AAPS J 2015 17(2)277-88

TheAAPSJournal,Vol.17,No.2,March2015(#2014)DOI:10.1208/s12248-014-9696-2

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WorkshopReport:CrystalCityV—QuantitativeBioanalyticalMethodValidationandImplementation:The2013RevisedFDAGuidance

BrianBooth,1MarkE.Arnold,2,12BinodhDeSilva,2LakshmiAmaravadi,3SherriDudal,4EricFluhler,5BorisGorovits,6SamH.Haidar,1JohnKadavil,1SteveLowes,7RobertNicholson,8MarieRock,9MichaelSkelly,1LaurenStevenson,3SriramSubramaniam,1RussellWeiner,10andEricWoolf11Received26September2014;accepted31October2014;publishedonline31December2014

Abstract.InSeptember2013,theFDAreleasedadraftrevisionoftheBioanalyticalMethodValidation(BMV)Guidance,whichincludedanumberofchangestotheexpectationsforbioanalysis,mostnotablytheinclusionofbiomarkerassaysanddata.Toprovideaforumforanopen,inclusivediscussionofthereviseddraftBMVGuidance,theAAPSandFDAonceagaincollaboratedtoconveneatwo-and-a-halfdayworkshopduringearlyDecember2013inBaltimore,MD,USA.Theresultingformatembodiedextensiveopendiscussionandeachthematicsessionincludedonlybrief,concisedescriptionsbyAgencyandindustryrepresentativespriortoopeningthe?oordiscussion.TheWorkshopwasbuiltaroundfourthematicsessions(CommonTopics,Chromatographic,Ligand-BindingAssays,andBiomarkers)anda?nalsessionwithinternationalregulators,concludingwithareviewoftheoutcomesandrecommendationsfromthethematicsessions.ThisWorkshopreportsummarizestheoutcomesandincludestopicsofagreement,thosewheretheFDAwillconsidertheIndustry’sperspective,andthosewheretheworkshopprovideda?rstopendialogue.Thisarticlewillbeavailabletothebioanalyticalcommunityathttp://www.aaps.org/BMV13.KEYWORDS:bioanalyticalmethodvalidation;CrystalCityV;FDAguidance.

INTRODUCTION

Thequantitativemeasurementsofdrugs,metabolites,andbiomarkersinnonclinicalandclinicalstudiesprovideessentialinformationintheassessmentofsafetyandef?cacyofdrugs.Drug

12U.S.FoodandDrugAdministration,SilverSpring,MD,USA.Bristol-MyersSquibbCo.,Princeton,NJ,USA.3BiogenIdec,Cambridge,MA,USA.4RocheInnovationCenter,Basel,Switzerland.5P?zerInc.,PearlRiver,NY,USA.6P?zerInc.,Andover,MA,USA.7QuintilesBioanalyticalandADMELabs,Ithaca,NY,USA.8PPD,Richmond,VA,USA.9WILResearch,Skokie,IL,USA.10MerckResearchLaboratories,Rahway,NJ,USA.11MerckResearchLaboratories,WestPoint,PA,USA.12Towhomcorrespondenceshouldbeaddressed.(e-mail:mark.arnold@bms.com)ABBREVIATIONS:A&P,AccuracyandPrecision;ADC,Antibody-DrugConjugate;BQL,BelowQuantitationLimit,

DEFINITIONS:UnconjugatedDrug,drugspontaneouslyreleasedinvivofromanADC;Antibody-ConjugatedDrug,drugconjugatedtotheantibodymoiety;TotalAntibody,AntibodywithDARequalorgreaterthan0.Includesconjugatedandfullyunconjugatedantibody;MockClinicalSample,Samplespreparedbypoolingsamplesorspikingoverendogenouslevels.UsedinthecontextofdemonstratingbiomarkerstabilityonstorageandmeasuredperiodicallywithaDx.

orbiomarkerconcentrationsfrequentlyserveastheprimaryorsecondaryendpointsofmanyclinicalstudiesindrugdevelopment.Consequently,thereliabilityorqualityofthatdataunderpinsthestudyoutcome.Forexample,inbioequivalencestudies,thepharmacokinetic(PK)comparisonisthebasisforapproval,thusthequalityoftheconcentrationdataisessential.Similarly,asbiomarkersandtheresultingpharmacodynamic(PD)interpreta-tionsareexploitedmoreextensivelyinestablishingef?cacyandlabelingclaims,thequalityofthesedeterminationswillrequiregreaterdemonstrationoftheassayqualityandusage.Inallcases,thesePKorPDmeasurementsarebasedonestablishedprinciplesandscientistscanutilizeacommon,vettedparadigmofpractices,independentoftheanalyticalplatformtodemonstratethattheassaysprovidereliabledata.

Theevolutionofthisbioanalyticalparadigmbeganwiththe?rstAmericanAssociationofPharmaceuticalScientists(AAPS)/FoodandDrugAdministration(FDA)BioanalyticalWorkshopin1990(1).Scientistsinthebioanalytical?eldworkedwiththeregulatorycommunitytoestablishacommonlanguageandexpectationsingeneratingpharmacokineticdatafordrugsandmetabolites.ThesevalidationprincipalswereintroducedintoregulationsbyHealthCanadain1992(2)andthenbytheFDAwhichpublishedthe?rsteditionofitsGuidanceonBioanalyticalMethodValidation(BMV)(3)in2001.Sincethen,thedialoguehasbroadenedsigni?cantlythroughscienti?cconferencesnotonlywithintheUSA,butglobally.ThelastdecadehasseentheadditionalintroductionofBMVregulationinBrazil(4),theEU(5),andJapan(6),with

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othercountriesconsideringnewregulationorapplyingexistingregulationsfromotherregions.InSeptember2013,theFDAreleasedadraftrevision(7)oftheBMVguidance,andthediscussioncontinued.Thecurrentdraftdocumentincludedanumberofchangestotheexpectationsforbioanalysis,mostnotablytheinclusionofbiomarkerassaysanddata.

Toprovideaforumforanopen,inclusivediscussionofthereviseddraftBMVGuidance,theAAPSandFDAonceagaincollaboratedtoconveneatwo-and-a-halfdayworkshopduringearlyDecember2013inBaltimore,MD,USA.AlthoughnotheldinArlington,VA,CrystalCityV(CCV)builtuponitshistoricprecedentstofacilitateanopendialoguebetweentheindustryandAgency.Anobjectivesetforthearlyintheplanningwastheneedformoreextensiveopencommentsessions.Theresultingformatembodiedthatconceptandeachthematicsessionincludedonlybrief,concisedescriptionsbyAgencyandindustryrepresentativespriortoopeningthe?oordiscussion.Thoseintroductorypresentationsfocusedonwhathadchangedandwhy,andithighlightedareasofindustryconcerntobediscussedintheopensessions.EachopensessionwasmoderatedbyAgencyandindustryrepresentatives,includedapanelofsubjectmatterexperts(SME)fromtheFDAandindustry,andutilizedadditionalSMEsfromtheFDAandindustrytoroamamongtheaudienceandstimulatethediscussion.

TheWorkshopwasbuiltaroundfourthematicsessions(CommonTopics,Chromatographic,LigandBindingAssays,andBiomarkers)anda?nalsessionwithinternationalregulators,concludingwithareviewoftheoutcomesandrecommendationsfromthethematicsessions.Allofthesesessionshighlightedthechangesinthepharmaceuticalindustry,inparticularthegrowthofbiotechnology-basedtherapies.Over450scientistsattendedtheWorkshop,with25%ofparticipantscomingfrom18countriesoutsidetheUSA.Inmanycases,theattendeesrepresentednotonlytheirowncompaniesbutalsothenumerousregionalandglobalbioanalyticalconsortia(e.g.,GlobalBioanalysisConsortium,InternationalConsortiumonInnovation&QualityinPharmaceu-ticalDevelopment,EuropeanBioanalysisForum,JapaneseBioanalysisForum,AppliedPharmaceuticalAnalysis—India,AcBio,CanadianForumforAnalyticalandBioanalyticalSciences,ChineseBioanalysisForum,GlobalCROCouncil),thusbringingtheconcerns,thoughts,andinterestofanevenlargerbioanalyticalcommunityintothediscussion.

AswithpreviousCrystalCitymeetings,partoftheobjectivewastodiscusschangesinregulatorythinkingandthescienceappliedtonewdrugdevelopmentthathadoccurredsincethelastWorkshop.ThisWorkshopincludeddiscussionsonsuchscience-driventopicssuchasthestatusofincurredsamplereanalysis(ISR)afterseveralyearsofapplication,newimmu-noassaytechnology,antibody-drugconjugates(ADCs),andtheapplicationofLC-MS/MSforproteinquantitation.Additional-ly,theAgencythroughtheguidanceanditspresentationshighlightedareasofadditionalfocusforchromatographicandligand-bindingassays,suchasstocksolutionexpiryandmostnotably,theinclusionofbiomarkerassayswithintheGuidance.Extensivediscussions,frommultiplepointsofviewweregeneratedaroundthetopicspresented.

ThesalientoutcomesofCCVcanbebrokendownintotwomajorcategories:topicsforwhichtherewasgeneralconsensusbetweenindustryandFDAbasedontheclari?ca-tionsanddiscussionswhichtookplaceandtopicsforwhichnoconsensuswasachievedbetweenindustryandtheAgency.

Boothetal.

Theissuesforwhichnoconsensuswasreachedwerecharacterizedbytwosituations:topicsonwhichviewpointsdifferedandtopicsinwhichthescienceisstilltoonew.Incaseswhereviewpointsdiffered,theAgencyagreedtoconsiderindustrypositionswithintheirdeliberationsleadingtothe?nalguidance.Inthelattercase,theAgencysoughtthecurrentthinkingandexperienceofindustryscientists,whichwillhelpguidefuturerecommendationsintheseareas.ThefollowingprovidesasummaryofallofthediscussionswhichtookplaceatCCVdelineatedbythesecategories.

CHROMATOGRAPHICASSAYS:CONSENSUSTOPICThebasicexpectationsforBMVofchromatographicassayshavebeenpreviouslyde?nedinthe2001FDAguidance(3)andsubsequentconferencereportsonthetopic(8,9).METHODVALIDATION

Severalaspectsofcrossandpartialvalidationreceivedextensivediscussion.Itwasnotpossibletofurtherre?nethecriteriaforeithertypeofvalidationduetothebreadthoftheissue.However,furtherclari?cationwasaddedtothefollowingissues:PartialValidation

Theneedtovalidateanewanalystbroughtintoastudywasdiscussed.Theconsensusthatwasreachedisthatnewanalystsareexpectedtobetrained/quali?edonrelevanttechniquespriortosupportingstudies.Thereisnoexpecta-tionthattrainingorquali?cationdatashouldbeincludedinavalidationreport,althoughitmustberetainedintrainingorstudy?les.Performingasingletestrunthatpassesacceptancecriteriawassuggestedasanexampleforanewanalyst.

Regardingtheneedtopartiallyvalidatechangesofananticoagulantcounter-ion,theconsensusviewwasthatvalidationisnotrequiredunlessaneffectisobservedduringthemethoddevelopment.CrossValidation

Duetovariedexperiencesandthewiderangeofscenarios,nofurtherdetailregardingacceptancecriteriawereelucidated,butitwasagreedthatcriteriashouldbede?nedina“standardoperatingprocedure”(SOP)orvalidationplandocumentapriori.Furthermore,itwasrecommendedthatcrossvalidationanalysesshouldbeperformedinbothlaboratories(orbetweenmethods),usingsharedspikedmatrixqualitycontrol(QC)samplesandnon-pooledincurredsubjectsamples.Pooledincurredsamplescanbeusedwheninsuf?cientvolumeexists.ReferenceStandards

ConsiderablediscussionofSectionIIIAofthedraftGuidance(7)centeredonuseofreferencestandardsinchromatographicmethods.Thesectionclari?estypesofreferencestandardsintermsoftheirsourceandcontinuestodesignateexpectationsaroundsupportingdocumentationincludingexpirationdate,certi?catesofanalysis(COAs)andevidenceofpurity.Theextensionofreferencestandarddocumentationtoincludeinternalstandards(IS)wasalso

The2013CrystalCityVConferenceReport

presented.TherewasagooddiscussionattheWorkshopontheproposeddocumentationfortheinternalstandardmaterial.IndustryrepresentativesnotedthatitisnotalwayspossibletoobtainaformalCoAforstablelabel(SLIS)oranaloginternalstandardsduetothelimitedamountofmaterialsynthesized.ItwasalsosuggestedthatpurityandstabilityoftheISisgenerallynotcriticalfortypicalchromatographicassays(e.g.,LC-MS/MS)aslongasthesuitabilitycanbedemonstrated(e.g.,nointerfer-encefromtheIStotheanalyte(s)ofinterest).Inresponse,theAgencyclari?edthatCoAsfortheISarepreferred,butotherdocumentationcharacterizingthepurityisacceptablewhenaformalCoAisnotavailable.Inanycase,thesuitabilityofthematerialforitsintendeduseshouldbedemonstratedpriortoand/orduringuse.ThisextendedtoanagreementthatthestabilityoftheISneednotbedemonstratedaslongassuitability(lackofinterference)isdemonstrated.MatrixEffectsandSelectivity

AtthecurrentWorkshop,clari?cationwassoughtonthetypesofmatrixeffectsthatshouldbestudiedduringvalidation.Thegoalofthematrixeffectassessmentistodemonstrateconsistentassayperformanceacrossmultiplesourcesofmatrixthatre?ecttheanticipatedpopulationfromwhichsamplesareexpected.Variationsoflipidandspeci?cortotalproteinconcentrationswhichmayresultfromthediseaseindicationshouldbeaddressed.Theimpactofhemolyzedandlipemicsamplesmaybeassessedduringmethoddevelopment;aspeci?cassessmentofthesefactorsisnotexpectedduringmostvalidations,unlesstheassayisintendedtosupportstudieswhereahighpercentageofsamplesmaybehemolyzedorarefromhyperlipemicsubjects.

Whilevalidationexperimentsshouldassessionsuppression/enhancement(matrixeffect),calculationofma-trixfactor(MF)isnotanexpectedcomponentofvalidation,althoughitisunderstoodthatproperinvestigationofionsuppression/enhancementprovidesdatanecessarytomaketheMFcalculationifapplicabletotheinvestigation.MFscanbecalculatedforboththeanalyteandinternalstandardinagivenmatrix.Ideally,thevariationinMFsfortheanalyteiscompensatedfor,atleasttosomedegree,bythevariationoftheMFoftheinternalstandard.Stablelabelinternalstandardstypicallyprovidethebestdegreeofcompensation,hencetheyarerecommendedforMS-basedassays.

Selectivityistheabilityofananalyticalmethodtodifferentiateandquantifytheanalyteofinterestinthepresenceofmatrixcomponentsinthesample.Assayselec-tivityassessmentisanimportantcomponentofvalidation.Theconsensusattheworkshopwasthatselectivityshouldbeassessedinsixindividuallotsofmatrix.Theselotsmayincludehemolyzedorhyperlipemicsamplesasdeemednecessary.Generally,genderassessmentsofselectivityarenotexpected,butconsiderationshouldbegiventoanyuniqueattributesoftheanalytethatcouldbeimpactedbygender.Ifitisanticipatedthatsamplesfromspecializedstudypopulations(e.g.,renallyimpaired)maybeanalyzedwithanassay,considerationshouldbegiventoassessingselectivityinblankmatrixfromthetargetpopulationorcontrolsamplesobtainedfromsuchsubjects.

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ValidationRunAcceptance

FeedbackonthedraftGuidanceindicatedalevelofconfusionaroundwhatisproposedregardingtheanticipatednumberofvalidationrunsthatencompassabioanalyticalmethodvalidation.Thiswasre?ectedintheWorkshopdiscussionswithspeci?cfocusonthereferencetothetotalnumberofruns(“minimumofsixrunsconductedoverseveraldays”)andthenumberofrunsrequiredtodemonstratewithin-runandbetween-runaccuracyandprecision(A&P).Aneedemergedduringthediscussiontoaddressafundamentalconcernthatscientists,auditors,andinspectorsshare.Thatis,thepotentialof“cherry-picking”onlyvalidationrunsthatmeetaprioriacceptancecriteriadoesnotprovideanaccuraterepresentationofmethodperformance.Fromthediscussion,aconsensuswasderivedtoaddressabetterestimateoftheA&Pofabioanalyticalmethodduringvalidationthancurrentlyemployed.

ItwasproposedthatmethodA&PshouldbedeterminedfromQCperformanceindependentofaprioricriteria.Toaccommodatethis,runacceptanceduringA&Pvalidationrunsshouldbebasedoncalibrationcurveperformanceonly.Backcalculatedcalibratorconcentrationsshouldmeetaccep-tancecriteria(75%ofcalibratorswithin±15%ofnominal,exceptatthelowerlimitofquantitationoftheassay(LLOQ)wheretheyshouldbewithin±20%)topassA&Pvalidationrunsonly.Usingaminimumofthreeruns,theA&PstatisticsshouldthenbecalculatedfromtheQCsusingatleast?vereplicatesinacceptedrunsatfourconcentrationsincludingtheLLOQ,aswellaslow(LQC),middle(MQC),andhigh(HQC)concentrations,typicallycorrespondingtothoseusedforrunacceptanceQCs.Aspeci?cassessmentofA&Pattheupperlimitofquantitationoftheassay(ULOQ)isnotrequiredforchromatographicmethods.Furthermore,atleastoneA&Prunshouldusecalibratorsandvalidationsamplespreparedfromindependentstocksolutions.Forallothervalidationruns,bothcalibratorsandsamplesmaybepre-paredusingasingleveri?edstocksolution.TheA&PresultsfortheQCsinallpassingrunsshouldbesubsequentlyreported.Toreiterate,thisapproachavoidsincludingQCcriteriainrunacceptanceinA&PvalidationrunsanddelineatesA&PperformanceasthetotalityofQCperfor-manceisdistinctfromthecalibrationcurveperformancethatdeterminewhetherarunpassesorfails.

Forothervalidationruns(i.e.,thosenotassociatedwithA&Pandtypicallywithstabilityassessments),runacceptanceshouldbebasedoncalibrationstandards(asde?nedabove)andtheperformanceofrunacceptanceQCs(i.e.,analyticalQCs)consistentwithcurrentregulatoryguidance.Speci?cal-ly,atleastthreeconcentrationsofQCsinatleastduplicateshouldbeincorporatedintoeachrun(LQC,MQC,andHQC).Fortheruntopass,75%ofcalibratorsshouldbewithin±15%ofnominal,exceptattheLLOQwheretheyshouldbewithin±20%andatleast67%(e.g.,atleastfouroutofsix)ofallQCsshouldfallwithin±15%oftheirrespectivenominalconcentra-tionsandatleast50%oftheQCsateachlevelshouldpass.Recovery

Basedonthedescriptionprovidedinthedraftguidance,aclari?cationwassoughtontheappropriateapproachfordeterminingrecoveryduringvalidation.Theconsensusviewwasthatrecovery,asappliedtochromatography-based

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bioanalyticalmethods,shouldbede?nedasacomparisonoftheinstrumentalresponseassociatedwithextractedsamplescomparedtothatofextractsofcontrolmatrixspikedwithanalytepostextraction.Theresponseofthesamplesspikedpostextractionrepresents100%recovery.Recoveryoftheanalyteneednotbe100%,buttheextentofrecoveryofananalyteandoftheinternalstandardshouldbeconsistentandreproducible.Recoveryexperimentsshouldbeperformedbycomparingtheanalyticalresultsforextractedsamplesatthreeconcentrations(low,medium,andhigh)withthoseofcorrespondingspikedcontrolextracts.MethodUse

Anumberoftopicswerediscussedrelatedtotheapplica-tionofvalidatedmethodsduringstudysampleanalysis.Forexample,theAgencyclari?editsconcernthatequipmentconditioningandsystemsuitabilitytestingwithcalibrators,QCs,andstudysamplesfromapendingruncouldriskbiasingthedecisionsonacceptanceandrejectionofthependingrun,orofthestudysampleresults.Therewasnoconcernaboutusingseparatelypreparedextractsorreusingextractsfromacom-pletedrunforpurposesofconditioningorsystemsuitability.

ThedraftguidancerecommendsaddingnewQCsatdifferentconcentrations,iftheoriginalQCconcentrationswerenotrepresentativeofstudysampleresultsfromearlyanalyses.TheconsensuswasthatpartialvalidationofnewcalibrationandQCconcentrationswouldbeappropriateonlyifthenewconcentrationswerenotbracketedbyearliervalidations.Ordinarily,demonstrationofthepreparedcon-centrationwouldsuf?cewhenaddingQCconcentrationsinvalidatedregionsofthecalibrationrangeforabetterdemonstrationofassayperformancenearthemostcriticalconcentrationsofstudysamples.

Duringsampleanalysis,whenQCsfailduringindividualrunsthatmeetacceptance,thedraftguidancerecommendsreportingthesevalues.TheAgencyindicatedthatitisinterestedinevaluatingvariabilityinperformance,includinganyoutliervalues.WhereresultsaregeneratedforQCsinfailingruns,documentationoftheirresultsinthereportisrecommendedbutitisnotnecessarytoincludethoseQCsintheaccuracyandprecisionstatisticalanalysis(seelaterdiscussiononDocumentingandReporting).

Duringmostbioequivalencestudies,andduringotherstudiesdesignedwithrepeatedmeasuresfromindividualsubjects,itisdesirabletominimizethebetween-runvariability.Thedraftguidancerecommendsthatsamplesfromagivensubjectshouldbeanalyzedinasinglerun.Theconsensuswasthatthisshouldbethepracticewhenpracticable.Productssuchasthosetherapeuticproteinswithextremelylongeliminationhalf-liveswasanexamplediscussedwherethisapproachisnotpragmatic.Whateverapproachistakenforsampleanalysis,itshouldincludeconsiderationandstepstominimizevariabilitybetweenperiods.CHROMATOGRAPHICASSAYS:NON-CONSENSUSTOPIC

ThissectiondescribesthetopicsforwhichtherewaslackofagreementatthecurrentAAPS/FDABioanalyticalMethodValidationWorkshopandispresentedtoprovidethereasonsfornoconsensus,andpromotefurtherdiscussion.

Boothetal.

AnalyteStabilityinPresenceofConcomitantMedicationsAmajorityofWorkshopattendeesmaintainedthathistoricaldatasuggestthatconcomitantmedicationsposelittleriskforin?uencinginstabilityofanalytes,and,therefore,assessingthestabilityofanalytesinthepresenceofconcomitantmedicationsaddslittlevalue.However,therewassomediscussionofrarecaseswheninstabilitywasobservedwithotheranalyte(s)butthiswasattributedtochemistryissues,andonlyinsuchcasesshouldadditionalstabilitybeconducted.TheAgency’sargumentforconductingadditionalstabilityevaluationswithconcomitantmedications,particularly?xed-dosecom-binations,wasthatthisisanacceptablepracticetoassurestabilityintheknownpresenceoftwo(ormore)analytes,sincestabilityisanintegralpartofmethodvalidation.Someconferenceattendeesindicatedthattheanalyticalsitesmaynotalwaysbeawareofalltheconcomitantdrugsusedinaparticularstudy,andtheymaydifferfromstudytostudy.WhiletheAgencyagreedthattheintentofsuchstabilityassessmentisnotanall-encompassingandexhaustiveinvestigation,itshouldberestrictedtoknownorcommonlyusedmedications.TheAgencyindicatedthatitdoesnotseeanyreasonfornotconductingsuchstabilityassessments,especiallyfor?xed-dosecombina-tions,butwillconsiderthedivergentperspectives.Multi-BatchRuns

Aruncanconsistofdistinctprocessingbatchesforvariousreasons(e.g.,severalmulti-wellplates,multipleanalysts,limitedcapacityforextraction).Forrunswithdistinctprocessingbatches,theutilityofseparateQCsineachdistinctprocessingbatchtodemonstrateaccuracywithinthosedistinctbatches,aswellastherun,wasdebated.TheAgencydescribedsituationswhereintherunpassedQCacceptancecriteria,althoughthemajorityofQCsinoneormoredistinctprocessingbatch(es)withintherunfailed.Therefore,theaccuracyofthedistinctprocessingbatcheswithintherunwasquestionable.ThisisthebasisfortheAgency’srecommendationtoincludeatleastduplicateQCsatallconcentrationsineachdistinctprocessingbatchwithinarun,andestablishacceptancecriteriaforthewholerun,aswellasthedistinctbatcheswithintheruns.WhilethevalueofadditionalQCsindistinctprocessingbatcheswasrecognized,someconferenceattendeesnotedthattheaddition-alQCscouldincreasethetotalnumberofQCswithinananalyticalrunbeyondthegenerallyacceptable5%.Additional-ly,itmayposeanundueburdenwhenthedistinctprocessingbatcheshavelimitedsamplecapacityorwhenthereareinsuf?cientnumbersofstudysamplesineachdistinctbatch.ItwasalsonotedthatthecreationofpartialrunacceptancecriteriahadbeenrejectedinpreviousWorkshopsduetothecomplexityofrulesandinterpretationofthedata.Therewasadiscussionona?t-for-purposeapproach;however,noconsensuswasreachedonanapproachtoassuretheaccuracyofmulti-batchruns.LIGAND-BINDINGASSAYS:CONSENSUSTOPICSThegroupdiscussedandclari?edanumberofligand-bindingassay(LBA)issues.Thetopicsforwhichconsensuswasreachedaredescribedbelow.

The2013CrystalCityVConferenceReportSelectivity/Specificity

Thegroupdiscussedtheparticularaspectsofselectivityandspeci?cityastheypertaintoLBAs;speci?cally,interferencesfromsubstancesstructurallysimilartotheanalyte,aswellasothermatrixcomponentsshouldbeevaluated.Theformeristypicallyrelatedtothespeci?cityoftheassayreagentsandthelattertotheabilityoftheassaytoselectivelymeasuretheanalyteofinterestinacomplexbiologicalmatrix.

Sincesmallmoleculeconcomitantmedicationsarenotstructurallysimilartolargemoleculetherapeutics,thesetypicallydonotneedtobetestedforinterferenceintheassays.Othersubstancesthatmaybindthetherapeuticmolecule(e.g.,solubletarget)shouldalsobeevaluatedasappropriate.Intheeventthatasigni?cantinterferenceisidenti?ed,developmentofanorthogonalmethodmayneedtobeconsidered.Itis,therefore,recommendedtotestforanticipatedinterferencesduringassaydevelopmentsothatmethodmodi?cations,ordevelopmentofnewmethods,maybeimplementedpriortoassayvalidationandimplementationforsampleanalysis.Thiswouldincludespecialpopulationswhenknownatthetimeofmethoddevelopment.

Theapproachfortheselectivityassessmenttoaddressmatrixeffectswasagreedaspreviouslypublished(10).Brie?y,matrixsamplesfrom10ormoreindividualsshouldbetestedunspikedandspikedwithanalyteatLLOQandHQCconcentrations.Eightypercentofsamplesshouldmeetthefollowingcriteria:unspikedsamplesshouldmeasureBQL,andmeasuredconcentrationsshouldbewithin25%ofnominalforLLOQspikesand20%ofnominalforHQC.CalibrationCurve

ThecalibrationcurveshouldbeassessedwithcalibratorsthatarepreparedindependentoftheQCs.Methodvalidationexper-imentsshouldincludeaminimumofsixrunsconductedoverseveraldayswithatleastsixnon-zerocalibratorconcentrationsthatspantheanticipatedquantitativeassayrange.TheseconcentrationsshouldincludeLLOQandULOQ,butshouldnotcoincidewithconcentrationsofthelow,mid,orhighQCs.Additionalcalibratorsoutsidethequantitativeassayrange,referredtoasanchorpoints,mayalsobeincludedtofacilitatecurve?tting.Acceptancecriteriaforthecalibrationcurveduringmethodvalidationalignwithpreviousrecommendations(11).Speci?cally,75%ofthecalibratorsinthecurveshouldhaveback-?ttedconcentrationswithapercentdeviation±20%(25%attheLLOQ)(versusthestatednominalconcentrations)inordertopassacceptancecriteria.Thenumberofcalibratorsshouldbeadequatetoaccommodatethecurve?tmodel(12).Inordertoaccommodatethesecriteria,eightcalibratorsarefrequentlyassessedtoensurethatrunswith75%passingcalibratorsstillretainsixpassingcalibrators.Itisacknowledgedthatitmaynotalwaysbepossibletoincludesixtoeightcalibratorswithintheassaycalibrationrange.RunAcceptance

Asdiscussedunder“ChromatographicAssay,”methodaccuracyandprecisionacceptancecriteriashouldbedeterminedfromQCperformanceduringvalidationindependentofaprioricriteria.Toaccommodatethis,runacceptanceduringA&Pvalidationrunsshouldbebasedoncalibrationcurveperfor-manceonly,wherecalibratorconcentrationsmeetacceptance

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criteriafor(75%ofthecalibratorsinthecurveshouldhaveback-?ttedconcentrationswithapercentdeviation±20%(25%attheLLOQ)versusthestatednominalconcentrationstopass).AllQCsfrompassingA&Prunsarethenevaluatedtodeterminetheassayaccuracyandprecisioncriteriatobeusedinsubsequentrunsandstudysampleanalysis.PrecisionandAccuracy

Toassessprecisionandaccuracy,?veQCconcentrationsshouldbeused(LLOQ,low,mid,high,andULOQ)(10,13).Sixindependentassayrunswithatleastthreereplicatesofeachconcentrationperrunshouldbeconductedoverseveraldays.Thisenablesamorerobustestimationofbothintra-andinter-assayA&P.Duringvalidation,theintra-runandinter-runaccuracyandprecisionofthemeanconcentrationsshouldbewithin20%ofthenominalvalueateachlevel(25%attheLLOQ).ThesesameQCsshouldalsobeusedtodeterminethetotalerror.Thetotalerror(i.e.,sumofabsolutevalueofthe%relativeerrorand%imprecision)shouldnotexceed30%(40%atLLOQ)(10).DilutionalLinearityandHookEffect

Dilutionallinearityandthehookeffectaretypicallyassessedseparatelyfromaccuracyandprecision.Dilutionallinearityexperimentsareperformedtodemonstratethathighconcentrationsoftheanalytecanbeaccuratelymeasuredbydilutingintothequantitativerangeoftheassayandmulti-plyingthemeasuredconcentrationbythedilutionfactor.Theprozone(hookeffect)istypicallyassessedinthesameexperimentbymeasuringsamplesspikedwithveryhighconcentrationsofanalytewithoutdilution.Aprozoneisidenti?edwhenincreasinganalyteconcentrationresultsinnochangeordecreasedsignalswhencomparedtotheprecedingconcentration.SampleAnalysis

CalibrationcurverangesforLBAsarefrequentlynarrow(1–3logs)andcannotcovertheanticipatedrangeofstudysampleconcentrationsformoststudies.Instead,sampledilutionisemployed,supportedbydemonstrationofdilutionallinearityduringpre-studyvalidation.Whiletheremaybecircumstanceswhereanalyzingallstudysamplesfromasinglesubjectinasinglerun(plate)maybedesirable,itisnotalwayspossibleorpracticalforligand-bindingPKassays.

Ligand-bindingassaysgenerallyinvolveduplicateanaly-sisofallstandards,QCsandsamples.Whennewertechnol-ogiesarenotasreproduciblecomparedtoawell-characterizedLBA,thetendencyistoperformtriplicateanalysistoincreasecon?denceintheresults.AsLBAshaveadvanced,particularlyinthequalityoftheassayreagents,itmaybejusti?abletomovefromduplicateanalysistosingletanalysisascurrentlyimplementedforLC-MS/MSmethods.Agreementonrunningsamplesinsingletfromtheindustryperspectivewasachieved,aslongasQCintra-andinter-plateprecisionisachievedaccordingtothemethodvalidation.Someproposalsontighteningthesecriteriatoallowahighercon?denceinrunningsingletswerediscussedwithoutanyconcreteconclusion.

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