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1、CaptoTMadhereAnovelmultimodalanionexchangemediumforlargescalepurificationofmonoclonalantibodies2/GE/2024/7/27OutlineMonoclonalantibodytiterdevelopmentCaptoTMadheredesignedforMAbpurificationScreening/optimizationofloadingconditionsApplicationsDifferentselectivitycomparedtotraditionalionexchangersSele
2、ctiveremovalofaggregatesTwosteppurificationofIgG1fromCHOcellcultutesupernatantViralclearancestudyProcesseconomySummary3/GE/2024/7/27MonoclonalantibodytiterdevelopmentFed-batchtiterof2,7g/Lreportedalreadyin1997(Zhouetal)usingGSgeneexpressionsystem1to2g/Lconsideredtobecurrentindustrialstandard10g/Lest
3、imatedtobeachievedinnext5yearsMid80s0.05-0.1g/LMid90s0.5-1g/LCurrently2-6g/LFuture10g/L?4/GE/2024/7/27HighertiterimplicationsondownstreampurificationHighercelldensitiesMorecelldebrisMorecontaminantshostcellproteinsDNAvirusesdimers/aggregatesGreaterquantityofMAbperbatchSupernatantMinlogreductionHCP10
4、6ng/mg 6logsDNA106ng/pg6logsMuLVn/a20logsMAbaggregates2-25%1logs J.Bonnerjea, IBC Dublin Oct 23 20065/GE/2024/7/27RemovalofcontaminantsHightitersHighcontaminantlevelsNeedforstrongpostProteinApolishingmediumCaptoTMadhere6/GE/2024/7/27OutlineMonoclonalantibodytiterdevelopmentCaptoTMadheredesignedforMA
5、bpurificationScreening/optimizationofloadingconditionsApplicationsDifferentselectivitycomparedtotraditionalionexchangersSelectiveremovalofaggregatesTwosteppurificationofIgG1fromCHOcellcultutesupernatantViralclearancestudyProcesseconomySummary7/GE/2024/7/27CaptoTMadhereThedesignofCaptoTMadhere+N-Benz
6、yl-N-methylethanolamineCaptoTMHighflowagarose8/GE/2024/7/27Isastrongmultimodalanionexchangerdesignedfor:IntermediatepurificationandpolishingaftercaptureonProteinARemovalofLeachedProteinADimersandaggregtesHostcellproteinsVirusesNucleicacids.preferablyinflowthroughmode.Improvesproductivityandprocessec
7、onomyHighloadinflow-throughmode(100-300mgMAb/ml)ContaminantremovaltoformulationlevelintwochromatographicstepsCaptoTMadhere9/GE/2024/7/27Other2-stepprocesses2-stepprocessessuggestedby:WyethLonza(includingCelltech)Tanox2-stepprocessesallcontainthefollowingchromatographysteps:ProteinAAIEX10/GE/2024/7/2
8、7EngineeredProteinA1)Time02)18hour3)Control123rProtein ASuReligandStability inCHOcell lysate1)Time02)18hour3)Control123rProtein ASuReligandStability inCHOcell lysateStabilityCIPMabSelectTMMabSelectSuReTM0.1M NaOH15minStabilityCIPMabSelectTMMabSelectSuReTM0.1M NaOH15minEADCBProteinAcapturewithMabSele
9、ctSuReTMGhoseetal.,(2005)BiotechBioeng92,665-673GenericelutionconditionsGenericelutionconditions11/GE/2024/7/27HighlyproductivetwostepprocessMabSelectSuRePoolforFinalFiltrationUF/DFCaptoadhereVirusInactivation&FiltrationCellremovalCellculture12/GE/2024/7/27MabSelectSuReAIEXCaptoQCIEXCaptoSPoolforFin
10、alFiltrationUF/DFCaptoadhereCaptoadhereAIEXCaptoQ*VirusInactivation&FiltrationCellremovalCellculture&Thetoolboxconcept*WO 2004/076485 (Lonza)13/GE/2024/7/27OutlineMonoclonalantibodytiterdevelopmentCaptoTMadheredesignedforMAbpurificationScreening/optimizationofloadingconditionsApplicationsDifferentse
11、lectivitycomparedtotraditionalionexchangersSelectiveremovalofaggregatesTwosteppurificationofIgG1fromCHOcellcultutesupernatantViralclearancestudyProcesseconomySummary14/GE/2024/7/27FlowthroughmodeonCaptoTMadhereStartWashstartmAUNon-bindingPartialbindingAntibodyyield&ContaminantclearanceYIELDPURITYOPT
12、IMIZATION15/GE/2024/7/27DesignofExperimentsforoptimizationFactors(inputs)Load(x1),pH(x2),Conductivity(x3)Responses(outputs)Yield(Y1),Dimer/aggregates(Y2)HCP(Y3),ProteinA(Y4)YieldD/AProteinAHCPhttp:/16/GE/2024/7/27OptimizationofloadingconditionsDesignofExperimentsFACTORSETTINGLoadSetaccordingtogoal:7
13、5-300mg/mlConductivity”Normal”rangechosen:2-15mS/cmpHInitialexperimentsarerequiredGOALWithaloadof100mg/mlYield90%Dimer/Aggregates1%ProteinA5ppmHCP50ppm17/GE/2024/7/27OptimizationofloadingconditionsLowerpHlimitinthedesignpH5.95-SampleloadedatpH7.8-Load1mg/ml-ElutioninpHgradientfrom7.8to4.0lowerpHinDo
14、E:6.018/GE/2024/7/27pH6.0,2mS/cm(green)LowerpHlimitinDoE,6.0pH8.0,2mS/cm(blue)UpperpHlimitinDoE,8.0Sampleload75mg/mlOptimizationofloadingconditionsUpperpHlimitinthedesign19/GE/2024/7/27LoadpHCond.6875300215OptimizationofloadingconditionsTheDesignLinearandinteractionmodels20/GE/2024/7/27LoadpHCond.68
15、75300215OptimizationofloadingconditionsTheDesignQuadraticmodels21/GE/2024/7/276543210-1-2-3-4-5-6-7-8%Conductivity2mS/cmConductivity8.5mS/cmConductivity15mS/cmYieldLoadpHCondLoad*pHLoad*CondGoal:HighYieldinflowthroughpoolpHCondLoad22/GE/2024/7/27Dimer/aggregateclearanceGoal1%LoadpH*pHpH-101%Goal:Low
16、dimer/aggregateconcinflowthroughpoolpHCondNotsignificantLoadD/Aconcinstartmaterial:3.2%23/GE/2024/7/27HCPclearanceGoal50ppm5-543210-1-2-3-4ppmLoadCondpHLoad75mg/mlLoad187.5mg/mlLoad300mg/mlGoal:LowHCPconcinflowthroughpoolpHCondLoadHCPconcinstartmaterial:206ppm24/GE/2024/7/27ProteinAclearanceGoal5ppm
17、-543210-1-2-3-4ppmpH5CondpH*CondGoal:LowProteinAconcinflowthroughpoolpHCondLoadNotsignificantProteinAconcinstartmaterial:36ppmYIELDConductivity15mS/cmDimer/AggregatesConductivity15mS/cmHCPLoad300mg/mlProteinALoad300mg/mlSweetspotanalysisGivesthearea(red)wherethethegoalsarefulfilled.Ataloadofatleast1
18、00mgMAb/mlresin:YieldinFT90%D/AinFT1%ProteinAinFT5ppmHCPinFT50ppmDoESummary26/GE/2024/7/27OutlineMonoclonalantibodytiterdevelopmentCaptoTMadheredesignedforMAbpurificationScreening/optimizationofloadingconditionsApplicationsDifferentselectivitycomparedtotraditionalionexchangersSelectiveremovalofaggre
19、gatesTwosteppurificationofIgG1fromCHOcellcultutesupernatantViralclearancestudyProcesseconomySummary27/GE/2024/7/27Differentselectivitycomparedtotraditionalionexchangers CaptoTMadhereElutionpH:6.2-4.9CaptoQElutionpH:9.96.2Elutionorder:MAb4MAb3MAb5MAb2MAb1pHgradientelutionof5MAbs28/GE/2024/7/270200400
20、6008001000mAU0.05.010.015.0mlClarifiedNS0cellculturesupernatant1.3mgIgG1/mlpI7.58.4CaptureonMabSelectSureElutionpoolD/Aconcentration6%6%aggregatesSuperdex20010/300Selectiveremovalofaggregates29/GE/2024/7/27Optimizationofloadingconditions1)Bindingmode,ElutionbypHgradient2)Flow-throughmode+2pHunitspH5
21、.5pH7Load100200mg/mlpH5.57Conductivity1050mS/cmOptimizationbyDoE050100150200250mAU4.06.08.010.012.0102030ml050100150200250mAU4.06.08.010.012.0102030ml5.130/GE/2024/7/27EffectofpHandconductivityonyieldYieldControlledbypHIndependentofConductivity(1050mS/cm)Load(100200mg/ml)31/GE/2024/7/27EffectofpH,co
22、nductivityandloadonD/A-clearanceD/AclearanceinfluencedbypHHigherpHCondHighercondLoadLowerloadInteractionpHcondConditionsforD/A-clearancepH6.5andconductivity30mS/cmHighD/Aclearance32/GE/2024/7/27Sampleload265mg/mlD/Acontentwas6%Regeneration0.1MHAcpH3.0050010001500200025003000mAU0.05.010.015.020.025.0
23、30.035.0mlInjectWashElutionCIPSelectiveremovalofaggregates33/GE/2024/7/27SEConSuperdex20010/300FlowthroughfractionsBoundmaterialD/Acontent60%D/AadsorbedtoCaptoTMadhere!34/GE/2024/7/27DimersandaggregatesclearanceSampleload120mg/mlD/Areducedfrom6to0.6%(10-fold)AccumulatedD/A1%atsampleloadupto200mg/mlT
24、otalyieldofmonomer94%CaptoadherehasahighpotentialtoselectivelyremoveD/AfromMAbpreparations*D/A=dimersandaggregates*35/GE/2024/7/27ViralclearancestudyIgG1poolfromMabSelectSuRespikedwithstocksolutionsof:MVM(MinuteVirusofMice)SinglestrandedDNAvirusNon-enveloped,20-26nmMuLV(MurineleukemiaVirus)Singelstr
25、andedRNAEnveloped,80-110nmAppliedtoCaptoadhereinflowthroughmode36/GE/2024/7/27VirusclearanceCaptoadhereVerygoodlog10reductionfactorsevenforconditionswheretraditionalionexchangersdonotwork!37/GE/2024/7/27TwosteppurificationofIgG1MabSelectSuReAIEX,CaptoQCIEX,CaptoSPoolforFinalFiltrationUF/DFCaptoadher
26、eAIEX,CaptoQ*CaptoadhereVirusInactivation&FiltrationCellremovalCellculture*WO 2004/076485 (Lonza)38/GE/2024/7/27CaptureonMabSelectSuReIntermediatewashcontainedmainlyD/AandHCPHCPreducedfrom128300to55ppmProteinAconcentration1ppmAggregatecontent0.7%.Wash25mMNaP,5%isopropanolAnnaGrnbergetal.“Screeningan
27、doptimizationofbufferconditionsforProteinAchromatographyusinga96-wellformat“IBCconferenceinSanFrancisco(2006)39/GE/2024/7/27ThreestepprocessMabSelectSuReCaptoSCaptoQ(bind/elute)(flow-through)40/GE/2024/7/27TwostepprocessMabSelectSuReCaptoadhere41/GE/2024/7/27Boththetwo-andthreestepprocesssuccessfull
28、ypurifiedMAbtoimpuritylevelswellacceptableforformulation*MabSelectSuRe+CaptoS+CaptoQ*MabSelectSuRe+CaptoadhereTwovsthreesteppurification42/GE/2024/7/27CaptoadhereclearanceProteinAeluateCaptoadhereClearanceHCP500-10000ppm2-3logsProteinA5-100ppm1-2logs*DNA10-1000ppb3-4logs*MuLVMVMn/a3.5-5.0logs5.5-6.5
29、logsMAbaggregates1-10%1-2logs*Inmostcasesbelowlimitofquantification,1ppb43/GE/2024/7/27Twosteppurificationwith90-98%yieldProteinAandCaptoadhereMAbCelllinepILoadingconditionsHCP50ppmPrA5ppmD/A1%YieldpHCondLoad1CHO978187.512(206)0(36)0.5(3.3)902CHO8.3-8.95.531502(10)2(260)0.6(2.1)953NS07.5-8.4621509(8
30、5)0(0)0.8(1.5)954SP2/07.7-8.072010030(500-2000)0(1)0.15(0.14)915CHO8.87.5202007.5(38)0(1)50%Processtimedownto2-3daysWorkingvolume10.000LTiter5g/L051015202530MabSelect+SPandQSepharoseFastFlowReferenceprocess1MabSelectSuRe+CaptoS+CaptoQReferenceprocess2MabSelectSuRe+CaptoQ+Captoadhere3-stepprocessMabS
31、electSuRe+Captoadhere2-stepprocessProductioncostcontributionUSD/kg010203040506070DSPprocesstimehTime49/GE/2024/7/27OutlineMonoclonalantibodytiterdevelopmentCaptoadheredesignedforMAbpurificationScreening/optimizationofloadingconditionsApplicationsDifferentselectivitycomparedtotraditionalionexchangers
32、SelectiveremovalofaggregatesTwosteppurificationofIgG1fromCHOcellcultutesupernatantViralclearancestudyProcesseconomySummary50/GE/2024/7/27SummaryCaptoadhereimprovesprocesseconomyby:Contaminantremovaltoformulationlevelsinonepost-ProteinAstepWideoperationalwindowofpHandconductivitySavingsintimeandopera
33、tingcostswithtwostepchromatographicprocessMabSelectSuRePoolforFinalFiltrationUF/DFCaptoadhereVirusInactivation&FiltrationCellremovalCellculture51/GE/2024/7/27StickwithCaptoTMadhereDesignedforMAbpurificationSelectiveremovalofkeycontaminantsAllowshighlyproductive2-stepprocess52/GE/2024/7/GEHealthcareB
34、io-SciencesAB,aGeneralElectricCompany.GEHealthcareBio-SciencesABBjrkgatan3075184UppsalaSwedenMabSelect,MabSelectSuRe,Capto,SuperdexandSepharosearetrademarksofGEHealthcarecompanies.GE,imaginationatworkandGEmonogramaretrademarksofGeneralElectricCompany.Allgoodsandservicesaresoldsubjecttothetermsandcon
35、ditionsofsaleofthecompanywithinGEHealthcarewhichsuppliesthem.GEHealthcarereservestheright,subjecttoanyregulatoryandcontractualapproval,ifrequired,tomakechangesinspecificationsandfeaturesshownherein,ordiscontinuetheproductdescribedatanytimewithoutnoticeorobligation.ContactyourlocalGEHealthcarerepresentativeforthemostcurrentinformation.2007GeneralElectricCompanyAllrightsreserved.
