新药基因毒性杂质风险分析Risk assessment of genotoxic impurities in NCE

RiskAssessmentofGenotoxicImpuritiesinNewChemicalEntities:StrategiesToDemonstrateControl

AndrewandIgnacioTeasdale,H.Sanchez*, DavidFloresElder,

#Sou-JenChang,§SophieWang,∥RichardThompson,⊥NancyBenz,¶

AstraZeneca,CharterWay,SilkRoadBusinessPark,Maccles eld,CheshireSK102NX,UnitedKingdom

GlaxoSmithKline,ParkRoad,Ware,HertfordshireSG120DP,UnitedKingdom

§AbbottLaboratories,200AbbottParkRoad,PA71,BuildingAP-50,AbbottPark,Illinois60064-6220,UnitedStates

∥AmgenInc.,1AmgenCenterDrive,ThousandOaks,California91320,UnitedStates

⊥BenVenueLaboratoriesInc.,BoehringerIngelheim,300North eldRoad,Bedford,Ohio44146,UnitedStates

¶AbbottLaboratoriesDepartmentR45T,BuildingR8-2,1401SheridanRoad,NorthChicago,Illinois60064,UnitedStates#TakedaGlobalResearch&DevelopmentCenter,Inc.,OneTakedaParkway,Deer eld,Illinois60015,UnitedStates

Syntheticprocessesusedtoproducepharmaceuticaldrugcategoriesrangingfromthosewithnostructuralalertsforsubstancestypicallyrequiretheuseofelectrophilicagentstogenotoxicitytothosethatareknowntobecarcinogenic.Thefacilitatecarbon carbon,carbon nitrogen,carbon oxygen,principlesofstructural

andcarbon sulfurbondformation.Examplesincludealkylating7alertswereoriginallyproposedbyAshbyandTennantonthebasisofstructure/Amesmutagenicity/agents,benzylhalidesandMichaelacceptors.Someofthesecarcinogenicitycorrelationsforcompoundscontaininganagentsmayalsopossesstheabilitytoreactwithbiologicalelectrophilicfunctionalgroup(oracompoundthatcouldbesubstratessuchasDNA,whichwouldraiseconcernsaboutmetabolisedtoonecontainingsuchareactiveentity,e.g.sometheirpotentialcarcinogenicity.Anyresiduesofacon rmedaromaticamines).

DNA-reactiveelectrophilicreagentorintermediateinadrugIn2008,theEMAadoptedthestagedTTCapproachinitssubstancewouldbecategorizedasgenotoxicimpurities(GTIs).publishedQ&Adocument,whilereducingtheallowedlimitstoExistingregulatory3guidelinessuchasICHQ3A(R2)/Q3BhalfofthoseproposedbythePhRMAtaskforce.8Asan(R2)/Q3C(R4)1 donotadequatelyaddresstherequire-mentsforcontrollingtracelevelsofGTIs.example,theEMAguidanceallowedalimitofdailyexposureof

60

μμgforclinicaltrialslastinglessthan30

marketingnalizedIn2004versiontheEUapplicationsin2006issuedforthatadraftpharmaceuticals.addressedguidancecontrollingfollowed4ThisEuropeanGTIsbyinastrategygasproposedwasproposedbythePhRMAbythetaskFDAforce.daysinsteadof120inAtheirsimilardraftstagedguidelineTTCMedicinesAgency(EMA)guidelinerecommendedthat,inissuedlaterin2008.9

caseswherespeci csafetydatawereunavailableforaGTI,theGTIsmaybegeneratedduringdrugsubstancesynthesis,GTIshouldbecontrolledtoalevelbasedonathresholdofdrugproductmanufacture,orduringstorageofdrugsubstancetoxicologicalconcern(TTC).Theguidelinerecommendedtheordrugproduct.Inmanycases,GTIscanbecontrolledduringTTCasadefaultlimitofdailyexposuretothepatientof1.5μgthedrugsubstancesyntheticprocessprovidedthattheGTIsinonthebasisofanincreasedcancerriskof10 5.questionarenotgeneratedaseitheradrugsubstanceAlsoin2006,aPhRMAtaskforceissueditswhitepaperdegradationproductorproducedduringdrugproductproposingastagedTTCwhereinexposuretohigherlevelsofmanufacturingorstorage.IncaseswheretheGTIisgeneratedGTIswouldbeacceptableincaseswheredurationofexposurethroughinteractionswithmanufacturingprocessingagentsislimited,asisthecaseduringclinicaldevelopment.5The(e.g.,excipients,residualsolvents,etc.)orduringstorageduringhigherlimitswerebasedonanextrapolationfromdailylifetimenormalconditions,controllingthelevelsoftheGTIinthedrugexposuretoshort-termexposuresoflessthan12months6butproductmayalsoberequired.ManyoftheprincipleswithanadditionalsafetyimplementedforthecontrolofGTIsindrugsubstancewill

6factorapplied,sothecalculatedexcessriskofcancerwas10insteadof10 5,sinceearlyclinicaltrialsoftenincludehealthyvolunteers,andevenforpatientstheReceived:September25,2012

pharmacologicalbene tofthedrugmaynotyethavebeenPublished:January14,2013

Table1.PhRMAsurveyresultsonGTIs

areasofgeneralconsensusareasofdiversity

evaluationoftheAPIsyntheticpathwayforGTIsatthepreclinicalstagepointmonitoringinthesyntheticforGTIsprocesstobeginconsidertheGTIthenumberofstepsbackfromthe nalAPIthattheGTIoriginateswhenprovidingarationalefornottestinguseofofGTIslimittestsvsquantitativereportinguseeliminatedofscienti incjustithedownstream cationinlieuchemistryoftestingorwhenprocessaGTIisintroducedearlyintheprocessandisreactiveenoughtobedegreecontrolofofvalidationGTIs

ofmethodsusedforalsoapplyfordrugproduct.However,thedrugproductprocesselectrophilicreagents.Theirinherentreactivenatureandisoftenconstrainedinitsabilitytopurge(orremove)GTIs.potentialtoreactwithnucleophiles(includingDNA)mayAcriticaltenetofdrugsubstanceprocessdevelopmentistoensurethatGTIsintroducedorgeneratedduringtheprocessraiseconcernsfromtheperspectiveofcarryoverintothe nalarecontrolleddrugsubstance.However,highlyreactiveelectrophilicreagents

4toacceptablelevels.OnthebasisofEMAguidancetheselimitsarecurrentlybasedonthestagedTTC,shouldbee ectivelypurgedfromtheAPIsynthesisbyanyTTC,orcompoundspeci csafetydataassociatedwiththesubsequentdownstreamchemistry.Therefore,anevaluationofimpurity(ies)concerned.GTIcontrolmaybeexecutedthroughtheriskposedbysuchimpuritiesisrequired.TheassessmentofimplementationofsimpleprocessoperationssuchaswashesGTIcarryoverinvolvesidentifyingthepotentialpresence/andcrystallizationstepsandinmanycasesindirectlythroughremovalofsuchentities,asthesyntheticreactionproceedstofurtherdownstreamsyntheticreactions.Thus,thedevelopmentthe nalAPI.ItiscriticalthatsuchanassessmentbalancestheofthedrugsubstanceprocessincludesconsiderationsthatareriskofobservingtheGTIinthe naldrugsubstancewiththebasednotonlyonscale-up,safety,economicandenvironmentalprobabilityofitsremoval(purge)basedonknowledgeofthefactorsbutalsoontheproductionofanAPIwithimpuritychemistryusedinthesyntheticprocess.Itisimpracticaltolevelsthatareincompliancewithregulatorystandards.evaluate/identifyeveryconceivableimpurity;hence,suchanTheevaluationandcontrolofGTIsinasyntheticprocessisaassessmentthereforeneedstobebasedonprocessunder-multidisciplinaryactivity.Considerationsfromtoxicological,standingoflikely/probableimpurities.IndeedtheEMAprocessing,andanalyticalperspectivesareinvolvedandmustguidelineadvocatessuchanapproach.4

alignwithregulatoryrequirements.Whilecurrentregulatory2.2.TheEvaluationProcess.EvaluationofgenotoxicguidancedoesprovideclearexpectationsregardingtheallowedpotentialisgenerallyperformedinitiallythroughacomparisonlimitsofGTIsduringclinicaldevelopmentandatmarketingofstructuresofreagents/startingmaterials/intermediatesintheapplication,therearestillanumberofareasopentosyntheticschemewiththoseofknowngenotoxins,eitherinterpretation.ExamplesofGTIregulatorygrayareasincludethroughsimplecomparisonwithaknownalertingfunctionality,de ningthescopeofthesearchforGTIs(numberofchemicale.g.Ashby Tennantalerts,7throughsearchesofpublishedstepsbackfromthe naldrugsubstanceinthesynthesisandinformation,orthroughassessingstructuresina(quantitative)considerationofhypotheticalbyproducts),howscienti cstructure/activityrelationshipSAR/QSARsoftwaredatabasejusti cation(basedonchemicalexpertiseandknowledgeofsuchaDEREK(deductiveestimationofriskfromexistingthechemistryofthesyntheticprocess)canbeusedinlieuofknowledge)eofotheranalyticaltesting,expectationsfortypeofanalyticalmethod-sourcesoftoxicologicaldata,e.g.TOXNET,canalsobeuseful,ologyandrequiredlevelofvalidation,anduniversalunder-thisbeingparticularlytruewhenaddressingrelativelycommonstandingofspeci ccontrolsasafunctionofstageofclinicalreagentsforwhichspeci csafetydatamayalreadyexist.development.Abenchmarkingsurvey,issuedbyPharmaceut-ItiscriticalthatanysuchassessmentisaugmentedbyhumanicalResearchknowledgeandexpertise.Forexample,thedatasetunder-

10andManufacturersofAmerica(PhRMA)in2009,re ectsthatmanyoftheseareasareaddressedpinningtheinsilicosystemshouldbeevaluatedinordertodi erentlybyindustryandregulatoryagencies(Table1).ensurethatthereissu cientdatabasecoveragetoallowtheTheproposalsdescribedinthispaperexpandupontheEMAreviewertohavecon denceineitherapositiveoranegativeguidelineandFDAdraftguidancetodescribestrategicresultfromtheSARanalysis.

approachesforevaluationofGTIsthroughoutthevariousThestructuresassessedtypicallyincludestartingmaterials,stagesofpharmaceuticaldevelopment.Itdetailsriskassessmentreagents,intermediates,andknownprocessimpurities.ThisisstrategiesthatcanbeimplementedtoevaluatewhichGTIsoftenfurtheraugmentedduringthedevelopmentprocessbyshouldbemeasuredanalyticallyandwheretestingcanbetheinclusionofadditionalstructuresthatarederivedfromeliminatedorreducedonthebasisofscienti crationale.eitherincreasedknowledgeofthesyntheticprocess(intermsof

2.AQUANTITATIVERISKASSESSMENTFORimpuritiesassociatedwiththeprocess)and/oridenti edEVALUATIONOFGENOTOXICIMPURITYdegradationproductsofthedrugsubstance(andproductCARRYOVERwhereapplicable).Compoundsthatdonotcontainstructuralalertsfor

2.1.ScopeofEvaluation.AsoutlinedintheIntroduction,genotoxicityaretreatedasconventionalimpuritiesthemajorityofGTIsarisefromthesyntheticprocessitself.3andarecontrolledinaccordancewithICHQ3A/3B/3C.1 Com-Syntheticdrugsubstancesaretypicallyconstructedthroughpoundswithstructuralalertsforgenotoxicityrequirefurthersystematicmodi cationofacompound’smolecularframeworkaction.Thesecompoundscanbetestedformutagenicitythroughtheformationandrecon gurationofcarbon carbon,throughinvitromethods,typicallyAmestesting.IftheAmescarbon nitrogen,carbon oxygen,andcarbon sulfurbonds.testisnegative,thentheimpuritycanbetreatedasaCurrentsyntheticmethodologyissuchthattheformationofconventionalimpurityandmanagedasperICHQ3A/3B/3Csuchbondsispredominantlyachievedthroughtheuseofguidelines.

2.3.RiskAssessmentofGTICarryover.Ifastartingempiricalapproachestosuchassessmentsbasedonthepointofmaterial,reagent,intermediate,orabyproductisidenti edasaintroductionoftheGTIinquestionandthenumberofstagespossibleGTI,itmayappearthatthebestwaytocontrolitremovedfromtheAPI.Suchanapproach,euphemisticallywouldbetoeliminateitspresencethroughanalternatereferredtoas‘stagecounting’,hasunfortunatelynotbeensynthesisroute.However,inmostcases,thisisanimpracticaluniversallyacceptedbyregulatoryauthorities.Severalregu-approachbecauseanalternatesynthesismayproducelowerlatoryagenciescontinuetoasktheSponsortoprovideyields,havepooreroverallqualitycontrol,andultimatelymayanalyticaldataevenincaseswherecompellingscienti cnotbeeconomicallyfeasible.Moreimportantly,itishighlyargumentswouldsuggestproofofabsence.Suchargumentslikelythatanyalternatesynthesiswillcontainotherimpuritiesshouldbesu cient,particularlywhentheassessmentaddressesthatrequiresimilarcontrolattheTTClevel,andsigni cantthechemistry(reactionsandpuri cations)speci ctoeachstepadditionale ortwillberequiredtodischargetheriskofthosethatisbeing“counted”inaparticularprocess.Nonetheless,itnewimpurities,withoutmateriallyimprovingthesafetyofthewouldbeusefuliftherewereanalternativeapproachthatAPIorreducingtheoverallrisktopatients.ControlofGTIscontainedaquantitative(orsemiquantitative)element,asisratherthaneliminationisthereforestronglyadvocated.Similarpresentedanddescribedinthefollowingsection.considerationsapplytoALARP(aslowasreasonablyItispossibletotakesuchanapproachtoriskassessmentonpracticable)andwereacceptedbyEMAintheQ&Athebasisofthespeci cphysicochemicalpropertiesofaGTI,supplements.8alongwithanunderstandingoftheprocessconditionstowhichTodemonstratecontrol,anassessmentofthepotentialriskitisexposed,toquantitativelyassesstherisk.Theserisk-basedofcarryoverofaGTIatalevelexceedingstagedTTCorTTCassessmentsrelyontheconceptofevaluatingpurgefactorslevelstothedrugsubstanceismade.Thisisalignedwithqualitywhichcanbede nedastheabilityofasyntheticprocesstobydesign(QbD)andtheriskassessmentprinciplesenshrinedpurge(eliminate)aparticularGTI.19,20

inICHQ8andQ9.11,12Aseeminglysimplebutpotentially2.4.PurgeFactors.Theprinciplesupportingthemyopicwaytoaddressthisistodevelopasensitiveanalyticalcalculationofapurgefactorisasimpleone.ItistoidentifymethodandtestforthepossibleGTIinquestionatthepointthekeyphysicochemicalparametersthatin uencetheremovalafterintroduction,atthe naldrugsubstance,oratsomeofaspeci cGTIinaprocess(inQbDterms:criticalqualityintermediatepoint,i.e.QualitybyTesting(QbT).Thisattributes(CQAs))andtousethese,incombinationwiththeapproach,however,canbeatechnicallychallengingandspeci cprocessconditions,todetermineapurgefactor,i.e.howresource-intensiveactivity,especiallywhenappliedtoallthemuchoftheGTIislikelytoberemoved.GTIsthatareassociatedwiththesyntheticprocessandrunsThekeyparametersidenti edarereactivity,solubility,contrarytothetenantsofQbD.Moreoverthisapproachfailstovolatility,ionisability,plusanyadditionalphysicalprocessrecognizethatreactiveGTIswilloftenbedestroyedordesignedtoeliminateimpurities,e.g.chromatography.Theeliminated nalinthesubsequentprocessstepsleadinglatterapproachisoftenemployedinmedicinalchemistrysyntheticroutesemployedforpreparingtestmaterialsfor rstThedrugtotheusesubstance.ofscienti cunderstandinginrelationtoevaluationhumanclinicaltrials.InordertoensurethataconsistentofGTI-relatedriskisgrowing.Severalarticles13,14haveoutlinedapproachistaken,ascoringsystemhasbeendevisedwhichistheuseofQbDprinciplesintheassessmentofGTI-relatedrisk,outlinedinTable2.thesebeingcenteredontheuseofspiking/purgefatestudiesto2.4.1.Reactivity.Thepurposeofthisspeci cparameteristoprovetheabilityofthesyntheticprocessinquestiontoremoveassessthereactivityoftheGTIinquestioninrelationtospeci cGTIs.Suchapproachesareextremelyusefulwhenprocessingconditionstowhichitissubsequentlyexposed.Onappliedinthecontextofa xedprocess.thebasisofreactivitytowardtypicalreagentsWorkconductedbytheProductQualityResearchInstituteencounteredduringworkuporstorage(e.g.,otherchemicals(PQRI)relatingtosulfonateesters15,16(Teasdaleetal.)usedwithintheprocess,water,acid,base,alcohols,etc.)itiscompellinglydemonstratedhowagoodscienti cunder-proposedthatGTIscouldbegenerallyplacedintooneofthreestandingofthepropertiesofacompound,combinedwithacategories(seeTable3).riskassessmentoftheimpactofprocessconditions,canbeusedItiscritical,though,thatanysuchclassi cationmusttakee ectivelytocontrol,oreveneliminatethepotentialGTIriskposed.Suchapproachesasthosedescribedabove,althoughoneforcomprehensive,areimpracticaltoapplytoallpossibleGTIsahighlyreactiveGTI,e.g.acylhalide,andthesecondforalesswithineachandeverysyntheticprocess.Whatissoughtinsteadreactivearylnitrocompound.isarisk-basedmechanismwherebythepotentialriskofThionylchlorideistypicallyusedtoconvertnonreactivecarryovercanbequicklyandsimplyassessed.Formanycarboxylicacidsintoreactiveacylhalides,whichcanthenbeimpurities,chemistry-basedargumentsalonemaybesu cientfurtherprocessed(e.g.,formationofesters,amides,etc.).21toconcludethatanimpuritywouldnotresideinthe nalAPIHowever,duetotheirhighreactivitytheyareverye cientlywithouttheneedforlaboratoryexperimentation.Thisapproachpurgedfromthesubsequentdownstreamchemistryreac-isoftenusedwhenprocessingconditionsforthedownstreamtion(s);thisalsoappliestothionylchlorideitselfwhichreactspurgeordestructionoftheimpurityareobvious.Aconsistentviolentlywithwater.22

frameworkforthistypeofrisk-basedassessmentwherebytheIncontrast,arelativelystablenitrocompoundwouldshowpotentialforcarryovercanbeassessedthroughconsiderationofhighreactivity,undermostreductionconditions,toformanthephysicochemicalpropertiesoftheGTIandtherelevantaromaticamine,butitmaywellbeunreactiveduringprocessingconditionsisrecommended,thepotentialbene tssubsequentstagesoftheprocess.toregulatoryreviewersbeingobvious.SuchanapproachisnotExperimentaldatarelatingtotheprocess(e.g.inprocessanewconcept.BothDobo17andPierson18havedescribedreactionmonitoring)canbeusedtore nethisparameter.For

Table2.Purgefactorsisolationoftheproductasasolid,thegenotoxicreagent/

physicochemicalintermediateshouldremaininthereactionmotherliquorsandparameterspurgefactoraberemovedwhentheliquorsare lteredo .Assignmentofareactivityhighlyreactive=100purgefactorforthisparameteristhusbasedonthesolubilityof

moderatelyreactive=10theGTIinquestionwithintheprocesssolvent.Table4

lowreactivity/unreactive=1outlinesthede nitionsusedwithintheUSPrelatingto

solubilitybfreelysoluble=10solubility.Forthepurposesofthepurgefactorcalculations

moderatelysoluble=3thesehavebeengroupedtomatchthethreeclassesde nedin

sparinglysoluble=1Table1.

volatilitycboilingThescaleusedforthesolubilityfactorhasbeensetovera

processpointsolvent>20°=Cthatofthereaction/range1 10.Experiencehasshownthatthesolubilityfactor

boilingreaction/processpointwithinsolvent.±10°C=of3thatofthecouldmuchhigher,perhapsjustifyingarangeof1 100aswas

boilingusedforreactivity.Howeverwebelievethatthatthemoreprocesspointsolvent>20°=C1thatofthereaction/conservativescaleof1 10shouldberetainedtocompensate

ionisabilityionisationforvarianceinprocessconditionssuchasuncontrolled

fromthatpotentialofthedesiredofGTIproductsigni cantlyddi erentcrystallization,poorwashingand/orine cientdeliquoringof

physicalchromatographyprocesses:chromatography:separation10 100basedonextentoftheisolatedproduct.

physical2.4.3.Volatility.Manylow-molecularweight,potentiallyotherscavengerprocesses:resinse.g.evaluatedonanindividualbasis.genotoxicimpurities(PGIs)suchasaldehydesandalkylhalidesaPurgefactor=concentrationbeforepurging/concentrationafterarevolatile;e.g.methylchlorideformedthroughthereactionofpurging.bmethanolandHCl(duringHClsaltformation)hasaboilingrecrystallisationThisprocessrelateswherebytosolubilitytheimpuritywithininthequestion,contextifhighlyofapointof 23°C.Manysyntheticprocessesemploysolventsoluble,distillationorsolventexchange.Asaresultofsuchaprocess,fromanyvolatileGTIpresentmayalsoberemoved,dependingonsolventthewill

throughdesiredremain

solventproduct.withinc

distillationThisthemotherrefersorsolventtoliquorsthedeliberateandhencebepurged

exchange.removaldThisrelatesofathevolatilityoftheGTIrelativetoeithertheboilingpointof

tothesolventorthetemperatureofthereactionprocess.aqueousadeliberate

ofpHandtochangeorganicattemptlayer,topartitionthedesiredproduct/GIbetweenan

theionised/un-ionisedtypicallyachievedthroughstatetheofmanipulationoneofthe2.4.4.Ionisability.WheretheionisabilityofaGTIandthat

components.ofthematrixinwhichitispresentdi er,forexampleanionisableGTIwithinanonionisableintermediateoraTable3.Genotoxiccompoundsclassi edonthebasisofnonionisableGTIwithinanionisabledrugsubstance,thereactivitypotentialexiststoreducetheleveloftheGTIthroughliquid/liquidextraction,byemployingatwo-phasesystemwithreactivityclassgenotoxicgroupsappropriatemanipulationofthepHoftheaqueousphase.highlyreactiveepoxides/aldehydes/sulfonateTheremovalofanalkylhalideGTIfromanionisabledrug

aziridines/hydrazinesesters/acylhalides/substanceprovidesoneexampleofsuchanapproach.pH

moderatereactivityNprimaryorSmustards/Michaelhalidesreactiveacceptors/halo-alkenes,adjustmentoftheaqueousphasecouldallowthedruglowreactivityaminosubstancetobeextractedintotheaqueousphase;thecarbamatesaryls,nitrocompounds,purinesorpyrimidines,nonionisablealkylhalideGTIremainsintheorganicphasethatcanthensimplybediscarded.Thereafter,thepHoftheaqueousphasecanbeadjustedandthedrugsubstanceback-examplewhereanin-processcontrolshowscompletionofextractedintoanappropriatefreshorganicsolvent.Inadditionreaction(<1%GTIremaining)thiscanbeusedtoclassifythetoclassicalliquid/liquidextractions,solid-phaseextractionGTIconcernedashighlyreactiveforthatstageoftheprocess.(SPE)canalsobeemployed.

2.4.2.Solubility.Whereagenotoxicreagent/intermediateisNote:Aspeci cpurgefactorisonlyassignedinrelationtointroducedintothesyntheticprocesstheprocessisgenerallythisparameterwheresuchanapproachisspeci callyapplied.optimizedtomaximizebothyieldandproductquality.One2.4.5.Chromatography.Althoughpotentiallycostlyincriticalfactorinanyprocessistherequirementthatthecomparisontothesimpleprocessingtechniquesalreadyintendedreactionactuallyoccurs.Thisgenerallymeansthatthedescribed,chromatographyneverthelessdoesprovideaverygenotoxicreagent/intermediateinquestionislikelytobehighlypowerfuland exibletoolforremovalofaGTIwhererequired,solubleinthesolventselectedfortheprocessinquestion.Thisparticularlyforearly-phase,nonoptimized,medicinalchemistry,solubilitywillmeanthatwheretheprocessconcernedinvolvessyntheticroutes.

Table4.Solubilityde nitions23

descriptivetermsolubilityclasspartsofsolventrequiredfor1partofsolutesolubilityrange(mg/mL)solubilityassigned(mg/mL)verysolublefreely<1>10001000freelysoluble1 10100 1000100solublemoderately10 3033 10033sparinglysolublesparingly30 10010 3310slightlysoluble100 10001 101veryslightlysoluble1000 100000.1 10.1practicallyinsolubleorinsoluble>10000<0.10.01

PreparativeHPLC,normallyinanormalphasemode,isanneeded.Inthiscontexttherequiredpurgefactorwouldbeestablishedtechniqueappliedtothereductionorremovalof5000,asbasedonthemaximumlevelofmethylchlorideimpurities,atamultikilogramscale,usuallyatthedrugpotentiallypresentis5%=50,000ppm.Thus,inthisinstanceasubstancestage.TheremovalofaGTIcouldsimplybepurgefactorof>500,000associatedwithmethylchlorideconsideredasubsetofthestandardchromatographicchallengeshouldprecludetheneedforanyfurtherinvestigation.ofimpurityremoval.Thisisexploredinfurtherdetailthroughaseriesofexamples.Analternativetotheuseofchromatographyistheuseofresins.Leeetal.(2010)recentlydemonstratedtheabilityof3.RISKASSESSMENTCASESTUDIES

certainnucleophiliccation-exchangeresinstoe ectivelypurgesulphonateestersfromthe nalstageofanAPI.24Thefollowingsectionoutlinesanumberofexamplesthatserve

Againaspeci cpurgefactorisonlyassignedinrelationtotoillustrateboththeutilityofthepurgetoolanditsrobustness.thisparameterwherechromatographyisspeci callyapplied.2.4.6.CalculationofPurgeFactors.Asoutlined,scoresforScheme1.SynthesisofBAY

43-9006

eachparameterareassignedonthebasisofthephysicochemicalpropertiesoftheGTIrelativetotheprocessconditions.ThescaleisbasedonthepremisethatahighpurgefactorequatestohighGTIclearance.Thus,ahighpurgefactorvalueindicatesalowprobabilitythataGTIwillbeobservedonthebasisofknowledgeofproduct’sphysicochemicalpropertiesandunderstandingofthesyntheticprocess.Foreachstagetheseindividualpurgefactorsarethensimplymultipliedtogethertodeterminea‘purgefactor’forthatstage.TheoverallpurgefactoristhensimplyamultipleofthefactorscalculatedpurgefactorwouldindicatethelevelofaGTItobe>100timesbelowtheappropriateTTClimit,thennofurtheractionshouldtypicallyberequired.Wherethevalueofthecalculatedpurgefactorwouldindicatetheleveltobebetween10and100timesbelowtheappropriatelimit,thenitshouldpotentiallybesupportedbyconductofappropriateanalyticaltesting/furtherprocessinvestigation.Thismaytaketheformof,forexample,periodictestingoruseofpurgeandspikeexperimentsascon rmationforremovinglong-termtesting.WherethecalculatedpurgefactorindicatesthatthelevelcouldexceedtheappropriateTTC,thenanalyticaltestingasaItshouldbenotedthattheexamplesincludebothknownminimumwouldberequired.Itmayalsoprovideanindicationmutagensandpotentialmutagens.Thereisnointenttoinferoftheneedtomodifytheprocessingconditionstofurtheranythingaboutthespeci cstatusoftheimpuritiesinquestion,reducetheriskofcarryover.merelytousethemasexamplesforthespeci cpurposeofHowthismightbeappliedisaddressedinthefollowingdemonstratingtheutilityofthepurgetool.

hypotheticalexamples.Theinitialcasestudiesdescribeindetailtheprocessby

2.4.6.1.Example1.Foraproductdosedat150mgadaywhichpurgevaluesareselectedservingtoillustratetherigorthatisintendedforchronicuse,theTTClimitof1.5ug/dayinvolvedinthisprocess.Casestudies3 5focusonthewould,inconcentrationterms,representalimitof10ppmforaccuracyofthepurgefactorcalculation,ineachcase,throughanyGTIpresent.Shouldtherebeagenotoxicintermediate,comparisonwithactualdata.

thenthecalculationofriskofcarryoverwouldstartatapointof3.1.CaseStudy1:PurgingofHighlyReactiveThionyl100%,i.e.,1,000,000ppmoftheintermediateinthestepinChloridefromSynthesisofBAY-43-9006.Thesyntheticwhichitisproduced.Thus,inthisinstanceapurgefactorofschemefortheproductionofBAY-43-9006isshowninScheme100,000wouldindicatethattheprocessislikelytoreducethe1.Thionylchloride(SOCl2)isintroducedintostage1(ofa4-leveloftheGTIinquestiontoalevelequivalenttotheTTC.stageprocess)toconvertnonreactivepicolinicacid(4)intothe(i.e.,[concentrationbeforepurging,1,000,000ppm]/[purgecorrespondingreactiveacylhalideintermediate(4-chloropyr-factor,100,000]=concentrationafterpurging,(10ppm).idine-2-carbonylchloride)(5).Although,thionylchlorideisThus,inthisinstancecalculatedpurgefactorsof>10,000,000reportedtoAmespositive,thereisincreasingevidencethatthisshouldprecludetheneedforanyfurtherinvestigation.isanartifactofthetestsystem.AmestestsperformedinDMSOCalculatedpurgefactorsbetween1,000,000and10,000,000givepositiveAmes ndings,whereasthesametestperformedinsignalthatfurtherinvestigationmayberequired,and nallyacetonitrileisAmesnegative.25Itiswell-knownthatsul nylchloridescalculatedpurgefactorsof<100,000wouldrequireasaminimumanalyticaltestingoftheGTIinquestion. de(CDMS),canreactaknownwithmutagen.DMSOto26producechlorodimethylsul-TheCDMSisprobablytheagentresponsiblefortheAmespositiveresultofthetestarticle2.4.6.2.Example2.Forthesameproductdescribedabove,inDMSO.reactionmonitoringhasshownthatduringasaltformationTheacylhalide(5)isreactedwithmethylaminetoproducestep,alevelofapproximately5%methylchlorideisformed.thecorrespondingamide(7)in88%yield.TheamideisthenThismeasurementcanbeusedtocalculatethepurgefactorcoupledwith4-aminophenol(8)inthepresenceofbasetorequiredandallowforadjustingthepurginge ciency,ifproducethepenultimateintermediate([4 4-aminophenoxy)-

Table5.PredictedpurgefactorsforthionylchlorideinBAY43-9006process

stagereactivity(R)solubility(S)volatility(V)processesphysical

(PP)comments

110010101SOClproposition.2isavery

vacuum.ThionylAnalytereactiveintermediate(R=100),andthehighyields(89%)supportahighreactione ciency

chloridehashighboilssolubilityat79°Cin(DMFV=10).(S=There10),isandnointermediatephysicalprocessing5isisolated,inthiswashedstage(PPand=dried1).under

21001013SOClAnalyte2will

though,hasreact

isdissolvedhighwithsolubilitymethylamine

inethylinTHFbase

acetate(Sand=and10).with

washed/extractedThereaqueousisnospecibrine

with c(useddryingto

aqueousstepextract

brine(V=isolatedintermediate)(R=100).

(PP1).=The3).isolatedintermediate(7),

31001013SOCl2willreactwithbases(K-tert-butoxide/K2CO3)andwithaqueousbrine(usedtoextractisolatedintermediate)(intermediateR=100).Analyte(7),though,hashighisdissolvedsolubilityininethylDMFacetate(S=10).andTherewashed/extractedisnospeci cwithdryingaqueousstep(brineV=1).(PPThe=3).isolated4110101Reactivitysolubilityisinpredicteddichloromethanetobelow,(asSthe=10).reactionAnyisresidualperformedthionylinachloride,nonaqueousthough,environmentislikelyto(Rbe=1).removedAnalyteonhasdrying.ahighScheme2.SynthesisofGSK183390A

a

aReactionsconditions:(a)NaOEt,0 80°C,yield99%;(b)CH3NHNH2,EtOH,90°C,yield22%(unwantedisomeryield45%);(c)NHCHroomtemperature,yield93%;ethyl(d)ammoniumbromoisobutyrate,formate,yieldPd/C69%;catalyst,(h)TFA,re ux,CHMeOH,yield50%;(e)aqHBr,yield97%;(f)2OH·HCl,NaOAc,EtOH,Boc2O,2Cl2,Et3N,yield96%;(g)K2COthen3,DMF,Et2Cl2,yield82%;(i)NaOH,roomtemperature,yield98%;(j)SOCl2,toluene,80°C,3N,10,roomtemperature,yield96%;(k)NaOH,80°C,yield73%.

Table6.TheoreticalpurgefactorsforGTIsinPPARα/γagonist(1)syntheticprocess

GTIstagereactivityM=10,HL==100,1solubilityM=3,FL==10,1volatilityM=3,HL==10,1totalpermultiplestagerationaleforpurgefactor

CH3NHNH221001033000bpdistillation/partialofCH3NHNH288drying 90°C,solventremovedby

31339productisolatedanddried

410103300likelyformedreaction(stepwithj);productSO2Clandisolatedalsowithandthedriedacylchloride

ALL8.1×106

SO2Cl4(j)100101010000highlyreactive,volatilebp79°C

4(k)100101010000reactsinstantaneouslyinaqueousenvironment

1.0×108

(2-pyridyl)]-N-methylcarboxamide)(9)in87%yield.This(1)Methylhydrazineisusedinstage2tocyclisetheintermediate(9)isthencoupledwithisocyanate(10)toyielddiketone(4)toproducethepyrazoleester(5).theAPI(3).(2)Thionylchlorideisusedtoactivatethepyrazole(10)viaThebasisbywhichpredictedvaluesareselectedandthetheformationofanacidchloridewhichisthenreactedassociatedrationalesaredescribedinTable5.withthebenzylamine(10)toformtheAPI.Theoverallpurgefactoris9×10.12Theextremelyhigh

reactivityofthionylchloridetohighpH/aqueousconditionsApurgefactorassessmenthasbeenappliedtothisroutetodominatesthepurgingfactorinthe rstthreestages.determinethelevelofriskfromagenotoxicimpurities

3.2.CaseStudy2:RetrospectiveEvaluationofaperspectiveaspartofanevaluationofthepotentialcontinuedPPARα/γAgonist(GSK183390A).Thesyntheticschemeuseoftheproceduredescribed(Table6).

(Scheme2)showstheoriginalmedicinalchemistryrouteusedEmpirically,theriskofcarryoverofGTIs/PGIsisoftentomanufactureaPPARα/γagonistindrugdevelopment.Fromassessedonthebasisofproximity(pointofintroductioninthesynthesis)tothe nalAPI.Thus,thionylchloridetheoreticallyaGRAperspectivetherearetwoknowngenotoxiccompoundsposesthehighestriskwithrespecttocarryoverintotheAPIasinvolvedinthesynthesis:itisintroducedintothe nalcouplingstage.Inpracticethisis

Scheme3.SynthesisofAZD9056

19Scheme4.Synthesisofstartingmaterial

(III)

API.Thetheoreticalpurgefactoris1.0×108,whichsupportsthecontentionthattherewillbenorequirementtotestforthionylchlorideinAPI.Similarly,methylhydrazine(theoreticalpurge8.1×108)willbee cientlypurgedfromthereaction.3.3.CaseStudy3:AZD9056.Thisexamplerelatestothe

synthesisofadevelopmentaldrugAZD9056.Withinthelaterstagesofthesynthesisthreepotentiallygenotoxicimpuritieswereidenti edasbeingofconcern(seeScheme3).ThethreepotentialGTIswerethefollowing:

(1)AZD9056aldehyde:anintermediatewithinthesynthesisofAZD9056;(2)isopropylchloride:formedduringtheisolationstagethroughthereactionbetweenisopropylchlorideandhydrochloricacid(HCl),(3)AZD9056chloride:formedthroughreactionbetweentheAPI(analcohol)andHCl.

Ariskassessmentwasconductedforeachoftheseinturn,andtheoverallresultsaredisplayedinTable7.notthecaseforthefollowingreasons.Thionylchlorideis3.3.1.AZD9056Aldehyde.Onthebasisofapurgefactor

highlyreactive,bothwiththeintendedreactant(11)andwithpredictionof10,000,thiswouldindicatethatthelikelywater°(stepk,Table6);this,combinedwithitscarryoverofthiscompoundintothe nalAPIislikelytobe<100ppm.Givenitscloseproximityto nalAPI,thepuri edanC)e andcientitspurgemiscibilityofanyintolueneunreacted(reactionmaterialsolvent),volatilityandensuresfacilitates(bp79thatAPIwasinvestigatedanalyticallyto ndtheactuallevelstherewillbenocarryoverintothe nalAPIand,importantly,parisonofpredictedandmeasuredpurgefactorsforAZD9056process

identityofpotentiallyimpuritygenotoxicstagereactivitysolubilityvolatilityfactor/stagepurgeoverallpurgefactormeasuredfactorpurge

AZD9056aldehydecrudenonisolated(freebase)10(moderate)a1b1involatile10

AZD9056aldehydecrudeisolated1unreactive101involatile10

AZD9056aldehydepure1unreactive101involatile10

AZD9056aldehyde10×10×10=112000AZD9056chlorideccrudenonisolated(freebase)N/AN/AN/AN/A

AZD9056chloridecrudeisolated1unreactive11involatile1

AZD9056chloridepure1unreactive3d1involatile3

AZD9056chloride1×3=10

isopropylchloridecrudenonisolated(freebase)N/AN/AN/AN/A

isopropylchloridecrudeisolated11010100

isopropylchloridepure11010100

isopropylchloride100×100=1000038500a

toAlthoughconversionessentiallygoesto

d100).bAlthoughhighlysoluble,thealdehydecompletion,isnotthepurgedreactionbecauseisonlythemoderatelycrudeisnotfast;isolated.hence,acChlorideconservativeimpurity gureisofgenerated10wasappliedinthe(ascrudeopposedstage.Solubility,althoughlow,isgreaterthanthatofAZD9056HClsalt.

parisonofpredictedandmeasuredpurgefactorsfornitro-anilineimpurity(I)

identity/structureofGIofconcernstagedetailsreactivity(HL==100,1)M=10,solubilityL(F==1)10,M=3,volatility(LH==1)10,M=3,purge/stagefactormeasuredfactorpurgenitro-anilinereduction1001nonisolated1100

nitro-anilinecyclisation10101100not<5detectedppm

overallcalculatedpurgefactor10,000

Scheme5.Synthesisofomeprazolepotassium

saltScheme6.SynthesisofanAPIinvolvingtheuseofhydrazineandasubstitutedhydrazineintermediate(compound

2)

counting’approachtaken.Againariskassessmentwasmade,andthisgaveapredictedpurgefactorof10,000(comparedtoameasuredpurgefactorof40,000).Putinthecontextofthelevelformed(5%=50,000ppm)thenwithapredictedpurgefactorof10,000,thiswouldindicateapredictedlevelof<5ppm,apredictionsupportedbytheactualanalyticalresults.3.3.3.AZD9056Chloride.Thisexampleisperhapsthemost

interestingasitprovidestestamenttotheabilityofthisriskassessmenttooltogainacriticalinsightintotheprocess.Inthisinstancetheimpurityformedisunreactive,relativelyinsoluble,andnonvolatile,suchthatthecalculatedpurgefactorof3indicatesthattheprocessisthereforeunlikelytoe ectivelypurgethisimpurity.Thisagaintallieswiththeobservedfactorof10.Thus,inthisinstancethepredictionindicatestheneedforadditionalcontrol,eithertocontrolformationoftheimpuritythroughprocesscontrolormodi cationoftheprocesstofacilitateitsremoval.underpredictsthepurgecapacityoftheprocessbyafactorof3.4.CaseStudy4:GIinaRegisteredStartingMaterial.

10.Hadalessconservativevalueof100beenappliedtotheThefollowingexampleisderivedfromthesynthesisofareactivetermforthereductiveaminationstep,thentheregisteredstartingmaterialandrelatestoanassessmentofthepredictivevaluewouldhavematchedtheactualobservedvalue.riskofGIsbeingpresentwithinitasaresultofitssynthesis3.3.2.IsopropylChloride(IPC).Theexperimentalmeasure-(Scheme4).Theimpurityofconcernisthenitroprecursor(I)mentsshowedthatlevelsofIPCinsolutionexceeded5%w/w.InthisexampletheoverallcalculatedpurgefactorwasGiventhefactthatsuchahighlevelofaPGIisformedinthe10,000,thusindicatingalowprobabilityofcarryoverintothelatterstagesofthesynthesis,thiswouldgenerallybeastartingmaterial(<100ppm).Thispredictionwasshowntobesigni cantcauseforconcern.Thisiscertainlytruewerea‘stepcorrectwhentheactualpurgefactorwasmeasured,parisonofpredictedandmeasuredpurgefactorsfornitropyridylN-oxideimpurity(A)

identity/structureGIofconcernofstagedetails(H=reactivity100,L=1)M=10,(F=solubility10,M=3,(H=volatility

L=1)L10,=1)M=3,totalpermultiplestagemeasured(ppm)level

nitropyridyl(A)stagechloride1:formation11nonisolated11

nitropyridyl(A)stage2:coupling11nonisolated1120003000(basedspike)onnitropyridyl(A)stageformation3:oxidation/salt110110<1

parisonofpredictedandmeasuredpurgefactorsforhydrazineandsubstitutedhydrazineintermediate(compound2)

identity/structureofGIofconcernstagedetails(H=100,reactivityM=10,L=1)(F=10,solubilityM=3,L=1)(H=10,volatilityM=3,L=1)totalpermultiplestagemeasuredlevelhydrazinestagehydrazide1:formation100101-nodistillation1000

stage nal2:intermediate10031300

stagecyclisation/isolation3:10031300

overallclearancecalculated9×108<0.1ppm(LOD)

identity/structureofGIofconcernstagedetails(H=reactivity100,L=1)M=10,(Fsolubility=10,M=3,(Hvolatility

L=1)=L10,=1)M=3,totalpermultiplestagemeasuredlevel

intermediate2

stage nal2:intermediate1001011000

stagecyclisation/isolation3:10031300

overallclearancecalculated3×105<1ppm(LOD)

impurity(I)notbeingdetected(<5ppm)(Table8).Asthisfactoris9×108.ThisisborneoutbytheexperimentalresultsrelatestoanimpuritywithinastartingmaterialthatitselfisthatshowlevelsintheAPItobenotdetected(LOD0.1ppm).introducedintotheoverallsyntheticprocessatanumberofWithrespecttothereactiveintermediate2,despiteitscloserstagesawayfromtheisolatedAPI,itisproposedthattheuseofproximitytothe nalAPI,thecalculatedpurgefactorisagainthisriskassessmenttoolcouldbeusedtoquicklyevaluatethehigh,3×105.Thisismirroredintheexperimentaldatathatriskofcarryover,inthisinstanceintoastartingmaterial,andshowed

thuseliminatetheneedforspeci canalyticaltesting.4.5.CaseStudy5:GIinaRegisteredStartingMaterial

(Example2).Inthisexamplethespeci cconcernrelatesagain

toapotentialGI(A)withinastartingmaterial(I).Inthis■levelstobe<1ppmatLODlevel(seeTable10).CONCLUSIONAthoroughevaluationoftheriskposedbyGTIsisnowainstancetheimpurityofconcern(A)isanitropyridylN-oxidecrucialpartoftheoverallprocessofevaluatingthequalityofderivative(Scheme5).medicines.SuchanevaluationinvolvesassessingthepotentialWhatisinterestingwiththisspeci cexampleisthattheforcarryoveroftheGTI(s)inquestionatlevelsofconcernintoimpurityofconcern(A)isunreactiveinbothstages1and2oftheAPIand/ordrugproduct.Thiscontributiondescribesatheprocess(seeTable9).Furthermore,asneitherstageisnumberofwaysinwhichsuchanevaluationcanbeconducted,isolated,thenthereisnoopportunitytopurgetheimpurityincludingthenovelapproachofpurgefactorcalculation.throughremovalbasedonsolubility,andtheimpurityisnotTheadvantageofsuchanapproachoverotherempiricalvolatile.Hence,thecalculatedpurgefactoris1;thatis,instagesapproachesisthatitprovidesaquantitativeassessmentofthe1and2oftheprocessthereisunlikelytobeanysigni cantrisk,basedonaneasilyunderstoodandstandardizedscale.NotreductioninthelevelofimpurityA.Thiswasmirroredonlycanthisapproachbeusedtoprovidecompellingevidenceexperimentallywhenaspikeof3000ppm(equivalenttothetobackupachemicalevaluation,itcanalso,asshownthroughspeci cationlimitof0.3%)wasmadeintostartingmaterial(I),therealcasestudyexamples,driveabetterunderstandingofanalysisatstage2withinthenonisolatedintermediate(IV)GTI-relatedrisksuchthatattentionisultimatelyfocusedonshowedlevelstohavereducedtoonly2000ppm,afactorofanalyticalcontrolofthosespeci cGTIsthatposeanactuallessthan2.Subsequentanalysisofthe nalisolatedratherthanatheoreticalriskofpresenceinthe naldrugintermediateshowedthatlevelshadbeenreducedto<1substance.

ppm;thiswasduetothehighsolubilityoftheimpuritywithinthesolventsystememployedforthisstage.Acomparisonoftheexperimentalresulttothepredictedpurgefactor(forthisstage■AUTHORINFORMATIONCorresponding

avalueof10)illustratestheconservativenatureofthesolubility*E-mail:andrew.teasdale@Author

termwhenassessingtheriskofcarryover.Notes

4.6.CaseStudy6:Hydrazine.Inthisexampletheriskof

carryoverofahighlyreactive(nonelectrophilic)reagent,hydrazine,andareactiveintermediate,substitutedhydrazine(2),wasassessed,asshowninScheme6.■Theauthorsdeclarenocompeting nancialinterest.REFERENCESThisexampleillustrateswelltheadvantageofthisfocusedQ3A(1)(R2):InternationalImpuritiesConferenceinNewDrugonSubstancesHarmonisation;October(ICH).2006.GuidelineapproachtocalculationofGI-relatedrisk.Hydrazine,ahighlyreactiveknowncarcinogen,isusedinthisprocess,threestagesQ3B(2)(R2):InternationalImpuritiesConferenceinNewDrugonProductsHarmonisation;2006.(ICH).GuidelinefromtheAPI.Aclassicalstage-countingapproachbasedonanassumedpurgeof10perstagewouldindicatea1000-foldQ3C(3)

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