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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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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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:

sales@biaseparations.comTel.:+38659699500

orders@monoliths.comFax.:+38659699599

tech-support@monoliths.comwww.biaseparation.com