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Testproceduresforassessmentofthequalityof pharmaceuticalproductssubjecttovarious requirements Usersofanalyticalmethodsdescribedinthe pharmacopeiasUSP/NF,IP,Phar.Eur.,arenot requiredtovalidateaccuracyandreliabilityof thesemethods,butmerelyverifytheirsuitability underactualconditionsofuse itisessentialthatproposalsforadoptionofnewor revisedcompendialanalyticalmethodoralternate inhousemethodsbeshownequivalentto,orbetter 2 than,thecurrentmethod
AspectsofAMVanditstestingquorum
VerificationofCompendialMethods
Acompendialprocedureisconsideredvalidatedifitis publishedasofficialtextinapharmacopeiaoran interimannouncement(addendums) Whenusingcompendialmethods,thefullvalidation isnotnecessary,butverificationoftheprocedureis veryimportant Verificationensuresthattheprocedureissuitablefor usewithaspecificingredientorproduct,inaspecific laboratory,withspecificlaboratorypersonnel, equipment,andreagents
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Examples...
titrimetricmethodsforwaterdeterminationshouldbe verifiedforaccuracy(andabsenceofpossible interference)whenusedforanewproductorraw material Forimpuritytesting,thesuitabilityofacompendial proceduremaybeanissueforseveralreasons(e.g., impurityprofilechangefromdifferentroutesof synthesis,compositionofformulation,orinterference fromexcipients). Itisrecommendedthattheprocedureforcertification ofsuitabilityofthemonographsofthepharmacopeia beused
CharacterizationofReference Standard
Duringmethodvalidation,awellcharacterized standardshouldbeused wellcharacterizedreferencestandardisacritical factorformethodvalidation Forpotencyassay,thepurityofthestandardmustbe assigned thereferencestandard(usedasprimarystandard) shouldalwaysbeacquiredfromarecognizedauthority, suchastheNationalInstituteforStandardand Technology(NIST),USP,EP,etc.
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StabilityIndicatingMethod
requiredpertheICHguidelines Tochecktheprocedureisabletodetectthechanges withtimeinthepertinentproperties(e.g.,active ingredient,preservativelevel)ofthedrugsubstance anddrugproduct,andshouldaccuratelymeasurethe activeingredientswithoutinterferencefrom degradationproducts,processimpurities,excipientsor otherpotentialsystemcomponents. comprehensiveforceddegradationstudyandHPLC coelutionevaluationconducted,inorderto demonstratethesuitabilityoftheproceduretodetect 11 anychangesthatareattributabletodegradation.
ForcedDegradationStudies(Stress Studies)
maintoolusedtopredictstabilityissues,develop analyticalmethods,andidentifydegradationproducts orpathways performedpriortotheothervalidationparameters (e.g.,accuracy,repeatability,intermediateprecision, specificity,DL,QL,linearityandrange,solution stability,androbustness) multiplestrengthsofdrugproductswiththesame excipientcomposition(includingdifferentformulation ratios),forceddegradationstudiescanbeperformed withonlyoneformulation 12
Formultiplestrengthsoraformulationofdrugproduct withdifferentexcipients,eachdifferentformulation compositionshouldbeevaluatedusingforced degradationstudies. ForINDphase1andINDphase2applications,the forceddegradationstudiesforthestabilityindicating natureoftheassaymethodismethodspecific, therefore,iftheassaymethodsfordrugsubstanceand drugproducthavedifferentconditionswhichcan causechangesinselectivity
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moreasampleisdegraded,themorelikelya lossinmassbalancewillbeobserveddueto secondarydegradation,lossofimpuritiesinthe solventfront,andlossofabsorptionduetoring openingorotherdegradationpathways Forceddegradationstudiesfordrugproduct shouldbeperformedbeforecommencing stabilitystudiesofregistrationbatches Duringdataacquisition,DADshouldbeused forHPLCandallspectraofpeaksshouldbe
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MethodValidationParameters
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Dataelementsrequiredfor analyticalmethodvalidation
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SpecialconsiderationsforAMVifdoneby chromatographicmethods...
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FilterBias
theanalysisistypicallycarriedoutbyspectroscopy(UVVis) utilizingachromatographicmethod(HPLC,GC) Filterorcentrifugeisutilizedtoremoveparticulatesthatmay clogthecolumnoraffectabsorbancereadings differenttypesofsyringefilters,suchasnylonorPTFEwitha sizeof0.45or0.2m,shouldbeinvestigateddependenton thesamples. Thefiltershouldbevalidatedbyfilteringaportionofworking standardsolutionthrougheachsyringefilter,discardingthefirst 23mL,andcollectingthefiltrateforanalysis. resultfromthefilteredsolutionshouldbecomparabletothatof unfilteredsolution. 20
SystemSuitability
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HPLCSystemSuitabilityParameters
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Injectionrepeatability
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acceptancecriteriaareasfollows,theRSDfor peakareaofinterestfromfiveorsixinjections ofworkingstandardsolutionshouldbe2.0%for potencyassay,10%forimpuritytestingand residualcleaningtesting,and3.0%dissolution testing. IftheproducthaslowstrengthorS/Nofthe activepeakislessthan50,theRSDofthe peakareaoftheactivefromthesixconsecutive injectionsof3%maybeacceptableforpotency 24 assay
Checkstandard
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Tailingfactor(T)
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Theoreticalplatenumber(N)
Where t is the retention time of active peak and w is the peak width of the peak, obtained by extrapolating the relatively straight sides of the peak to the baseline. Appropriate requirements for this parameter should be derived from validation data
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Systemdrift
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percentagerecoveryofsystemdriftinjectionthroughout therunshouldbewithin98102%. Ifthesystemdriftmeetsthisrequirement,theaverage peakresponsefromthefirstconsecutiveinjectionscanbe usedforthecalculationofthesamples,otherwisea bracketingprocedureshouldbeusedforcalculationofthe samples. Inadditiontothepeakarea,theretentiontimeshouldalso beevaluated. Foridentificationtheretentiontimesshouldnotvaryby morethan2%.
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Resolution(R)
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ExplanationsforDataelementsrequiredfor analyticalmethodvalidation...
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Accuracy
determinedbyspikingknownamountsofthe activeatsuitablelevelsofthelabelclaimed amounttothecorrespondingplacebopowder (oranamountofplacebomixturecontainingall theingredientsfortheformulationexcept active),andthencalculatingthepercent recoveryoftheactive Forphase3accuracyexperiments,triplicate samplepreparationsarerequiredateach spikinglevel,andaminimumofthreelevels shouldbeassessed.
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Percentagerecoveryiscalculatedbytheassayed amountdividedbyaknownamountofanalytespiked inthesample Percentagerecovery,averagerecoveryfromeach levelandoveralllevels,andconfidenceintervals shouldbeevaluated potencyassays,RSDofrecoveriesforeachspiked levelNMT2.0%. Forlowstrengthofdrugproducts,e.g.,1mg,wider ranges(3.0%forpotency,5.0%fordissolution)maybe 36 applied.
Accuracyandacceptancecriteria
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Precision
Intermediateprecision
measureofthemethodssensitivitytominor changesinequipmentperformance,and/orto variationintheoperatorstechniqueonany givenday secondanalystshouldperformtheassay,using differentequipment,andonadifferentdayto confirmthatacceptableresultscanbeobtained Absolutedifferencebetweenthemean percentagelabelclaimsoftheactivegenerated 39 bythetwoanalystsshouldbe<3.0%
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Reproducibility
absolutedifferencebetweenthemean percentagelabelclaimsoftheactivegenerated bythetwoanalystsshouldbe<3.0% exactcriteriaisbasedonthetypeoftest,and theultimatespecification e.g.,drugsubstanceassayspecificationis98 102%,thenthedifferencebetweenlaboratories shouldbe1.5% butforadrugproductwithaspecificationof 90110%awidercriteriacouldbeused
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Linearity
Alinearrelationshipbetweentheconcentration andrespectiveresponsecanbeobtainedby analyzingaseriesofstandardsolutions Atleastfivestandardsolutionswithaspecific rangeshouldbepreparedOneinjectionof eachofthelinearitystandardsolutions sufficient Thepeakareaoftheactivewillbemeasuredat differentconcentrationlevels,andplotted 44 againstthecorrespondingconcentrations
Therangeintheregressionline
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Specificity
toconfirmthatananalyticalprocedureisspecificfor theanalyteofinterestinthepresenceofcomponents suchasimpurities,degradants,andmatrix components(excipients) HPLCspecificitydemonstratedbyseparationof criticalpairsofthetwocomponents(theactiveand impurityortwoimpurities)thateluteclosesttoeach other bymakingindividualinjectionofdiluent,eachimpurity, theactive,andtheplacebo(oranalyticalprepared placebo) 49
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StabilityofStandardandSample Solutions
potency assay, the standard & sample solutions considered to be stable if the % difference between initial values of standard and samples, and those at specific times, is NMT2.0%,butanydownwardtrendinthedata shouldalsobeevaluatedforpossibleimpacton theanalysis.
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impurityassay,thesamplesolutionconsidered stableifthefollowingconditionsaremet:
DetectionLimit(DL),and QuantitationLimit(QL)
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Visualevaluation
Signaltonoiseratioapproach
signaltonoiseratioofpeakofanalyteofinterestin thesampleshouldbeatleast3:1fromDLsolution, and10:1fromtheQLsolution Chromatographictechniques,thesignalofthepeak andthebaselinenoisecanbemeasuredmanuallyor usingbuiltinsoftware. ThedetectionlimitandQLofanalytemaybe determinedbyserialdilutionofastandardsolution withdiluent,andinjectingontotheHPLCsystemfor assay.ThenQLandDLwillbedeterminedbysignalto noiseratio. 58
Standarddeviationoftheresponse andslope
is the standard deviation of the response; S is the slope of the calibration curve. may be estimated based on standard deviation of blank (measurement of the magnitude of analytical background response using six replicate blank samples) or residual standard deviation of regression line or the standard deviation of Y(gamma) -intercepts.
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Robustness
RobustnessExperimentaldesignfor chromatographicparameters
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Thedesignofexperiment(DOE)couldalsobe usedtoallowmultipleparameterstobevaried ineachexperiment,andthusreducethe numberofexperiments. Theinstrumentsystemmustbeequilibrated undereachtargetandrobustnesscondition. Thesystemsuitabilityrequirementsshouldbe evaluatedineachexperimenttoensurethe appropriatesystemsuitabilitycriteriaaresetfor 64 themethod.
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Samplepreparationforpotencyassay
NonchromatographicMethodValidation
AdditionalQCDrugsubstances,excipients,and/or drugproductsparticlesizedistribution,opticalrotation methodologiessuchasDSC,PXRD,Raman spectroscopy,andnearinfraredspectroscopyshould bevalidatedpriortouse validationparameterslessextensivethan chromatographicmethods includerepeatability,intermediateprecision,and robustness,butspecificityandaccuracymayalsobe applicable
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FailureandRevalidation
Incaseofafailureofcomparisonduring repeatabilityandintermediateprecision,all aspectsshouldbetakenintoconsideration(i.e. chemistskills,labequipmentvariation,and samplevariation) Foranyfailuresduringvalidationexperiments,a thoroughevaluationisrequiredtoensurethat thefailureistrulyduetothemethod,andnot duetolaboratoryerrororotherunexpected issuessuchassamplehomogeneity 70
Theneedtorevalidatethemethodwillbe evaluatediftherearechanges,suchascolumn vendor,drugsubstancesrouteofsynthesis,and drugproductscomposition Somechangesmaynotrequirerevalidationor mayonlyrequirepartialrevalidation(e.g.,new excipientswouldrequirespecificexperiments), buttheevaluationshouldalwaysbemade,and thejustificationfornotrevalidatingshouldbe documented. 71