FingerprintyourbioprocessformorerobustproductionAlesStrancar,August 2017
AboutBIASeparations
• Incorporated in September 1998 in Ljubljana, Slovenia
• An OEM partnership with Agilent (former HP) in 2008
• In 2011 moved to new dedicated facility in Ajdovščina
• In 2012 strategic partnership with SDK Corporation (Shodex), 9B USD multinational company with HQ in Japan
• In 2016/2017 first projects to supply CIM product for biopharmaceutical drug manufacturing
ConvectiveInteractionMedia(CIM®)Pre-packedmonolithiccolumns
CIMac™AnalyticalandCIMmultus™/CIM®Preparativecolumns
Services,ProcessdevelopmentandTechnicalSupportDevelopmentofprocessesandmethodsfor
separation/concentration/purificationoflargebiomolecules
Customimmobilisations,productdevelopment
ProcessanalyticaltechnologyAt-linePATHPLCsuiteforimprovingprocesscontroland
understanding
BIASeparations productsandservices
BioprocessKnowledgePackages
Basedon20yearsofexperiencewithover500bioprocessprojects,BIASeparationsispleasedtoofferBioprocessKnowledgePackages thatincludepublished/unpublisheddata,andexperience-based,expert recommendationsfor purificationandanalyticalmethodsthatutilizeour
uniquemonolithchromatographycolumnsandotherbest-in-classtechnologiestoenabledevelopmentofthemostefficientandcost-effectivebioprocesspossibleforyour
biomolecule.
(Pleasenote:eachpurificationstrategymayvarybasedonthespecificbiomolecule,biologicdrugspecificationsandupstreamproductionmethods.BioprocessKnowledgePackagesaremadeavailableto
potentialclientsonaroyalty-freebasis)
BioprocessKnowledgePackages available
• BioprocessKnowledgePackage-pDNA(worksfor>30kbp)
• BioprocessKnowledgePackage-mcDNA
• BioprocessKnowledgePackage-RNA
• BioprocessKnowledgePackage-Adenovirus
• BioprocessKnowledgePackage-AAV(allserotypes)
• BioprocessKnowledgePackage-Fluvirus(allserotypes)
• BioprocessKnowledgePackage-IVIG
• BioprocessKnowledgePackage-IgM
BIASeparationsState-of-the-ArtProductionFacility>30MUSD investment
Certifications&Approvals• DMFforDEAE,QAandSO3andC4HLDCIM®monolithswere
filed,otherspending• Partnersaudits(Baxter,Novartis,Octapharma,Boehringer
Ingelheim,Teva,Agilent,.....)• FDAaudited(accordingtoUSAGMPregulations)• JAZMPaudited(accordingtoEUGMPregulations)• ISO9001:2008
IP• 4USpatentsandtheirforeignequivalentsgranted,more
pending:– CIM®technologyandmanufacturing– Differentgeometriesincludingscale-up
ConvectiveInteractionMedia(CIM®)monolithic
columns
Novelapproach– Monolithiccolumns:• Convectivemasstransport– flow
independentresolution andcapacity–veryfastprocesses
• Accessiblesurfaceforlargemolecules– highcapacity
• Laminarflow- Noshearforces– betteryieldsofe.g.IgM
Traditionalapproach- Porousparticle:• Diffusivemasstransport– slow
processorlowerresolution• Porestoosmall– verylowcapacity• Countercurrentflow– shearforces
– loweryields
AdvantagesofCIM® monolithicresins(membraneis„thinsliceofthemonolith“)
1mL 8mL 80mL 800mL 8L 40LI.D.(mm) 6.4 6.5 16.2 65 243 636O.D.(mm) 18.3 14.4 33 105 285 680
Thickness(mm) 5.95 3.95 8.4 20 21 22
DimensionsofCIM®radialmonoliths
Tubularformatenablesshortmonolithiccolumndesignatlabandindustrialscale
80,800,and8000mlCIMmonoliths
IntroductionofcompositematerialstocombineadvantagesofSSandplastics
• Epoxythermosetcomposite• Re-enforcedwithcarbonfibers• Coatedpin-holefreewith-
USPClassVIParyleneC
• Disposable butmultiuse• Stainlesssteelperformancecharacteristics• cGMPcompliant
allowsforrobustcontinuousoperations
CIMmultusTM compositematerials– matchingstainlesssteelcharacteristics
BUT:• 3timescheaper• 5timeslighter• allowforpre-packedcolumntransport• customer decidestousedisposable
columnassingleormultiuseunit
MainApplications– moleculetype
CIM®Columns
Viruses&VLPs
PlasmidDNA
DNAdepletion
Largeproteins
Endotoxins
BindingcapacitiesofCIM®columnsMolecules Dynamicbindingcapacity
influenza 2E+12 vp/mL
T7phage 1E+13pfu/mL
M13phage 4.5E+13pfu/mL
lambdaphage 1E+13pfu/mL
PRD1phage 6E+13pfu/ml
adenoviruses 2E+12vp/mL
baculovirus 2.4E+11pfu/ml
pDNA 8mg/mL
genomic DNA 15mg/mL
IgM 25– 50mg/mL
endotoxins >115mg/mL
MembraneversusCIM®monolithproductionofcanineadenovirusType2– yielddoubled
Fernandes,Petal,Bioprocessdevelopmentforcanineadenovirustype2vectors,GeneTherapy(2012),1–8
Membrane versusCIM®monolith productionoflentiviralvector - yielddoubled
V.Bandeiraetal.,DownstreamProcessingofLentiviralVectors:ReleasingBottlenecks,HumanGeneTherapyMethods23:1-9(August2012)
EvaluationofdifferentsupportsforpurificationofliveinfluenzaA - yielddoubled
Averagevalues QA monolith Q membraneQ porousparticles
semi-affinityporousparticles
VirusRecovery 54% 35% 35% 27%
DNADepletion 96% 95% 95% 91%
ProteinDepletion 95% 94% 98% 99%
DynamicBindingCapacity
10.3 log10TCID50/mLSupport
10.3log10TCID50/mLSupport
9.0 log10TCID50/mLSupport
8.4log10TCID50/mLSupport
Maurer etal.,PurificationofBiologicalProducts,Waltham,MA/USA,2007
50%betterrecoveryresultsine.g.1,5Mdosesofvaccineinsteadof1Mdoses,atthesamecostsoftheprocess=0,5Mdosesarepureprofit
PlasmidDNApurificationusingCIM®DEAEcolumns: 15-foldincreaseinproductivity
Urthaleretal.,J.Chrom. A,1065 (2005),93-106
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CIM® DEAE Q Ceramic Hyper D 20 Fractogel EMD DEAE (S) Source 30 Q Toyopearl DEAE 650-M DEAE Sepharose
CIM®DEAEbindingcapacity=~8 mg/ml
BoehringerIngelheim: „15-foldincreaseinproductivity“• Highbindingcapacityatrelevantflowrates• Highelutionconcentration- pDNAelutedinlowervolume(importantforSEC!)• Fastprocess(noproductlossduetooxidativedegradationorenzymaticattack)
UsedforCPIIItrials
EconomicbenefitforthecustomerusingCIM®MonolithPlasmidDNApurificationpack
1mlCIMmonolith– BIASep ParticlebasedCalculations
Buffer 76,3mlbuffer/mgpDNA
Time 23,6min/mgpDNA
Recovery 85%
Purity cGMP gradeCosts usingcolumnfor1runQuantityofpurifiedpDNA 5,10mgPDNA
€(Columncosts) 114€/mgpDNA
€(Column+buffer) 114€/mgpDNA
€ (column+buffer+work) 123€/mgpDNACostsusingcolumnsfor10runsQuantityofpurifiedpDNA 51mgpDNA
€(Columncosts) 11,4€/mg pDNA
€(Column+buffer) 11,8€/mgpDNA
€ (column+buffer+work) 21,1 €/mgpDNACostsusingcolumnsfor20runsQuantityofpurifiedpDNA 102mgpDNA
€(Columncosts) 5,7€/mg pDNA
€(Column+buffer) 6,1€/mgpDNA
€ (column+buffer+work) 15,4 €/mgpDNA
CalculationsBuffer 108,0 mlbuffer/mgpDNA
Time 70,0min/mgpDNA
Recovery 79%
Purity cGMP gradeCosts usingcolumnfor1runQuantityofpurifiedpDNA 4mgPDNA
€(Columncosts) 227€/mgpDNA
€(Column+buffer) 228 €/mgpDNA
€ (column+buffer+work) 257€/mgpDNACostsusingcolumnsfor10runsQuantityofpurifiedpDNA 40 mgpDNA
€(Columncosts) 23€/mg pDNA
€(Column+buffer) 24€/mgpDNA
€ (column+buffer+work) 53€/mgpDNACostsusingcolumnsfor20runsQuantityofpurifiedpDNA 79mgpDNA
€(Columncosts) 11€/mg pDNA
€(Column+buffer) 12€/mgpDNA
€ (column+buffer+work) 42€/mgpDNA
CIMmonolithic
columnsoffer3timescheaper
purificationcostsof pDNA
forgenetherapy
Aggregates,damaged
emptyfull
Separationsofempty,fullanddamagedAAV capsidsusingAnionexchangeCIMmultusTM QAcolumn
Column:CIMacTM SO3MfA:20mMacetate,pH4MfB:20mMHEPES+1MNaCl,pH8
IsfullAAVparticleonlyonespecies?CheckwithpHgradient- unmatchedresolution
FullAAV
SeparationofexosomesusingCIM®largechannelanionexchangecolumn– enablingfeature
Buzzietal.,MSS2014,Portoroz
PATfixTM
In-processcontrolHPLCsystemwithunique
software
WhatisProcessAnalyticalTechnology(PAT)?ProcessAnalyticalTechnologyis„asystemfordesigning,analyzing,andcontrollingmanufacturingprocessesthroughtimelymeasurementsofcriticalqualityandperformanceattributesofrawandin-processmaterialsandprocesses,withthegoalofensuringfinalproductquality.“(FDAPATGuidance,2004)
Monitoring
ProductQuality
ProductContentProductImpuritiesBetweenprocess
steps
Finalproductcontrol
Properprocessunderstandingandgoodprocesscontrol=Robustprocess
Failuresduetopoorcontrol,butdetectedHighfailurezone
RobustzoneLimitedflexibilityduetonarrowOWs
Processunderstanding
Processc
ontrol
Providesanopportunitytoapplyacontrolclosertothesourceofvariabilityintheprocess
UpstreamProcessing
DownstreamProcessing
Realityinbioprocessing
Integratedsystemusedtodetectchangesandquantifycomplexanalytes
CustomtailoredtomeetrequirementsofbioanalyticalHPLCtechniques
BIASeparationsPATfixTM
• Easytousedatamanagement• Massvisualizationof
chromatograms• Automaticdetectionof
changes• PredictionofcomplexCQAs
• Columnwithoutcarry-over• Immediatesampleanalysis
BIASeparationsPATfixTM
UseoftheHPLCismandatoryforaccuratemassbalancecalculation
10ml/min=4500cm/h=360CV/min(res.time:0,1s)=fasterthanbiosensor
CIMacTM /Bio-monolithTM HPLCColumns
Noentrapmentinthecolumn,nocarry-over
AdvantagesofCIM® monolithicresins –Noentrapmentinthecolumn,nocarry-over
CIMacTM analyticalcolumnsforPATHPLC– nocarryoveroflargemoleculesorviralparticles
Available:• CIMac™QA• CIMac™DEAE• CIMac™SO3• CIMac™EDA• CIMac™pDNA• CIMac™Adeno• CIMac™AAVempty/fullSoontocome:• CIMac™AAVtotal• CIMac™Lenti• CIMac™Vaccinia
OverlayofUV280traces
UV:RSDof∆𝒕𝒓𝒆𝒕(𝒇𝒖𝒍𝒍 − 𝒆𝒎𝒑𝒕𝒚):6%;FLD:RSDof∆𝒕𝒓𝒆𝒕(𝒇𝒖𝒍𝒍 − 𝒆𝒎𝒑𝒕𝒚):4%
RetentiontimedifferenceoffullandemptyAAVcapids,bothUVandFLD.
Minutes
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mAU
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CIMacTM AAVempty/fullcolumns; Intra-batchreproducibility(Batch15-003-AV01)
AAVisstickingtoallplastics – samplepreparationisthekeystepforaccurate results
Setup• Validation• Informationacquisition
Analyze• Visualization• Detectionofchanges
• Prediction
Act• Optimaltime-pointofharvest
• Celllysismonitoring• Poolingdecisions• Development
Report•Massvisualizationofdata
BIASeparationsPATfixTMworkflow
PATfixTM Optimalpointofharvest
Unitoperationisahighlydynamicsystem.
Case:mAbproduction
Howitisevolving?
Whatisinside?• IgG,• Nucleicacids,• Hostcellprotein,• Mediacomponents,• CHOcells,• Productagglomerates,...
RajamanickamV,SagmeisterP,SpadiutOandHerwigC.ImpuritymonitoringasnovelPATtoolforcontinuousbiopharmaceuticalprocesses,submitted.
Determinationofoptimaltime-pointofharvest(PichiaPastoris,proteinexpression)
• Samplestakenatregularintervals,centrifuged,bufferadjustedandinjecteddirectlyontothecolumn.
• Chromatogramalignmenttoincreasetheaccuracyofprediction
Determinationofoptimaltime-pointofharvest(PichiaPastoris,proteinexpression)
T2 r
ange
pointofdecision
optimaltimeofharvest
Deviationanalysis
Determinationofoptimaltime-pointofharvest(PichiaPastoris,proteinexpression)
Cohn(I+)II+III
Dissolution24h@4°C
Centrifugation20min
5%EtOHaddition,centrifugation
IEX:CIMmultusQA
Viralinactivation16h
IEX:CIMmultusSO3
TFF,finalformulation
Intravenousimmunoglobulin(IVIG)purificationprocessscheme
IVIGpurification– firstchromatographystepusingstrongAEXCIMmultuscolumn
Column:8mLCIMmultusQA
Loadingbuffer:20mMNa-acetate,pH5.0Elution:loadingbuffer+1MNaClCIP:1MNaOH+2MNaCl
Sample:1gCohnII+IIIpastein10mLloadingbuffer
ProductIGIVinflowthroughfraction(s)
Initialsam
ple
Flowthrough
Washing
Elution
CIP
IgG IgA,IgMandotherimpurities
HPLCfingerprintusingstrongAEXCIMaccolumninlineargradient
CIMacAEXfingerprintchromatogram- sample
Preparativechromatogram
SECchromatogram- sample
IgG
IgA,IgMandotherimpurities
IgG
IgA,IgMandotherimpurities
Preparativechromatogram
CIMacAEXfingerprintchromatogram– FTfract.1
SECchromatogram– FTfract.1
HPLCfingerprintusingstrongAEXCIMaccolumninlineargradient
CIMacAEXfingerprintchromatogram– FTfract.2
Preparativechromatogram
SECchromatogram– FTfract.2
HPLCfingerprintusingstrongAEXCIMaccolumninlineargradient
CIMacAEXfingerprintchromatogram– FTfract.3
Preparativechromatogram
SECchromatogram– FTfract.3
HPLCfingerprintusingstrongAEXCIMaccolumninlineargradient
CIMacAEXfingerprintchromatogram– FTfract.4
Preparativechromatogram
SECchromatogram– FTfract.4
HPLCfingerprintusingstrongAEXCIMaccolumninlineargradient
CIMacAEXfingerprintchromatogram– FTfract.5
Preparativechromatogram
SECchromatogram– FTfract.5
Fraction 5 should be discharged due to
the too high level of IgA, FXI and aggregates
– by SEC not visible
HPLCfingerprintusingstrongAEXCIMaccolumninlineargradient
CIMacAEXfingerprintchromatogram– Elutionfract.
Preparativechromatogram
SECchromatogram– Elutionfract.
IgA IgMIgG
HPLCfingerprintusingstrongAEXCIMaccolumninlineargradient
Normalizedchromatograms
Averagerelativestandarddeviationofallareanormalizedfingerprintsis5.6%(includingthesampleobtainedwithfermentationatdifferentscales(50Lvs200L)).
Onecanconcludethefermentationisveryrobust.
PATfixTMFingerprintapproachtostudyrobustnessoftheAAVfermentationscale-up
PATfixTM cheaperinfoastraditionalmethods
DNATargetmolecule
Hostcellproteins
PATfixfingerprintinjection=about20€(column+buffers+labor),takesabout20min
(crio)TEM:125€,30minAUC:200€,1hSDS-PAGE:20€,4hInfectivity:<10€,3-7days
ELISA:5€/well,4hSDS-PAGE:20€,4h
qPCR:20€/well,3-4hssPCR:30€/well,3-4hAGE:20€,2h
ToSUMup:
Traditionalmethodspersample:100- 400€Time:1dayto1week
PATfixpersample:20€Time:20minBUTnotmagicbox– needsworktounderstand
Adenoviruspurificationprocessmonitoringusingfingerprintapproach
Lysis
Nucleasetreatment
Clarification
Ultrafiltration/diafiltration
AEXChromatography
BufferExchangeFingerprintingColumn:CIMacTM AdenoFlowrate:1mL/minBufferA:50mMTris,pH8.0BufferB:50mMTris+1MNaCl,pH8.0
CIMac™pDNAAnalyticalColumn
Product number Product name Description150.8501-1.4 CIMac pDNA column DEAEmonolithicmatrixwitha
controlledliganddensityandstructuralcharacteristics
• DEAEmonolithicmatrixwithacontrolledliganddensityandstructuralcharacteristics‒ 5.2mmIDx15mmL,V=0.32mL
• Flowrates:0.2– 2mL/min• Maximumpressureoverthecolumn:100bar
OptimizationofprecipitationwithCaCl2
0 0.1 0.3 0.5 1.0 MM C S
PATHPLCtobalancebetweenRNAremovalandpDNAyield
CIMmultusDEAEpreparativecolumn:
MA123456
Figure 1: Agarose gelelectrophoresis - Molecular weightmarker (lane M), sample alkalinelysate plasmid pEGFP-N1 (lane A),peak 1 (lane 1), peak 2 (lane 2),peak 3 (lane 3), peak 4 (lane 4),peak 5 (lane 5), pDNA open circularform standard (lane 6)
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0 5 10 15RelativeAb
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E.coli culturewithplasmid
Cellharvest
Alkalinelysiswithadjustmentto
0.5MCaCl2
Clarification
CIM® DEAE
CIM® C4
Bufferexchange
Adjustmenttobindingconditions
Adjustmentwith(NH4)2SO4
CIMac™pDNAAnalyticalColumn– alkalinelysisoptimisation
CIMac™pDNAAnalyticalColumn– 1stchromatographystep
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Alkalinelysiswithadjustmentto
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Clarification
CIM® DEAE
CIM® C4
Bufferexchange
Adjustmenttobindingconditions
Adjustmentwith(NH4)2SO4
Conditions:Flowrate– 1ml/min;BufferA– 200mMTrispH8.0andbufferB– 200mMTRIS+1MNaClpH8.0;Injectionvolume– 20µl;Samplewasdiluted1:3withwater;UVdetection– 260nm;Peak1andPeak2– otherimpurities,Peak3– RNA,Peak4– OCpDNA,Peak5– SCpDNA.
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Conditions: Flow rate – 1 ml/min; Buffer A – 200 mM Tris pH 8.0 and buffer B – 200 mM TRIS+ 1 M NaCl pH 8.0; Injection volume – 5 µl; UV detection – 260 nm; Peak 1 – OC pDNA form;Peak 2 – SC pDNA form;
Topoisomers
OC 2%
SC 98%
E.coli culturewithplasmid
Cellharvest
Alkalinelysiswithadjustmentto
0.5MCaCl2
Clarification
CIM® DEAE
CIM® C4
Bufferexchange
Adjustmenttobindingconditions
Adjustmentwith(NH4)2SO4
CIMac™pDNAAnalyticalColumn– 2ndchromatographystep
Onlinenucleasetreatmentmonitoring
Loopvolume:1mL,injected1mLof3timesdilutedsamples;flowrate:1mL/min.
• HSNuclease(MoBiTec;CatNo1070-01;Lot#202222;250units/µL)dilutedwithChromatographyBufferAto1unit/µL,1.5units/µLand2units/µL.
• FinalNucleaseconcentrations:50units/mL;100units/mL;150units/mL.
• Aliquots incubatedinwaterbathat37°C;every30minutesonealiquotdrawnandimmediatelyanalyzedbyHPLC.
Lysis
Nucleasetreatment
qPCR:20€/well,3-4hssPCR:30€/well,3-4hAGE:nonumericresultTotallabor:1000€Total:upto3000€for3reactions
PATfix:Preplabor:100€&gohomeCIMacrun:2- 5€Total:250€for3reactions
Onlinenucleasetreatmentmonitoring– costcomparisonPATfixTM – traditionalmethods
Conclusions• HPLCfingerprintingisconvenienttechniquetomeasuremultiplesampleparameterssimultaneously:+ Reproducible+ Highresolution+ Flexible± Fastbutnotyeton-line- Difficulttoevaluate (needsexperience)
• PATfixTM algorithms+ Systemverification+ Samplestabilitycontrol+ Dilutioncontrol+ Advancedmathematicalmanipulationofchromatograms+ Fast,reliableandsimultaneouspredictionofmultiple
samplecomponents
BIASeparations - industrystandardforproductionofGeneTherapyproductsandExosomes
Ø PlatformprocessesforpDNA,AAV,FluandAdeno,....Ø FirstdrugpurifiedusingCIM®monolithsonthemarket,onepassed
CPIIItrial(pDNAforgenetherapy),5projectsinCPIII.Ø Morethan100projectsinCPI– CPII trials(variousInfluenza,various
Adenovirus,variousAAV,bacteriophages,variousIgMs,Inter-alpha-inhibitors,...).
Ø Morethan500projectsinpre-clinicaltrials(InfluenzaAandBvirus(eggs,VeroandMDCKcells),Rabiesvirus,Rotavirus,AAV,variousAdenovirussubtypes,HepatitisA,Vaccinia,Mulv,MVM,Felinecalicivirus,Japaneseencephalitis,Crimean-Congohemorrhagicfever,Hantaanvirus,VLP(HepatitisB,HPV,Influenza,Adenovirus),bacteriophages(Lambda,T4,VDX10,Pseudomonasphage),TomatoandPepinoMosaicvirus,pDNA,IgM,variousproteins).
Foranyadditionalinformationpleasecontactus:
[email protected].:+38659699500
[email protected].:+38659699599
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