6th November 2002 Analytical Development, PAR&D (AW)Method Validation in the Pharmaceutical Industry Kate Arnot

OverviewThe drug development processGeneral recommendationsExamplesAssay Dissolution test for an immediate release tabletIdentity Identification test for a drug substanceImpuritiesQuantitative test residual solvents in a drug substanceLimit test residual solvents in a tablet

DRUG DEVELOPMENT PROCESS10,000 compounds1 new medicine

A phased approach to analytical validationPhase I Limited validation, focussing on key method attributes eg.specificity, limits of quantitation and linearityPhase II Starting to include accuracy and precision data to support specificationsPhase III Full validation according to ICH guidelines will be completed for all analytical methods prior to submission of marketing applications

General recommendationsDesign your validation studies to reflect the way that the method will be applied in routine use as far as possibleEnsure that you have considered the specification you want to achieve in designing your studiesKnow your method before you start to validate!

Assay - Dissolution test for an immediate release tabletDissolution testing is used to evaluate the release of drug substance from tabletsDissolution apparatus 2 (paddles), 50rpm, 37C, aqueous surfactant as dissolution medium, UV analysis, sampling at a single 45 minute timepointTypical Specification: Q = 80 at 45 minutes (ie each of 6 units should achieve 85% dissolution)

Dissolution specification USP acceptance tableS1, 6 units Each unit is not less that Q+5%S2, 6 units Average of 12 units (S1+S2) is equal to or greater than Q and no unit is less than Q-15%S3, 12 units Average of 24 units (S1+S2+S3) is equal to or greater than Q, not more than 2 units are less than Q-15%, and no unit is less than Q-25%

Dissolution apparatus

Typical dissolution profileTime (mins)% Dissolution

data

PAR&D data

BatchP/1427/339090C

Nbk RefP/4299/03

timen=80sd2sdn=80sd2sd

000

15546.713.4514.59.1

30822.34.7712.14.2

45901.42.8791.93.8

60941.42.8841.73.4

QA data

P/1427/33 (3+3 crossover)Summary data

Bath 1Bath 2Bath 1 meanBath 2 meanOverall meansdRSD

time123456n=3n=3n=6

0000

155845485756575057545.510.4

308380808383838183821.51.9

458989889091918991901.21.4

609394939395949394940.80.9

BH153X (3+3 crossover)Summary data

Bath 1Bath 2Bath 1 meanBath 2 meanOverall meansdRSD

time123456n=3n=3n=6

0000

154748434552434647463.47.4

308381818580818282821.82.2

459190909092899090901.01.1

609494949293969494941.31.4

BH153X (original QA analysis)Overall meansdRSD

time123456n=6

00

15514955585051523.46.6

30828082787882802.02.4

45898889918789891.31.5

60939193959192931.51.6

BE batches

0

5413.424272648413.4242726484

824.67651820614.6765182061

902.81052231732.8105223173

942.81736999182.8173699918

BE BFC (PAR&D n=80)

time (mins)

% dissolved

Sheet2

Sheet3

Validation parameters evaluatedSpecificityLinearityAccuracy and rangePrecisionRepeatability Intermediate precisionRobustness

SpecificityDemonstrate absence of interference from excipients and reagents at the monitoring wavelengthFor example calculate the absorbance of a placebo solution as a percentage of the absorbance of a standard solutionTypically results of less that 2% would be considered acceptableSpecificity may also be inferred from linearity and accuracy data

LinearityRange studied 0 to 150% of nominal (7 solutions, including a blank solution)Linearity experiments conducted in the presence and absence of excipientsDetermine equation of the line, correlation coefficient, 95% confidence interval of the slope and the intercept. Verify that the 95% confidence interval of the intercept includes zero, or that the result is insignificant in the context of the experimentCheck that the response for both experiments is the same calculate the slopes as a % of each otherTypically results of 98 to 102% would be considered acceptable

Linearity in the presence of placebo y = 42.983x + 0.004 r = 0.99989 95% CI of slope 0.0730 95% CI of intercept 0.016Concentration (g/100ml)Absorbance

Linearity in the absence of placeboy = 43.033x + 0.006 r = 0.99990 95% CI of slope 0.695 95% CI of intercept 0.009Concentration (g/100ml)AbsorbanceComparison of slopes: (42.983/43.033) x 100 = 99.9%

Accuracy and Range3 samples at each of 5 different concentration levels were evaluated: 50%, 85%, 100%, 115% and 125% of nominalSamples were analysed against standards as per the method and % recoveries calculated Typically results in the range 95 to 105% would be considered acceptableThe overall standard deviation and relative standard deviation may be used as an estimate of precision

Example Accuracy DataExperiment 150%97.385%96.8100%96.5115%96.0125%95.8Mean96.5sd0.7Experiment 250%99.385%99.4100%99.6115%98.7125%98.9Mean99.2sd0.5

Example Accuracy Data - continuedExperiment 1Samples mixed, and allowed to stand overnightExperiment 2Samples stirred continuouslyTime1Recovery98.8%(hours)499.1%2199.2%2799.3%

Accuracy/Linearity how do you prepare your samples?Prepare samples containing drug substance plus an appropriate quantity of ground placebo tabletPrepare samples containing drug substance plus appropriate quantities of an excipient mixPrepare samples by using different weights from a ground active tablet preparation, making a correction for nominal concentrationMake sure that you are confident in your sample extraction technique

Precision - RepeatabilityOne analyst conducting multiple replicates of the same sample, on the same day using the same equipment, and applying the method as written.For dissolution experiments correcting for tablet weight may be appropriateDetermine the mean, the range, the standard deviation and the relative standard deviation.

Precision Intermediate precisionA second operator conducts the repeatability experiment on a different day using different equipment where possible.Determine the individual data and overall mean, range, standard deviation and relative standard deviation.Consider testing statistically that there is no significant difference between operators before combining the data set.

RobustnessA number of attributes were evaluated prior to validation:Standard and sample solution stabilityEffect of pH and the requirement to buffer and/or degass the dissolution mediumReagents from different suppliers were evaluatedSampling tubes, filters and sampling and filtration speeds were evaluatedSingle point vs profile testing

Identification Infrared method for a drug substanceInfrared spectroscopy is frequently used in identification testingSample is dispersed in KBr, analysed using the DRIFTS technique, and the spectrum obtained visually compared with that of a fully characterised reference standard analysed under the same conditions.Typical Specification: Result consistent with reference standard

Validation parameters evaluatedSpecificity

Identification testing should optimally be able to discriminate between compounds of closely related structure which are likely to be present (ICH Q6A) The choice of such potentially interfering materials should be based on sound scientific judgement with a consideration of the interferences that could occur (ICH Q2B)

SpecificityDemonstrate specificity between drug substance and other potentially interfering compounds eg. starting materials, isolated intermediates and other materials manufactured or formulated in the same plantConsideration should also be given to enantiomers, counter ions/salts, solvates and polymorphs if necessary.

Drug substanceCompound formulated in same plantDevelopment compoundIsolated intermediate

Impurities Quantitative test for residual solvents in drug substanceA range of solvents are used in the manufacture of a drug substance. It is necessary to quantify these, and demonstrate that they comply with ICH Q3C requirementsCapillary GC method with flame ionisation detection.Typical Specification: 0.5% w/w maximum

Validation parameters evaluatedSpecificityLinearityAccuracyPrecisionReproducibilityIntermediate precisionRangeLimits of detection and quantitationRobustness

SpecificityDemonstrate method is specific for all the solvents used in the registered synthetic processCan use linearity data for solvents in the presence and absence of the drug substance to show that the drug substance does not interfere

Specificity chromatogram

LinearityRange studied 0 to 150% of nominal (9 solutions, including a blank solution and a solution at the LOQ)Drug substance required without solvent, or low levels of solvent and then make a correctionDetermine equation of the line, correlation coefficient, 95% confidence interval of the slope and the intercept. Verify that the 95% confidence interval of the intercept includes zero, or that the result is insignificant in the context of the experiment

AccuracyCalculate recoveries from the linearity experiments by analysing the solutions against an external standard as per the method.Typically results in the range 80 to 120% would be considered acceptableThe overall standard deviation and relative standard deviation may be used as an estimate of precision

Intermediate precision - Japanese styleExpt No12345678Operator11112222GC12211221Column12121212

Experiments are conducted on different days with each analyst using each instrument/column combination in turnReport results, overall mean, overall standard and relative standard deviation, and 95% CI of sd

RangeDetermined from the linearity, accuracy and precision data

Limit of Quantitation and DetectionLimit of quantitation established as 0.05% w/w Samples at this concentration included in linearity and accuracy experimentsLimit of detection estimated as 0.02% w/w by visual inspection of chromatograms

LOQ chromatogram

RobustnessA number of attributes were evaluated prior to validation using an experimental design approach:Injector temperatureDetector temperatureInitial temperatureRamp rateHold timeSplit flowColumn head pressure

Impurities Limit test for residual solvent in a tabletAn alcohol is used as a preservative in a purchased film coating concentrate. It is removed during the manufacturing process, but this needs to be demonstratedFast GC method with flame ionisation detection.Need to demonstrate that tablets comply with requirements for Class 3 solvents ie less than 0.5% w/w

Validation parameters evaluatedSpecificity Limit of quantitationLinearityFor solvents arising from the drug substance and tablet manufacturing process0.05% w/w0.05% to 0.5% w/w

ReferencesICH www.ICH.orgInternational Pharmaceutical Product Registration Aspects of Quality, Safety and Efficacy, eds Cartwright and Matthews, Ellis Horwood, 1994, Chapter 8, Analytical validationThe validation of analytical methods for drug substances and drug products in UK pharmaceutical laboratories, G S Clarke, J. Pharm. Biomed. Anal., 12. 643 652 (1994)FDA www.FDA.govTechnical review guide: Validation of chromatographic methods