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PublishedonUSPMedicinesCompendium()TrastuzumabFinalAuthorizedVersion1.0ANTI-HER2LIGHTCHAIN1DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECANTI-HER2HEAVYCHAIN1EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKC6470H10012N1726O2013S42Molecularweight,approx.1,45,423DaImmunoglobulinG1(human-mousemonoclonalrhuMAbHER2³1-chainantihumanp185c-erbB2receptor),disulfidewithhuman-mousemonoclonalrhuMAbHER2lightchain,dimer[180288-69-1].TrastuzumabisarecombinantDNA-derived,humanizedmonoclonalantibody(IgG1kappa)thatcontainshumanframeworkregionswiththecomplementarity-determiningregionsofamurineantibody(4D5)thatbindstoHER2.Thehumanizedantibodyselectivelybindswithhighaffinityinacell-basedassay(Kd=5nM)totheextracellulardomainofthehumanepidermalgrowthfactorreceptorprotein.TherecombinantproteinisproducedinCHOcellculture.Performance-BasedMonograph(Containstests,procedures,andacceptancecriteriaforthematerialundertest.Italsoincludesthecriteria-basedprocedurestodemonstratethatanAcceptableProcedureisequivalenttotheReferenceProcedures.)DEFINITIONTrastuzumabcontainstherecombinantDNA-derivedhumanizedmonoclonalIgG1kappaantibodyhavingameasuredpotencyofNLT80.0%andNMT125.0%ofthestatedpotencyinalowbioburdensolution.IDENTIFICATIONA.BIOASSAYStandardsolution:USPTrastuzumabRSinanappropriatediluentSamplesolution:TrastuzumabdilutedinanappropriatediluentsimilartothatoftheStandardsolution.SystemperformancerequirementsandAnalysis:ProceedasdirectedintheAssayforPotency.Acceptancecriteria:ThedoseresponseoftheSamplesolutionshouldbesignificantwithdefinedlowerasymptoteandupperasymptoteandsimilartothatoftheStandardsolution.Ratioofthehighestresponseandlowestresponseofthedoseresponsecurveshouldbee1.3.B.PEPTIDEMAPPINGUseachromatographicprocedure.(SeeBiotechnology-DerivedArticlesPeptideMapping1055.)AnalyzethematerialtobetestedbyasuitabletechniquecapableofresolvingpeptidesgeneratedfromaTrypsindigest.ThedigestisconductedunderreducingconditionsandprovidesNLT80%digestion.Theanalyticalmethodusedprovidesaminimumof90%coverageofthesequence.Standardsolution:DigestanddiluteaquantityofUSPTrastuzumabRSinanappropriatediluent.Samplesolution:DigestanddiluteaquantityofTrastuzumabinanappropriatediluenttoobtainanominalconcentrationoftrastuzumabsimilartothatoftheStandardsolution.Controlsolution:Digestanddiluteaquantityofanappropriatenegativecontrol(non-trastuzumabmonoclonalantibody)inanappropriatediluenttoobtainanominalconcentrationsimilartothatofStandardsolution.[NOTEThedigestsdescribedintheStandardsolution,Samplesolution,andControlsolutionareconductedatthesametime,usingthesamestockandconcentrationofreagents.]Analyticalsystem:UseaprocedurevalidatedasdescribedinMCgeneralchapterAssessingValidationParametersforReferenceandAcceptableProcedures10.SystemperformancerequirementsSpecificity:IntheprofileoftheStandardsolution,CDRregions1,2,and3shouldbeidentifiedusingasuitableprocedure.ThepeptideprofileoftheControlsolutionisdistinctlydifferentfromthatoftheStandardsolution.AnalysisSamples:StandardsolutionandSamplesolutionThepeptideprofilesoftheStandardsolutionarevisuallycomparedtotheSamplesolution.Acceptancecriteria:TheprofileoftheSamplesolutioncorrespondstotheprofileoftheStandardsolution.TherelativeretentiontimeofthepeakscorrespondingtotheCDRregions(withrespecttoareferencepeak)intheSamplesolutionshoulddifferfromtheStandardsolutionbyNMT±0.03.C.ISOELECTRICFOCUSINGAnalyzeTrastuzumabusinganisoelectrophoreticfocusingprocedureusingabroadrangeampholyte(isoelectricpoint(pI)rangeof3.010.0).(SeeCapillaryElectrophoresis1053).Standardsolution:USPTrastuzumabRSinanappropriatediluentSamplesolution:DiluteaquantityofTrastuzumabinanappropriatediluenttoobtainanominalconcentrationsimilartothatoftheStandardsolution.Controlsolution:Diluteaquantityofanappropriatenegativecontrol(non-trastuzumabmonoclonalantibodywithknownpIrangebetween7.5and10.0)inanappropriatediluenttoobtainanominalconcentrationsimilartothatoftheStandardsolution.[NOTEThepIofnegativecontrolshouldnotfallbetween8.5and9.0.]Analyticalsystem:Useavalidatedprocedure.RefertoMCgeneralchapterAssessingValidationParametersforReferenceandAcceptableProcedures10.(Althoughgeneralchapter10isdirectedtochromatographicmethods,conceptsintheguidelinearegeneral.)SystemperformancerequirementsSpecificity:ThepIoftheStandardsolutionisbetween8.5and9.0.ThepIoftheControlsolutionshouldbedifferentfromthepIoftheStandardsolution.AnalysisSamples:StandardsolutionandSamplesolutionComparethepIfromth