, such as impurities, degradation products, and matrix components

Lack of specificity of an individual analytical procedure may becompensated by other supporting analytical procedure(s)Other international authorities (IUPAC, AOAC) have preferred the term “Selectivity” reserving “Specificity” for those procedures that are completely selective

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Specificity

Identification• to ensure the identity of the analyte

Purity Test• accurate statement of the content of impurities of an analyte

(related substances, heavy metals, residual solvents, etc.)Assay

• an exact result which allows an accurate statement on the content of potency of the analyte in a sample

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Specificity

Some analytical procedures are not sufficiently specific for theintended purpose

• Assay by titration• Assay of enantiomer by achiral method• Identification by UV absorbance

A combination of two or more analytical procedures is recommended to achieve sufficient specificity

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Specificity

Should be one of the first validation itemsWhen the criteria are not met, this indicates that the method isnot sufficiently developedPoor specificity can impact accuracy, precision and linearity

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Linearity

The ability to generate responses which are directly proportional to the concentration of an analyte in a sample

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Linearity

A linear relationship requires fewer standards to define the response curve than a non-linear onejustifies the use of a single point standard to quantitate across the range

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Linearity

USP <1225>• Evaluated across the RANGE• Visual examination of a plot of signals as a function of

concentration• Statistical evaluation of correlation coefficient, y-intercept, slope,

and residual sum of squares of regression line or after mathematical transformation

ICH Guidance• Performed directly on the drug substance (dilution of standard

stock solution)• Separate weighing of synthetic mixtures of the drug components• Minimum of five concentration is recommended

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Linearity

Required for• Assays (Category I)• Impurity tests (Quantitative Category II)• May be for Performance Tests (Category III-Dissolution, etc.)

Not required for • Limit tests (Category II- Impurity Test)• Identification

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Range

UPS <1225>• The interval between the upper and lower quantitation levels

of analyte (including these levels) demonstrated by suitable precision, accuracy, and linearity

ICH Guidance• The following minimum specified ranges should be

considered• Assay of drug substance or finished drug product

– from 80% to 120% of the test concentration• Content Uniformity

– minimum of 70% to 130% of the test concentration, unless otherwise justified

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Detection Limit/ Quantitation Limit

Characteristic for low level impurity assaysDL (LOD)

• the lowest amount of any analyte which can be detected but not necessarily quantitated

QL (LOQ)• the lowest amount of analyte that can be determined with

acceptable precision and accuracy

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Determination of Detection Limit

Non-instrumental Methods• analysis of samples with known concentrations of analyte to

establish the minimum concentration at which the analyte can be reliably (the ability to reproducibly obtain a signal for the analyte) detected

• Examples– TLC, titrations, color compositions

Instrumental• Same as for non-instrumental methods, plus estimation of the DL

can be made at a value at which the signal to noise ratio (S/N) is 2:1 or 3:1

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Estimation of DL (ICH)

DL = 3.3 σ/S• Where: σ = Standard deviation (“noise”) of response• S = slope of the calibration curve for linearity

DL can be based on any of the following• σ of the blank• σ of the lowest level linearity point• the calibration plot (regression)

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Quantitation Limit Definition

USP <1225>• the lowest amount of analyte in a sample that can be determined

with acceptable precision and accuracy under the stated experimental conditions

Essentially identical to ICH Q2AQuantitation Limit (QL) = Limit of Quantitation (LOQ)

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Determination of QL

Non-Instrumental• from analysis of samples with appropriate minimum level of

analyte quantified with acceptable accuracy and precisionInstrumental

• same as non-instrumental, plus estimation of the QL can be made at a value at which the signals to noise is 10:1

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Estimation of QL (ICH)

QL = 10 σ/S• where σ = standard deviation of response• S = slope of the calibration curve for linearity• σ can be based standard deviation of the blank or of the lowest

level linearity point

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Case Study

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Accuracy

Definition• USP

• “The closeness of the result obtained by the method to the true value.”

• ICH• “The closeness of the result obtained by the method to a value

that is accepted as conventionally true value or as a reference value.”

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Accuracy

The accuracy of an analytical method should be established across its range

ICH recommends a minimum of 9 determinations over a minimum of 3 concentrations covering the stated quantitation range (3 test at 3 conc.)Accuracy is calculated as

• the percentage of recovery by the assay of the known added amount of analyte in the sample

• the difference between the mean and the accepted true value, together with confidence intervals

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Accuracy

5 ways to determine the Accuracy• Testing of a Reference Standard• Mixture with Excipients (Spiked placebo)• Standard addition (Spiked sample)• Inferred from linearity and specificity data• Comparison with a method known to be accurate (reference

method)

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Accuracyassay of a drug substance

Testing of a Reference StandardComparison with a reference standard method

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Recovery of Drug F Ref Std at different levels

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Comparison with Reference Method

There is no difference with p < 0.05

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Precision

The degree of agreement among results from individual samplings of a homogeneous sample

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Precision

Precision is expressed as RSDPrecision should be evaluated across the specified Quantitation range of the methodDetermination

• Assay individual samples of a homogeneous preparation• Calculate Standard Deviation or Relative Standard Deviation

Method precision should include all sources of variation from sample preparation to rounding the final test result

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Precision

ICH recommends a minimum of 9 determinations over a minimum of 3 concentrations covering the stated quantitationrange (3 test at 3 conc.)

OR

Using a minimum of 6 determinations at 100% of the test concentration

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ICH-USP Level of Precision

Repeatability• agreement within a short period or the same analyst and

instrumentationIntermediate precision

• agreement in results intra-laboratory but from different days, analysts and equipment (as appropriate)

Reproducibility• agreement in results between laboratories (as in a collaboration

study)

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Precision vs. Ruggedness

In USP, Reproducibility and intermediate precision are also considered together with Ruggedness

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Precisionchoice of acceptance criteria

Practical Considerations• Ability to meet criteria

• may be instrument dependent• Maximum acceptance criteria for precision should be linked to

acceptance criteria for claim or specification to be able to obtain a reliable result (reduce Out-Of Specifications)

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Precision

Repeatability (5 replicate injections)Assay Limit (%) Max, % RSD99.0 – 101.0 0.37 98.5 – 101.5 0.55 98.0 – 102.0 0.73 95.0 – 105.0 1.9 90.0 – 110.0 3.9 Impurities (depending on level) 5 to 25

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Precision choice of acceptance criteria

Intermediate Precision• FDA recommends a minimum of 2 different days with different

analysts• Acceptance criteria

• Perform F-test• results between labs should not be statistically different• Typically about 2x repeatability (2 x RSD)

– However, a statistical difference may still not be significant (practical consideration). Need to evaluate suitability for methods intended use

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Reproducibility Precision

FDA• at least 2 laboratories within the company

FDA• for multi-laboratory follow ASTM 691

• Standard practice for conducting an laboratory study to determine the precision of a test method

AOAC Protocol • 8 samples, 8 laboratories

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Ruggedness

Reproducibility with the same samples using different laboratories, analysts, days, reagent lots (same brand), and environmental conditionsPrecision should be evaluated across the specified Quantitation range of the method

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Ruggedness vs. Robustness

Reproducibility of the test results under conditions normally expected from laboratory to laboratory and from analyst to analystMethod parameters are kept constant (difference with robustness)

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Ruggedness

Determination• Analysis of an homogeneous sample in different laboratories, by

different analysts, under prevalent environmental conditions using the specified parameters

• The degree of reproducibility of test results is determined as function of the variables

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Ruggedness

study normal variables• Analyst A, B, C• Day 1, 2, 3• Lab L, M N• Column X, Y, Z

Selected comparisons are evaluated since evaluating all possible variations would require 81 experiments (3 x 3 x 3 x 3)The influence of each variable may be evaluated using a multi-factor experimental design (Plackett- Burman, Hendrix, etc.)

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Ruggedness

Degree of reproducibility of test results as a function of the variables

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Robustness

The ability to remain unaffected by small but deliberate variations in method parameters- evaluates reliabilityProvides an indication of the reliability of the method during normal usage

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Robustness

Evaluated by varying method parameters and determining the effect (if any) on the results of the methodIt should be considered early in the development of a methodIf the results are susceptible to parameter variations, these parameters should be adequately controlled and a precautionary statement included in the method

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Robustness

Two approaches• Uni-variable

• Systematically varying each parameter sequentially• Typically, each parameter may be varied by 5 – 10% above

and below the value set in the method• Multi-variable

• Use of the statistical design– Central Composites– Fractional factorials,– Two-factor Plackett-Burman statistical approach– Hendrix

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System Suitability

Ensures that both methodology and instrumentation are performing within expectation prior to the analysis of the test samples

USP General Chapter <1225>• “System suitability tests are based on the concept that the

equipment, electronics, analytical operations, and samples to beanalyzed constitute an integral system that can be evaluated as such. System suitability test parameters to be established for aparticular procedure depend on the type of procedure being evaluated. They are especially important in the case of chromatographic procedures.”

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System Suitability

Provides assurances that the system is working at the time of analysisEnsures that both methodology and instrumentation are performing within expectations prior to the analysis of the test samplesShould be monitored during run time to verify that criteria remain realistic and achievable

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System Suitability

Parameters for chromatographic method• Resolution – specificity• Column efficiency – specificity• Relative standard deviation – precision• Tailing factor – accuracy and precision• QL – sensitivity• Capacity factor – specificity• Reference Standard Check - analyte

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USP Method Categories

Category I• Quantitative test for major active ingredients (chromatographic

and titration)Category II

• Impurities or degradation compounds; quantitative or limit testsCategory III

• performance tests (dissolution, drug release, tapped density, particle size)

Category IV• Identification tests (TLC, IR, UV, Chemical ID <191>

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USP <1225>Data Elements for Validation

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ICH Q2BData Elements for Validation

May be required, depending on the nature of the specific testIs not needed if reproducibility has been performed

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Choice of Performance Characteristics

Not all Performance Characteristics are needed to validate an analytical method or validated in the same way

• Determine the expected use of the test procedure (e.g., assay, dissolution, limit test, ID test)

• Take into account the type of analytical technique used (chromatographic, spectrophotometric, titration, etc.)

Establish the requirements of the analytical application• Degree of precision and accuracy needed• The concentration range of the analyte• The level of tolerable interference, if any

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Protocol for Final Validation Studies

Initial analytical methods are often developed in R&D areas with some preliminary validation dataThese procedures can be slightly modified when transferred to QC areas for complete validation including multi-site collaborative studies

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Validation Protocols

The validation protocol document contains• Description of the analytical method and its intended use

(purpose)• List of applicable performance characteristics• Appropriate acceptance criteria• Review and approval signatures

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Pre-validation Assessment

Equipment• Suitable for expected accuracy?

Reference Materials• Suitability characterized?

Analytical Method• Is procedure finalized?

Validation Protocol• Management / QA approved?

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Equipment

• Demonstrate that equipment used in validation studies is suitable for use and is comparable to equipment used for routine analysis

• Calibrated (as applicable)• Qualification should have been performed

– Installation Qualification– Operation Qualification– Performance Qualification

• Routine maintenance performed- proper working order

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Reference Materials• evaluate suitability of use

• FDA requires no additional evaluation of USP Reference Standards

• Standardization of reagents vs. Primary standards when available(i.e., use of reagent grade chemical need qualification)

• Principal analyte (purity and homogeneity)• Impurity Standards

– chemical purity is very critical if used to determine relative response

– Solution stability of standard preparations

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Analytical Method

• must be final before validation• Individual procedure steps may impact overall method accuracy

or precision• Solution preparations

– concentration of analyte in solution for analysis (suitable range)

• System suitability– necessary parameters and acceptance criteria must be

specified– Changes may negatively impact validation studies

previously performed– Later changes in tailing factor or resolution criteria from

robustness evaluation may have a significant impact on integration in accuracy evaluations

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Standard and Sample Solution Preparation

Overall method precision and accuracy are also impacted by sampling, weighing, and quantitative dilutionsNo instrumental technology can overcome these errors

• The precision and accuracy of a final analytical method can be better than the precision and accuracy of the preparation of thetest solutions

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Revalidation

Revalidation may be necessary in the following cases• a submission to the USP of a revised analytical method• use of an established general method with a new product or

raw material

The ICH documents give guidance on the necessity for revalidation in the following circumstances

• changes in the synthesis of the drug substance• changes in the composition of the drug product• changes in the analytical procedure

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RVSRI deffects in Analytical Method Validation

EquipmentPersonnelReference MaterialsAnalytical MethodsValidation ProtocolChange controlWeak links between different chain to final product be completely assured

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Quick QuizCumulative errors in serial dilution

To prepare 100 fold dilution of a stock solution, what is the most precise procedure when considering the necessary individual Class A pipette and volumetric flask tolerances?

A. 1.0 mL/10 mL; 1.0 mL/10 mLB. 5.0 mL/50 mL; 5.0 mL/50 mLC. 10.0 mL/100 mL; 10.0 mL/100 mLD. 1.0 mL/100 mLE. 2.0 mL/200 mLF. 5.0 mL/500 mL

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Tolerance of Class A Glassware(see USP <31> Volumetric apparatus)

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Quick QuizCumulative errors in serial dilution

Sum of step errors

0.6 + 0.2 + 0.6 + 0.2 = ± 1.6%0.2 + 0.1 + 0.2 + 0.1 = ± 0.6%0.2 + 0.08 + 0.2 + 0.08 = ± 0.43%0.6 + 0.08 = ± 0.68%0.3 + 0.05 = ± 0.35%0.2 + 0.03 = ± 0.23%

A. 1.0/10; 1.0/10LB. 5.0/50; 5.0/50C. 10.0/100; 10.0/100D. 1.0/100E. 2.0/200F. 5.0/500

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Analytical Method ValidationICH Q2A and USP • the ability to assess unequivocally the analyte in...

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