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Birgit Schmauser | April 20082 |

Analytical Method DevelopmentAnalytical Method Development

Presented by:Presented by:

Birgit Schmauser, PhDBirgit Schmauser, PhD

Federal Institute for DrugsFederal Institute for Drugsand Medical Devices (BfArM)and Medical Devices (BfArM)

[email protected]@bfarm.de
http://www.travelphoto.net/photos/pictures/indien/in10.jpg

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In this presentation:In this presentation:

Standards in developing analytical methods for Standards in developing analytical methods for Originator and multisource generic FPPsOriginator and multisource generic FPPs

SpecificationsSpecifications

StabilityStability

Parallel development of analytical methods for Parallel development of analytical methods for cleaning validationcleaning validation

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Originator Originator ,, First-timeFirst-time GenericGeneric and Multisource Genericand Multisource GenericOriginator Originator First-timeFirst-time GenericGeneric Multisource GenericMultisource Generic

API qualityAPI qualitystandardsstandards

OriginatorsOriginatorsspecificationsspecifications

Information fromInformation fromregulatory agenciesregulatory agencies(publicly available) &(publicly available) &

literature dataliterature data

PharmacopoeiasPharmacopoeias

FPP qualityFPP qualitystandardsstandards

OriginatorsOriginatorsspecificationsspecifications

Information fromInformation fromregulatory agenciesregulatory agencies(publicly available) &(publicly available) &literature dataliterature data

PharmacopoeiasPharmacopoeias

AnalyticalAnalyticalmethodsmethods

EstablishEstablish identity,identity,potency, purity of APIpotency, purity of APIand FPP byand FPP byin-house methodsin-house methods

DeriveDerive identity, potency,identity, potency,purity of API and FPP bypurity of API and FPP byin house methodsin house methods

VerifyVerify identity, potency,identity, potency,purity of API and FPP bypurity of API and FPP bypharmacopoeial methodspharmacopoeial methodsand in-house methodsand in-house methods

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HPLC-method to assay potency and purity risk assessmentHPLC-method to assay potency and purity risk assessmentOriginator Originator First-timeFirst-time GenericGeneric Multisource GenericMultisource Generic

SelectivelySelectively screenscreen /detect any/detect anyimpurity or degradantimpurity or degradantEstablishEstablish potencypotency

IdentifyIdentify impurities/degradantsimpurities/degradants

CharacteriseCharacterise allallimpurities/degradants

impurities/degradants CalculateCalculateResponse factorsResponse factors(qualification by clinical use)(qualification by clinical use)

DeriveDerive impurities/degradants fromimpurities/degradants fromOriginator Originator Characterize in-houseCharacterize in-houseimpurities/degradantsimpurities/degradantsCalculate response factorsCalculate response factors

Verify impurities fromVerify impurities fromPharmacopoeiaPharmacopoeiaCharacterise in-houseCharacterise in-houseimpurities/degradantsimpurities/degradants(Response factors)(Response factors)

EstablishEstablish reference materialsreference materials ExtractExtract (&(& reproducereproduce ) reference) referencematerialsmaterials

Use pharmacopoeial referenceUse pharmacopoeial referencematerialsmaterials

AdaptAdapt to routine useto routine use AdaptAdapt /modify to/for routine use/modify to/for routine use Implement for routine useImplement for routine use

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Interchangeability (IC) of Interchangeability (IC) of multisource generic FPPsmultisource generic FPPs((Essential similarityEssential similarity with Innovator FPP)with Innovator FPP)

PharmaceuticalPharmaceutical ++ BioequivalenceBioequivalenceEquivalenceEquivalence

IC =IC = PEPE ++ BEBE

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Pharmaceutical equivalencePharmaceutical equivalence FPPs meet theFPPs meet the samesame or or comparablecomparable standardsstandards by use of

by use of equivalent analytical methodsequi

valent analytical methods SameSame APIAPI ((chemicalchemical andand physicalphysical equivalence)equivalence) SameSame dosage formdosage form andand route of administrationroute of administration SameSame strengthstrength ComparableComparable labelinglabeling

Equivalence inEquivalence in pharmaceutical developmentpharmaceutical development Equivalence inEquivalence in stabilitystability

Equivalence inEquivalence in manufacture (WHO-GMP)manufacture (WHO-GMP)

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Prequalification requirementsPrequalification requirements Validation of analytical methods is aValidation of analytical methods is a prerequisiteprerequisite for for

prequalification of product dossiersprequalification of product dossiers Non-compendial APIs and FPPsNon-compendial APIs and FPPs are tested withare tested with methodsmethods

developed by the manufacturerdeveloped by the manufacturer ForFor compendial APIs and FPPs the applicability of compendial APIs and FPPs the applicability of

pharmacopoeial methodspharmacopoeial methods to particular productsto particular products mustmustbe demonstrated (verification)be demonstrated (verification)

Analytical methods must be developed and validatedAnalytical methods must be developed and validatedaccording to TRS 823,according to TRS 823, Annex 5,Annex 5, Validation of analytical Validation of analytical

procedures used in the examination of pharmaceutical procedures used in the examination of pharmaceutical materialsmaterials ; ICH Q2 (R1); ICH Q2 (R1)

To be used within GLP and GMP environmentsTo be used within GLP and GMP environments

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CLINICALCLINICAL PHARMACEUTICALPHARMACEUTICAL METHODSMETHODS

AtAt initialinitial phase of pharmaceutical developmentphase of pharmaceutical developmentToTo determinedeterminebioavailabilitybioavailability in healthyin healthyvolunteersvolunteers

To develop a stable andTo develop a stable andreproducible formulation for thereproducible formulation for themanufacture of bioequivalence,manufacture of bioequivalence,

dissolution, stability and pilot-scaledissolution, stability and pilot-scalevalidation batchesvalidation batches

To understand theTo understand the profileprofile of relatedof relatedsubstances and to studysubstances and to study stabilitystabilityTo startTo start measuringmeasuring the impact of the impact of keykeyproductproduct andand manufacturing processmanufacturing processparametersparameters onon consistentconsistent FPP qualityFPP quality

AtAt advancedadvanced phase of pharmaceutical developmentphase of pharmaceutical developmentTo proveTo prove bioequivalencebioequivalence after after critical variationscritical variations totothe prequalified dossier the prequalified dossier

ToTo optimiseoptimise ,, scale-upscale-up andand transfer transfer aa stablestable andand controlledcontrolledmanufacturing process for themanufacturing process for theprequalification productprequalification product

To beTo be robust, transferable, accuraterobust, transferable, accurate andand preciseprecise for specification setting,for specification setting,stability assessment and QC release of stability assessment and QC release of prequalified product batchesprequalified product batches

Use of analytical methods -Use of analytical methods - genericsgenerics

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PrerequisitesPrerequisites for analytical method validationfor analytical method validation Six Ms

Quality of theQuality of theanalytical methodanalytical method

MManan MMachineachine

qualifiedqualified

calibratedcalibrated

robustrobust

qualifiedqualified

MMethodsethods

suitablesuitable

characterisedcharacterised

documenteddocumented

MMilieuilieuMMaterialaterial MManagementanagementQualityQuality

ReferenceReferencestandardsstandards

Tempe-Tempe-raturerature

AnalystsAnalystssupportsupport

skilledskilled

HumidityHumidity

VibrationsVibrations TimeTime

SuppliesSupplies

Irradi-Irradi-ationsations

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Method development life cycleMethod development life cyclePlanning

Development and Validation Policy

Objectives/Requirements of Method

Information Gathering Resource Gathering

Method developmentInitital Method Development

Pre-Validation EvaluationMethod Optimization

RobustnessSystem Suitability

DevelopmentPlan Project

Customer EvaluationTesting

Validation Experiments

Method Transfer Experiments Filed Method in Use

PeriodicallyMonitoring/Review

of Methodsin Control Labs

From: Analytical Chemistry in a GMP Environment. Edited by J.M. Miller and J.B. Crowther, ISBN 0-471-31431-5, Wiley & Sons IncFrom: Analytical Chemistry in a GMP Environment. Edited by J.M. Miller and J.B. Crowther, ISBN 0-471-31431-5, Wiley & Sons Inc..

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Validation should verify the suitability of anValidation should verify the suitability of ananalytical methodanalytical method for its intended purposefor its intended p urpose

Validation should be founded onValidation should be founded on methodmethod

development performed beforehanddevelopment performed beforehand thatthatsuggest thesug gest the suitability and robustnesssuitability and robustness of theof themethodmethod

Validation may be performed in different waysValidation may be performed in different ways(individual purpose) according to(individual purpose) according to commoncommonstandardsstandards

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Validation protocolValidation protocol Method principle / objectiveMethod principle / objective Listing of responsibilitiesListing of responsibilities

Laboratories involved and their role in the validationLaboratories involved and their role in the validation Method categorizationMethod categorization List of reagents (including test lots) and standardsList of reagents (including test lots) and standards Test procedures to evaluate each validation parameter and proposedTest procedures to evaluate each validation parameter and proposed

acceptance criteriaacceptance criteria Plan or procedure when acceptance criteria are not metPlan or procedure when acceptance criteria are not met Requirements for the final reportRequirements for the final report

The validation process cannot proceed until the protocol andThe validation process cannot proceed until the protocol andall parties involved approve the acceptance criteriaall parties involved approve the acceptance criteria

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Innovator versus GenericsInnovator versus GenericsInnovator Innovator GenericsGenerics

R & D on APIR & D on API ++ --

Preclinical trialsPreclinical trials ++ --

Clinical trials phase I and IIClinical trials phase I and II Method validationMethod validationsummarysummary

--

Clinical trials phase IIIClinical trials phase III Method validationMethod validationcompletedcompleted

--

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

Entering of Generics; PharmaceuticalEntering of Generics; Pharmaceuticaldevelopment, Comparability withdevelopment, Comparability withInnovator Innovator

Validated methodsValidated methods Validated methods:Validated methods:GMP and GLPGMP and GLP

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Validation CharacteristicsValidation CharacteristicsIdentificationIdentification ImpuritiesImpurities AssayAssay

quantitativequantitative limitlimit

AccuracyAccuracy -- ++ -- ++

PrecisionPrecision -- ++ -- ++SpecificitySpecificity ++ ++ ++ ++

Detection LimitDetection Limit -- -- ++ --

Quantitation LimitQuantitation Limit -- ++ -- --

LinearityLinearity -- ++ -- ++

RangeRange -- ++ -- ++RobustnessRobustness ++ ++ ++ ++

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Accuracy and precisionAccuracy and precision

Accurate &Accurate &

preciseprecise Accurate &Accurate &impreciseimprecise

InaccurateInaccurate&&preciseprecise Inaccurate & impreciseInaccurate & imprecise

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PrecisionPrecision Expresses theExpresses the closeness of agreementcloseness of agreement between a series of between a series of

measurements obtained from multiple sampling of the samemeasurements obtained from multiple sampling of the samehomogenous samplehomogenous sample

Is usually expressed as theIs usually expressed as the standard deviation (S),standard deviation (S), variancevariance (S(S 22)) or or coefficient of variationcoefficient of variation (RSD)(RSD) of a series of measurementsof a series of measurements

Precision may be considered at three levelsPrecision may be considered at three levels RepeatabilityRepeatability (intra-assay precision)(intra-assay precision) Intermediate PrecisionIntermediate Precision (variability within a(variability within a

laboratory)laboratory)

ReproducibilityReproducibility (precision between laboratories)(precision between laboratories)

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Analytical Method DevelopmentAnalytical Method DevelopmentNormal distribution,Normal distribution, probability function [P(x)]probability function [P(x)]and confidence interval [CI]and confidence interval [CI]

Probability (P)Probability (P) , that measurements from a normal distribution fall within [-x, that measurements from a normal distribution fall within [-x nn , +x, +x nn]]for xfor x nn = n= n is described by theis described by the erf erf -function-function (( = mean)mean) :

An interval of 3An interval of 3 coverscovers 99.73% of values99.73% of values N

umberoftimes eachvalueoccursxxnn PP

0.68268950.6826895

22 0.95449970.9544997

33 0.99730020.9973002

44 0.99993660.9999366

55 0.99999940.9999994Values23 2 3

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Normal distribution, probability function [P(x)] andNormal distribution, probability function [P(x)] and

confidenceconfidence

interval [CI]interval [CI]

Probability-PProbability-P Confidence interval [CI]Confidence interval [CI]centered around the mean []centered around the mean []in units of sigma [in units of sigma [ ] described by] described by

inverseinverse erf erf -function:-function:

A CI of 95% includes valuesA CI of 95% includes values 1.95 1.95 around the meanaround the mean

PP xxpp

0.8000.800 1.281551.28155 0.9000.900 1.644851.64485

0.9500.950 1.959961.95996

0.9950.995 2.575832.57583

0.9990.999 3.290533.29053

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Relationship of variability, probability and reliability of dataRelationship of variability, probability and reliability of data High variability of data (largeHigh variability of data (large ) generate large confidence intervals and) generate large confidence intervals and

thus lower the reliability of the meanthus lower the reliability of the mean

Low variability of data (smallLow variability of data (small )) generate small confidence intervals andgenerate small confidence intervals andthus increase the reliability of the meanthus increase the reliability of the mean

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RepeatabilityRepeatability Six replicate sample preparation steps from a homogenously prepared tabletSix replicate sample preparation steps from a homogenously prepared tablet

mixture (nominal value of API 150 mg)mixture (nominal value of API 150 mg)

InjectionInjection Peak areaPeak area AssayAssay

11 173865173865 147.10 mg/98.06%147.10 mg/98.06%

22 174926174926 148.00 mg/98.66%148.00 mg/98.66%33 172933172933 146.32 mg/97.54%146.32 mg/97.54%

44 175011175011 148.08 mg/98.72%148.08 mg/98.72%

55 179557179557 151.95 mg/101.30%151.95 mg/101.30%

66 176425176425 149.28 mg/99.52%149.28 mg/99.52%

MeanMean 175453175453 148.45 mg/98.96%148.45 mg/98.96%SD (SD ( )) 23292329 1.98 mg/1.32%1.98 mg/1.32%

RSDRSD 1.32%1.32% 1.32%1.32%

MeanMean 3 SD = 3 SD =Confidence interval of 99.73%Confidence interval of 99.73%

98.9698.963x1.32% =3x1.32% = 95%95% - 102.92%- 102.92%

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Intermediate precisionIntermediate precision Expresses within-laboratories variations (different days, different analysts,Expresses within-laboratories variations (different days, different analysts,

different equipment etc.)different equipment etc.)

InjectionInjection Peak areaPeak areaanalyst 1analyst 1

Peak areaPeak areaanalyst 2analyst 2

Peak areaPeak areaanalyst 3analyst 3

11 173865173865 175656175656 17796517796522 174926174926 175878175878 17855617855633 172933172933 176004176004 17734217734244 175011175011 176344176344 17801117801155 179557179557 175332175332 17946617946666 176425176425 174959174959 179688179688

MeanMean 175453175453 175695175695 178504178504SD (SD ( )) 23292329 495495 918918

RSDRSD 1.32%1.32% 0.28%0.28% 0.51%0.51%

Analyst 1: 98.96%Analyst 1: 98.96% 3 x 1.32% 3 x 1.32%

Analyst 2: 99.12%Analyst 2: 99.12% 3 x 0.28 3 x 0.28

Analyst 3: 100.70%Analyst 3: 100.70% 3 x 0.51 3 x 0.51

Average of 3 analystsAverage of 3 analysts 3SD 3SD ::

95%95% - 102.23%- 102.23%

MeanMean 3 SD: (177252 3 SD: (177252

100%100%))

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ReproducibilityReproducibility Expresses the precision between laboratoriesExpresses the precision between laboratories

Collaborative studies, usually applied toCollaborative studies, usually applied to

standardisation of methodologystandardisation of methodology

Transfer of technologyTransfer of technology Compendial methodsCompendial methods

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AccuracyAccuracy Expresses theExpresses the closeness of agreementcloseness of agreement betweenbetween the value which isthe value which is

accepted either as a conventionalaccepted either as a conventional true valuetrue value or or an acceptedan acceptedreference valuereference value and theand the value foundvalue found

Sometimes referred to as Sometimes referred to as TRUENESSTRUENESS

truetruemeanmean

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To find out whether a method is accurate:To find out whether a method is accurate:Drug substance (assay)Drug substance (assay)

Application of the method to an analyte of known purity (e.g. referenceApplication of the method to an analyte of known purity (e.g. referencesubstance)substance)

Comparison of the results of one method with those of a second well-Comparison of the results of one method with those of a second well-

characterised method (accuracy known)characterised method (accuracy known)

Drug product (assay)Drug product (assay) Application of the method to synthetic mixtures of the drug product componentApplication of the method to synthetic mixtures of the drug product component

to whichto which known quantitiesknown q uantities of the analyte have been addedof the analyte have been added Drug product may exceptionally be used as matrixDrug product may exceptionally be used as matrix

Drug substance/Drug product (Impurities)Drug substance/Drug product (Impurities) Application of the method to samples spiked withApplication of the method to samples spiked with known amountsknown amounts of impuritiesof impurities

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AccuracyAccuracy :: Application of the method to synthetic mixtures of theApplication of the method to synthetic mixtures of thedrug product componentsdrug product componentsto which known quantitiesto which known quantitiesof the analyteof the analytehave been addedhave been added

RecoveryRecovery reducedreducedbyby ~10 15%~10 15%

From: Analytical Method Validation and Instrument Performance Verification, Edited byFrom: Analytical Method Validation and Instrument Performance Verification, Edited byChung Chow Chan,Herman Lam, Y.C. Lee and Xue-Ming Zhang, ISBN 0-471-25953-5, WileyChung Chow Chan,Herman Lam, Y.C. Lee and Xue-Ming Zhang, ISBN 0-471-25953-5, Wiley& Sons& Sons

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When to expect Accuracy problemsWhen to expect Accuracy problems Insufficient selectivityInsufficient selectivity of the methodof the method

Impurity peaks are not resolved and account for assay valueImpurity peaks are not resolved and account for assay value RecoveryRecovery is < 100%is < 100%

Irreversible adsorption of analyte to surfaces of the systemIrreversible adsorption of analyte to surfaces of the system

IncorrectIncorrect assay value of aassay value of a reference standardreference standard Due to decomposition of reference standardDue to decomposition of reference standard

Incorrect assay value due toIncorrect assay value due to change in matrixchange in matrix Analytical laboratory still uses the preceding matrix as standardAnalytical laboratory still uses the preceding matrix as standard

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SpecificitySpecificity Is the ability to assess unequivocally the analyte in the presence of componentsIs the ability to assess unequivocally the analyte in the presence of components

which may be expected to be present (impurities, degradants, matrix)which may be expected to be present (impurities, degradants, matrix)

Identity testingIdentity testing To ensure the identity of an analyteTo ensure the identity of an analyte

Purity testingPurity testing To ensure accurate statement on the content of impurities of an analyteTo ensure accurate statement on the content of impurities of an analyte

AssayAssay To allow an accurate statement on the content of an analyte in a sampleTo allow an accurate statement on the content of an analyte in a sample

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Specificity:Specificity: Overlay chromatogram of an impurity solution with aOverlay chromatogram of an impurity solution with asample solutionsample solution


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Specificity andSpecificity and stabilitystabilityStress stability testing to ensure theStress stability testing to ensure the stability indicating potentialstability indicating potential of anof ananalytical methodanalytical method Apply diverse stress factors to the APIApply diverse stress factors to the API Apply diverse stress factors to the FPPApply diverse stress factors to the FPP

Stress conditions: e.g. Supplement 2 of Generic Guideline; TRSStress conditions: e.g. Supplement 2 of Generic Guideline; TRS929, Annex 5929, Annex 5

Assure that the API can be assessed specifically in the presence of knownAssure that the API can be assessed specifically in the presence of knownand unknown (generated by stress) impuritiesand unknown (generated by stress) impurities

Assure that known impurities/degradants can be specifically assessed in theAssure that known impurities/degradants can be specifically assessed in thepresence of further degradantspresence of further degradants

ByBy peak purity assessmentpeak purity assessment and (overlay of)and (overlay of) chromatogramschromatograms

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StressStress stability studies versusstability studies versus forced degradationforced degradation studiesstudiesStressStress

parameter parameter Forced degradationForced degradation Stress stabilityStress stability

(5 15% decomposition)(5 15% decomposition)

AcidAcid 0.2 ml0.2 ml 1N HCl1N HCl / 5 ml API-solution / 3h,/ 5 ml API-solution / 3h,6h, 12h, 24h7d (RT & 60C)6h, 12h, 24h7d (RT & 60C)

pHpH 2 (2 weeks) 2 (2 weeks)

BaseBase 0.2 ml0.2 ml 1N NaOH1N NaOH / 5 ml API-solution // 5 ml API-solution /3h, 6h, 12h, 24h7d (RT & 60C)3h, 6h, 12h, 24h7d (RT & 60C)

pHpH 10 (2 weeks) 10 (2 weeks)

HH22OO 22 / Oxygen/ Oxygen 0.2 ml 5% or 0.2 ml 5% or 35%35% HH22OO 22 / 5 ml API-/ 5 ml API-solution (RT, to 7d & 60C, 3h)solution (RT, to 7d & 60C, 3h)

1 g/ml oxygen bubbled through (8 hours)1 g/ml oxygen bubbled through (8 hours)0.1 2% H0.1 2% H 22OO 22 (24 hours)(24 hours)

HeatHeat 60C60C / 5 ml solution (3h, 6h7d)/ 5 ml solution (3h, 6h7d) --

HeatHeat 105 C105 C / solid API (1d and 7d)/ solid API (1d and 7d) 60C (4 weeks)60C (4 weeks)

UV or LightUV or Light 365 nm or white fluorescent light / solid365 nm or white fluorescent light / solidAPI (1d and 7d)API (1d and 7d)

HumidityHumidity -- 50C / 80% RH (4 weeks)50C / 80% RH (4 weeks)

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Limit of Detection (LOD, DL)Limit of Detection (LOD, DL) The LOD of an analytical procedure is the lowest amount of analyte in sampleThe LOD of an analytical procedure is the lowest amount of analyte in sample

which can bewhich can be detected but not necessarily quantitated as an exact valuedetected but not necessarily quantitated as an exact value

Determination is usually based onDetermination is usually based on

Signal to noise ratio (~3:1) (Signal to noise ratio (~3:1) ( baseline noisebaseline noise ))or or

Standard deviation of response (Standard deviation of response ( ) and Slope (S)) and Slope (S) 3.33.3 /S/S

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Limit of Quantitation (LOQ, QL)Limit of Quantitation (LOQ, QL) The LOQ is the lowest amount of analyte in a sample which can beThe LOQ is the lowest amount of analyte in a sample which can be

quantitativelyquantitatively determined with suitable precision and accuracydetermined with suitable precision and accuracy The quantitation limit is used particularly for the The quantitation limit is used particularly for the

determination of impurities and/or degradationdetermination of impurities and/or degradation

productsproducts

Determination is usually based onDetermination is usually based on Signal to noise ratio (~10:1) (Signal to noise ratio (~10:1) ( baseline noisebaseline noise ))or or

Standard deviation of response (Standard deviation of response ( ) and Slope (S)) and Slope (S) 1010 / / SS

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NoiseNoise

LODLODSignal toSignal to NoiseNoise = 3:= 3: 11

LOQLOQ

Signal toSignal to NoiseNoise = 10:= 10: 11

LOD, LOQ and Signal to Noise Ratio (SNRLOD, LOQ and Signal to Noise Ratio (SNR))

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LOQLOQ Quantitation by SNR is acceptedQuantitation by SNR is accepted Quantitation by Standard deviation of response (Quantitation by Standard deviation of response ( ) and Slope (S)) and Slope (S)

(10(10 / / S) is more adequate as it involves theS) is more adequate as it involves the responseresp onse of theof theactual analyteactual analyte

Best to calculate in the region close to y-interceptBest to calculate in the region close to y-intercept

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LOQ and impuritiesLOQ and impurities In determination of impurities in APIs and FPPs the LOQ should beIn determination of impurities in APIs and FPPs the LOQ should be

determineddetermined in the presence of APIin the p resence of API

LOQ should beLOQ should be NMT reporting levelNMT reporting level

LOQ should be givenLOQ should be given relativerelative to the test concentration of to the test concentration of APIAPI

Specificity of impurity determination should always be demonstratedSpecificity of impurity determination should always be demonstratedin the presence of APIin the presence of API at API specification levelsat API sp ecification levels

Spiking of test concentration (API/FPP) with impurities atSpiking of test concentration (API/FPP) with impurities atlevels of their specification rangelevels of their specification range

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SpikingSpiking API test concentration (normalised)API test concentration (normalised)

0.1 mg/ml (100%)0.1 mg/ml (100%) Impurity spiking concentrationsImpurity spiking concentrations

0.001 mg/ml (1%) specification limit0.001 mg/ml (1%) specification limit 0.0001 mg/ml (0.1%)0.0001 mg/ml (0.1%) limit of quantitation limit of quantitation(minimum requirement)(minimum requirement)

API at test concentrationsAPI at test concentrations

API below test concentrationsAPI below test concentrations

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LinearityLinearityof an analytical procedure is its ability (of an analytical procedure is its ability ( within a givenwithin a given rangerange ))to obtain testto obtain test resultsresults which are directlywhich are directly proportional to theproportional to theconcentrationconcentration (amount) of analyte in the sample(amount) of analyte in the sample

If there is aIf there is a linear linear relationshiprelationship test results should betest results should beevaluatedevaluated by appropriateby appropriate statistical methodsstatistical methods

Correlation coefficient (r)Correlation coefficient (r) Y-intercept Y-intercept Slope of regression lineSlope of regression line Residual sum of squaresResidual sum of squares PLOT OF THE DATAPLOT OF THE DATA

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Usual acceptance criteria for a linear calibrationUsual acceptance criteria for a linear calibrationcurvecurve r > 0.999r > 0.999 ;; y-intercept a < 0 to 5% of target concentrationy-intercept a < 0 to 5% of target concentration

RSD (wrt calibration curve) < 1.5-2%RSD (wrt calibration curve) < 1.5-2%

r > 0.997 r < 0.997

From: Analytical Method Validation and Instrument Performance Verification, Edited by Chung ChowFrom: Analytical Method Validation and Instrument Performance Verification, Edited by Chung ChowChan,Herman Lam, Y.C. Lee andChan,Herman Lam, Y.C. Lee andXue-Ming Zhang, ISBN 0-471-25953-5, Wiley & SonsXue-Ming Zhang, ISBN 0-471-25953-5, Wiley & Sons

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RangeRange TheThe rangerange of an analytical procedure is the intervalof an analytical procedure is the interval

between the upper and lower concentration (amounts) of between the upper and lower concentration (amounts) of

analyte in the sample for which it has been demonstratedanalyte in the sample for which it has been demonstratedthat the analytical procedure has athat the analytical procedure has a suitable level of suitable level of precision, accuracy and linearityprecision, accuracy and linearity

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RangeRange AssayAssay 80 to 120%80 to 120% of test concentrationof test concentration

Content uniformityContent uniformity 70 to 130%70 to 130% of test concentrationof test concentration

DissolutionDissolution Q-20% to 120%Q-20% to 120%

ImpuritiesImpurities Reporting level 120% of specification limit (with respect toReporting level 120% of specification limit (with respect to

test concentration of API)test concentration of API)

Assay & ImpuritiesAssay & Impurities Reporting level to 120% of assay specificationReporting level to 120% of assay specification

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Linearity is limited to 150%of shelf life specification of impuritiesLinearity is limited to 150%of shelf life specification of impurities

Test concentration can beTest concentration can beused to determine impuritiesused to determine impurities

To determine drug substanceTo determine drug substance(assay) the test concentration(assay) the test concentrationmust be dilutedmust be diluted

The range is 0 ~ 150% of The range is 0 ~ 150% of impurity specificationimpurity specification


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RobustnessRobustness Robustness of an analytical procedure should showRobustness of an analytical procedure should show

thethe reliabilityreliability of an analysisof an analysis with respect towith respect to deliberatedeliberatevariations in method parametersvariations in method parameters

The evaluation of robustness should be consideredThe evaluation of robustness should be consideredduring theduring the development phasedevelop ment phase

If measurements areIf measurements are susceptiblesusceptible to variations into variations inanalytical conditions theanalytical conditions the analytical conditions shouldanalytical conditions shouldbe suitably controlledbe suitably controlled or aor a precautionary statementprecautionary statement should be included in the procedureshould be included in the procedure

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Influence of buffer pH and buffer concentration in mobile phaseInfluence of buffer pH and buffer concentration in mobile phaseon retention times of API and impuritieson retention times of API and impurities

Conclusion: The buffer composition should be maintained in a range of Conclusion: The buffer composition should be maintained in a range of

8585 0.5% 0.5% Missing:Missing: Acceptance criterion for maximal deviation of retention time should beAcceptance criterion for maximal deviation of retention time should bedefined unless justifieddefined unless justified

APIAPI Impurity AImpurity A Impurity BImpurity B Impurity CImpurity C

As isAs is 10.4610.46 3.863.86 7.437.43 8.268.26

buffer pH 5.9buffer pH 5.9 10.4510.45 3.943.94 7.517.51 8.388.38

buffer pH 6.9buffer pH 6.9 10.4610.46 3.943.94 7.497.49 8.348.34Buffer conc. 83%Buffer conc. 83% 7.847.84 3.433.43 6.166.16 6.666.66

Buffer conc. 87%Buffer conc. 87% 15.2615.26 4.774.77 9.619.61 11.1811.18

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System suitability testingSystem suitability testing Based on the concept that equipment, electronics,Based on the concept that equipment, electronics,

analytical operations and samples to be analysedanalytical operations and samples to be analysedconstitute an integral systemconstitute an integral system that can be evaluated asthat can be evaluated assuchsuch

Suitability parameters are established for each analyticalSuitability parameters are established for each analyticalprocedureprocedure individuallyindividually

Depend on theDepend on the type of analytical proceduretype of analytical procedure

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MethodMethod stabilitystability System suitabilitySystem suitability over timeover time

Sample solution stabilitySample solution stability A solution of stavudine is stable for ~ 2 h, then it starts to degrade toA solution of stavudine is stable for ~ 2 h, then it starts to degrade to

thyminethymine

Impurity-spiked sample solution stabilityImpurity-spiked sample solution stabilityA solution containing stavudine spiked with its impurityA solution containing stavudine spiked with its impuritythyminethymine does not allow to clearly distinguish betweendoes not allow to clearly distinguish betweendegradation and spikedegradation and spikeA solution containing stavudine of a FPP-stability sampleA solution containing stavudine of a FPP-stability sample

solution does not allow to clearly distinguish between FPP-solution does not allow to clearly distinguish between FPP-stability degradation and sample solution degradationstability degradation and sample solution degradationShould be analysed immediatelyShould be analysed immediately

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When to be surprised about validation data:When to be surprised about validation data: Precision of Precision of

impurity determinationimpurity determination

Precision of Precision of API determinationAPI determination

Method precision of Method precision of released API (dissolution)released API (dissolution)

System precisionSystem precision % RSD 0.33 2.25% RSD 0.33 2.25

Method precisionMethod precision % RSD 0.0% RSD 0.0

Average peak areaAverage peak area % RSD 0.08% RSD 0.08Acceptance criterionAcceptance criterion % RSD 2.0% RSD 2.0

Average peak areaAverage peak area % RSD 0.4% RSD 0.4

Acceptance criterionAcceptance criterion % RSD 10.0% RSD 10.0

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Specification range (USL-LSL)Specification range (USL-LSL) Process variability (usuallyProcess variability (usually 2 SD) 2 SD) Analytical variability (Analytical variability ( 3 3 ))

~ NMT 30% of total specification range~ NMT 30% of total specification range

Analytical variabilityAnalytical variability Process variabilityProcess variability Reliability of evaluation of major process variablesReliability of evaluation of major process variables by analyticalby analytical

proceduresprocedures depends ondepends on analytical variabilityanalyt ical variability

ImpuritiesImpurities

LOQLOQ and specification limit (e.g. qualification limits NMTand specification limit (e.g. qualification limits NMT0.15%)0.15%) Response factorsResponse factors (LOQ modified by response factor)(LOQ modified by response factor)

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Methods for cleaning validationMethods for cleaning validation Method for assay and related substances used in stability studies of API andMethod for assay and related substances used in stability studies of API andFPPFPP

SpecificitySpecificity (in(in samples taken from a cleaning assessmentsamples taken from a cleaning assessment )) LLinearity of responseinearity of response (from(from 50% of the cleaning limit to50% of the cleaning limit to 11 0x this0x this

concentrationconcentration ; R; R 22 0.9900)0.9900) PrecisionPrecision

RepeatabilityRepeatability (RSD(RSD 5%)5%) iinternter mediate precision [ruggedness (USP)]mediate precision [ruggedness (USP)] ReproducibilityReproducibility

LLimits of detection and quantitationimits of detection and quantitation

AAccuracy or recovery from rinsateccuracy or recovery from rinsate (( 80%)80%) , swabs, swabs (( 90%)90%) , and, andprocess surfaceprocess surface (( 70%)70%) RRangeange (( lowest level is at least 2x higher thanlowest level is at least 2x higher than LOQLOQ))

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SummarySummaryAnalytical procedures play a critical role in pharmaceuticalAnalytical procedures play a critical role in pharmaceuticalequivalence and risk assessment/managementequivalence and risk assessment/management

Establishment of Establishment of product-specificproduct-specific acceptance criteriaacceptance criteria

Assessment of Assessment of stabilitystability of APIs and FPPsof APIs and FPPs

Validation of analytical procedures should demonstrate thatValidation of analytical procedures should demonstrate thatthey arethey are suitable for their intended usesuitable for their intended use

Validation of analytical procedures deservesValidation of analytical procedures deserves special attentionspecial attentionduring assessmentduring assessment of dossiersof dossiers for prequalificationfor prequalification

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analy meth dev 06.12.09

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