Contents...were asked what types of bioreactor they would implement for a new facility comingonlinein 2 years,as expected,over two-thirdscitedbatch-fedsingleuse bioreactors,while32%

Contents

01 Introduction to Continuous Manufacturing: Technology Landscape and TrendsEric S. Langer &Ronald A. Rader, BioPlanAssociates

02 A Brief History of Perfusion BiomanufacturingJerry Shevitz&John Bonham-Carter,RefineTechnology

03 Bioreactor Configuration and OperationChristel Fenge, Jörg Weyand, Gerhard Greller, Thorsten Adams,

SartoriusStedimBiotech

04 How to Develop a Perfusion ProcessVéronique Chotteau,RoyalTechnicalUniversity,KTH

05 Case Study: Optimized PerfusionShaun Eckerle,GallusBioPharmaceuticals

06 Process Intensification Approaches for Cost Sensitive Protein ApplicationsWillem Adriaan de Jongh,Expres2ionBiotechnologies

07 Impact of Single-use Technology on Continuous BiorocessingWilliam G. Whitford,ThermoFisherScientific

08 Continuous Multicolumn Chromatography ProcessesMarc Bisschops,TarponBiosystems

09 Continuous Processes: Economic EvaluationAndrew Sinclair&Andrew Brown,BioPharmServices

10 Vision: Integrating Upstream and Downstream in a Fully Continuous FacilityTim Johnson,Genzyme

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IntroductiontoContinuousManufacturing:TechnologyLandscapeandTrends

Eric S. Langer,President,BioPlanAssociates

Eric S. Langer is president and managing partner at BioPlanAssociates, Inc., a biotechnology and life sciences marketingresearchandpublishingfirmestablishedinRockville,MDin1989.He iseditorofnumerousstudies, including“BiopharmaceuticalTechnologyinChina,”“AdvancesinLarge-scaleBiopharmaceuticalManufacturing”,andmanyotherindustryreports.

Theclassicandlargelypredominantapproachtobioprocessing,bothupstreamanddownstream,remainsbatchprocessing,withmanufacturingbatchfluidsessentiallymovingincrementallyenmassasabolusfromoneprocessstepandsetofequipmenttothenext.Thisassemblyline-like,finish-one-stepthenmovealltheprocessfluidstothenext,approachcertainlyworkswellbutanumberoftechnologicaladvancesandrelatedtrendsaremakingcontinuousbioprocessingattractive. Continuous bioprocessing strategies are making advances andare being adopted or considered for many new drug bioprocesses beingimplemented.Meanwhilesomeestablishedbioprocessing facilitiesarebeingretrofittedandupgradedformorecontinuousoperations.Continuousupstreambioprocessingisactuallynotnew,withfiber-basedperfusionbioreactorswidelyusedforclassicfused-cellhybridomaculture,e.g.,inthe1980s,whenitwasreplacedbyrecombinantantibodymanufacturingmethods.

We can expect higher future adoption of bioprocessing by continuousmethods1,2. Already, about a dozen or more marketed recombinant proteinproductsaremanufacturedusingperfusionorothercontinuousbioprocessingtechnologies.LeadingadoptersincludeGenzymeandBayer.Mostadoptionofcontinuous bioprocessing has involved upstream processes, with continuousdownstreampurification tending to lagbehind.Thus, it iscurrentlycommonfor new bioprocesses being implemented to combine continuous upstreamprocessing with conventional batch purification. Continuous chromatographytechnologies,suchassimulatedmovingbed(SMB)andperiodiccounter-currentchromatography,aregenerallynotyetreadyyetforcommercial-scaleadoption.Regulatorybarrierstocontinuousbioprocessing,suchhowtodefinelots,havebeenresolved,andcontinuousprocessingfitsbetterthanbatchprocessingwithautomation,QbDandPAT.Theseaspectsaremakingthebenefitsofcontinuousprocessingincreasinglyattractivetobiopharmamanufacturers.

Ronald A. Rader,SeniorDirector,BioPlanAssociates

Mr.Raderhasover25yearsexperienceasabiotechnology,andpharmaceutical information specialist and publisher, includingEditor/PublisherofAntiviralAgentsBulletin,Editor-in-ChiefofthejournalBiopharmaceuticals,andmanydataresourcesincludingBiopharmaceuticalProductsintheU.S.Market,nowinits12thEd,andthefirstbiosimilarsdatabase.

01AbouttheAuthors:

54

culture stage-related loss of cell viability or altered glycosylation, isreduced,withcontinuousbioprocessinginherentlymoreconsistentandrobust.Problemsassociatedwithproteolyticorotherdegradationovertimeinbioreactorsandothervesselscanbeavoidedorminimized.Andifanyproblemsdooccur,onlypart,nottheentire,productionrunlikelyneedsberejected.

d) Increased flexibility:Continuousmanufactureenablesmoreadaptabilityandefficientfacilityutilization,similartotheadvantagesofsingle-usedevices. Bioprocessing becomes much more portable, and facilitiesmore clonable. Couple this with the trend for adoption of modularbioprocessing systems, multiple smaller continuous bioprocess linesin smaller facilities worldwide, and we expect this approach will beincreasinglyadoptedforcommercialmanufacturing.

The BioPlan 10th Annual Report and Survey of the BiopharmaceuticalManufacturingevaluateskeytrendsandaspectsofthebioprocessingindustry.Wesurveyedtheattitudesof300industryprofessionalstowardsperfusionandcontinuous processing in 2013 3,4. Attitudes are common with relatively newbioprocessingtechnologies.Overall,respondentssawmoreproblemsassociatedwithperfusion/continuousvs.fed-batchprocessing.“Processcomplexity”wastheprimaryconcern,citedby69%(%indicatingthisfactoreither“muchbigger”ora “somewhatbigger”concern), followedcloselyby “Processdevelopmentcontrol challenges” noted by 64.7%. Other issues included “Contaminationrisk”at58.6%and“abilitytoscale-up”at54.3%.Incomparison,forthesameaspects, concerns over batch fed processes were noted by very few (single-digitpercentages)respondents.Muchofthisperceptionwill likelychangeastheindustryisincreasinglyexposedtothesuccessfulapplicationofcontinuoustechnologiesinclinicalandcommercialscalebioproduction.

Continuous upstream bioprocessing generally involves retaining productioncellswithinthebioreactoratafixedvolumeandfixedcellconcentrationonacontinuousbasis,suchasfor30-90daysorevenlonger.Thebioreactorfluidhasamuchhighercellconcentration,withcellsretainedwithinthebioreactorbyvariousmethods.

Thecurrentleadingmethodinvolvesuseofspecializedfilter-basedequipment.Othermethodsforcellretentionaredonebycentrifugationanduseofcapillaryorotherfiber-basedandmicrocarrierreactorswherecellsself-attachtofiberorparticlesubstrates.

Therearemanybenefitstooperatingbioprocessescontinuouslyratherthaninbatchmode,withmanyofthesesimilarandcomplementingthoseofsingle-useandmodularsystems.Thesebenefitsinclude:

a) Reduced costs: Operating continuously allows use of much smaller-scaleequipment,withasmallervolumebioreactor(andsmallersizesfor most other equipment) operating over time resulting in as muchproductasmuchlargerequipmentoperatedinfed-batchmode.Besidessmaller-scale equipment generally costing less, this allows muchsmallerfacilitiesandequipmentfoot-print,withlessspaceandutilitiesrequired,particularlywhensingle-usesystemsareused.

b) Increased productivity:Becausemuchofthebioprocessingequipmentisoperatedcontinuously,thereislittleneedforlargetransfer/storagevesselsandnohaltsbetweenprocesses.Bioprocessingthustendstomovemuchmoresmoothly.Muchhigherbioreactorcelldensitiescanbeattained,providinghigherproductyieldandconcentration.Also,thenumberofbioprocessingstaffrequiredisdecreased,andtheirworkatlargescaleislessphysicallydemanding.

c) Improved quality: Biological molecules are naturally producedcontinuously,andcomparedtobatchculture,continuousculturetendstobemorecontrollable,lessintenseandstressful,includinglessshearandmedianutrient levelskeptconstant.Productvariability,e.g., later

IntroductiontoContinuousManufacturing:TechnologyLandscapeandTrends01 COnTInUOUS BIOPROCESSInG CURREnTPRACTICE&FUTUREPOTEnTIAL

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IntroductiontoContinuousManufacturing:TechnologyLandscapeandTrends

References:1) Langer, E.S., “Trends in Perfusion Bioreactors: The next Revolution in Bioprocessing?” BioProcess

International,nov.2011,p18-22.

2) Bonham-Carter,J.andShevitz,J.,“ABriefHistoryofPerfusionManufacturing:HowHigh-ConcentrationCulturesWillCharacterizetheFactoryoftheFuture,”BioProcessInternational,Oct.2011,p.24-30.

3) Langer,E.S.,10thAnnualReportandSurveyofBiopharmaceuticalManufacturing,April2013

4) Langer, E.S., “Perfusion Bioreactors Are Making a Comeback, But Industry Misperceptions Persist,”BioprocessingJournal,Winter2011/12,p.49-52.

5) Rader,R.A.andLanger,E.S.,“UpstreamSingle-UseBioprocessing:FutureMarketTrendsandGrowthAssessment,”BioProcessInternational,Feb.2012,p.12-18.

01 COnTInUOUS BIOPROCESSInG CURREnTPRACTICE&FUTUREPOTEnTIAL

In fact, continuous processing equipment manufacturers and users ratheruniformly report that many of these problems have been resolved withapplicationofcurrenttechnologies,includingsingle-useequipment.Perfusion/continuousprocessingisnowgenerallysignificantlylesscomplex,lesspronetocontaminationandmorereadilyscalablethanfed-batchmethods.Industryperceptionsofperfusion/continuousvs.fed-batcharelagging,andlikelyreflectalackofdirectexposureorexperiencewiththetechnology.Whenthosesurveyedwereaskedwhattypesofbioreactortheywould implementforanewfacilitycomingonlinein2years,asexpected,overtwo-thirdscitedbatch-fedsingleusebioreactors,while32%and25%citedsingleuseperfusionbioreactorsatclinicalandcommercialscales,respectively.

7

BioPlan Associates expects increased and rapid adoption of continuousbioprocessingatallscales,includingcommercialmanufacture.Theimperativesofcost-savings,flexibilityandproductqualitywillincreasinglydrivetheindustrytoexplorecontinuousprocessing.This,inturn,willexpandtheindustry’scurrentknowledgeandexperiencebase,whenmakingmajorchangesinmanufacturingplatforms. Particularly, as perfusion and other continuous bioprocessingtechnologiesareimprovedandincreasinglyadaptedforsingle-useequipmentand modular systems, adoption will further accelerate. Many upcomingcontinuousbioprocessingtechnologiesareactuallyverynovel.Forexample,asingle5Lbioreactorcurrentlyindevelopmentwillbeabletomanufacturethesamequantityofproduct,oftenatbetterquality,comparabletoa5,000Loverthesametimeperiodusingthesameamountofmedia1.Casestudiesandotherreportsofsuchperformancewillfurtherpromoterapidadoption.

Wepredictincreasinglyrapidadoptionofsingle-usesystemsforthemajorityof new commercial manufacturing facilities over the next 5 years, and weexpect continuous bioprocessing, particularly for upstream processing, tofollowasimilartrajectory.Useof theseproducts is likely to further increasewithhybridsystemsthatusebolt-on-typetechnology,thatretrofitcomponentsunitoperationsforexistingsystems.Otherconventionaltechnologies,suchascentrifugation,willalsoseenincreasingadoptionincomingyears.Potentiallyrevolutionarycapillaryfiberperfusionbioreactorsandothernewtechnologies,includingthosefordownstreamprocessing,willbelikelycomingonlineandbemorewidelyadoptedforcommercialmanufactureoverthenext10years.

Source:10thAnnualReportandSurvey,BiopharmaceuticalManufacturing,April2013,BioPlanAssociates,Inc.Rockville,MD

Batch fed bioreactors, Single use

Batch fed bioreactors, Stainless steel

Perfusion bioreactors, Single use

Concentrated batch fed bioreactor

Perfusion bioreactors, Stainless steel 13.0%

18.3%

22.9%

22.1%

32.1%

25.2%

39.7%

58.0%

65.6%

42.0%

CommercialClinical

Fig.1: Likelihood of Implementing Bioreactor,

by Type

‘When specifying bioreactor types for a new [Clinical] or [Commercial] scale biologics facility, how likely are you to specify”%“verylikely”or“likely”

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ABriefHistoryofPerfusionBiomanufacturingHowHigh-ConcentrationCulturesWillCharacterizetheFactoryoftheFuture

Today’srenewedinterestinperfusioncultureisduetoanincreasedawarenessof its advantages, some general improvement in equipment reliability, anda broadening of operational skills in the biomanufacturing industry. Somemisperceptionspersist,however,accordingtoa2011reviewbyEricLanger.1Our view here of the history of perfusion and fed-batch processes includessomediscussionoftechnologicalprocessimprovementsandchallengesthatthebioprocessindustryfaces.

A team of authors at Serono in Switzerland wrote in 2003:The major advantage of the perfusion mode is high cell number and highproductivity inarelativelysmall-sizebioreactorascomparedwithbatch/fed-batch.Inordertosustainhighcellnumberandproductivity,thereareneedstofeedmediumduringthecellpropagationphaseandtheproductionphase.Incontrast tobatchand fed-batchprocesses,where there isnometabolitesremoval, in continuous processes medium is perfused at dilution ratesexceedingthecellulargrowthrate.Forthis,agoodseparationdeviceisneededtoretaincellsinthebioreactor.2

Manycellretentiondevicesperformwell,toagreaterorlesserdegree,atsmallscale,includinggravity-basedcellsettlers,spinfilters,centrifuges,cross-flowfilters,alternating tangential-flowfilters, vortex-flowfilters,acousticsettlers(sonoperfusion),andhydrocyclones.Allaredescribedwell in the2003papermentionedabove.Butonlyafewtypesarereliableatlargerscalesandscalableenoughforbioindustrialuse.

HerewecomparetheATFSystemfromRefineTechnologywithspinfilters,cellsettlers,andcentrifuges.Weamnotincludingothertechnologiesherebecauseofscalabilitylimitationsandalackofprovenmarketacceptance.

02

John Bonham-Carter,VicePresident,RefineTechnology

A serial entrepreneur who has worked with many differentupstream technologies, and has propelled Refine Technologytobecomethe industry leader incontinuouscultureequipmentsupply. A regular international speaker on bioprocessing, SMEentrepreneurshipandbusinesscoaching.

Jerry Shevitz,PresidentandChiefResearchOfficer,RefineTechnology

Jerry Shevitz, Ph.D. received his degree in biochemistry fromCUnY,newYork,n.Y.,subsequentlyfocusingoncellbiologyandImmunology.Jerryhasanextensivebackground,spanningaboutthreedecades,inthebiomanufacturingindustry,fromsmallscaleresearch to large scale process development, with companiesinvolvedindrugdevelopmentandalsowithequipmentsuppliers.Jerryisaproductiveinventoralwayslookingforimprovedsolutionsandnewapproaches.HeisPresidentofthecompanyandheadstheRefineresearchdepartment.

AbouttheAuthors:

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However,despitethedominanceoffed-batchasanindustrystandard,perfusioncontinued to be championed. Perfusion offered an excellent solution forproductionwithunstableproteinsthatcouldnotremaininthetoxicenvironmentofanever-deterioratingfed-batchculture.Withperfusion,suchproductscouldbe removed rapidly from a vessel and stored appropriately to preserve theirstability.Manypeoplechoseperfusiontobypassconstraintsofspaceandcostfactors.Furthermore,ascultureproductivityincreased,andalthoughitgreatlybenefitedfed-batchprocesses,perfusionpromisedevengreateroutputfromacontinuousculture.

Sotheuseofperfusionneverdied;infact,astheuseofspin-filtersdeclined,othercellseparationdevicesslowlyemerged.Thosewerebasedonfiltration,gravity settling, and centrifugation. Continued development of numerousproductsthatheldoutthepromiseofcommercializationprovidedthedrivingforce to experiment with new culture technologies. Occasionally a perfusionprocess,wasscaledtocommercialproduction.

Perfusion’s Early PotentialThe advantages of using perfusion for enhancing production of cell-derivedproductswererealizedinthelate1980sandearly1990s.Inthoseearlydaysofthemodernbiotechnologyindustry,productioncelllineswerenotfullydeveloped,andtheirproductexpressionwasverysmall—fromafewmicrogramstoafewhundredmilligramsperliterinbatchorfed-batch.Attainablecellconcentrationswereonlyafewmillionpermilliliter.

Spin Filters: Perfusion offered a way to derive more product from such lowproducers.Itwaswellknownthatperfusioncouldincreasecellconcentrationbyasmuchasanorderofmagnitude3.Thespinfilterwasthemostcommonperfusiondeviceused; itwasthebestcell-separatingdeviceavailableat thetime,supportedbyreputableequipmentmanufacturers.

Spinfiltersremaininuseatafewsitesbuthavebeenlargelyphasedout,largelybecauseoftheirlimitedscale-uppotentialandunreliability:Whenabioreactor’svolumescalesupbythecubeofitsradius,thesurfaceareaofitsspinfilterscreenscalesbythesquareofitsradius.Aninternalspinfiltercantakeupasignificantportionofproductionspacewithinavessel,andonceitsscreenfouls,therunisterminated.Anexternalproductionspinfiltermaysolvethisshortcoming,butithasdrawbacksrelatedtocost,maintenanceandsterilizationdifficulties.

A more important factor behind the lackluster acceptance of perfusion inthoseearlyyearswastherapidevolutionofcellbiology.new,moreproductiveexpressionsystemsandimprovedmediadevelopmentpermittedlargeincreasesin culture productivity; product concentrations were increasing from severalhundredmilligramstoaboutagramperliter.Productionneedscould,therefore,beachievedwiththewell-understoodfermentationtechnologies,batchandfed-batch.Scaleupwasaccomplishedsimplybymovingtobiggervessels.

The success of batch and, more important, fed-batch, not only inhibited thewider acceptance of spin filters, but also of other evolving cell-separationtechnologies. The difficulties associated with spin-filter operations and theundevelopedstateofnewperfusiontechnologiesstigmatizedtheprocess.Thedominanceoffed-batchcontinuedwellintothenextdecade.

ABriefHistoryofPerfusionBiomanufacturingHowHigh-ConcentrationCulturesWillCharacterizetheFactoryoftheFuture02 COnTInUOUS BIOPROCESSInG CURREnTPRACTICE&FUTUREPOTEnTIAL

Perfusion performed at CMC Biologics

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theoptionofcontractingoutacontinuousbiomanufacturingplatform.Outsidetheestablishedbiomanufacturinginfrastructure,biosimilarandotherrelativelynewbiologicalmanufacturerssuchasBioconandKanghongBiopharmaarealso lookingfavorablyontheperfusionmodelbecauseof itsassociatedcostefficiency.Perfusionisback.

Simplicity and reliability have long been key factors to consider in biologicsproduction, especially where manufacturing involves high-value products inalarge-batchenvironment.Theindustryisnowbeingchallengedasitmovesforwardtorealizethemuch–touted“factoryofthefuture,”whichwillincorporateseveralplatformtechnologies.Onesuchtechnologyiscertainlytheadoptionofdisposablesthroughoutproductionfacilities.

Perfusionisabroadterm,whichmanypeoplemaystillviewunfavorably.Althoughmany,infact,useperfusionatsomelevel,noteveryoneadmitstoit—nortohowtheydoit,norhowoften.Companiesareexperimentingwithperfusiontosolvechallengesorimplementnovelsolutionsatmanyprocessstages:highdensity,large-volumecellbanking11;seedexpansion8;n–1perfusion12;andofcourse

High cell concentrations are a game-changer: From the early 2000s andparticularlyinthepastfewyearsanothercriticaltransitioninbiopharmaceuticalmanufacturingoccurred.Furtheradvancements indevelopmentofcell lines,expressionsystems,andmediaformulationsresultedinanimpressiveabilitytogrowcellstoveryhighconcentrationsandachieveproductconcentrationspreviously inconceivable. Using fed-batch as a reference, in the mid 1990sattainablecellconcentrationswereabout5×106cells/mL,withrecordproductconcentrationsof1–2g/L;todaythosearegreaterthan15×106cells/mL,withproductconcentrationsofupto10g/L.Althoughthoseconcentrationsarestillnottypical,theyindicatewherethefieldisheading.Thoseresultsareamplifiedbytheuseofperfusion,throughwhichsubstantiallyhighercellconcentrationsandproductoutputcanbeachieved.4,5

Perfusion Returns to ManufacturingAgenerallackofmanufacturingcapacityforecastatthebeginningofthiscenturywasovercomethroughbothbiologicalinnovationandengineeringconstruction.Today’s overcapacity places most of the available space in the hands ofrelatively few companies. Even as some large biofacilities are mothballed,newercompaniesbuildmodernfacilitiesbasedonthelatesttechnologies.Feworganizationswouldnowconsiderbuildinganew,multiple–20,000-Lbioreactorfacility.Risingcompetitioninthehealthcaresector,whetherthroughgenerics/biosimilarsormultipledrugswiththesameindication,requiresthevastmajorityofbiopharmaceuticalproductstobemoreeasilyproducedinsmallerandmoreflexibleplants—eveninmultiplelocations.newultrahigh-densitycellcultureprocesses such as concentrated fed-batch and concentrated perfusion arewellsuitedtothisnewmanufacturingenvironmentandfacilitateashifttowardsingle-use technologies. That helps companies reduce both risk and capitalinvestment,allowingthemtodelaymakingmajorfacilitydecisions.

Sothefaceofbiomanufacturingtodayisverydifferentfromthatofjustadecadeago.nearlyeveryoneusesperfusioninsomeway—fromlargebiopharmaceuticalcompanies such as Pfizer, Medarex, and Genentech 6–8 to small biotech andnovelvaccinemanufacturerssuchasShireandCrucell9,10.Inaddition,today,contractmanufacturers,suchasGallusBiopharmaceuticalsandRentschler,runseveralcommercialperfusionprocessesallowingcompaniesachoiceand


Microcarrier-based perfusion in a single-use ATMI bioreactor

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Astandardhollow-fibermoduleisusedtoseparatecellsandproduct.However,unlikesystemsthatrecirculateaculturethroughafilterinonedirection,thealternatingtangential-flowactionconstantlycleansthefiberseveryfivetotensecondswithabackflushaction.Withonlyasingleconnectiontothebioreactor,cellsandmediaenterandleavetheATFSystem,flowingreversiblythroughthehollowfibers.FlowiscontrolledbythediaphragmmovingupanddownintheATFSystem’spump.Thisgeneratesarapid low-shearflowbetweenvesselandpump,ensuringrapidexchangeandpromptreturnofcellstothereactorandminimizingtheirresidenceoutsidethebioreactor.Thechoiceofporesizeforthehollowfiberdetermineswhatelementsareretainedandwhichonespassthroughtothepermeate.

From Research to Manufacturing — the Scale-Up Challenge:For companies requiring increased protein production in preclinical work,manyperfusiontechnologiescanquicklydeliver.

Onecommonapproachistochooseasmall-scalecell-retentiondevicethatoffersahighdegreeofconfidenceforscalingtoacommercialmanufacturingprocess.Scalingupabioreactorintroducesitsownissues,soengineersdon’twantperfusionequipmenttoaddfurthercomplications.Severaltechnologieshavebeenusedat largescale,andeachsystembrings itsownlimitations.Forexample,well-knownspin-filtertechnology,previouslydiscussed,usesatwo-dimensionalscreentoretainthecells.Limitationsofthesystem(whetherinternalorexternal)ariseduringscaleupandatelevatedcellconcentrationswhenrapidfeedratesarerequired.Consequently,toreducerisksofscreenblockage,theprocessdurationmustbeshortenedortheculturemaintainedatlowcellconcentrationtopreventexcessiveaccumulationofcelldebrisonthescreen.Thelatterisusuallywhatoccurs.

Different but familiar problems occur with inclined or gravimetric settlers.Cells spendsignificant time inanexternal, suboptimalenvironmentwithinthe settler (particularly) as the size of a system is increased. Additionally,asasystemisincreased,whengreaterperfusionratesarerequired,raisingrecirculationflowratescanleadtoinefficientcellseparationandsignificantcellloss,whichlowersoutputandincreasescosts.

finalproductionreactors13.Perfusionhasevolvedtoo:Itisnolongersolelyatwo-orthree-monthprocess,butcanbeasshortasathree-dayboosttoastandardfed-batchprocess.Perfusionhasbecomeaspecialistoperation.Implementationdependsonthenatureofdifferentfacilities,celllines,processes,andproducts—aswellaseachcompany’sownoperatingphilosophy.Successdependsonmany factors,not leastofwhich isacompany’schoiceofperfusionsystem.But one challenge — that of producing a reliable cell-retention device —may have been solved to a great degree by a relatively new hollow-fiberperfusiondevice.

Case Study

The ATF System (Fig.1)offersnearlylinearscale-upforsimplicityofoperationandvalidation.Generally,conventionalfiltrationsystemswillfailrapidlywhenusedtoseparatemediafromacomplexsuspensionofacellculturewithahighbioburden.Bycontrast,thisparticularsystem,duetoitsflowdynamics,hasaninherentself-cleaningabilitytoallowitsrangeoffiltermaterialsandporesizestoperformsignificantlylongerthanmightotherwisebeexpected.


Fig. 1: The ATF System

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An Xcellerex bioreactor used in an intensified

perfusion process with ATF System equipment

For companies that require simpler systems that can be operated by anonspecialistor thatdonotwant todevote years tobuilding those requisiteskills, the ATF System can provide a robustly scalable process platform formostcelllines.Laboratory-scaledevicesarerunasstandardtoproducethesameconditionsandflowsthatcommercialscaledeviceswilluse.Twokeyparameterstokeepconstantarethefiltrateflowratioandtheflowthrougheach individualhollowfiber.Otherparameters thatwouldnormally requireattention—e.g.,filtersurfaceareaandresidencetime—are factored intotheequipmentconfigurationdesigntolimitvariabilitypotential.Scale-upisthereforestraightforwardtohelpteamsbuildtheirconfidenceandexperiencerapidly.Additionally,unliketheoldersystems,afailureintheATFSystemdoesnotmeanfailureoftherun.Theperfusiondevicecanbeeasilyexchangedwithanother inasterilewaytocontinuetheprocess.Bioreactor issuesactuallycometothefore:Canalarge-scalebioreactorhandletheoxygendemandsofacellconcentrationthatisabout10timeshigherthanusual?

A Factory of the (near) FutureAstablecell line isaprerequisite foraperfusionprocess if it is intended toproduceahigh-qualityproductforanextendedtime.Consideringthestateofbiologicalmanufacturing todayand industry trendsof thepast twodecades,somefeaturesofthefactoryofthefuturecanbeanticipated:

A Continued Move Toward Single Use: Innovations in disposable bioreactordesigns have moved the industry toward their increased use. That trend isreflected by the large number of companies that are currently supplyingsingleuseBioreactors(SUBs).InnovativeSUBsfromsub-oneliterto2,000Lare readilyavailable today.AlongwithSUBs,significant improvementshavebeenmadeinprocessingequipment,sensors,andothercomponents,allwithdisposabilityinmind.

A Shortened Bioreactor Train:Theabilitytogeneratehigh-cell-concentrationculturescombinedwiththeabilitytofreezelargevolumesofsuchcultureshasmadeitpossibletocreatehigh-volumecellbanks.Asinglesamplecanbeusedto inoculate a relatively large bioreactor directly, eliminating multiple steps,savingtime,andgreatlyincreasingreliability.14

Centrifugeshavebeenscaledupsuccessfullyforseveralperfusionprocesses,oftentoveryhighflowrates.However,thehighleveloffine-tuningrequiredtomaintainthereproduceabilityofsuchsystems—particularlyduringscaleup—aswellastheircostgreatlydiscouragetheiruse.

Despitethose issues,eachcell-retentiondevicehasasolid followingamonga number of companies. Skilled and experienced individuals maintain suchsystems.Theyassessandimprovescale-upandscale-downperformance.


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Simplified Product Stream:Generatingafilteredproductstreambyfiltrationperfusioncanshortenthestepsbetweenvesselandcolumn.

Concentrated Perfusion:Although1g/L/dayisroutinelyachievabletodayusingconcentratedperfusion,2-3g/L/day15hasbeenreported,and5g/L/daycanberegardedasthenextstep.Thevolumetricproductivityofconcentratedperfusionmeansthatat5g/L/day,one500-Lreactorwouldproduce2.5kgofproteineveryday,andover500kg/year.

Ifthesegoalsareachievedintheforeseeablefuture,thereislittlereasonforevenahigh-doseblockbustertobemanufacturedinanythinglargerthana500-Lvessel,whereasmostotherproductscouldbehandledwithcurrentlaboratory-scaleequipment.Thefuturesizeofthefactory,forupstreamprocessesatleast,looksverysmallindeed.


References:1) Langer ES. Perfusion Bioreactors Are Making a Comeback, but Industry Misperceptions Persist.

BioProcessJ.9(2)2011:49–52.

2) VoisardD,etal.PotentialofCellRetentionTechniquesforLarge-ScaleHigh-DensityPerfusionCultureofSuspendedMammalianCells.Biotechnol.Bioeng.82(7)2003:751–765.

3) ShevitzJ,etal.StirredTankPerfusionReactorsforCellPropogationandMonoclonalAntibodyProduction.AdvancesinBiotechnologicalProcesses,Vol.11.A.Mizrahi(Ed).AlanR.Liss.,Inc.:newYork,1989;p81.

4) CarstensJ.Perfusion!JeopardyortheUltimateAdvantage?BioProcessInt.webinar,2009Sept.

5) Bonham-CarterJ,etal.WhichProcessOptionIsRightforMe?BioresearchOnline18October2010.

6) YuanH.CellCultureProcessScale-Up,TechnologyTransfer,andAssessmentofProcessComparabilityUsingMultivariateAnalysis:ACaseStudy.BioProcessInternationalConferenceandExhibition,October2009,Rayleigh,nC.

7) AlahariA.ImplementationofCost-ReductionStrategiesforHuMAbManufacturingProcesses.BioProcessInt.7(7)2009:s48–s54.

8) TezareR.RunningInoculumCulturesinPerfusionModetoEnableHigherSeedingDensitiesofProductionCultures.BioProcessInternationalConferenceandExhibition,2010,Providence,RI.

9) ComptonB,etal.UseofPerfusionTechnologyontheRise.Gen.Eng.news27(17)2007.

10) DeVochtM.IntensificationofaPER.C6®-BasedRecombinantAd35ManufacturingProcesstoPrepareforCommercialScaleProduction.FifthAnnualOptimisingBiomanufacturingProcesses,Brussels,Belgium,3December2008.

11) noe W. Paradigm Changes and Technology Gaps in the Biopharmaceutical Industry. BioProcessInternationalConferenceandExhibition,October2009,Raleigh,nC.

12) Yuan H. A Platform for Late-Stage Monoclonal Antibody Process Development: Robust High SeedingDensity CHO Cell Culture Process Utilizing Perfusion at n-1 Stage. IBC Antibody Development &Production,Bellevue,WA,16–18March2011

13) Muller,D.ATFPerfusion-BasedProductionPlatformsforBiopharmaceuticals.ESACTConference,Vienna,Austria,15–18May2011.Assessment,”BioProcessInternational,Feb.2012,p.12-18.

14) SethGetal.DevelopmentofanewbioprocessschemeusingfrozenseedtrainintermediatestoinitiateCHOcellculturemanufacturingcampaigns.BiotechnologyandBioengineering,WileyOn-lineaccepted3December2012

15) JohnsonT,11April2013.ProgressionoftheContinuousBiomanufacturingPlatformfortheProductionofBiologics.PresentationatMassachusettsBiologicsCouncilConference.

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BioreactorConfigurationandOperation

Throughthehighercelldensitiesandtitersachievedinconcentratedfed-batchandperfusionculturestypicallysmallerculturevolumesandbioreactorscalesare required to produce the therapeutic protein or antibody. This is a greatadvantageasthefootprintofaproductionfacilitycanbereducedandscale-upissuesaremitigatedduetothemarginalscale-upfactorbetweenclinicaland commercial production. Intensified cell culture processes are especiallybeneficial in the context of single-use facilities as they provide productioncapacitiesat1000Lscalethatinthepastwhereonlyachievablewithtentimeslarger bioreactors.1,2 Furthermore, modern high-end cell culture processesaimtomaintainthecellsinadefinedmetabolicstateinordertoensurestableproduct quality through controlling protein folding and glycosylation. In thiscase,themainaimisnotnecessarilytoreachveryhighcelldensities,buttoensureasteadystateofnutrientsandmetabolitesinthebioreactor.

How to perform concentrated fed-batch or perfusion operationAfterinoculationofthebioreactorandaninitial1–2daybatchgrowthphase,theremovalofcellfreesupernatante.g.withtheRefineATFSystemisstartedataconstantharvestflowrate.Atthesametime,thecultureisreplenishedwithfreshmedium.Whenapplyingsingle-usebioreactorssuchastheBiostat®STR,theadditioniscontrolledviaafeedpumpthatreceivesasignalfromloadcellsoraplatformbalancemaintainingadefinedbioreactorweight.Asthecelldensitygrowsandthenutrientconsumptionandmetaboliteformationincreases,theharvest rate is subsequently increased to maintain a certain exchange rateof freshmediumpercelloralternativelyagivenmediumexchangerateperday.3On-linebiomassmeasurement,e.g.withtheBioPAT®ViaMassprobethatwill soon be available for single-use Biostat®STR and RM bags, provides anautomatedoptiontocontroltheperfusionratebasedoncelldensity.Usingat-lineglucoseandlactatemeasurement,e.g.withtheBioPAT®Trace,anadditionalconcentratedfeedcanbeappliedtocontroltheglucoseconcentration.

03

Dr. Christel Fenge,VicePresident,SartoriusStedimBiotech

Dr.ChristelFengeisVicePresidentofMarketingforFermentationTechnologiesatSartoriuswithglobalresponsibility formanagingtheproductportfolioofmulti-andsingle-usebioreactors.

PriortojoiningSartorius,ChristelheldthepositionoftheGeneralManagerofRecipharmBiologics,aSwedishCMO.Previouspostsincluded senior management positions in biopharmaceuticaldevelopmentatAstraZeneca.ShestartedherindustrialcareerincellcultureprocessdevelopmentatPharmacia&Upjohninvolvedin the development of Refacto™ and in product managementat B.Braun Biotech. Christel Fenge received her doctorate inBiochemistryfromtheUniversityofHanover,Germany.

AbouttheAuthors:

Acknowledgementsto: Jörg Weyand, Gerhard Greller, Thorsten Adams,SartoriusStedimBiotech

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UsingSartoriusstirredtanksingle-usebioreactorsincombinationwithdifferentsizesoftheRefineATFSystem,concentratedfed-batchandperfusionprocessescanbedevelopedatthe2Lbenchscale,e.g.usingtheUnivessel®single-useincombinationwithourBiostat®BorB-DCUcontrollerandsubsequentlyscaledto500Lto1000LscaleintheBiostat®STR.At500Land1000Lscale,theATFSystem filter modules might be connected via side ports of the single usebioreactorbagusinguptotwo1́ ́sterileconnectorsandoperatedinanexternalloopofthebioreactor.Itiscriticalthatthisexternalloopisasshortaspossibletoavoidthatthecellcultureisexposedtouncontrolledconditions,e.g.differenttemperatureandpotentialoxygenlimitations.

Single-use bioreactor configurations suitable for intensified cell culturesKeytosuccessfulconcentratedfed-batchandperfusionoperationisanefficientaerationsystemthatprovidesklavaluesabove10-15h-1tosupplytheculturewithsufficientoxygen (Fig.2).At thesame time,excessivecarbondioxide isformed in the intensified culture which needs to be removed to avoid anyinhibitoryeffectonproductivityorevenproductquality.

klavaluesdeterminedindifferentsingle-usebioreactorbagvolumesoftheBiostat®STR,equippedwith2x3bladesegmentimpellers,usingthegassingoutmethodinphosphatebufferedsaline,aerationrate0,1vvm,150µmholesofCombisparger,temperature25°C.

Figure1providesaschematicdepictionofatypicalconcentratedperfusionorfed-batchset-upbasedontheBiostat®STR.

Key considerationsTypicalperfusionratesare intherangeof1–2bioreactorvolumesperday.Applyingasmallcellbleedstreamenablestheestablishmentofadefinedcellgrowthrateandbythatahighviabilitycanbemaintainedwhichinturnmitigatescloggingofthecellretentiondevice.4Dependentontheporesizeorcut-offofthecell retentionmembrane,either theproduct isrecovered in thecell freeharvest (concentrated perfusion) or in the bioreactor content (concentratedfed-batch).Asmostantibodiesareratherstable,concentratedfed-batchwithaccumulationoftheproductinthebioreactorisasimpleandstraightforwardapproachtoincreasespacetimeyieldsofagivenfacility.Concentratedperfusionisthemethodofchoiceforrecombinantproteinsthatinmanycasesarepronetodegradationormightshowfeedbackinhibitionandshouldthereforeberemovedfromthecellcultureintoachilledharvesttankandsubsequentlypurified.

BioreactorConfigurationandOperation03 COnTInUOUS BIOPROCESSInG CURREnTPRACTICE&FUTUREPOTEnTIAL

Fig. 1: Set-up of a concentrated fed-batch

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Modernsingle-usebioreactordesignssuchasoftheBiostat®STRallowadvanced,intensified cultivation strategies whilst providing tools to mitigate operationalrisksassociatedtoacomplexbioprocessingstrategyandthusenablingrobustsingle-useproductionforclinicaltrialsandcommercialdrugmanufacturing.

Thiscanbeachievedwith theCombisparger thatmicrospargescompressedairorpureoxygenthroughdefined150µmholesandprovidesastrippinggasflowthrough0,8mmholesatthesametime(Fig.3).Thissingle-usespargerdesign emulates a successful aeration strategy applied since many years inconventionalstainlesssteelbioreactors.

Aproblemthatshouldnotbeunderestimated isexcessiveaerosol formationintheexhaustgasduetothehighgasflowratesandthehighproteincontentintheconcentratedcellcultures.Aspecificallydevelopedsingle-useexhaustcooler design based on the well-known principle of plate heat exchangers(Fig. 4) mitigates the risk of blocked filters and increases process reliabilitydramatically.Additionalsafetylocksinthebioreactorcontrolsoftwarepreventbioreactoroverflowincaseofcloggingofthecellretentiondevice.Asaworstcasesafetylock,allfeedpumpsandgasflowsareinterruptedifthepressureinthebioreactorexceedsthemaximumdefinedoperatingpressure.

03 COnTInUOUS BIOPROCESSInG CURREnTPRACTICE&FUTUREPOTEnTIALBioreactorConfigurationandOperation

Fig. 3: Combisparger with 150µm defined

micro-holes and 0,8mm holes.

Fig. 4: Exhaust cooler

References:1) J.Lim,etal.,BioPharmInt.24(2),54–60(2011)

2) J.Pollocketal.,Biotechnol.andBioeng.110(1),206-219(2013)

3) T.Adamsetal.,BioPharmInt.SupplementMay2011,S4-S11

4) C. Fenge et al., in “Animal cell technology: Development, processes and products”, Butterworth-Heinemann,365-370(1992)

27

HowtoDevelopaPerfusionProcess

1. Introduction In a continuous process, the culture medium is continuously renewed byremovalofconditionedmediumandfeedingoffreshmediumwhilethecellsaretotallyorpartiallyretainedinthebioreactorbyacellseparationdevice.Thevolumeoffreshmediumisidenticaltotheoneofspentmedium,whichispreferablycell-free.

The process development can be divided in two main parts, the selection/optimizationoftheparametersandfeaturesoftheperfusion process(excludingthecellseparationdevice)andthedevelopment of the cell separation process itself.TheselectionoftheculturelengthismadefrominformationofbothpartssupplementedwithCostofGoods(COGS)andfailureriskconsiderations.

The efforts of perfusion process development may vary depending on thepurpose of the process: in several cases where the production yield is notcritical, theeffortscanbe limitedwhilemoreeffortsmightbeneededwhendevelopingacommercialproductionprocesswheretheproductionyieldandtheproductqualityarehighlyimportant,andlikelyevenmoreeffortswillbeneedediftheproductofinterest(POI)isunstableorsensitive.

Limitedeffortsarerequired in thecaseofproduction for researchpurposes(i.e.casewheretheproductionyieldislessimportantthanthetimeneededtoobtainthetargetproduct),fortheproductionofcellsaimedatcellbankingorforinoculationofalargerbioreactor.Inthesecases,theaccentisputoncellhealthiness,i.e.highviability,andincreasedcelldensitywhilethecellstabilityismaintained.Inthecaseofcellbankingorcellseedmanufacturing,highviabilityis required both for the success of the next operation step, respectively cellcryopreservationandseedingoflargerbioreactor,andtoavoidcellselectionbycelldeath,whichcouldresultincellpopulationshift.

04

Véronique Chotteau,HeadofCellTechnologyGroup,RoyalInstituteofTechnology(KTH)

Véronique Chotteau has more than 25 years of experience inmammaliancellculturecultivation(suspensionandadherent)andmorethan10yearsofexperienceinbiopharmaceuticalindustryincludingdifferentresponsibilitiesasprojectmanager,pilotplanthead,expertinanimalcellculturedevelopment(small,pilotandcommercialsize/GMP),businessdevelopmentsupport.VéroniquejoinedPharmaciaUpjohn,Stockholm,(nowadaysSwedishOrphanBiovitrum) in 1996. She worked with the process developmentof factorVIIIRefacto,otherperfusionprocessesaswellas fed-batch processes. She left the industry in 2008 when she wasofferedtotakeoverthegroupofCellTechnology,Dept.IndustrialBiotechnology/Health, School of Technology, Royal Institute ofTechnology(KTH),Sweden.Theactivitiesofhergrouparefocusedon established cell-based processes (perfusion, metabolic fluxanalysismodeling)andstemcellbioprocessing.

AbouttheAuthor:

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Forthecaseofproductionforresearchpurposes,oftenthecelllinesarerapidlyobtainedbutsub-optimal,i.e.withlowcellularproductivity.Inthatcaseworkingwithahighcelldensitycancompensatethelowcellspecificproductivitywhilea high viability gives some insurance that the product quality is sufficient –howeverthisisofcoursedependingontheproducedmolecule.

2. Systems for development of perfusion processes Generally speaking, the main trends observed in batch culture will remaintrueinperfusionculture,e.g.afavorableeffectofaplanthydrolysateonthePOIproductionobservedinbatchculturewillmostlikelybeconfirmedwhenappliedinperfusionprocess.Itisadvisabletoconfirm(andpossiblyrefine)theobservationsmadeinbatchcultureinperfusionsystembeforetheirapplicationinaprocess.

2.1. Screening modelSmall vessel cultivation systems aimed at screening larger numbers ofconditions can be used for a pre-determination of parameters, followed byconfirmationor refiningatbioreactorscale.Typically,mediumselectionandeffectofmediumcomponentscanbescreenedsavinglaborandtime.

Apseudo-perfusionprocess (alsocalledsemi-perfusionorquasi-perfusion),usingshakeflasks,spinnersor50mLtubeswithventedcapscanbeusedtosimulateperfusion.Dailymediumrenewalisoperatedmanually:thecultureiscentrifuged,thesupernatantisdiscardedpartiallyortotallyandthecellsarere-suspendedinfreshmedium.

Amaindifferencebetweenthepseudo-perfusionsystemandperfusionistheresidencetimeofthecomponents,whichisasymptoticallyevolvingtotheinputvalueinthelattercase.Forinstance,inaperfusionittakes3daysatperfusionrate1reactorvolume/day(RV/day)foracompletemediumrenewalsincethefreshmedium isconstantlydiluted in theculture (seeFig.1).Contrary, inapseudo-perfusion,theentiremediumvolumeisrenewedatonceforthesameapparentrateof1RV/day.Duetothisdifference,apartialmediumrenewalissometimesadoptedinsteadofcompletemediumrenewal.

COnTInUOUS BIOPROCESSInG CURREnTPRACTICE&FUTUREPOTEnTIAL

Evolutionwithtimeoftherelativeconcentrationofanewcomponentpresentatconcentration1inthefreshmediumofaperfusionprocessoperatedinbioreactorat1RV/dayperfusionrate

2.2 Bioreactor and scale-down modelBioreactorsystemsareusedforthedevelopmentoftheperfusionprocessesand most of the parameters can easily be studied in scaled-down models.Exceptionsareparameterssuchastheshearstressandthedeleteriouseffectofbubble/gassing,forwhichthescaled-downstudyismorechallenging.Thestudyofthecellseparationdeviceitselfhastotakeintoaccountthelimitationsofthetargetedlargescaleforparametersliketheliquidflowsorthepower.

3. Development of the perfusion process

3.1 Medium selectionAculturemediumneedstoincludeallthenecessarycomponentstosustainthecellgrowthandproductionofPOI,e.g.incasemetal(s)oravitaminare

04 HowtoDevelopaPerfusionProcess

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crucialforthePOIactivity.nowadaysserum-freeandchemicallydefinedmediaprovidenotonly thesenecessarycomponentsbutmoreovergiveenhancedcellgrowth,cellsurvivaland/orproductionfromadditionalcomponentsandoptimizedformulation.

Abasemediumcanbeadvantageouslysupplementedwithafeedconcentrate,whichhasbeendevelopedforfed-batchprocess,toimprovethePOIproductionforinstance.Startingthedevelopmentofaperfusionprocesscanbeinitiatedbytheevaluationof5to10commercialmediainabatchshakeflaskproductivityteststudy leading to theselectionof2 to3basemedia.Supplementationofthesewithdifferentfeedconcentratescanthenbestudied.Fromthisstudyabasemedium,potentiallysupplementedwithfeedconcentrate,canbeselectedandtestedinperfusionmode.

Subsequent to this, the medium can be further refined/optimized if it isnecessary to improve the POI cell specific productivity for the goal of theprocess.Anotheraspectisthatsinceperfusionmoderequireslargevolumesof liquidhandling,minimizingtheperfusionratewithoutcompromisingtheprocessperformancescanbedesirable.Thiscanbeachievedbytuningthemedium composition and use of concentrated media (Konstantinov et al.2006;Ozturk1996;Runstadler1992).

3.2 Perfusion rate strategyTwo main strategies can be distinguished to determine the perfusion rate:eitherbasedonthecelldensityorbasedontheavailabilityofamainsubstrateintheculture.Sometimes,itisevendesirabletoincreasetheperfusionratetoreducetheby-productaccumulation.

3.2.1 Perfusion rate strategy based on CSPRAnestablishedstrategyistoadjusttheperfusionrateasalinearfunctionofthecelldensity(Ozturk1996;Konstantinovetal.2006;Clinckeetal.2013b),i.e.toapplyacellspecificperfusionrate,CSPR,whereCSPR=perfusionrate/celldensityorD/celldensity.

Thisallowsavoidingthedepletionofcomponent(s)inthecultureandhasbeendemonstratedtosustainupto200x106cells/mL(Clinckeetal.2013b).Inordertosavemedium,identifyingtheminimalCSPR(CSPR_min)iscritical.AmethodtoselectCSPR_minis:

• inoculatethebioreactoratcelldensity0.3to1x106cells/mL,initiatethecultureinbatchmodeandstarttheperfusionatD=1RV/daywhenthecelldensityhasreached2to3x106cells/mL–importantlystarttheperfusionwhilethecellsarestillinexponentialgrowthphase

• allowthecellstogrowexponentiallyuntile.g.20x106cells/mL,bydailymonitoringthegrowthrate,whileincreasingDto2RV/day(orhigher)incasethegrowthwouldslowdown

• establishaculturearound20x106cells/mLofexponentiallygrowingcellsby performing daily cell bleeds compensating for the cell growth – thiscultureisanexcellentsystemtotestvariousparameterslikeCSPR,pH,etc.

• intheculturestabilizedat20x106cells/mL,identifytheCSPR_minforthegivencelllineandmediumwiththefollowingstepsappliedat1to3daysintervals(durationrequiredtoobservetheeffectsofanimplementedmodification):

increaseDof0.5RV/daystepandgotoeitheri)orii)dependingoftheoutcome

i)ifthegrowthisincreased(byincreasingD),theactualCSPRistoolowandDhastobeincreased(ofe.g.0.5RV/daystep).RepeatincreasingDby0.5RV/daystepsuntilfurtherincreaseofDdoesnotresultinimprovedgrowth.ThenexttolastDgivesCSPR_min.

ii) if the growth is not increased (by increasing D), CSPR_min is nothigherthanCSPRinuseandispossiblylower.TesttoreduceDby0.2RV/daystepandobserveifthegrowthremains(ornot)unchanged.Inthepositive,continuetodecreaseD;inthenegativethenexttolastDgivesCSPR_min.

noticethatafteraslowergrowthhasbeenobserved,itrequiressometime(atleast3days)forthesystemtorecoverfromdepletion(dependinghowseverethedepletionwas).

COnTInUOUS BIOPROCESSInG CURREnTPRACTICE&FUTUREPOTEnTIAL04 HowtoDevelopaPerfusionProcess

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Itisrecommendedtocontrolthefeedofglucoseandglutamineseparately,atastoichiometric rateand tomaintain theirconcentrationat low levelswhenapplyingaCSPRduringtheprocessdevelopmentphase.Asamatteroffact,the need of these substrates can be different from the need of the othermediumcomponents.Whentheprocessisestablished,thesesubstratescanadvantageouslybedeliveredtogetherinthefreshmediumformulation.

3.2.2 Perfusion rate strategy based on main substrate measurement Controlof theperfusionratecanbebasedonamainsubstrate likeglucose(Dowdetal.2001).Glucoseispresentataselectedconcentrationinthemedium.Fromdailyglucoseconcentrationmeasurement,theperfusionrateisincreasedordecreased inorder tomaintain theglucoseconcentrationconstant in theculture.Thiscanbebasedondailymanualglucoseconcentrationmeasurementoronamoresophisticatedon-linemeasurementofglucose.

3.3 Removal of toxic by-productsAmmoniaand lactateareknown for theirnegativeeffecton thecell growthandproductivity.Highammoniaconcentrationsarealsoreportedtoaffecttheglycosylationprofile(Goocheeetal.1991;Jenkinsetal.1996).Usingadialysissystemwith10kDacut-off(Buntemeyeretal.1992)showedthatspentmediumcould be re-used however this is not today an industrial practice. They alsoshowed that other (un-identified) low molecular weight components thanlactateandammoniahadatoxiceffect.

Incasethelactateorammoniaconcentrationsarereachingunfavorablelevels,theperfusionratecanbeincreasedtoremovetheseby-products.Agraphicalrepresentationoftheeffectoflactateorammoniaconcentrationsonthegrowthrateorthecellspecificproductionratecanprovideguidelinesfortheselectionoflimitsoftheseby-productsintheprocess.

3.4 Cell density – target, monitoring and controlSeveralstrategiestoperformaperfusionprocesscanbeadopted.

• Stable cell density with growing cells:Maintainingthecellviabilityashighaspossibleandthecellsingrowingstageisoneofthemainstrategiesusedinperfusionfield.Afteracultureperiodofincreasingthecelldensitytoatargetlevel,thecelldensityismaintainedstableatthislevelinasystemwherecellremoval(automaticallyormanuallyoperated) isperformedataratecompensatingthecellgrowth(Konstantinovetal.2006).Industrialprocessesareoperatedonthisprincipleformonths.

• Increased cell density: Another strategy based on growing cells is toincreasethecelldensityuntilaphysicallimitationofthecelldensityitselfortheequipmentisreached–orclosetobereached-(Clinckeetal.2013a;Clinckeetal.2013b).

• Stable cell density with arrested cells:Athirdstrategyconsistsofafirstcultureperiodofincreasingthecelldensitytoatargetlevel,thentoslowdownorcompletelyarrestthecellgrowth,whichisknowntobepotentiallyassociatedwithahighercellspecificproductivityinacellspecificway.

3.4.1 Inoculation cell densityThanksto themediumrenewalappliedassoonasthecellshavereachedacoupleofmillionscells/mL(seeSection3.2.1foranexample),theinoculationcell density has not the same major impact as in a fed-batch process. Theculturecanbeinitiatedasabatchcultureintheconditionsmimickingshakeflaskscale.Theperfusionisthenstartedwhenthecellsarestillinexponentialgrowthphase.Ahigherinoculationcelldensityallowsshortingdownthetimerequiredtoachievethetargetcelldensity.Anoptioninthislattercaseistostarttheperfusionthesamedayastheinoculation.

3.4.2 Selection/optimization of the cell densityTwodecadesago,thecelldensityinperfusionreachedafewmillionscells/mLinmanycases.Astandardinindustrytodayistotargetaround20x106cells/mLbutthereisatrendtowardsmuchhighercelldensitieswherethebenefitofperfusioncan be fully exploited. It is probable that today many industrial processes aretargeting50to80x106cells/mL(Clinckeetal.2013a,JohnsonT2013).


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3.4.3 On-line cell measurementBesides manual sampling, the cell density can also be measured on-line based on the dielectric properties of the cell, i.e. permittivity and/orcapacitance,bycommercialprobes.Recentlyin-situmicroscopetechnologyhasalsobeendeveloped.

Anotherwayisacelldensityevaluationobtainedbymonitoringtheconsumptionofoxygenorglucose(Kyungetal.1994;Meuwlyetal.2006).

3.4.4 Cell bleeding Cell bleeding is operated to partially remove the cells from the bioreactor,typicallybypumpingout thecellbroth fromthebioreactor.Thisoperation issystematicallyincludedinastrategywherethecellsaremaintainedatastablecelldensity(seeabove).Threemethodscanbeused:

• The more accurate method is to use a continuous pump automaticallycontrolledbasedontheon-linecelldensitymeasurementinordertotrackthecelldensitysetpoint.

• Thecontinuouspumpcanalsobemanuallytunedbasedondailyoff-linecelldensitymeasurementachievingsatisfactoryresults.

• Dailymanualcellremovalcanalsobeusedduringtheprocessdevelopmentphase:theperfusionismomentarilystopped,cellbrothisremovedandthennewfreshmediumisaddedtocompensatetheremovedculturevolumebeforere-startingtheperfusion.

Manualcellremovalsarealsooperatedadhoctoreducethecelldensity,e.g.torespectgivencelldensitylimitsofaregisteredprocess.Duringtheprocessdevelopment,thestudiedconditionscanresultindegradedcellpopulationwithlowviabilityand/orabsenceofgrowth;amanualcellremovalisthenappliedtohelpthecellrecovery.Thiscanbeaccompanied(ornot)byamomentarilyincrease of the perfusion rate in order to speed up the medium renewal,providingmorefavorableenvironmentalconditions.

3.4.5 Cell arrest Likewiseinfed-batchprocessescellarrestbyphysicalorchemicalmeanscanbeused,giventhatthecellspecificproductivityisincreasedandthattheproteinqualityiscorrect-orevenimproved-(Angepatetal.2005;Chotteau2001;Ohetal.2005).CellarrestinGO/G1phasecanbereversiblyobtainedforinstancebyreducingthetemperature(Angepatetal.2005;Zhangetal.2013)oraddingachemicallike(toxic)butyrate(Ohetal.2005),seeSection3.4,paragraph‘Stablecell density with arrested cells’. The (more abundant) knowledge reported forbatch and fed-batch processes like the ranges of temperatures or butyrateconcentrationscanbeapplied inperfusionprocesses.Theseparametershavetobeoptimisedonacelllinespecificbasis.Typicallyalotofcelllinesarestillgrowingat34˚Cor35˚Cbutslowerthanat37˚Candarebarelygrowingat31˚C.Aspreviouslymentioned,theseapproachescanleadtoincreasedcelldamage.

3.4.6 Cell viabilityLow cell viability can affect the POI quality due to the associated proteolyticactivityreleasedbythelysedcells.Anotherimportanteffectofthepresenceofdeadcellsisthereleaseofnucleicacidandcelldebris,reportedtoplayamajorroleinfilterclogging(Escladeetal.1991;Mercilleetal.1994).Itisthereforehighlyadvantageoustomaintainthecellviabilityashighaspossible.Finally,aconsequenceofdeadcellsistheaccumulationinthecultureofcelldebris,whichmayberemovedthroughbleedingorviasomeperfusiondevices.

3.5 Protein qualityThePOIpresentintheharvestisstoredinacooledharvesttankduringtheculture(unlesscontinuouspurificationisemployed),allowingagoodpreservationofthePOIquality.For instance theproteolyticactivity ishighlyreduced.During theprocessdevelopment,theevolutionwithtimeofthePOIqualityinthecooledtank is studied according to the quality attributes important for the POI, i.e.analyses/characterisation.ThisstudytogetherwithlogisticsandCOGSfactorswill contribute to the decision of the harvest frequency for the process. Theconstantenvironmentoftheperfusiongreatlycontributestothestabilityofthequalityattributeswithtime.Anotherfactorofattributeprofilevariationwithtimeistheapplicationofcellarrest(seeSection3.4.5).


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IfsmallvariationsinthePOIqualitylikeminorvariationsinthedistributionofspeciesoccur,acommonprocedureinindustryistopooldifferentPOIbatchesissuedfromdifferentharvestsfromdifferentculturerunsinordertoreconstitutethePOIaccordingtothespecifications.

3.6 Parameter optimizationDuringtheprocessdevelopment,theeffectoftheparametersontheprocesscanbepreliminarystudiedinbatchmodeorinpseudo-perfusion(seeSection2.1).Thisisthenconfirmed/furtherstudiedatbioreactorscale.Duringabioreactorrun,severalparametervaluescanbetestedsequentiallyintime(Milleretal.2000;Hilleretal.1993):

• acultureatagivenconstantcelldensityisestablished(seeSection3.4)

• theeffectofagivenparametervalueistestedduringseveraldays,e.g.4to7days,bymonitoringthecellgrowth,viability,metabolism,POIproductionandquality(ifrelevant)

• thenthisparameterischangedtoanewvaluetobetested

• afteratransitionperiod,e.g.2to3days,theeffectofthisnewvaluecanbemonitoredasdescribedabove.noticethatincasethepreviousparametervaluewasextremeanddamageableforthecells,thetransitionperiodhastobelongeruntilthecellgrowthandviabilityarebacktotheirnormalvalues.

A factorial analysis, also called Design of Experiment, approach canadvantageously be adopted to study the effect of several parameters on theprocess as commonly used in the whole culture process development field(Pintoetal.2008;Bollinetal.2011;Sandadietal.2006).

3.6.1 Environmental parametersDifferentphysicalparametershavelargeormoderateinfluenceontheprocess.

• pH: The pH has a major effect on the cell growth, POI, glucose/lactatemetabolismandthereforealkaliaddition,osmolalityandpCO2level.Often,differentpHvaluesareoptimalforthecellgrowthandthePOIproduction.

• Dissolved oxygen concentration (DO): The DO has often a much minoreffectonthePOIproductiongiventhatDOisbetweensome20and80%ofairsaturation(Linketal.2004).Highercellspecificglutamineandglucose

uptakerateshavebeenreportedforhybridomawithincreasingDO(Janetal.1997;Thommesetal.1993).Below20%DO,thecellsaresubmittedtostressaffectingthemetabolicandproductionratesaswellasthegrowthrate.

• partial pressure of carbon dioxide,(pCO2): pCO2 values in the range of15 to105mmHg (2 to14kPa)havenomajor influenceon theprocess.Largervaluescanaffect thecellgrowth, thePOIproductionandquality.Anadvantageofperfusioncomparedtofed-batchislowervaluesofpCO2providedbythemediumrenewal.

• Temperature:seeSection3.4.5.

• Scale-upfactors,i.e.shearrate/shearstress,gassing-bubbledamage:see‘Scaling-upandTechtransfer’Ozturk,SS

3.6.2 Substrate concentration optimizationItisrecommendedtomaintaintheglucoseandglutamineatstablevaluesduringaproductionprocesstomaintainthecellmetabolismconstant.Lowlevelsofglucose,i.e.≈5mM,andglutamine,i.e.≈0.5mM,resultinlowproductionoflactateandammoniasothisisavaluablestrategyforperfusionprocesses.


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9) Goochee CF, Gramer MJ, Andersen DC, Bahr JB, Rasmussen JR. 1991. The oligosaccharides ofglycoproteins: bioprocess factors affecting oligosaccharide structure and their effect on glycoproteinproperties.Bio/technology9(12):1347-55.

10) Hiller GW, Clark DS, Blanch HW. 1993. Cell retention-chemostat studies of hybridoma cells-analysisof hybridoma growth and metabolism in continuous suspension culture in serum-free medium.Biotechnologyandbioengineering42(2):185-95.

11) JanDC,PetchDA,Huzeln,ButlerM.1997.Theeffectofdissolvedoxygenonthemetabolicprofileofamurinehybridomagrowninserum-freemediumincontinuousculture.Biotechnologyandbioengineering54(2):153-64.

12) Jenkinsn,ParekhRB,JamesDC.1996.Gettingtheglycosylationright:implicationsforthebiotechnologyindustry.naturebiotechnology14(8):975-81.

13) JohnsonT,11April2013.ProgressionoftheContinuousBiomanufacturingPlatformfortheProductionofBiologics.PresentationatMassachusettsBiologicsCouncilConference.

14) KonstantinovK,GoudarC,ngM,MenesesR,ThriftJ,ChuppaS,MatanguihanC,MichaelsJ,navehD.2006.The“push-to-low”approachforoptimizationofhigh-densityperfusionculturesofanimalcells.Adv.Biochem.Eng./Biotechnol.101:75-98.

15) KyungYS,PeshwaMV,GryteDM,HuWS.1994.Highdensitycultureofmammaliancellswithdynamicperfusionbasedonon-lineoxygenuptakeratemeasurements.Cytotechnology14(3):183-90.

16) Link T, Backstrom M, Graham R, Essers R, Zorner K, Gatgens J, Burchell J, Taylor-Papadimitriou J,HanssonGC,nollT. 2004.Bioprocessdevelopment for theproductionofa recombinantMUC1 fusionproteinexpressedbyCHO-K1cellsinprotein-freemedium.JournalofBiotechnology110(1):51-62.

17) Mercille S, Johnson M, Lemieux R, Massie B. 1994. Filtration-based perfusion of hybridoma culturesin protein-free medium: Reduction of membrane fouling by medium supplementation with Dnase I.Biotechnologyandbioengineering43(9):833-46.

18) MeuwlyF,PappF,RuffieuxPA,BernardAR,KadouriA,vonStockarU.2006.Useofglucoseconsumptionrate(GCR)asatooltomonitorandcontrolanimalcellproductionprocessesinpacked-bedbioreactors.JournalofBiotechnology122(1):122-9.

19) MillerWM,BlanchHW,WilkeCR.2000.Akineticanalysisofhybridomagrowthandmetabolisminbatchandcontinuoussuspensionculture:effectofnutrientconcentration,dilutionrate,andpH.ReprintedfromBiotechnologyandBioengineering,Vol.32,Pp947-965(1988).Biotechnologyandbioengineering67(6):853-71.

20) OhHK,SoMK,YangJ,YoonHC,AhnJS,LeeJM,KimJT,YooJU,ByunTH.2005.Effectofn-Acetylcysteinonbutyrate-treatedChinesehamsterovarycellstoimprovetheproductionofrecombinanthumaninterferon-beta-1a.Biotechnologyprogress21(4):1154-64.

21) OzturkSS.1996.Engineeringchallengesinhighdensitycellculturesystems.Cytotechnology22(1-3):3-16.

22) PintoRC,MedronhoRA,CastilhoLR.2008.SeparationofCHOcellsusinghydrocyclones.Cytotechnology56(1):57-67.

23) RunstadlerPW.1992.Theimportanceofcellphysiologytotheperformanceofanimalcellbioreactors.AnnalsofthenewYorkAcademyofSciences665:380-90.

24) SandadiS,EnsariS,KearnsB.2006.Applicationoffractionalfactorialdesignstoscreenactivefactorsforantibodyproductionbychinesehamsterovarycells.Biotechnologyprogress22(2):595-600.

25) ThommesJ,GatgensJ,BiselliM,RunstadlerPW,WandreyC.1993.Theinfluenceofdissolvedoxygentensiononthemetabolicactivityofanimmobilizedhybridomapopulation.Cytotechnology13(1):29-39.

26) ZhangY,Stobbe,P,Orrego,CS,Chotteau,V.2013.PerfusionatveryhighcelldensityofCHOcellsanchoredinaCFD-designednon-wovenmatrixbasedbioreactor.manuscriptinpreparation.

41

CaseStudy:OptimizedPerfusion

AbstractImplementationofaperfusionprocessallowsfor improvedandreproducibleproductivitywithina low tomid-rangeproducingsystem.Gallusoptimizedabioreactor process for a Sp2/0 cell line producing a monoclonal antibody.The previously developed process was not providing optimal results.Theunderstandingofmetabolicneedandproperperfusionoptimizationallowedfora56%increaseincumulativebioreactoroutput.

IntroductionThe perfusion bioreactor process is useful for generating high celldensity cultures and when properly executed yielding improved bioreactorperformanceandcumulativeoutput.Inordertoproperlydevelopaperfusionprocess,understandingthemetabolicneedsofthecultureisarequirement.Withoutproperunderstanding,theperfusionratesandbleedratesmaynotbe operated to best maintain the metabolic need of the culture. Throughunderstanding and adjustment, a perfusion process can be optimized andexecutedtomaintainhighcelldensity,highviabilitycultureswithconsistentandsustainedantibodyproduction.Thiscasestudydemonstratesunderstandingthemetabolicneedandadjustingtheperfusionparameterstobetteroptimizeapreviouslyestablishedprocess.

05

Shaun Eckerle,SeniorScientist,GallusBioPharmaceuticals

ShaunEckerlejoinedCentocorBiologicsLLCin2004aspartofcellculture manufacturing. Subsequently he moved to ManufacturingTechnical Support for non-conformance resolution, technologytransfer and manufacturing process improvement. Following theacquisition of the facility by Gallus BioPharmaceuticals, ShauntransitionedtoProcessDevelopment leveraginghisexpertisewithperfusionbioreactorsystemstonewprocessesdevelopedatGallus.Shaun has worked closely with Xcellerex to establish expertise oftheXcellerexdisposablebioreactortechnologytoGallus.CurrentlyShaunisdevelopingprocessestoconvertfromtraditionalstirredtankbioreactortoXcellerexDisposabletechnology,developingscalabilityfromtheXDR-10toXDR-2000aswellasATFSystemperfusionwiththeXcellerexplatform.ShaunhasaB.S.BiologicalSciencesandisworkingonaM.S.Biochemistry.

AbouttheAuthor:

4342

Materials and Methods • CellLine:Sp2/0cellexpressingmonoclonalantibody • Perfusion–ATFSystem • Glassvesseloperations:Applikon5L/SartoriusDCUII • MetaboliteAnalysis:nOVABioProfile400 • CellCounting:Manual • IgGAnalysis:HPLC • GlucoseFeed • GlutamineFeed

Procedure • InoculumscaledupinT-flasks,expandedtodisposablecellbags • Performanceof5LGlassVesselwithATF2toconfirmprocess • OptimizationofPerfusionProcessin5LGlassVesselwithATF2 • Scaleupto50LStainlessSteelwithATF6

05 COnTInUOUS BIOPROCESSInG CURREnTPRACTICE&FUTUREPOTEnTIALCaseStudy:OptimizedPerfusion

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4544

ConclusionsByfurtherunderstandingthemetabolicneedoftheculture,theprocesswasadaptedtoremoveexternalfeeds.Theoptimizationoftheperfusionprocesshas lead to a more rapid increase in viable cell density and titer resultinginanaverageof56%increaseincumulativebioreactoroutputat labscale.Perfusion processes when optimized can lead to significant increases inproductyieldandultimatelyreductionincostofgoods.

05 COnTInUOUS BIOPROCESSInG CURREnTPRACTICE&FUTUREPOTEnTIALCaseStudy:OptimizedPerfusion

Fig. 3

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47

ProcessIntensificationApproachesforCostSensitiveProteinApplications

Thechoiceofamanufacturingplatformandproductionmodearesomeofthemostimportantstrategicdecisionsinrecombinantsubunitvaccinedevelopment.Drosophila S2 insect cell expression is less known than the extensively usedSpodoptera(Sf9)orTrichoplusiani(Hi-5)insectcellbasedBaculovirusexpressionsystem(BEVS).neverthelessS2cellshavebeenusedinresearchforalmost40years.ThecelllinewasderivedfromlatestageDrosophilamelanogaster(Fruitfly)embryosbyDr.Schneiderintheearly1970s,whonamedthecelllineDrosophilaSchneider line 2 (synonyms: S2, SL2, D.mel. 2). The S2 system has uniqueadvantagesforlow-costproductioncomparedtoBEVSasitisastablecellline,non-viral andanon-lytic system.Thisallows forawide varietyofupstreamprocessingoptionscomparedtotheobligatorybatchprocessapproachofthehigh-yielding,butlyticBEVSsystem.

Thefieldofneglecteddiseasesisspecificallyrelevantfortheapplicationofprocessintensifying and cost reducing processing production modes. Particularly,thegeographicdistributionofmalariaandthephilanthropic fundingsourcesinvolved require production to be as cost-effective as possible. Single-usebioreactorscombinedwithperfusionproductionmodeprovidemanufacturingflexibilityandeconomicadvantages,bothhighlydesirableinthistypeofprocess.ExpreS2ionBiotechnologiesaimtodevelopcost-effectiveDrosophilaS2basedproductionprocessescombiningitsExpreS2constitutiveinsectcellexpressionsystemwithsingle-usebioreactorandperfusiontechnology.

ExpreS2ion has established collaborations with The Jenner institute, OxfordUniversity and The Center for Medical Parasitology, Copenhagen University, todevelop the protein production processes for the blood-stage malaria vaccineantigenprotein(referredtoasProtein2inthetext)andtheplacentalmalariavaccineantigenVAR2CSA,respectively.Theproductionofthesecomplexproteinvaccineantigensprovidesanidealopportunitytoapplyadvancedprocessingtechnologies.

06

Willem Adriaan (Wian) de Jongh, CSOandco-founder,ExpreS2ionBiotechnologies

Dr.deJongh(SouthAfrican)obtainedaBachelordegreefollowedby a M.Sc. in Chemical Engineering from the University ofStellenbosch,SouthAfrica.Thereafter,hewasawardedadoctorateinBiotechnologyfromtheTechnicalUniversityofDenmarkin2006.Dr.deJonghhassevenyears’experienceinthepharmaceuticalindustry in molecular biology; project management; processdevelopment;andprocesstransfertocGMPmanufacturing.

AbouttheAuthor:

4948

Methods:Batch,fed-batchandperfusionmodeswerecomparedforgrowthprofilesandproductyield.AtruncationvariantoftheVAR2CSAplacentalmalariavaccineantigen and full-length Protein2 were cloned into a pExpreS2 vector andtransfectedintoDrosophilaS2insectcells.Stablecelllineswereestablishedin three weeks using antibiotic selection in T-flask culture. The cells wereexpandedandinoculatedatbetween5E6and8E6cells/mlforbatch,fed-batch,orconcentratedperfusionin1LDasGip,2LBBraunorCellReady3Lbioreactors.Thebatchproductionrunswereharvestedafter3days,fed-batchafter7daysandperfusionculturesafter6or9days.AnATFSystem(alternatingtangentialflow) from Refine Technology was employed for concentrated perfusionproduction.Thebioreactorconditionswere25°C,pH6.5,and110-150rpmstirrerspeedusingaMarineimpeller.Theperfusionratesweresetto0.5to3ReactorVolumes(RV)perdayandwasincreasedsignificantlyfasterfortheCellReady3LperfusionruncomparedtotheBBraunruns,with3RVperdayreachedbyday6vs.day9fortheBraunruns.

ThePerfusion-Biosep runwasperformedusing the10LBioSep (Applikon) ina2LBBraun bioreactor and the Perfusion run was performed using the ATF2 (Refine) in aCellReady3L(Merck-Millipore).ThePerfusion-Filterexperiment(Wangetal.2012)wasperformedusingtheWavesystem(GEHealthcare)ina2LCellBagusingafloatingfilterwithnominalporesizeof7µm.


Results:Cell counts achieved using perfusion technology

S2cellsnormallygrowtocelldensitiesof40–50E6cellspermLinbatchmode.A fed-batchapproachcan increase thecellcounts to60-80E6cellspermL.However, further increasesofup to104E6cellspermLhavebeenreported(Wangetal.2012)whenusingafloatingfilterinawavebioreactor.ExpreS2ionhasachieved140E6cellspermLusingtheBiosepperfusiontechnologyin2Land5LBBraunbioreactors.Recently,theapplicationofATFSystemperfusiontechnologyhasimprovedthecelldensityto300-350E6cellspermLinbothBBraun2LandCellready3Lbioreactors(Fig.1).

Effect of feed strategySignificant effects on growth and production were seen depending on feedstrategy. The increased growth rate observed for the CellReady3L perfusionruncomparedtotheBBraunbioreactorwasduetoafeedprofiledesignedtoallowmaximumgrowthrateintheCellready3L(seeFig.2A).ThefeedprofilefortheBBraunrunwasdesignedtoobtainlineargrowth.Anexponentialgrowthrateupto350E6cells/mLwasachieved,andtheproductionwasonlystoppedbecausethemaximumflowrateofthefilterwasreached.However,itisclearthatthespecificproductivityoftheS2cellsunderexponentialgrowthconditionswassignificantlylowerwhencomparedtothelineargrowthconditions.Similaryieldswereachievedondays1through6inbothbioreactors,eventhoughthecellcountswereuptothreefoldhigherintheexponentialgrowthexperiment(seeFig.3).Similarly,alineargrowthprofilewasmaintainedfortheVAR2CSAconcentratedperfusionrun.However,onday9theperfusionratewasincreasedfrom2to3RV/day,whichleadtoalargeincreaseincellnumber.AsthiswasthemaximumpossibleperfusionratewiththeATF2,theincreasedcellcountcouldnotbemaintainedwithanincreasedperfusionrate,whichledtoadrasticdecreaseincellviability.Thisdemonstratestheneedtomaintainaminimumperfusion rate to achieve high viability. ExpreS2ion estimates the neededperfusionrateusingthestandardapproachofattemptingtomaintainaconstantflowratepercellperdaythroughouttherun.

ProcessIntensificationApproachesforCostSensitiveProteinApplications06

Fig. 1: Maximum cell counts achieved in

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Yield improvements achieved using Fed-batch and Concentrated PerfusionTheVAR2CSAtruncationvariantwasexpressedinbatchandfed-batchculturein1LDasGipBioreactors.Ahigherthan30%yieldincreasewasachievedwhenusingafed-batchapproachcomparedtobatchproduction.

ConcentratedperfusionwasalsoperformedonthecelllineinaBBraun2Lbioreactor,andextremelyhighcellcountsof350E6cells/mLwereachieved.Unfortunately,noquantitativeanalysistechniquewasavailabletodeterminetheyieldincreasesachieved.AnELISAmethodiscurrentlyunderdevelopment,butfromwesternblotanalysisitcouldbeseenthatsignificantyieldincreaseswereachieved.

The production of Protein2 was also compared in batch, fed-batch andconcentratedperfusionusingbothCellReady3Landglassbioreactors.Significantyieldincreaseswereobtainedgoingfrombatchtofed-batchproduction,andagainfromfed-batchtoconcentratedperfusion.ComparableyieldswereobtainedinbothCellReady3LandBBraunbioreactors(seeFig.3).Furthermore,350E6 cells/mlwereachievedinconcentratedperfusionmodeusingtheATFSystemandCellReady3L.ConcentratedperfusionleadtofinalProtein2yieldsof210mg/Land500mg/Lafter6or9dayproductionruns.


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Protein stability:Strikingly,itcouldbeobservedthatdecreasedcellviabilityonthelasttwodaysoftheVAR2CSAperfusionrunduetoatoolowperfusionrateledtoextensiveproductdegradation.Clearly,thisdegradationcouldbeavoidedbymaintainingcellviability.Similarly,SDS-pageanalysisofthepurifiedofProtein2fromaday8harvestfromFed-batchculture,oraday10harvestfromtheperfusionculture,showedincreasedintensityofbandscorrespondingtotwodegradationsproductofProtein2whencomparedtothefed-batchculture(seeFig.4).Inthislattercasetheproductcleavagewaslesssevere,althoughitwasalsopresentevenwhiletheculturewasmaintainedathighviability.

SDS-pageanalysisofpurifiedProtein2fromaday8harvestfromFed-batchculture,oraday10harvestfromtheperfusionculture.

Conclusion:The protein stability issues observed for Protein2 (and to a lesser extent forVAR2CSA)demonstrateoneofthekeyweaknessesofconcentratedperfusiontechnology,namelytheneedforproductstabilitytoenableextendedproductresidencetimesinthebioreactor.However,fordegradationproneproteins,theoptionofperformingstandardperfusionusingtheATFSystemoffersasimplesolution by reducing the product residence time to less than 24 hours withdirectharvestat4oC.Itisthereforenecessarytoevaluateeachproteinbasedon


stabilitybeforedecidingonapplyingeitherconcentratedperfusionorstandardperfusion.Forinstance,theVAR2CSAtruncationvariantcouldbesuccessfullyproducedusingconcentratedperfusionon theconditionofhighcell viability.Significant yield increases through increased cell counts, and consequentproductionscalereductionsarepossibleinbothcases.ConcentratedperfusionorstandardperfusionusingtheATFSystemthereforeoffersattractiveprocessintensificationapproachesforcostsensitiveproteinproductionneeds.


Fig. 4

References:1) WangL,HuH,YangJ,WangF,KaisermayerC,ZhouP,2012,MolBiotechnol.2012Oct;52(2):170-9

FurtherReading:Moraes,AngelaM.;Jorge,SoraiaA.C.;Astray,RenatoM.;Suazo,ClaudioA.T.;CalderonRiquelme,CamiloE.;Augusto,ElisabethF.P.;Tonso,Aldo;Pamboukian,MarilenaM.;Piccoli,RosaneA.M.;Barral,ManuelF.;Pereira,CarlosA.,DrosophilamelanogasterS2cellsforexpressionofheterologousgenes:Fromgenecloningtobioprocessdevelopment,BIOTECHnOLOGYADVAnCES;v.30,n.3,p.613-628,MAY-JUn2012

Dyring C., Optimizing the Drosophila S2 Expression System for Production of Therapeutic Vaccines,BioProcessingJournal;Winter2011/2012,Vol.10Issue2,p28

DeJonghW.A.,SalantiA.,Proteinonthefly,EuropeanBiopharmaceuticalReview,2012

Dyring C., De Jongh W.A., Salanti A., A Robust, Scalable Platform for Recombinant Protein Expression,InternationalPharmaceuticalIndustry,2012

Acknowledgements:M.dosSantosMarquesResende*,L.Poulsen§,C.Leisted§,A.Strøbæk§,B.Berisha*,M.nielsen*,A.Salanti*,K.Hjerrild2,K.Wright3,M.Higgins3,S.J.Draper2,C.Dyring§

§ExpreS2ionBiotechnologiesApS,AgernAllé1,Hørsholm,DK-2970,Denmark,[email protected]

*CentreforMedicalParasitology,FacultyofHealth,CopenhagenUniversity,Denmark

2TheJennerInstitute,OxfordUniversity,UnitedKingdom

3DepartmentofBiochemistry,UniversityofOxford,UnitedKingdom

Fed-Batch Perfusion

Protein2:Full-length

Protein2:Degradationproduct1

Hostcellprotein

55

ImpactofSingle-useTechnologyonContinuousBiorocessing

Single-use in BioprocessingSingle-useinbioprocessingreferstomaterialsorequipmentthatcanbeusedinoneprocessingbatchorcampaign,andusuallyhavingaproductcontactsurfaceelementthatisdisposable.Suchequipmentrangesfromsinglematerial,verysimplestand-aloneitemssuchasatubing−tocomplexandcontrolledsystemsof many components and materials, such as a bioreactor 1. Relatedly, theapplicationofsuchequipmentrangesfromaninstrumentwithasingle,simplefunctiontoskidshousingentireorevencombinedunitoperations.Mostofthemore complicated single-use (SU) systems contain re-usable non-product-contactelements,forsuchpurposesassupport.SUsystemshavebeentakenup in the biopharmaceutical industry in general because of the numerousfeaturestheyprovide (Table1A).Over thepast10yearsorsothenumberofindividualprocessactivities,upstreamoperationssupport−aswellasentiresystems available has grown substantially (Table 1B). Some of the newerproductsavailableforupstreamapplicationsincludedisposablepumps,single-useflowpathauto-samplingandmicrocarrierseparators2.

Table1.Upstreamsingle-usetechnologyfeaturesandsystems

07

William G. Whitford,ThermoFisherScientific

BillWhitfordisSr.Manager,EmergingTechnologiesforThermoScientific Cell Culture and Bioprocessing in Logan, UT withover20yearsexperienceinbiotechnologyproductandprocessdevelopment. He joined the company nine years ago as ateam leader in R&D developing products supporting biomassexpansion,proteinexpression,andvirussecretioninmammalianand invertebrate cell lines. Products he has commercializedinclude defined and animal product-free hybridoma media,fed-batch supplements, and aqueous lipid dispersions. HehadpreviouslyservedasManufacturingManagerforPHASE-1Molecular Toxicology in Santa Fe, new Mexico producingcustom and standard DnA microarrays. An invited lecturerat international conferences, Bill has published over 200articles,bookchaptersandpatentsinanumberoffieldsinthebioproduction arena. He now enjoys serving on the EditorialAdvisoryBoardforBioProcessInternational

AbouttheAuthor:

A: Features Provided by SU B: Operations Supported by SU

Reducedcontaminationrisks Cellcultureforseedexpansionandproduction

Lowerinitialinvestmentcosts Media,bufferandprocessliquidpreparation

Lowerfacilityandoperatingcost Liquidpumping,filtration,collection,shipping

Reducedoperatorrequirement On-linecontentsmonitoringsensors/samplers

Processefficiencyandflexibility Transport/storageofintermediateandproduct

Timetomarketandeaseofuse Cryopreservationofseedsandintermediates

5756

Continuous Processes in Upstream BioproductionBy far the most common approach to continuous processing in upstreamanimalcell-basedbioproduction isthroughperfusionculture3,4. Inperfusionculture medium is added at rates exceeding the cell mass expansion rateand theexcessmedium is removedusingsomedevice to retaincells in thebioreactor 5, 6. A number of such research- and production-scale perfusionbioreactorsystemshavebeendevised7.Althoughmanyperfusionprocessesfor either suspended or adherent Animal cells are known to be used inmanufacturing-scaleproduction,detailsontheirdesignandoperationarenotalwayspublicallyavailable.Terminologyinthisdynamicfieldcangetfuzzy,forexample,continuousprocessingisalsoreferredtoascontinuousproduction,continuous flow processing or continuous manufacturing. Minor distinctionsaresometimesmadewiththem.Dependingontheperiodicityofeitherentireproductionepisodesorofmorediscreteindividualcomponentoperations,someevenapplysuchtermsassemi-continuousorpseudo-continuousoperation8.nevertheless, interest in the field is growing 9-12 significant stakeholderinvestment is occurring 13 and commercial instrumentation to support itsincorporationinsingle-useorhybridapplicationsisnowappearing.

Single-use in Continuous BioproductionSUtechnologiessupplyanumberofvaluestoanymodeofbioprocessing,butcanprovidesomespecificandenablingfeaturesincontinuousbioprocessing(CB)implementations14-17.CBhas introducedaninterestingtwistonthestandardparadigmof theconceptof iterationsofequipmentusage.Therehasalwaysbeenabitofwiggleinthedistinctionbetweentheconceptof“single-use”andsuchtermsas“disposable”or“limited-use”.PresentedhereisanintroductionofhowCBhasdeterminedare-examinationofafewrelatedconceptsinthisregard(Table2)andhowSUandhybridequipmentsupportssuchupstreamCBapproachesasintensifiedperfusionculture.


Table2.CB-modifiedqualifiersofusageinbiotechnology

MosteveryoperationinaCBprocesstrainisnowsupportedbyacommerciallyavailablesingle-use,orat leasthybrid,solution.Firstofall,manyof theSUequipment and solutions being developed for batch bioproduction have thesame or related application in CB systems. Examples here include simpleequipmentsuchastubingsandconnectors,tomorecomplexapplicationssuch

ImpactofSingle-useTechnologyonContinuousBiorocessing07

Concept Definition CB-specific Modification

Reusable Equipmentormaterialintendedforuseinaprocessforanindefinitenumberoftimes:especiallyindifferentproductioncyclesorbatches,andaftersalvagingorpreparationbyspecialtreatmentorprocessing.

none

Multi-orlimited-use Equipmentormaterialintendedforuseinaprocessforalimitednumberoftimes:determinedbyvalidatedprocedureorsubsequenttesting.

AsCBbydefinitioncanincreasethetimeandthroughputvolumesinvolvedineach“use”,reviewofthenumberofiterationsaddressedisadvised.

Single-use Equipmentormaterialintendedforuseinaprocessforonetimeandthenretiredfromuse.

AsCBbydefinitioncanincreasethetimeandthroughputvolumesinvolvedineach“use”,reviewofthevalidationrequirementisadvised.

Hybrid Equipmentormaterialcomposedofbothreusableandsingle-usecomponents.

none

Disposable Equipmentormaterialintendedforuseinaprocesseitherforonetimeorforuseinaprocessinalimitednumberoftimes,andthenretiredaswasteorgarbage.

Samealterationaseither“Single-use”or“Multi-use”,dependingupontheintent.

5958

asthecryopreservationof largeworkingstockaliquots inflexiblebioprocesscontainers(BPCs).ThelistofCB-supportingSUtechnologiesbeingdevelopedislargeandgrowing(Table3)

Table3.ContinuousBioproductionRelatedSingle-useTechnologies

ASUadvantageinprocessdevelopmentisitssupportsofanopenarchitectureapproach and a number of hybrid designs. Such designs include combiningreusableandsingle–usesystems,orbetweendivergentsuppliersofparticularequipment.Especiallyinbioproduction,themanyflexibilitiesofSUsupportamanufacturingplatformofexceptionalefficiency,adaptability,andoperationalease18.AdvancedsolutionsinSUtransfertubing,manifolddesignandcontainerportingalsosupportscreativity inprocessdesign.This isofparticular valueindesigningaprocesswithsuchdemandsasentirelynewflowpathsor lotdesignations,suchforCB.


SUsystemsupstreamprovideareduced footprintandeliminateof theneedfor cleaning and sterilization service. This complements perfusion culture’sinherentlysmallersizeandindependencefromcleaningforextendedperiodsoftime.

SeveralnewerapproachestoformulatingprocessfluidssupporttheconceptofCB.Single-usemixingsystemsaretypicallyconstructedofarigidcontainmentsystemwithamotorandcontrolsdrivingradiation-sterilizedsingle-usebagsequippedwithdisposableimpellerassemblies.Fromavarietyofmanufacturersthere are a number of distinct approaches to motor/disposable impellerassemblylinkages,tubinglinesandconnections.AlsoappearingareanumberofexcitingSUsampling,sensing,andmonitoringsolutions.Single-usepowdercontainerspermitseamless transferbetweenpowderand liquid formulationsteps,andtheridgedmixingcontainersareavailableinjacketedstainlesssteelforheatingandcoolingrequirements.Surprisingly,the“topping-up”oflarge-scalesingle-usefluidcontainerswithnewlypreparedbuffertoprovideavirtuallyunlimitedandconstantsupplyofeachbuffer/mediatypecanbevalidatedforGMPmanufacturingprocedures.

Continuous,automatedin-lineculturemediaandbufferdilutionandconditioninghavebeenattemptedfordecades,andinterestinthemremainshigh19.Oflate,advancements in the in the mass flow technology, monitoring and feedbackcontrol required to establish and maintain process fluid specifications arenowallowingsuchapproaches tobecomea reality20. Thecompactsizeandportabilityof theequipment involvedallows it toproducefluidsat the“pointofuse”andissupportedtheincorporationofSU.So, in-linepreparationandfluidconditioningprovidesbenefitstobioprocessingingeneral,supportsCBinparticularandcontributesspecificfeaturessupportingsingle-usetechnologyapplicationinCB.Forexample,itsdemandforsignificantlyreducedbufferpreptanksizessupportsapplicationofsingle-useBPCscontainersandmanifolds.

ProcessflexibilityisakeyfeatureinbothSUandCB.CBcontributestooverallprocess flexibility in that equipment tends to be easy to clean, inspect andmaintain−andgenerallypromotessimpleandrapidproductchangeover.SUsystemscanprovidesimilarflexibilityandeaseproductchangeoverbecause


Preparationandstorageofmedia/buffersinSUmixers

SUliquidandgasfiltrationofmanytypes,includingTFF

StorageofmediaandbuffersforCPfeedinginSUBPCs

DistributionofprocessfluidsinmeteredSUmanifolds

SUstorageandmetereddistributionofdrypowders

SUorhybridbioreactorcellcultureinseedgeneration

ProductioninSUorhybrid-SUperfusionbioreactors

ContinualappearanceofnewSUprobesandsensors

SUreal-timeautomatedonlinemulti-analysisinterface

SUflow-pathon-linereal-timecontrolledfeedporting

BulkharvestbySUcentrifugationorfiltrationintoBPC

PurificationinSUtraditionalorPCCchromatography

FinalfillinSUand/orautomatedandclosedapparatus

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theytendtobemoremodularandtransportablethanmuchoftheolderbatchequipment.Infactthesize,configurationandreducedservicerequirementsofSUsystemsactuallyencouragediversityofphysicallocationwithinasuiteorplant,aswellasre-locationtoothermanufacturingsites.

Single-useintensifiedperfusion-basedcontinuousbioproductionusingasingle-useThermoScientificHyClonebioprocesscontaineranddrum(right)supportinglarge-scalecultureinaFiberCellSystemsLS-HFBRhollowfiberperfusionbioreactor(left)

Duetoitsinherentdemandforimmediateprocessdataandcontrolcapabilities,CB supports initiatives in continuous quality verification (CQV), continuousprocessverification (CPV),andreal-timerelease (RTR)21-23.AlthoughCBwillnot be feasible for all products and processes, many implementations well-supporta“platform”approach,inwhichasingleprocesssupportsmorethanoneproduct.CBmostalwaysshortenstheprocessstream,reducesdowntime,andgreatlyreduceshandlingofintermediates.ThesefeaturessynergisewiththeoperationalefficienciesofSUsystems,contributing toagreatly reducedcumulative processing time for the API. Furthermore, they greatly simplifyproductiontrainsandinherentlyfacilitateapplicationofclosedandintegratedprocessingapproachestoindividualoperationsandevenprocesses.Especiallyin bioproduction, the modularity and integral gamma irradiation sterility ofSUcombinedwiththesustainedoperationofCBpromisetheappearanceofplatformsofunparalleledoperationalsimplicityandconvenience.


TheheartofaCBapproachisthebioreactor.Perfusionbioreactorshavebeensuccessfullyemployedinbioproduction,evenbiopharmaceuticalproduction,fordecades.And,ratherremarkably,disposablebioreactorshavebeenavailablefornearly20years.Attheresearchscaletherehaveevenbeensingle-usehollowfiberperfusionbioreactorsavailablefromavarietyofvendorsforover40years.However,onlyrecentlyhavecommerciallyavailableSUandhybridproduction-scaleperfusion-capableequipmentbecomeavailable24-26.

Hybrid continuous bioproduction accomplished in a Thermo Scientific HyPerformaS.U.B.TK250L(left)supportedbyaRefineTechnologyATFSystem(right).

Theproduction-scaleCBenablingSUbioreactortechnologiesnowappearingincludesingle-useandhybridperfusion-capablereactors(Fig.1,2);agrowingvarietyofSUandhybridmonitoringprobesandsensors;SUpumpsandfluiddelivery automation of various design; and automated SU online sampling,interface,valvingandfeedingtechnologies.Theircoordinatedimplementationinactualproductionsettingswithappropriatecontrolisnowbeginning.

Justifiedornot, concerns in the implementationofCB includeperformancereliability (incidence of failure), validation complexity, process control andeconomic justification27-29.But formanyprocesses,suchpreviouslimitations— or their perception — are being alleviated by advances in CB processing


Fig. 1

Fig. 2

A B

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technology and OpEx driven advances bioprocess understanding, reactormonitoring and feedback control 30, 31. However, while some CB attributesinherentlyprovideimmediateadvantages(suchasreducingreactorresidencytime)othersdopresentchallenges(suchascell-linestabilityconcerns).

Due to the limited contribution of API manufacturing to small-moleculepharmaceuticalcost,thelimitedbottom-linefinancialsavingsofCBhasbeenaconcern.However,biopharmaisadifferentanimalingeneral,andassuchtrends as globalization and biosimilars alter the picture even further, thefinancialbenefitsofCBarebecomingevenstronger32.

The fact thatmanySUsystemsareconstructedofstandardscompliantandanimal product-free materials supports CB applications in a wide variety ofproduct types and classification. In fact, SU systems are available for mostprocess formats (eg,microcarriersandsuspension),platforms (eg,cell line,vectors, culturemedia),modes (eg,dialysisorenhancedperfusion)orscale(eg,throughrapid,inexpensivescale-out).“Futureproofing”,orsupportingthesustainabilityofanewCBprocessinthefaceofproductlifecycleoremergingtechnology imperative, is supported by many SU features 33. Examples hereinclude SUs low initial facility, service and equipment cost and especiallyundedicatedmanufacturingsuitsandeaseofprocesstrainreconfiguration.

Asadvancedprocessingsolutionsareappliedtosingle-useperfusionmode-capablereactors,thedesignofclosed,disposable,integratedandcontinuousupstreambioproductionsystemsarefinallybeingrealized.



References:1) Whitford WG. Single Use Systems as Principal

Components in Bioproduction. BioProcess Int. 8(11)2010:34–42.

2) Whitford WG, Single-use Systems Support ContinuousProcessing in Bioproduction, World Biopharm Forum,Continuous Processing in Biopharm. Mfg, RobinsonCollege,Cambridge,UK,Jul1–2,2013.

3) Whitford WG, Cadwell JJS. Interest in Hollow-FiberPerfusion Bioreactors Is Growing. BioProcess Int.7(9)2009:54–63.

4) Bonham-Carter,JohnandShevitz,JerryA.BriefHistoryofPerfusionBiomanufacturing.BioProcessInternational9(9)October2011.

5) Shevitz J, et al. Stirred Tank Perfusion Reactors forCellPropagationandMonoclonalAntibodyProduction.Advances in Biotechnological Processes, Vol. 11. A.Mizrahi(Ed).AlanR.Liss.,Inc.:nY,1989;p81.

6) Langer ES, Trends in Perfusion Bioreactors: WillPerfusion Be the next Revolution in Bioprocessing?BioProcessInt.9(10)2011:18–22.

7) VoisardD,etal.PotentialofCellRetentionTechniquesfor Large-Scale High-Density Perfusion Culture ofSuspendedMammalianCells.Biotechnol.Bioeng.82(7)2003:751–765.

8) Pollock J, Ho SV, Farid SS. Fed-Batch and PerfusionCulture Processes: Economic, Environmental, andOperational Feasibility Under Uncertainty. BiotechnolBioeng.110,2013:206–219.

9) Warikoo V, et al. Integrated Continuous Production ofRecombinantTherapeuticProteins.Biotechnol.Bioeng.109(12)2012:3018–3029.

10) Palmer E. GSK Commits to Continuous Processing.FiercePharmaManufacturing 19 February2013; wwwfiercepharmamanufacturing.com/story/gskcommits-continuous-processing/2013-02-19.

11) Whitford WG. Continued Progress in ContinuousProcessingforBioproduction.LifeScienceLeaderJune2012:62–64.

12) Karen W. Biotech Firms in Race for ManufacturingBreakthrough.MITTechnologyReview30January2013;www.technologyreview.com/news/509336/biotechfirms-in-race-for-manufacturing-breakthrough

13) novartis-MITCenter forContinuousManufacturing,50AmesStreet,E19-502b,Cambridge,MA.

14) WhitfordWG.Single-UseSystems’SupportofPATandQbD in Bioproduction. LifeScience Leader December2010.

15) WhitfordWG.Single-UseSystemsSupportContinuousProcessing in Bioproduction. PharmaBioWorld 10(10)2012.

16) Whitford WG, Supporting Continuous Processingwith Advanced Single-use Technologies, BioProcessInternational,AprilSingle-useSupplement,April,2013.

17) W.Whitford, “UsingDisposables inCell-Culture-basedVaccineProduction”.BioProcessInternational,Vol.8,no.S4,P.April,2010.P.20-27

18) WhitfordWG.Single-UseBioreactorFlexibilityReduces

Risk,Costs,andDelaysinBioproduction.BiosimilarsandSingle-UseBioreactors,30Oct–3nov2012,London,England.

19) Matthews T, et al. An Integrated Approach to BufferDilutionandStorage,PharmaManufacturing.com,2009

20) Langer, ES. Improved Downstream Technologies Areneeded to Boost Single-Use Adoption, BioProcessInternational,SupplementMay2011.

21) EMAGuidelineonRealTimeReleaseTesting(formerlyGuideline on Parametric Release) http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2012/04/WC500125401.pdf

22) USFDA,AdvancingRegulatoryScienceatFDA:AStrategicPlan,August2011www.fda.gov/regulatoryscience

23) BioProcess International11(4)sApril2013Supplementy13WoodcockJ.FDA:Thenext25Years.AAPSAnnualMeeting and Exposition, 23–27 Oct 2011, Washington,DC.

24) Bonham-Carter J. Implementing ContinuousProcessinginaSingle-UseFacilityoftheFuture.SecondBiotechnology World Congress, 18–21 February 2013,Dubai,UAE.

25) WhitfordWGandCadwellJJS,“ThePotentialApplicationofHollowFiberBioreactorstoLarge-ScaleProduction”,BioPharm International Supplements, Volume 24, pp.s21-s26,May2,2011.

26) Cadwell JJS. novel Large-Scale Bioreactor SupportsContinuousManufacturing.SecondBiotechnologyWorldCongress,18–21February2013,Dubai,UAE.

27) Langer ES. Perfusion Bioreactors Are Making aComeback, But Industry Misperceptions Persist,BioProcessingJournal,Volume9,Issue2(Winter2010-2011).

28) RichardsonnE.,Chiang,Barbara.Equipmentdesignandprocesschallengeswithrunningaperfusionprocessinadisposablestirred-tank:Acasestudy,CentocorR&D,2007.http://www.refinetech.com

29) nasr M, Muzzio F and Cheuh A. Challenges forContinuous Processing, Main Stage Panel Discussion,Interphex2011,JacobJavitsCenter,nYnY,March29-312011.

30) European Commission. EUR 24282 – Factories of theFuture PPP, Strategic Multi-annual Roadmap 2010,Luxembourg:PublicationsOfficeof theEU, ISBn978-92-79-15227-6.

31) Acuna J, et al. Modeling Perfusion Processes inBiopharmaceuticalProduction.BioProcessInt.9(2)2011:52–58.

32) JungbauerAandPengJ.Continuousbioprocessing:AninterviewwithKonstantinKonstantinov fromGenzyme,Biotechnol.J.2011,6,1431–1434.

33) WhitfordWG.Single-UseSystemsSupportContinuousProcessing in Bioproduction. Second BiotechnologyWorldCongress,18–21February2013,Dubai,UAE.

65

ContinuousMulticolumnChromatographyProcesses

IntroductionChromatographyhasbeen,andwillprobablyremainfortheforeseeablefuture,themostimportantworkhorseinthepurificationofbiopharmaceuticalproducts.Awidevarietyofchromatographyproductsarecommerciallyavailable,offeringthepossibilitytoseparatetheproductofinterestbasedonaffinityinteractions,electrostatic interactions (ion exchange), hydrophobic interactions, size andcombinationsofthese.Inthebiopharmaceuticallandscape,thereisnotasingleproductthatisnotpurifiedusingatleastonechromatographicpurificationstepand most biopharmaceutical products require at least two chromatographicpurificationsteps.

Although powerful in terms of removing contaminants, chromatographicprocesses have a few disadvantages. In general, multi-step batch processesexhibitpoorproductivityandoftenthisleadstoscalabilitylimitations.Evencaptureprocesseswithnewhighcapacitychromatographicmediacannotalwayscopewiththehightitersthatarebecomingmoreprevalentincellcultureprocesses.Forexample,afedbatchcellculturebioreactorof2000literwitha5gm/Lexpressionlevelproducesmoreantibodythancanbeboundonacolumnwithaonemeterdiameter,evenifthatcolumniscycledtwiceperbatch.1

Inotherprocess industries, theselimitationshavebeensuccessfullydealtwithby implementing continuous multicolumn chromatography processes. notableexamplesofthisapproachistheuseofsimulatedmovingbed(SMB)technologyfor separating fructose from glucose and many chiral separations common inpurifyingAPI’smadethroughorganicsynthesis.Althoughthetraditionalsimulatedmoving bed technology is mainly applied for binary fractionations, continuousmulticolumnchromatographysystemshavealsofoundlargescaleapplicationsin capture processes. Examples of these are the purification of L-lysine andantibioticsfromfermentationbrothandtheproductionofascorbicacid(vitaminC).

08

Marc Bisschops,CSO,TarponBiosystems

Marc Bisschops is the Chief Scientific Officer of TarponBiosystems.TarponBiosystems isabiotechstart-upcompanybased in Boston that develops and commercializes a fullydisposablecontinuousmulticolumnchromatographytechnology.

Marc has 15 years of experience in the (bio-) pharmaceuticalindustry. He has been involved in process development andprocessimprovementprojectsthroughoutmostofhiscareer.Heheldscientificandmanagementpositionswithincontractserviceorganizations that focus on process development and processimprovementprojectsfor(bio-)pharmaceuticalproducts.Heisoneoftheworldexpertsinthefieldofcontinuouschromatographyand simulated moving bed (SMB) technologies and has beeninvolvedinmorethan150projectsinthisfieldworldwide.Marcis(co-)inventoronmanypatentsrelatedtoprocessimprovementsand/or innovative biomanufacturing technologies and is aninvitedguestspeakeratvariouspost-graduatecourses.

Before joining Tarpon Biosystems, Marc has headedbiopharmaceutical process development and processimprovement departments in Batavia Bioservices, Xendo andUnivalid.MarcholdsanMScinChemicalEngineeringandaPhDinBiochemicalEngineeringfromDelftUniversityofTechnology(nL).

AbouttheAuthor:

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With increasing cell culture expression levels, the capacity bottleneck inbiomanufacturinghasshiftedfromtheupstreamprocesstothedownstreamprocess. This has generated a need to address the limitations of batchchromatography in biopharmaceutical applications. This has resulted invariousdesignsformulticolumnchromatographysystemsforthepurificationofbiotherapeutics.

Key FeaturesThe principle of multicolumn chromatography is to create a (simulated)movementofthechromatographycolumnsinoppositedirectionoftheprocesssolutions.Thisresultsinacountercurrentcontactbetweentheliquidandthechromatography media, which allows overloading the columns beyond thedynamic binding capacity without suffering loss of material. When productbreaksthroughfromthefirstcolumn,itwillbecapturedonasecondcolumnintheloadzone.Withthis,countercurrentchromatographyprocessescanofferasignificantgainincapacityutilization.

Anotherbenefitofthecountercurrentcontactapproachisthatiteliminatesidlezones in theprocess. Inabatchchromatographycolumn, themasstransferzoneonlycoversasmallportionof theoverallchromatographyvolume.Themediaabovethemasstransferzoneisinequilibriumwiththefeedsolutionandhasnoadditionalcapacity tobindmoreproduct.Themediabelowthemasstransferzoneisincontactwithdepletedfeedsolutionandhenceiswaitingforthefirstproduct toarrive. Inacountercurrentprocess, these idle zonesareeliminatedandtheloadzonecanbedesignedtoonlycoverthelengthofthemasstransferzone.Thisisgenerallycorrespondstoasmallpartofthebatchcolumnvolume.Thesetwofeaturesareschematicallydemonstratedinfigure1.

Inbatchprocesses, thecolumnsize isproportional to thetotalmassofproteinthatneeds tobepurifiedandhence there isadirect relationshipwith the feedconcentration.Inacontinuouschromatographyprocess,theloadzoneismainlydesignedaroundthecontacttimeassociatedwithmasstransferzone.Thetotalvolumeofchromatographymedia in the loadzone thushardlydependson thestaticbindingcapacityandthefeedconcentration.Instead,theprocessisdesignedaroundthevolumethatneedstobeprocessed,ormoreprecisely,thefeedflowrate.


Schematiccomparisonbetweenabatchprocess(left)andamulticolumncountercurrentchromatographyprocess(right).

Thenumberofcolumnsthatisrequiredtorunacontinuousprocessdoesdependontiter. Inordertotransformtheloadstepintoacontinuouscountercurrentstep,at least twocolumnsareneeded.Thisbringstheminimumnumberofcolumnsforacontinuousprocesstothree,providedthatonecolumnprovidessufficienttimetodoallwashsteps,elution,regenerationandre-equilibrationsteps.Assoonastheloadvolumebecomesrelativelylow,whichisthecaseformediumandhighertiters,theloadtimebecomesproportionallysmallerandonecolumnisnolongersufficient.Forthesescenarios,theabilitytoconnectextra columns to the system without adding complexity to the valve systemis a valuable attribute. For polishing processes, where the chromatographicresolutionisnotasstraightforwardasinaffinityseparations,additionalcolumnsmayalsobeneededintheelutionzoneand/orwashzones.

Continuous and disposableThereisasubstantialgaininspecificproductivityoverbatchofferedbycontinuouschromatographywhilethesizeoftheoverallchromatographysystem–includingitscolumns–becomessignificantlymorecompact.Thecolumnsarecycledmanytimesthroughouteachbatch,usuallyuptothelifetimeofthechromatographicmedia.Thisprocessdesignenablesaviabledisposablechromatographyprocess.

08 ContinuousMulticolumnChromatographyProcesses

Fig. 1

Wash,Elution,

RegenerationEquilibration

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TheBioSMB®technologydevelopedbyTarponBiosystemsisdesignedaroundacompletelydisposableproductcontact/fluidpath.Mostimportantly,theTarponBioSMB®valvecassette,asingle-useacrylicblockcontainingallthevalvingandintegratedfluidconnectionstorunamulti-columnprocess,canoperateupto16columnsorothersingleusedevicessuchasmembranesormonoliths.Eachofthevalvesinthecassettecanbeindividuallyaddressed,therebyprovidingalltheflexibilitythatisrequiredtooperatevirtuallyanychromatographyprocessinamulticolumnconfiguration.Inadditiontothis,thepumps,tubingandsensorsarealsoavailableindisposableformat.

When theBioSMBsystem iscombinedwithprepackedcolumns,membraneadsorbers or any other chromatographic devices designed for single-useapplications,theentirechromatographyprocesscanbetranslatedintoaviablesingle-use option. With this, the BioSMB® technology provides a promisinganswer for those companies who are developing completely disposablestrategiesfortheentirebiomanufacturingprocess.

A BioSMB system for process development use with five prepacked chromatography columnsconnectedtothesystem.


Optimization StrategiesContinuous chromatography processes have more degrees of freedom thanbatchchromatography.Thisoffersmoreflexibilityinoptimizingtheprocesstomeetthespecificrequirementsofeachmanufacturingsituation.Forinstance,incontinuousprocessing,thebatchprocessingtimebecomesachoiceratherthananoutcomeofthedesignprocedure.

Clinical ManufacturingInclinicalmanufacturing,thecostcontributionoftheconsumablessuchasthechromatographymediatothetotalCOGs(CostofGoods) isquitesignificant.Thisismainlyduetothefactthattheseconsumablescannotbeexploitedtotheirfullextent.EvenexpensivechromatographymediasuchasProteinAaffinitymedia are depreciated within a single clinical manufacturing campaign. Theoptimizationstrategyforthissituationshouldtargetthetotalinstalledvolumeofchromatographymediaorthespecificproductivity(expressedasgramsofproteinpurifiedperliterofchromatographymediaperhour).

InaBioSMBprocess,thistranslatesintoprocessconditionsthattargetashortcontacttimebetweentheliquidandthechromatographymedia.Thiscanbeachieved by operating below the highest possible capacity utilization and intheprocessacceptingsub-optimalsavings inbufferconsumption. Inclinicalmanufacturing,thebufferconsumptionisgenerallynotthelimitingfactorandtheimpactofthetotalcostsofthecampaignisnegligibleinmostcases.

Commercial ManufacturingIncommercialmanufacturing,chromatographicmediaisdepreciatedovermanymorecyclesthaninclinicalmanufacturing.Itisnotuncommontovalidatemedialifetimeupto100oreven200cycles.Inthesesituations,thecostcontributionofthechromatographymediaisnolongerrelatedtothespecificproductivityoftheprocess,buttotheamountofproductthatispurifiedineachcycleperliterofchromatographymedia.Thisoptimizationstrategyforcontinuousmulti-columnchromatographythustargetscapacityutilizationoptimization.Withthis,thesavingsinbufferconsumptionwillalsobeoptimized.


Fig. 2

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Inordertoachievethemaximumcapacityutilization,theloadstepwillrequirea certain contact time, which has an impact on the specific productivity. Incommercialmanufacturing,however,thespecificproductivitydoesnotaffecttheCOGotherthanthroughcapitalcosts.

SchematicrepresentationofthetypicaloperatingpointsforclinicalmanufacturingandcommercialmanufacturinginaProteinAbasedBioSMBprocess.

Application AreasThe flexibility offered by the BioSMB disposable valve system makes it avery versatile technology, allowing a wide range of applications. For variouschromatographicprocesses,theimpactofthetechnologyhasbeeninvestigated.AbriefsummaryofsomeofthecasestudiesislistedinTable1.


Table1:SomeexamplesforwhichBioSMBtechnologyhasbeensuccessfullytested2,3,4,5,6.

Thecasestudieslistedabovewereperformedwithtraditionalchromatographymedia inprepackedcolumns. Inadditiontothis, thecombinationofBioSMBtechnology with alternative chromatography formats has been successfullydemonstrated. This includes the use of monolithic columns and membraneadsorberstoestablishacontinuouscaptureprocess.Thishasbeendonewithmembraneadsorbersionexchangeandwithaffinityligands.


Case study Chromatographic mode Specific productivity

Captureofantibodies ProteinAchromatography 2–6xbatch

Aggregateremoval Hydrophobicinteraction(HIC) 2–3xbatch

Aggregateremoval IonExchange 4–8xbatch

Captureofrecombinantproteins IonExchange 2–5xbatch

CaptureofVLPvaccines IonExchange 3–7xbatch

PolishingofaVLPvaccines SizeExclusion(SEC) 6–14xbatch

References:1) noyes,A.,Coffman,J.,GodavartiR.andBisschopsM.:“DevelopmentofaProteinASMBStepforamAb

with up to 10g/L Titers” Presented at Biopharmaceutical Manufacturing and Development Summit,Boston,november2010

2) Allen, L.: “Developing Purification Unit Operations for High Titre Monoclonal Antibody Processes”,presentedatIBCAntibodyDevelopmentandProductionconference,BellevueWA,March2011

3) Brower, M.: “Working Towards an Integrated Antibody Purification Process”, Presented at IBCBiopharmaceuticalManufacturingandDevelopmentconference,SanDiegoCA,September2011

4) Pieracci,J.,Mao,n.,Thömmes,J.,Pennings,M.,Bisschops,M.andFrick,L.:“UsingSimulatedMovingBedChromatographytoEnhanceHydrophobicInteractionChromatographyPerformance”,PresentedatRecoveryofBiologicalProductsXIV,LakeTahoe,August2010

5) Jiang,H.:“PurificationofH5n1andH1n1VLPbasedvaccines”,PresentedatIBCSingle-Useconference,LaJollaCA,June15,2010

Fig. 3

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73

ContinuousProcessesEconomicEvaluation

Introduction to cost modellingProcessmodelsaretoolstoanalyzeprocessesandmanufacturingoptionsandsupportdecisionmaking.Theyhavebeenusedinourindustryto:

• assessthecostofoutsourcing; • evaluateandscreenprocessdevelopmentoptions; • helpdevelopcapacityandexpansionstrategies; • compareexistingandnovelprocessing/manufacturingtechnologies

Inthissectionweexaminetheuseofcostmodellingincontinuousbioprocesses,focusing on the approaches adopted for the evaluation of batch and continuousoperations.Mostcostmodelsdrawontheprinciplesoffinancialandmanagementaccountingtoassessthecostimpactofdifferentinvestmentandoperatingdecisions.Formanufacturing,themostsignificantlineitemontheincomestatementisthecostofproducinggoodsforsale(referredtoasCostofGoodsSoldorCostofSales)whichisshowndirectlybelownetsalesrevenue.Subtractingthecostofgoodssoldfromthesalesrevenuegivesacompany’sgrossprofit,makingitpossibletoevaluatemanufacturing performance as a distinct measure that contributes to overallbusinessperformance.Thisisimportantfromanexecutivemanagementperspectivebecauseimprovementsinmanufacturingperformancethatresultinincreasedgrossmarginaremadevisible.Arobust,well-structuredcostmodelenablesmanagerstohaveabetterinsightintothekeycostdriversofthemanufacturingprocessaswellasthesensitivityofoverallcostofgoodstochangesinthesekeyparameters.

Cost of Goods (CoG) isby far themostcommonlyusedmethod.While ithasthe merit of being the most familiar to people in the industry, it is not themostrigorous.CoGshouldnotbeusedwherethere isaneedtounderstandthe interplay between the expenditures and project risk. net Present Value(nPV) methodology is the best technique to analyze alternative technologiesand manufacturing strategies, as it can account for the impact of delays inexpendituresandproperlyaccountforthetimevalueofmoney.

09

Andrew Sinclair,FounderandPresident,BiopharmServices

Andrewhasover30yearsdesignandoperationalexperienceinthebiopharmaceuticalindustry.HefoundedBiopharmServicesin1999todevelopatechnology-basedservicesbusinessfocusedonallaspectsofbioprocessing.Sincethenhehashelpedawiderangeofclientstobetterunderstandthevalueofnewtechnologytotheirmanufacturingbioprocesses.

AbouttheAuthors:

Andrew Brown,HeadofConsultancy,BiopharmServices

Andrewhassignificantexperienceinthesimulationandeconomicmodelling of biochemical and chemical processes and hasworkedwithawiderangeofclienttoassesstheeconomicimpactuponbioprocessesofnewtechnologies,processdefinitionsandmanufacturingstrategies.PriortojoiningBiopharmServices,heattainedanEngineeringDoctorate inBiochemicalEngineeringfromUniversityCollegeLondon.

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The industry has traditionally employed batch processing to manufacture thebiologicaldrugsubstanceandhassometimeusedcontinuousperfusionoperationfortheproductionbioreactor.Inperfusionfreshmediaiscontinuouslysuppliedtothebioreactorwhilstdrawingoffthecellfreecontentfromthebioreactoratthesamerate.Historically,thedecisiontogothisroutehasbeenlinkedtotheproduct: where the protein is inherently unstable, perfusion operation is thepreferredmethodology.Perfusionadoptionislargelydrivenbycustomarypracticeratherthanevidence.Althoughtherehasbeendiscussiononwhetherperfusionorfedbatchisthemoreproductive,therehasbeenlittlepublishedquantitativecostanalysistosupporteitheroptionforcommercialproduction.

Wearenowatastagewherethereisstronginterestintheuseofcontinuoustechnologiesforbioprocessing,basedonperceivedadvantagesrelatingto

• Cost • Smallerfacilityfootprints • Flexibility • Betterprocesscontrol/productquality

In this section we investigate the comparative cost of goods and the capitalrequirementofcontinuousprocessversusbatchprocessing.ThiswillbeanalysedusingtheBioSolveProcess(BSP)costmodellingpackage.BSPisusedasithasthecapabilityofmodellingbothcontinuousflowandbatchoperationsinthesameframework,allowingeasycomparisonoftheoperatingmodes.

BSP generates cost of goods estimates through the scaling and costing ofresources from a process description that includes recipe components andscalingrules.Thisisillustratedinfigure1.Theoperationmodesdictatehowthemodeldealswithequipmentandresourceallocations

Fig.1:Structureofacostofgoodsmodel

Batch processing.Thedownstreamprocessessizingisbaseduponthepooledharvested product processed within a defined period. The batch scale isdetermined by bioreactor volume, bioreactor numbers, product titre and thebottleneckbatchcycletime(forcellculturethisisthebioreactor).Thismeansthateachoperationisrequiredtoprocessthebatchinasettimedictatedbytheharvestbottleneck.Onlyasmallproportionofthebatchtimeisusedtoprocessproduct: the remainder is associated with activities such as preparation,cleaningandregeneration.

Fully continuous processing.Whensteadystateproductionhasbeenachieved,theprocesswillbecontinuouslyfedwithaproductflow,scaleisdeterminedbyrateandproducttitre.Therateofproductgenerationisthebasisforsizingtheunitoperationsandtheresourcesrequired.Typicallywearerunningoperationswith a finite capacity in a continuous line (filters, chromatography resins)membraneabsorbers,TFF,etc.).Thelifetimeisdeterminedbythecapacityoftheconsumable.Theunitoperationisdesignedsothat itcanalwaysreceiveflow.Thiscanbeachievedtwoways.

• Switchovertoafreshdevice(twodevicesrunninginparallel) • Putinsurgecapacitywhilstafreshdeviceisbeinginstalled

Hybrid processing. At some point in the continuous line, a product may becollectedasabatch.Thiscouldbeafterthecapturecolumnoratendoftheprocesspriortoformulation.Thereisarequirementtoswitchfromcontinuous


ContinuousProcessesEconomicEvaluation09

UserInterface Process Definition USP,Recovery,DSP

EquipmentListConsumables Materials

SiteReference

COG Capital

Utilities

Production Labour

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modetobatchmode.BSPallowstheusertodeterminethispointbyuseofaswitchoperation,allowingtheusertospecifytheamountmaterialthatformsthebatch forsubsequentprocessing. InadditionBSPprovidesaswitch thatallowsupstreambatchoperationstobeconvertedintoaflowforprocessinginacontinuousline.Thisapproachallowsthemodelusertoevaluatemanydifferentoperatingscenarios,forexample

• Runperfusionandcapturecontinuouslyandruntherestofpurificationasbatchoperation

• HavebatchbioreactorsfeedacontinuousDSPoperation

• Runacontinuousbioreactoranddownstream,batchingpriortoformulation

• Runoneunitoperationcontinuouslyinbatchprocess(chromatographyforexample)

BSP allows the user the flexibility required to fully evaluate continuous andbatchoperationsfromaneconomicperspective.

Fig.2:Scenariodefinitionforbatch,hybridandcontinuousprocess

Modelling Batch vs. ContinuousThe biopharmaceutical industry was developed around the concept of batchprocessing.Thoughsomecompanieshaveexperienceofoperatingperfusionbioreactors, there is little experience in the operation of downstreamcontinuously.Thedevelopmentoffullycontinuousprocesses,withaperfusionbioreactor downstream running in parallel has become of interest, and weseemanycompaniesbeginningtoestablishtheseset-upsatapilotscaletoinvestigatethefeasibilityofrunninginthisoperationalmode.

ModellingpackagessuchasBSParebasedoncommercialscalecostdatasetsandarewidelyusedtoevaluateprocessandtechnologychoicesintheindustry.This makes them an ideal tool for evaluating new innovative technologiesfromaneconomicperspective,providingvaluableinsightintotheimpactofanewtechnologyandhowbesttomaximiseitsvalue.ToillustratethiswehaveusedtheBSPpackagetoestimatecostofgoodstomanufactureamonoclonalantibodybulkdrugsubstanceforarangeofscenariostobetterunderstandtheimpactofcontinuousoperationonMAbproductioncosts.Themanufacturingscenariosconsideredaresummarisedinfigure2.Theobjectivesofthisstudyaretounderstandtheimpactofscaleontheeconomics,toevaluatetherelativecontributions of upstream and downstream processing and to gain insightintotheimpactofcontinuousprocessingonthecoststructure.Threeprocessconfigurationswereconsideredfigure3:

• Batchwithfedbatchupstreamandbatchdownstream • Hybridwithfedbatchupstreamandcontinuousdownstream • Continuouswithperfusionupstreamandcontinuousdownstream

Scenarioschematicflow


Fig. 3


Configuration Bioreactor500 kg/yr 2000 kg/yr

Rate VVD

Titre g/L

DSP Recovery

Batch 4x1600 4x6500 n/A 4.5 60%

Hybrid 4x1600 4x6500 n/A 4.5 61%

Continuous 2x875 2x3750 2 0.9 74%

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Twomanufacturingscaleswereconsideredforeachprocessscenariobasedupon an annual requirement to manufacture 500kg or 2000kg. Multiplebioreactorswereusedforeachscenariowiththescaleofequipmentvaryingwithannualthroughputasillustratedinfigure2.Eachbioreactorisharvestedinparalleltofeedtherespectivedownstreamline.Thetitrefromthefedbatchbioreactorwasassumedtobe4.5g/Linlinewiththecapabilitiesofmoderncelllines.Theperfusionculturewasassumedtogenerate0.9g/Lataperfusaterateof2vesselvolumesperday.

An overview of the BioSolve Process estimate of the manufacturing coststructureatascaleof500kg/yr.isgiveninfigure4.Thecostisbrokenintothekeycostcategoriesthatmakeuptheoverallcostofgoods:

• Capital • Materials • Consumables • Labour • Other(waste,maintenanceetc.)

The first interesting observation is that the upfront capital investment issignificantly reduced by about 60% for continuous operation at both scales(Fig. 4). The reduction is seen in both upstream and downstream: on closeexaminationwefindthatthisisdrivenbythemuchsmallerscaleofoperationrequiredforthecontinuousfacility.Accordingtothemodel,thepeakflowontothebatchproteinAcolumnisabout2800L/hr.;thisreducestoabout85L/hr.forthecontinuousoperation.Thesmallerscaleresultsfromthehighereffectiveutilisationofthecontinuousoperationcomparedtobatch.

Lookingattheoverallcostofgoodsthereisareductionatthe500kg/yr.scaleasonegoesfromthehybridtothefullycontinuousscenario.Thispattern isnotseenwhenscalinguptothe2000kg/yr.case.Inthiscase,themovetotheperfusionbioreactoroperationhasanegativeimpactonCoGresultinginthehybrid operation being the more attractive. So what is happening? The CoGmodelprovidesthatinsight,thecostbreakdownshowninfigure5providesaclue.Intheupstreamperfusionoperation,mediacostsdominatetheCoGandasprocessscale increasesrawmaterialcostsplayamoredominantrole in

overallCoG.Thereforewhenlookingatperfusionmediaconsumption,producttitreandmediacostsareimportantcostdriverswhencomparingthisapproachtofedbatchoperation.

Fig.4:Comparisonofcostofgoodsacrossthescenariosconsidered.

Looking at the overall downstream costs, we see again a reduction at the500kg/yr.scalewhenmovingfrombatchtocontinuousoperationofaround8%.Howeverasweincreasescaleto2000kg/yr.theCoGreductionislargeratabout20%oftheDSPoperatingcosts.Lookingintothedetailofthecostdistributionforbothformats,theProteinAunitoperationcostisadominantcostdriver.Inthebreakdownofthedownstreamcostsweseethattherearekeydifferencesbetween the batch and continuous processes. If the continuous processesrepresentamoreheavilyutilisedassetwewouldexpecttheconsumablesandmaterialcoststodominateandcapitaltodiminishwhencomparedthebatchprocess. Infigure5wesee thiseffect.What issurprising is that there isnorealreductionintheproportionofthelaborcosts.Inassessinglaborcostsaconservativeapproachhasbeentakentoassigninglabortooperations.Inrealitytheexpectationforhighlyautomatedoperationsisforthelaborcomponenttobelowergivingscopeforfurthersavingsinthisarea.



500 kg/yrCapital CoG$106 $/g

2000 kg/yrCapital CoG$106 $/g

Batch

FedBatchbioreactor

BatchDSP

72.6

52.8

58%

42%

31.3

63.7

33%

67%

93.4

83.2

53%

47%

11.1

28.2

28%

72%

Total 125.4 100% 0% 95.0 100% 0% 176.6 100% 0% 39.3 100% 0%

Hybrid

FedBatchbioreactor

ContinuousDSP

72.6

23.2

76%

24%

31.3

57.0

35%

65%

93.4

29.4

76%

24%

11.1

22.5

33%

67%

Total 95.8 100% 24% 88.3 100% 7% 122.8 100% 30% 33.6 100% 15%

Continuous

Perfusionbioreactor

ContinuousDSP

21.4

24.3

47%

53%

24.4

58.8

29%

71%

33.7

32.2

51%

49%

16.7

23.3

42%

58%

Total 45.7 100% 64% 83.2 100% 12% 65.9 100% 63% 40.0 100% -2%

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In this limitedexampleofusingascalablecostmodel,wehaveshown thatmodelling the different technologies provides valuable insight into the keycost drivers. This allows the user to identify potential areas of savings andexaminethedynamicsofscale.Althoughthisisonlyoneexamplewithatwoscaleofoperations,itisneverthelesspossibletodevelopinsightsthathighlightthepotentialofcontinuoustechnology.Basedonthisstudy,wecandrawthefollowingconclusionsaboutcontinuousprocessing:

• Lowercapitalinputs

• Upstreamneedstobeconsideredseparatelyfromdownstream

• Upstreamcostbenefits(batchvs.perfusion)dependonmediavolumerequirements,mediacosts,producttitre

• Fordownstreamsavingsareseenwithmorecostsmovingtovariablecostssuchasmaterials,consumables

Costbreakdownforbatchandcontinuousoperationsformanufactureat500kg/yrscale

Thereismuchmoreworkrequiredtobetterunderstandtheimpactofcontinuoustechnologyandtooptimiseitsuse.Theshiftoffixedcosts(capital)tooperationalcosts is important in termsofoperationalflexibilityandresponsiveness.Thefullbenefitof this isnotcaptured in traditionalCoGmodelling; furtherworkisneededusingnPVanalysis toquantify this.Using themoresophisticatedmodelling approach afforded by BSP, we can identify potential cost andoperationalbenefitsforcurrentandfuturemanufacturing.



References:1) BioSolveProcessandproprietaryprocessmodellingpackagebyBiopharmServicesLtd

Fig. 5

Fed Batch Batch Downstream

Perfusion Continuous Downstream

CapitalMaterialsConsumablesLabourOther

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36%

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83

Vision:IntegratingUpstreamandDownstreaminaFullyContinuousFacility

IntroductionAs the biotechnology industry continues its maturation, it is now faced withchallengesfromitsownsuccesses.Examplesincludeincreasingcompetitionandassociatedconcernswithspeedtomarket,increasinglydiversifiedproductportfolios that include stable products (e.g. antibody) and complex, lessstableproducts(e.g.recombinantenzymes),alongwithhighandlowproductvolumedemands.Additionally,currentcompaniesrequirerapidadjustmentofproductioncapacity toaccommodatefluctuatingmarketdemands(Kamarck,2006).Thesechallengescanbefurthercomplicatedbytheconceptofregionalmanufacturing throughout the globe. Moreover, there is a growing focus onproductbatch-to-batchandsite-to-siteproductqualityandconsistencypartlydue to enhanced analytical techniques as well as regulatory oversight. It istheauthor’sperspectivethatonepotentialsolutionthatcollectivelyaddressesthesediverseconcernsliesintheconversionoftraditionalbatchmanufacturingtothatofacompressed,integrated,andcontinuousmodel.

Process intensification through conversion from batch to continuousmanufacturing has long been applied in other industries, including steelcasting (Tanner, 1998), petrochemical, chemical, food and pharmaceutical(Reayetal.,2008;Anderson,2001;Thomas,2008;Fletcher,2010;Laird,2007).Despitethedifferencesbetweentheseindustries,theadvantagesofcontinuousmanufacturingarealwaysthesame,includingsteadystateoperation,smallerequipment size, higher volumetric productivities, streamlined process flows,lowcycletimes,andreducedcapitalcosts(Utterback,1994).

Currently, there are two dominant platforms for biopharmaceuticalmanufacturing:(1)perfusionbioreactors,typicallyusedforproductionoflessstableproteins(Fig.1,PanelA),and(2)fed-batchbioreactorsforproductionofstableproteins,suchasMAbs(PanelB).Inbothcases,thebioreactoroperation

10

Timothy Johnson,Genzyme

Dr.TimothyJohnsonisanAssociateDirectorintheCommercialCell Culture Development department at Genzyme, A SanofiCompany,inFraminghamMA.HereceivedaPh.D.inChemicalEngineeringfromtheUniversityofWashington,Seattlein1999.WhileatGenzymeCorp.,Timhasbeenresponsibleforsupporting,understanding, and improving the production of commercialcell culture processes; for bioreactor process development,optimization, scale-up, scale-down, and technology transfer.Most recently, he is the technical lead for developing andscaling-up Genzyme’s continuous manufacturing platform fortheuniversalproductionofbiopharmaceuticals.

AbouttheAuthor:

8584

is followed by multiple batch unit operations, including clarification, capture,polishingchromatographyandholdsteps.Thecontinuouscapture technologydiscussedbyWarikooetal.(2012),whenintegratedwithupstreamreactorsallowsforasignificantlystreamlinedprocesstrain(PanelC)duetoeliminationofnon-value-addedholdsteps,dramaticallyshorterresidenceandcycletimes,reductionof equipment size, and overall facility minimization. For example, when highproducingclonesandrobustchemicallydefinedmediaareutilized,thisplatformcanachieveveryhighcelldensitiesandvolumetricproductivitieswhileoperatingatsteadystate.Asaresult,sufficientproductioncapacitycanbeachievedwithsmallerbioreactors(<500L)vs.traditionalprocesseswherereactorscalesmayexceed10,000L.Theuseofcell separationdevices thatsimultaneouslyclarifytheharvestof cellsandcelldebriseliminates the traditional clarificationunitoperation.Mostimportantly,thedirectintegrationofthecontinuouscapturestepmakesharvestholdtanksobsolete,andreplacesthelargebatchcapturecolumnwith up to 2 orders-of-magnitude smaller columns used in the continuoussystem.Furthermore,continuousprocessingof theharvestconferssignificantadvantageswithrespecttoproteinquality.Specifically,eliminationoftheharvestandotherholdstepsdecreasestargetproteinexposuretoenzymatic,chemical,andphysicaldegradationandtherebymitigatesproductstabilityrisks.Withtheseobjectives inmind,additionalcorporationshavebegun topursuevariationsoftheirowncontinuousprocessingplatform(Daszkowski,T.)

Schematic of traditional and future manufacturing platforms for perfusion bioreactor process. (A)Traditional perfusion manufacturing process; (B) Traditional 10-20kL fed-batch manufacturingprocess;(C)newintegratedcontinuousmanufacturingplatform.

CriticalSystemsforContinuousProduction

Upstream SystemsThe success of upstream continuous perfusion reactor operations forcommercial production has been reported throughout the literature. Whiletraditionalstainlesssteelreactorsystemsarean industrymainstay,progressin the development and robustness of single use bioreactors provides for anattractivealternative.MostnotablevendorsinthesingleusearenaareSartorius,Hyclone,andXcellerex,andwhicharecapableofsupplyingreactorsinexcessof1000L.Whilereactortypeandvendortendstobeamatterofcorporateandprocess preference, several cell separation devices also exist upon which tofacilitatecontinuousoperations.Systemsinusebymajorcorporationsincludeinclinedplatesettlers(BiotechnologySolutions),tangentialflowfiltration(variousvendors), alternating tangential flow filtration (Refine Technology), acousticresonance (BioSep–ApplikonBiotechonology),andcentrifugation (Centritech–PneumaticScaleAngelus).Eachsystemwillhaveitsvariousprosandconsthatshouldbeconsidered,especiallytheneedforadditionalclarificationshouldincompletecellseparationresult.

Downstream SystemsSeveral continuous chromatography systems have been made available bynovasep (Pompey, France), Tarpon (Worcester, MA), Semba (Madison, WI),MassimoMorbidelli(Zurich,Switzerland),andGEHealthcare(Piscataway,nJ),which open up novel opportunities for the implementation of the integratedcontinuousbioprocessingconcept.Foran in-depthdiscussionof theperiodiccounter-current (PCC) chromatography (GE Healthcare) methodology, seeWarikoo et al. (2012). While there are numerous technical features of thesesystemsthatwillaffectlong-term,robustperformance,themostcriticalmaybetheabilitytofunctionallyclosethesystemsandprotectthemfromadventitiousagents throughout the process duration. To this end, some of the availablesystemsprovideoptionsforincorporatinggammairradiateddisposablesand/ortraditionalstainlesssteelfabricationwithsteamsterilizationmethods.


Vision:IntegratingUpstreamandDownstreaminaFullyContinuousFacility10

Fig. 1

8786

Integrated Continuous BioprocessingIntegrated continuous bioprocessing is a novel solution that offers uniqueadvantagesovertraditionalapproachesforrecombinantproteinmanufacturing.Thisnewplatformhasbeensuccessfullyappliedatdevelopmentscaletodrugswithdiverseproperties,suchasahigh-volumestableprotein(MAb)andalow-volume lessstableprotein (rhEnzyme),whichdefine theboundariesof real-worldproductionscenarios(Warikooetal.2012).Atlargescale,thesuccessfulimplementationoftheplatformrequiresafunctionallyclosedsystemthatcanbemaintainedfreefromforeignorganismsforprolongedperiodsoftime.

Ourvisionofthebiomanufacturing“facilityofthefuture”basedontheintegratedcontinuousplatformisoutlinedinfigure2.Thisgeneralfloorplanutilizesmultipleparallelandindependentcontinuousproductionlinesdesignedasafunctionallyclosed system that offers multi-product and multi-purpose manufacturingcapabilitywithreducedroomclassifications.Theflexibilityofthisschemeenablesrapid increase or decrease of production capacity based on real-time marketdemandusinga“numberingup”approachratherthanthetraditionalvolumetricscaleup.Theflexibilityofthesystemmaybefurtherenhancedbyincorporatingdisposablesolutions,bothupstreamanddownstream.Astheequipmentfootprintisdramaticallysmaller, thesizeoftherequiredmanufacturingfacilityandtherelatedcapitalcostaresignificantlyreduced(≥50%).Thisreductioninsize,andcost,alsofacilitatestheabilitytohavethepilotandclinical-scalemanufacturingprocess at the same scale as final production, therefore nearly eliminatingtechnicalandtimelineriskstraditionallyassociatedwithtechnologytransferandscale-up.AdditionaladvantagesofthisplatformarethatLarge-orsmall-volumedrugs,andtheproductionofeitherstableorunstableproteinscanbeachievedwhileoperatingatahighlevelofstandardizationandmobility, thusfacilitatingthe visionary concept of decentralized and portable regional manufacturingthroughouttheglobe.

While the proof-of-concept demonstration of continuous biopharmaceuticalmanufacturing focused on continuous operations from media feed throughto product capture (Warikoo et al. 2012), there have already been significantadvances towards continuous processing through to drug substance(Konstantinov, K. (2013), Daszkowski, T. (2013)). These ideas for continuous

bioprocessingaretakingholdthroughoutthe industry.Assuch, it isexpectedthatnumerousconceptualandpilotdesignswillbeunveiledinthenearfuturewithvariousdegreesofcontinuouscadencesuitabletothestrategiesassociatedwiththerespectivecorporations.Whiletherespectiveunitoperationsmaydifferslightly,thecoreconceptsoutlinedherewillmostlikelyremainthroughoutthevariousdesigns.

General floor plan of a multi-product biomanufacturing facility utilizing the integrated continuousbioprocessingplatform,implementedassixindependentandparallelprocesstrains.Thekeyfacilitydesignconceptsare: functionallyclosedsystems,modularity,smallequipmentfootprint,flexibility,“numberingup”insteadofscaleup.



Fig. 2

88

References:1) Anderson nG. 2001. Practical use of continuous processing in developing and scaling up. Organic

processresearch&Development.5(6):613-621.

2) Daszkowski, T. Continuous Processing in Biotech Production as an Alternative to a Modern Batch,Single-UseFacility.IBCFlexibleFacilities,April02-04,2013.SanFrancisco,CA

3) Fletchern.2010.Turnbatchtocontinuousprocessing.ManufacturingChemist.June201024-26.

4) KamarckM.2006.Buildingbiomanufacturingcapacity–thechapterandverse.natureBiotechnology.24(5):503-505.

5) Konstantinov, K., Integrated Continuous Manufacturing of Therapeutic Protein Drug Substances.ProvisionU.S.Patent61775060,2013.

6) Laird, T. 2007. Continuous processes in small-scale manufacture. Organic process research &Development.11(6)927.

7) TannerHA.1998.ContinuousCasting:ARevolutionInSteel.WriteStuffSyndicate

8) ThomasH.2008.Batch-to-continuous–comingoutofage.TheChemicalEngineer.805:38-40.

9) ReayD,RamshawC,HarveyA.2008.ProcessIntensification:EngineeringforEfficiency,SustainabilityandFlexibility.Butterworth-Heinemann.

10)Utterback,JM.1994.MasteringtheDynamicsofInnovation.HarvardBusinessSchoolPress.

11)Warikoo,V.,Godawat,R.,Brower,K.,Jain,S.,Cummings,D.,Simons,E.,Johnson,T.,Walther,J.,Yu,M.,Wright,B.,McLarty,J.,Karey,K.P.,Hwang,C.,Zhou,W.,Riske,F.,Konstantinov,K.2012.IntegratedContinuousProductionofRecombinantTherapeuticProteins.BiotechnologyandBioengineering.109(12),3018-3029


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Contents...were asked what types of bioreactor they would implement for a new facility comingonlinein 2 years,as expected,over two-thirdscitedbatch-fedsingleuse bioreactors,while32%