capto adhere纯化工艺DOE实验设计

CaptoTMadhereA

novelmultimodalanionexchangemediumfor

largescalepurificationofmonoclonalantibodies

OutlineMonoclonalantibodytiterdevelopmentCaptoTMadheredesignedforMAbpurificationScreening/optimizationofloadingconditionsApplications Differentselectivitycomparedtotraditionalionexchangers Selectiveremovalofaggregates TwosteppurificationofIgG1fromCHOcellcultutesupernatant ViralclearancestudyProcesseconomySummaryMonoclonalantibodytiterdevelopmentFed-batchtiterof2,7g/Lreportedalreadyin1997(Zhouetal)usingGSgeneexpressionsystem≥1to2g/Lconsideredtobecurrentindustrialstandard10g/Lestimatedtobeachievedinnext5yearsMid80’s0.05-0.1g/LMid90’s

0.5-1g/LCurrently

2-6g/LFuture

10g/L?HighertiterimplicationsondownstreampurificationHighercelldensitiesMorecelldebrisMorecontaminants hostcellproteins DNA viruses dimers/aggregatesGreaterquantityofMAbperbatchSupernatantMinlogreductionHCP~106ng/mg~6logsDNA106ng/pg~6logsMuLVn/a20logsMAbaggregates2-25%>1logs

J.Bonnerjea,IBCDublinOct232023RemovalofcontaminantsHightitersHighcontaminantlevelsNeedforstrongpostProteinApolishingmediumCaptoTMadhereOutlineMonoclonalantibodytiterdevelopmentCaptoTMadheredesignedforMAbpurificationScreening/optimizationofloadingconditionsApplications Differentselectivitycomparedtotraditionalionexchangers Selectiveremovalofaggregates TwosteppurificationofIgG1fromCHOcellcultutesupernatant ViralclearancestudyProcesseconomySummaryCaptoTM

adhere

ThedesignofCaptoTMadhere

+N-Benzyl-N-methylethanolamineCaptoTM

Highflowagarose

Isastrongmultimodalanionexchangerdesignedfor:

IntermediatepurificationandpolishingaftercaptureonProteinARemovalofLeachedProteinADimersandaggregtesHostcellproteinsVirusesNucleicacids

...preferablyinflowthroughmode.

Improvesproductivityandprocesseconomy

Highloadinflow-throughmode(100-300mgMAb/ml)ContaminantremovaltoformulationlevelintwochromatographicstepsCaptoTMadhereOther2-stepprocesses2-stepprocessessuggestedby:WyethLonza(includingCelltech)Tanox2-stepprocessesallcontainthefollowingchromatographysteps:ProteinAAIEXEngineeredProteinA1)Time02)18hour3)Control123rProteinASuReligandStabilityinCHOcelllysate1)Time02)18hour3)Control123rProteinASuReligandStabilityinCHOcelllysateStabilityCIPMabSelectTMMabSelectSuReTM0.1MNaOH15minStabilityCIPMabSelectTMMabSelectSuReTM0.1MNaOH15minEADCBProteinAcapturewithMabSelectSuReTMGhoseetal.,(2023)BiotechBioeng92,665-673GenericelutionconditionsGenericelutionconditions

HighlyproductivetwostepprocessMabSelectSuRe™PoolforFinalFiltrationUF/DFCapto™adhereVirusInactivation&FiltrationCellremovalCellcultureMabSelectSuRe™AIEXCaptoQCIEXCaptoSPoolforFinalFiltrationUF/DFCapto™adhereCaptoadhereAIEXCapto

Q*VirusInactivation&FiltrationCellremovalCellculture&Thetoolboxconcept

*WO2023/076485(Lonza)OutlineMonoclonalantibodytiterdevelopmentCaptoTMadheredesignedforMAbpurificationScreening/optimizationofloadingconditionsApplications Differentselectivitycomparedtotraditionalionexchangers Selectiveremovalofaggregates TwosteppurificationofIgG1fromCHOcellcultutesupernatant ViralclearancestudyProcesseconomySummaryFlowthroughmodeonCaptoTMadhereStartWashstartmAUNon-binding

PartialbindingAntibodyyield&ContaminantclearanceYIELD

PURITYOPTIMIZATIONDesignofExperimentsforoptimizationFactors(inputs)Load(x1),pH(x2),Conductivity(x3)Responses(outputs)Yield(Y1),Dimer/aggregates(Y2)HCP(Y3),ProteinA(Y4)YieldD/AProteinAHCPOptimizationofloadingconditions

DesignofExperimentsFACTORSETTINGLoadSetaccordingtogoal:75-300mg/mlConductivity”Normal”rangechosen:2-15mS/cmpHInitialexperimentsarerequiredGOALWithaloadof≥100mg/mlYield>90%Dimer/Aggregates≤1%ProteinA≤5ppmHCP≤50ppmOptimizationofloadingconditions

LowerpHlimitinthedesignpH5.95SampleloadedatpH7.8Load1mg/mlElutioninpHgradientfrom7.8to4.0lowerpHinDoE:6.0pH6.0,2mS/cm(green)LowerpHlimitinDoE,6.0pH8.0,2mS/cm(blue)UpperpHlimitinDoE,8.0Sampleload75mg/mlOptimizationofloadingconditions

UpperpHlimitinthedesignLoadpHCond.6875300215Optimizationofloadingconditions

TheDesignLinearandinteractionmodelsLoadpHCond.6875300215Optimizationofloadingconditions

TheDesignQuadraticmodels6543210-1-2-3-4-5-6-7-8%Conductivity2mS/cmConductivity8.5mS/cmConductivity15mS/cmYield

LoadpHCondLoad*pHLoad*CondGoal:HighYieldinflowthroughpoolpHCondLoad

Dimer/aggregateclearanceGoal≤1%LoadpH*pHpH-101%Goal:Lowdimer/aggregateconcinflowthroughpoolpHCondNotsignificant

LoadD/Aconcinstartmaterial:3.2%HCPclearanceGoal≤50ppm5-543210-1-2-3-4ppmLoadCondpHLoad75mg/mlLoad187.5mg/mlLoad300mg/mlGoal:LowHCPconcinflowthroughpoolpHCondLoadHCPconcinstartmaterial:206ppmProteinAclearanceGoal≤5ppm-543210-1-2-3-4ppmpH5CondpH*CondGoal:LowProteinAconcinflowthroughpoolpHCondLoadNotsignificant

ProteinAconcinstartmaterial:36ppmYIELDConductivity15mS/cmDimer/AggregatesConductivity15mS/cmHCPLoad300mg/mlProteinALoad300mg/mlSweetspotanalysisGivesthearea(red)wherethethegoalsarefulfilled.Ataloadofatleast100mgMAb/mlresin:YieldinFT>90%D/AinFT≤1%ProteinAinFT≤5ppmHCPinFT≤50ppmDoESummaryOutlineMonoclonalantibodytiterdevelopmentCaptoTMadheredesignedforMAbpurificationScreening/optimizationofloadingconditionsApplications Differentselectivitycomparedtotraditionalionexchangers Selectiveremovalofaggregates TwosteppurificationofIgG1fromCHOcellcultutesupernatant ViralclearancestudyProcesseconomySummaryDifferentselectivitycomparedtotraditionalionexchangers

CaptoTMadhereElutionpH:6.2-4.9

Capto™QElutionpH:9.9–6.2Elutionorder:MAb

4>MAb

3>MAb5>MAb2>MAb1pHgradientelutionof5MAbs02004006008001000mAU0.05.010.015.0ml

ClarifiedNS0cellculturesupernatant1.3mgIgG1/mlpI7.5–8.4CaptureonMabSelect™SureElutionpool

D/Aconcentration6%6%aggregatesSuperdex™20010/300SelectiveremovalofaggregatesOptimizationofloadingconditions1)Bindingmode,ElutionbypHgradient2)Flow-throughmode+2pHunitspH5.5pH7Load100–200mg/ml

pH5.5–7Conductivity10–50mS/cmOptimizationbyDoE050100150200250mAU4.06.08.010.012.0102030ml050100150200250mAU4.06.08.010.012.0102030ml5.1EffectofpHandconductivityonyield

YieldControlledbypHIndependentofConductivity(10–50mS/cm)Load(100–200mg/ml)EffectofpH,conductivityandload

onD/A-clearance

D/AclearanceinfluencedbypH HigherpHCond HighercondLoad LowerloadInteractionpH–condConditionsforD/A-clearancepH6.5andconductivity30mS/cm

HighD/AclearanceSampleload265mg/mlD/Acontentwas6%Regeneration0.1MHAcpH3.0050010001500202325003000mAU0.05.010.015.020.025.030.035.0mlInjectWashElutionCIPSelectiveremovalofaggregatesSEConSuperdex™20010/300

FlowthroughfractionsBoundmaterial

D/Acontent~60%D/AadsorbedtoCaptoTMadhere!

Dimersandaggregatesclearance

Sampleload120mg/ml→[D/A]reducedfrom6to0.6%(10-fold)Accumulated[D/A]<1%atsampleloadupto~200mg/mlTotalyieldofmonomer94%Capto™adherehasahighpotentialtoselectivelyremoveD/AfromMAbpreparations*D/A=dimersandaggregates*

Viralclearancestudy

IgG1poolfromMabSelectSuRe™spikedwithstocksolutionsof: MVM(MinuteVirusofMice) SinglestrandedDNAvirus Non-enveloped,20-26nm

MuLV(MurineleukemiaVirus) SingelstrandedRNA Enveloped,80-110nm

AppliedtoCapto™adhereinflowthroughmode

VirusclearanceCapto™adhereVerygoodlog10reductionfactorsevenforconditionswheretraditionalionexchangersdonotwork!

TwosteppurificationofIgG1MabSelectSuRe™AIEX,CaptoQCIEX,CaptoSPoolforFinalFiltrationUF/DFCapto™adhereAIEX,CaptoQ*Capto

adhereVirusInactivation&FiltrationCellremovalCellculture*WO2023/076485(Lonza)CaptureonMabSelectSuRe™IntermediatewashcontainedmainlyD/AandHCPHCPreducedfrom128300to55ppmProteinAconcentration<1ppmAggregatecontent0.7%.Wash25mMNaP,5%isopropanolAnnaGrönbergetal.“ScreeningandoptimizationofbufferconditionsforProteinAchromatographyusinga96-wellformat“IBCconferenceinSanFrancisco(2023)ThreestepprocessMabSelectSuRe™

→

Capto™S→Capto™Q(bind/elute)(flow-through)TwostepprocessMabSelectSuRe™

→

Capto™adhereBoththetwo-andthreestepprocesssuccessfullypurifiedMAbtoimpuritylevelswellacceptableforformulation*MabSelectSuRe™+Capto™S+Capto™Q**MabSelectSuRe+CaptoadhereTwovsthreesteppurificationCapto™adhereclearanceProteinAeluateCapto™adhereClearanceHCP500-10000ppm2-3logsProteinA5-100ppm1-2logs*DNA10-1000ppb3-4logs*MuLVMVMn/a3.5-5.0logs5.5-6.5logsMAbaggregates1-10%1-2logs*Inmostcasesbelowlimitofquantification,<1ppb

Twosteppurificationwith90-98%yield

ProteinAandCapto™adhereMAbCelllinepILoadingconditionsHCP<50ppmPrA<5ppmD/A<1%YieldpHCondLoad1CHO~978187.512(206)0(36)0.5(3.3)902CHO8.3-8.95.531502(10)<2(260)0.6(2.1)953NS07.5-8.4621509(85)0(0)0.8(1.5)954SP2/07.7-8.072010030(500-2023)0(<1)0.15(0.14)915CHO8.87.5202007.5(38)0(<1)<0.1(0.7)92Valueswithinparenthesiscorrespondtotheinitiallevels(startvalues)MAb6(CHOantibodyfeed):HCPwasreducedfrom9000ppmto2ppmwith98%yield!OutlineMonoclonalantibodytiterdevelopmentCapto™adheredesignedforMAbpurificationScreening/optimizationofloadingconditionsApplications Differentselectivitycomparedtotraditionalionexchangers Selectiveremovalofaggregates TwosteppurificationofIgG1fromCHOcellculturesupernatant ViralclearancestudyProcesseconomySummaryProcesseconomystudyThemodelprocesscoverstheunitproceduresfromharvestingtofinalformulation

One10,000Lfermentor Titer5g/L Resinsandmembraneslistprices Laborsetto$69/h Buffercost,average,setto$2.0/LProcesseconomy,templateMabSelectSuRe™Capto™adhereSuperProDesignerIntelligen,Inc.Harvest/clarificationVirusinactivationVirusfiltrationUF/DFSterilefiltrationCellcultureProcesseconomyandcostofproductionChromatographyresinLoad(g/L)Yield(%)Lifetime(cycles)CV(L)Cycles/batchTotaltime(h)Unitproductioncost(USD/kg)Twostepprocess

MabSelectSuRe™28922003814

Capto™adhere16095502491279607Threestepprocess

MabSelectSuRe28922003814

CaptoQ160965024912911305Captoadhere16095502271

Referenceprocess1

MabSelect24901004444

SPSepharose™FF24905040044318121QSepharose™6096505081Referenceprocess2MabSelectSuRe28922003814CaptoS96905040912611130CaptoQ16096502211

Combinationeffect,usemodernresins

(capacity,flow,andlifetimeimprovements)Costreduction>50%Processtimedownto2-3daysWorkingvolume10.000LTiter5g/L051015202530MabSelect™+SPandQSepharose™FastFlowReferenceprocess1MabSelectSuRe™+Capto™S+CaptoQReferenceprocess2MabSelectSuRe™+CaptoQ+Captoadhere3-stepprocessMabSelectSuRe+Captoadhere2-stepprocessProductioncostcontribution[USD/kg]010203040506070DSPprocesstime[h]TimeOutlineMonoclonalantibodytiterdevelopmentCapto™adheredesignedforMAbpurificationScreening/optimizationofloadingconditionsApplications Differentselectivitycomparedtotraditionalionexchangers Selectiveremovalofaggrega