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4th BioProScale Symposium

6 to 8 April 2016Berlin – Germany

Institute for Biotechnology and Fermentation in Berlin (IfGB) Seestrasse 13, 13353 Berlin, Germany

In cooperation with

Institute for Biotechnology and Fermentation in Berlin

Organisers Technische Universität Berlin – Chair of Bioprocess Engineering & Institute for Biotechnology and Fermentation in Berlin (IfGB)

Location Technische Universität Berlin, Institute for Architecture, Lecture Hall A151 Strasse des 17. Juni 152, 10623 Berlin (Charlottenburg), Germany

Three-day symposium about industrial scale bioprocess intensification from process development to large-scale understanding

Bioprocess intensification through Process Analytical Technology (PAT) and Quality by Design (QbD)

Industrial-scale bioprocess operation Manufacturing at large scale from process to product, robustness vs. repeatibility,

scaleability of operations, single-use techniques, process analytical technologies and process control

Scale down approaches and process analytical technologies for advanced process design

Comprehensive process monitoring, non-invasive sensors for all process scales, mul-tiparameter and soft-sensors, addressing inhomogeneities by sensor applications and multiposition sampling, modelling and control approaches, influence of early process steps on later down-stream operations, understanding heterogeneity and population development

Quality by design in bioprocess development High throughput applications, model based DoE, in-line process analytics, data

handling and analysis, multivariate data processing, scale down methods, evolutio-nary strategies for strains and processes, computation based process development

Chair of Bioprocess Engineering

• Manifold expression and production strategies

• Record-breaking productivities

• MeOH-induced and MeOH-free processes

• Tailor-made glycoproteins – Pichia Glyco Switch®

www.vtu-technology.com

Pichia pastoris Protein Expression Excellence

AutomAting your imagination

#imagineWithHamilton

Where will your imagination take your science?

4th BioProScale Symposium 2016

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About the organisers

Technische Universität Berlin: Department of Biotechnology – Chair of Bioprocess Engineering

TheresearchattheChairofBioprocessEngineeringattheTUBerlinisdirectedtothedevelopmentandapplicationofnewmethodsforfasterbioprocessdevelopment,includinggenetic,cultivation,andanalyticaltoolswithaspecialfocusontheindustrialscale.Itaimsspecificallyinunder-standingtheimpactofreactorinhomogeneitiesonthemicrobialmetabolismandadaptation,bothaffectingprocessrobustness.Thisknowledgeisappliedtodesignmolecularbiologicalandprocessengineeringsolutionsandthuscontributestotheunderstandingandimprovementofmicrobialprocessesofbothfundamentalandindustrialinterests.Bycombiningstate-of-the-artcultivation,sensoranddataanalysis,automation,andmechanisticmodellingtechnologieswithmolecularbiologicalandphysiologicaltechniques,theactivitiesattheChairofBioprocessEngineeringcontributetoimprovetheefficiencyofbioprocessesandthustothesocietaladvancementofIndustrialBiotechnology.

www.bioprocess.tu-berlin.de

IfGB – Institut für Gärungsgewerbe und Biotechnologie zu Berlin

Foundedin1874,undertheumbrellaoftheInstituteofFermentationandBiotechnologyinBerlin(IfGB)fermentationorientedresearchandeducationhasbeenconductedformorethen140years–alwaysinclosecooperationwiththeTechnischeUniversitätBerlin(resp.itspredesessorinstitutions).Since2003theVersuchs-undLehranstaltfürBrauereiinBerlin(VLB)e.V.isthesouleholderofIfGB.

Since2003,underthebrandnameIfGBservicesandtrainingforthespiritsindustryanddistillershavebeenoffered.Startingin2009ourserviceandtrainingprogrammeswillbeexpandedintothefieldofbiotechnology–againinclosecooperationwiththeInstituteofBiotechnologyofTUBerlin.

www.ifgb.de

IfGB is a brand of VLB Berlin e.V.www.vlb-berlin.org

Scientific advisory boardFrankDelvigne(UniversityofLiège,Belgium)

KristV.Gernaey(DTU,Denmark)

StefanJunne(TUBerlin,Germany)

ClausLattemann(Sanofi,France)

Welcome addressDearColleagues,LadiesandGentlemenDearGuestsandStudents

Myco-workersandIwouldliketowarmlywelcomeyoutoBerlintothe4thBioProScaleSymposium.Thissymposiumisdedicatedto100yearsofthefedbatchtechnique,whichwasinitiallydiscoveredanddevelopedbyFriedrichHayduckin1915inBerlinatthe“VereinderSpiritusfabrikanteninDeutschlandinBerlin”,apredecessororganizationoftheIfGB.Wealsocelebrate40yearsofBioprocessEngineeringinBerlinwiththeappointmentofMatthiasReussasprofessorforBioverfahrenstechnikattheTUBerlinin1976,and30yearsofthedegreeofBiotechnologyatouruniversity.

Withmorethan200participants,theBioproscaleSymposiumhasbecomeabroadlyacceptedimportantplatformforthediscus-sionofindustrial-scalebioprocessissueswithafocusoncellphysiology.Thespecificcharacterofthissymposiumistheviewonthemethodologyofcharacterizationandcontrollinglarge-scalebioprocessesindependentfromthespecificprocess,andthuswewillhavetalksrangingfrombioenergytobiopharmaceuticalproduction.Importantaspectsforabettercontrolofindustrialscaleproductionarenovelprocessanalyticaltools,modelandsoftsensorapproaches.Keysforthesuccessfulimplementationofbioprocessestowardsabio-basedeconomyarethecomprehensiveunderstandingofscaleupphenomenaandtheavailabilityofeffectivetoolstosimulatelargescaleconditions,andfinallytotesttherobustnessofanewprocessalreadyinalaboratoryenvironment.Currently,weseeanincreasingacceptancefortheideatoconsiderscaleissuesalreadyattheearlyproductdevelopmentstage.Thisprovokesmanynewdevelopmentsinthebiore-actorfield,butalsoneedsparallelprogressinsensoranddatahandlingtechnologies.Integrationofthisknowledgeprovidesaparadigmchangeinthewayofhowtotranslatebiomolecularresearchintonewbioproducts.

Weareverydelightedtohaveawealthofpromisinglecturesinourprogramandwouldliketoextendourgratitudeespeciallytothespeakerswhofollowedourinvitationandwillshareanddiscusstheirexpertisewithus,aswellastoourexhibitorsandsponsors,whoprovidedasubstantialbasisforapleasantatmosphere.IwishyouallaveryinterestingsymposiumandagreatstayinBerlin!

Professor Dr. Peter NeubauerTechnische Universität Berlin – Chair of Bioprocess Engineering

www.bioproscale-conference.org

PeterNeubauer(TUBerlin,Germany)

HenkNoorman(DSM,TheNetherlands)

MatthiasReuss(UniversityofStuttgart,Germany)

RalfTakors(UniversityofStuttgart,Germany)

RolandWeis(VTUTechnology,Austria)

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13:00 Welcome address and introductionPeterNeubauer(DepartmentofBioprocessEngineering,TUBerlin,Germany)

13:30 Plenary lecture: Reflections of a 40-years ripening process for modeling of bioreactors – from well mixed towards more reality (L01)MatthiasReuss(StuttgartResearchCenterSystemsBiologySRCSB,UniversityofStuttgart,Stuttgart,Germany)

Industrial scale operation

Chair AlvinNienow(UniversityofBirmingham,Birmingham,UK),PeterNeubauer(TUBerlin,Berlin,Germany)

14:15 Keynote lecture: Continuous improvements in the understanding of large scale aerobic fermentation processes (L02) StuartMichaelStocks(Novozymes,Bagsvaerd,Denmark)

14:45 Keynote lecture: Large-scale production of two molecules of pharmaceutical interest in Saccharomyces cerevisiae (L03)ClausLattemann(SANOFI,Paris,France)

15:15 Keynote lecture: Microbial GMP manufacturing at large-scale – challenges and considerations (L04)TorstenSchmidt(Lonza,Visp,Switzerland)

15:45 Sponsor Talk: Bioprocess scale-up from small to large pilot scale using Eppendorf fermentation systems (L05)ChristofKnocke(EppendorfBioprocessCenter,Jülich,Germany)

15:55 Coffeebreakwithpostersessionandexhibition

10:05 Fermentation process gradient effect on Corynebacterium glutamicum 1945 ΔACT3 PTUF-LDCCOPT producing cadaverine (L18)WilliamsOlughu(LoughboroughUniversity,Loughborough,UK)

10:20 Performance loss of Corynebacterium glutamicum cultivations under scale-down conditions (L19)AnjaLemoine(TUBerlin,Germany)

10:35 Studies on the clavam biosynthetic pathway: A controling step for clavulanic acid production (L20)HowardRamirez-Malule(UniversidaddeAntioquiaUdeA,Medellín,Colombia)

10:50 Sponsor Talk: Single-use online capacitance biomass measurement supporting process scale-up from 10 L to 1000 L in cell culture application (L21)StuartTindal(SartoriusStedimBiotech,Göttingen,Germany)


Metabolic adaptation and population dynamics

Chair MarcoOldiges(ForschungszentrumJülich,Germany),ChristianReitz(TUBerlin,Germany)

9:00 Keynote Lecture: Impacts of large-scale gradients mirrored on metabolic and transcriptional regulation (L15)RalfTakors(UniversityofStuttgart,Germany)

9:30 Rapid feast-famine cycles for studying metabolic response and adaptation representative for large-scale (L16)LiangWu(DSMBiotechnologyCenter,Delft,TheNetherlands)

9:50 Metabolic regulation of carbon metabolism: Key element for robustness to bioreactor inhomogeneities (L17)MichaelLimberg(ForschungszentrumJülich,Germany)

Programme at a glance, Wednesday, 6 April 2016

Programme

Bioprocess and bioreactor modelling

Chair ChristophHerwig(TUWien,Vienna,Austria),NicolasCruz-Bournazou(TUBerlin,Berlin,Germany)

16:40 Keynote lecture: Hybrid modelling and multi-parametric control of bioprocesses (L06)ChristophHerwig(TUWien,Vienna,Austria)

17:00 Industrial case study: Data science investigations to improve bioprocess scale-up (L07)PatrickSagmeister(ExputecGmbH,Vienna,Austria)

17:15 Multivariate data- and knowledge-driven tools for robust and efficient cell culture process development (L08)MichaelSokolov(ETHZürich,Zürich,Switzerland)

17:30 Implementation of computational fluid dynamics (CFD) as a Quality by Design (QbD) tool for industrial process development (L09)ThomasWucherpfennig(BoehringerIngelheimPharma,Biberach,Germany)

17:50 Scale down of industrial scale fermentors using Euler-Lagrange CFD: A penicillin case study (L10)CeesHaringa(DelftUniversityofTechnology,Delft,TheNetherlands)

18:05 A modelling framework for bioreactor simulations connecting physical and biological heterogenities (L11)JérômeMorchain(Laboratoired‘IngénieriedesSystèmesBiologiquesetdesProcédés,Toulouse,France)

18:20 Investigation of scale-up effects on mammalian cell culture process performance and critical quality attributes (L12)MatthiasBrunner(TUWien,Vienna,Austria)

18:30 Keynote lecture: Modelling populations of microorganisms – The Anaerobic Digestion Model No. 1 as an example (L13)KristV.Gernaey(TechnicalUniversityofDenmarkDTU,Lyngby,Denmark)

19:00 Keynote lecture: Need for speed: Tools for enhanced microbial bioprocess development (L14)MarcoOldiges(ForschungszentrumJülich,Germany)

19:30 Poster session, Exhibition & Welcome Reception 21:30 End

Programme at a glance, Thursday, 7 April 2016


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11:40 Keynote Lecture: Exploiting the heterogeneity of biotic and abiotic phase in order to increase bioprocess robustness: Toward new bioreactor design (L22)FrankDelvigne(UniversityofLiège,Belgium)

12:10 Multiparameter monitoring of gradients in the liquid phase of fermentation processes (L23)AnikaBockisch(TUBerlin,Germany)

12:25 Quantification of spatial heterogeneity in large bioreactors (L24)AndersNørregaard(TechnicalUniversityofDenmarkDTU,Lyngby,Denmark)

12:40 Sponsor Talk: Connected in situ measurement system (L25)YoannGasteuilandCélineVinson(smartINST,Lyon,France)

12:50 Sponsor Talk: Optical sensors for bioprocess development and control (L26)GernotJohn(PreSensPrecisionSensing,Regensburg,Germany)

12:55 Lunchbreak,postersessionandexhibition


Chair:KristGernaey(TechnicalUniversityofDenmarkDTU,Lyng-by,Denmark),StefanJunne(TUBerlin,Berlin,Germany)

14:00 Keynote Lecture: Bioprocesses under magnification: Learning from single cells about large-scale processes (L27)AlexanderGrünberger(ForschungszentrumJülich,Germany)

14:30 Development of a perfusion system in a microbioreactor using sedimentation as a scale down tool for ATF perfusion bioreactors (L28)SteffenKreye(Glycotope,Berlin,Germany)

14:45 Keynote Lecture: Issues that are relevant when scaling-up hMSC microcarrier production processes in stirred bioreactor (L29)RegineEibl(ZürichUniversityofAppliedSciencesZHAW,Wädenswil,Switzerland)

15:15 Sponsor Talk: Evaluation of a stirred small scale single-use bioreactor for microbial applications (L30)ThomasDreher(SartoriusStedimBiotech,Göttingen,Germany)

15:25 Scalability of cultivations in the 2-dimensional ro-cking single-use bioreactor CELL-tainer (L31)Anna-MariaMarbà-Ardébol(TUBerlin,Germany)

15:40 Towards efficient microaerobic processes using engineered Escherichia coli strains (L32)AlvaroR.LaraandKarimJaen(UniversidadAutónomaMetropolitana-Cuajimalpa(UAM),México)


16:50 Keynote Lecture: Novel fluorescence-based online monitoring of cell cycle and growth rate for the control of mammalian cell culture (L33)An-PingZeng(TUHamburg,Harburg,Germany)

17:20 Multiposition sampling, multiparameter monitoring and electrooptical tracking of microbial activity: New tools for better understanding biogas production (L34)ErichKielhorn(TUBerlin,Berlin,Germany),JörnBeheim-Schwarzbach(IASP,Berlin,Germany)

17:45 In-line photon density wave spectroscopy for bioprocess monitoring (L35)RolandHass(UniversityofPotsdam,Potsdam,Germany)

18:00 Keynote lecture: Stirred, not shaken – Unsolved issues and new problems in scale-down for scale-up (L36)AlvinW.Nienow(UniversityofBirmingham,Birmingham,UK)

19:30 Conference Dinner RestaurantZillemarkt,Bleibtreustr.48a,10623Berlin(SBahnhofSavignyplatz)

Programme at a glance, Friday, 8 April 2016

Quality by design in bioprocess development

Chair:RalfTakors(UniversityofStuttgart,Stuttgart,Germany),ErichKielhorn(TUBerlin,Berlin,Germany)

9:00 Keynote lecture: Next generation mAb production: Continuous manufacturing, PAT, and real time release (L37)DouglasRichardson(Merck,NewJersey,USA)

9:30 Keynote lecture: High-level protein production with Pichia: Case studies of development path from strain generation over bioreactor (L38)RolandWeis(VTUTechnology,Grambach,Austria)

10:00 Keynote lecture: ESETEC 2.0, a new, secretory Escherichia coli strain for the production of complex recombinant proteins at industrial scale (L39)GuidoSeidel(WackerBiotech,Jena,Germany)

10:30 Different process strategies for the Glutathione overproduction in Saccharomyces cerevisiae (L40)MartinSenz(ResearchandTeachingInstituteforBrewinginBerlinVLB,Berlin,Germany)


11:40 Keynote lecture: Parallel small-scale bioreactors with on-line monitoring to prove the consistency of medium ingredients and pre-cultures and to efficiently assess process characteristics (L41)JochenBüchs(RWTHAachen,Germany)

12:10 Towards intensifying design of experiments: An industrial Escherichia coli feasibility study (L42)MoritzvonStosch(NewcastleUniversity,Newcastle,UK)

12:30 Robot based bioprocess development by automated on-line optimal re-design in parallel bioreactors (L43)M.NicolasCruz-Bournazou(TUBerlin,Berlin,Germany)

12:45 Plenary lecture: From advanced PAT to QbD-compliant quasi-continuous integrated pharmaceutical production (L44)ReinerLuttmann(HamburgUniversityofAppliedSciences,Hamburg,Germany)

13:30 Concluding remarks PeterNeubauer(TUBerlin,Germany)

13:45 End of symposium

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Opening Session

13:00 Welcome address and introductionPeter NeubauerTechnische Universität Berlin, Institute for Biotechnology, Chair of Bioprocess Engineering, Berlin, Germany, [email protected]

13:30 Plenary lecture: Reflections of a 40-years ripening process for modeling of bioreactors – from well mixed towards more reality (L01)Matthias ReussStuttgart Research Center Systems Biology (SRCSB), University of Stuttgart, [email protected]

Thelectureaimsata“Tourdeforce”througha40yearsperiodofdevelopmentofbioreactormodeling.Outofmypersonalperspectiveitisobvioustoselecttheyear1976asastartingpointforthisretrospective-theyearinwhichIstartedwiththeinstallationofthedepartmentofBioprocessEngineeringwithintheInstituteofBiotechnologyattheTUBerlin.Themodelsusedatthattimewerebasedonthewell-knowntextbookassumptionofwellmixedsystemappliedtosinglephaseandgas-liquidapplications.Thefirstmilestonesforconsiderationofmorerealisticmixingcon-ditionsatlargerscaleweretackledwiththeaidofsocalledmicro-macromixermodelsforsimulatingglucosegradientsduringfed-batchoperationsofbaker´syeastproductioninstirredtankreactors.Thesemodelswerelaterextendedtocouplingmasstransfergas-liquidandmixinginhighlyviscousnon-Newtonianfermentationbroths.Themodelingac-tivitieswerelinkedwithextensiveexperimentalinvestigationsofrecirculationtimedistributionsforsingleandmultipleimpellersystems.Inthemidoftheeightieswestartedwiththeapplicationofcompartmentmodels,whichwerelaterextendedtomultiphaseapplicationsattheUniversityofStuttgart.WesoonrealizedthelimitationofthesemodelsafarastheproblemsofquantitativepredictabilityofthemanyparametersatdifferentscaleofoperationisconcernedandconsequentlysteppedintotheapplicationofCFD.Inthebeginningoftheninetiestheseapplicationswereperformedwiththeaidofhybridmodelingapproaches-anintegrationofcompartmentmodelsandCFD.Afteraperiodofstudyingalternativestothek-Emodelsthetoolboxofsimulatingthefluiddynamicconditionsinsingleandmultipleflowwascoupledtoagent-basedsimulationsofthestructuredbiophaseviaEuler-Lagrangemodelingstrategies.Forthefirsttimeitwaspossibletosimulatethelifelinesofsinglecellsandpopulationsinlargescaleoperations.Thepresentationiswrappedupwithadiscussionoffutureperspectivesofmodel-basedscaledownstrategiesandapplicationsofparallelprocessingforsimulationsofverylargebioreactorsusedinbio-industry.

Industrial scale operationChair AlvinNienow(UniversityofBirmingham,Birmingham,UK),PeterNeubauer(TUBerlin,Berlin,Germany)

14:15 Keynote lecture: Continuous improvements in the understanding of large scale aerobic fermentation processes (L02)Stuart Michael StocksNovozymes A/S, Bagsvaerd, Denmark, [email protected]

NovozymesisalargeandgrowingbiotechnologyconcernheadquarteredinDenmark,producingtensofthousandsoftonsoffinishedenzymeproductseachyearwithfood,feedandtechnicalapplications,aswellasasmallinterestinpharmaceuticalproduction.ScaleupandscaledownofproductionprocessesisacoreactivitysupportedbyanumberofengineersandscientistsbasedinR&D,pilotplantsandproductionfacilitieslocatedaroundtheglobe.Weaimtoimproveourunderstandingofscaleupandscaledown,sowecanbesmarterabouthowwedoit.Muchofwhatwedoisconfidential,butovertheyears,incollaborationwiththeDanishTechnicalUniversity(DTU),andotherinstitutions,wehavebeenactiveinpursuitofnewknowledgeandmodelswhichcandescribefermentationprocessperformanceacrossanumberofscalesandhavemadesomeprogresstowardsthisgoal.Whilethisjourneyisbynomeanscomplete,resultsofworkdoneonagitatorgeometry,oxygentransferandmodellingoftheprocesseshastakenusagooddis-tance;muchofthisdatacannowbepresented.Inaddition,therearenowsomefurtheropportunitiestobecomewiser,namely,thedevelopmentoflowcostofftheshelfprogrammableelectronicspackagesandthematurationoffluorescencespectroscopygivesusanunprecedentedopportunitytopeerintootherwiseopaquestainlesssteelreactors,andvalidateourprogresstowardsbetterunderstandingwhatwearedoing.

14:45 Keynote lecture: Large-scale production of two molecules of pharmaceutical interest in Saccharomyces cerevisiae (L03)Claus LattemannSANOFI - C&BD Biochemistry, Paris, France, [email protected]

Reportsontheproductionofheterologousmetabolitesinmicrobialsystemshavebeenpublishedrecentlydemon-stratingthatvariouscompoundclassescomprisingnaturalandnon-naturalmoleculescanbeproducedinmicroorgan-isms.Productionofsuchcomplexheterologousmoleculesinmicroorganismsinlargevolumefermentationhowever,requiresthecoordinateddevelopmentofproductionstrainsandthecorrespondingfermentationprocess.Twoexam-plesoflargevolumeproductionofactivepharmaceuticalingredientsinbaker’syeastwillbediscussed.

Wednesday, 6 April 2016

Programme


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15:15 Keynote lecture: Microbial GMP manufacturing at large-scale – challenges and considerations (L04)Torsten SchmidtHead of Microbial Manufacturing, Lonza AG, Visp, Switzerland, [email protected]

Microbialprocessesforproductionofbiopharmaceuticalsarecharacterizedbyahugevarietyofdifferentprocesssteps.Basedontheselectedhoststrainandexpressionsystemtherearemultipledifferentoptionsforprocessingthetargetprotein.Fermentationtiter,stepyieldsandproductpurityarethetypicalselectioncriteriainthisprocessdevelopmentphase.Lacksinscalabilityandprocessrobustnessareconsideredwithalowerpriority.Forthefirstproductionofearlyclinicalmaterialthosepotentialissuescanbeeasilysolvedbytheflexibilityofsmall-scalefacilitiesandoversizing.Whenitcomestolarge-scaleothersolutionsareneeded.Basedonthedesignofthefacilitylarge-scaleplantsforlate-phaseclinicalorcommercialmanufacturingarelessflexible.AlsoCostofGoods(COGS)haveamoreimportantroleatlarge-scale.

15:45 Sponsor Talk: Bioprocess scale-up from small to large pilot scale using Eppendorf fermentation systems (L05)Ulrike Becken1, Bin Li2, Christof Knocke3, Jens Schiffler1, Bruno Sommer Ferreira4, Ma Sha2

1Eppendorf AG Bioprocess Center, Jülich, Germany, 2Eppendorf Inc., USA, 3Vaudaux-Eppendorf AG, Schönenbuch, Switzerland, [email protected], 4Biotrend, Portugal

Theultimategoalinbioprocessdevelopmentistherealizationofcommercialproduction.Currently,thescale-upoffermentationprocesses,whichiscriticaltothesuccessofindustrialfermentationforbioproduction,isreceivingmuchattention.Eppendorffermentationsystemscoverawiderangeofworkingvolumesfromlessthan1Ltoaslargeas2,400L.Weinvestigatedtheirscale-upcapabilitiesfromsmalltopilotscale.Withinthispresentation,wediscussengi-neeringparameterscriticalforscale-up,suchasvesselandimpellergeometry,tipspeed,mixingtime,oxygentransferrate(OTR),kLa,andpowernumber.Furthermore,wepresentrecentcustomerdataontheproductionofthebiopoly-merpolyhydroxybutyrate(PHB)usingBacillussacchari.Successfulscale-upoftheprocessfrom2Lto200Lexemplifiesthescale-upcapabilitiesofEppendorffermentationsystems.


Bioprocess and bioreactor modellingChair ChristophHerwig(TUWien,Vienna,Austria),NicolasCruz-Bournazou(TUBerlin,Berlin,Germany)

16:40 Keynote lecture: Hybrid modelling and multi-parametric control of bioprocesses (L06)Christoph HerwigChristian Doppler Laboratory for Mechanistic and Physiological Methods for Improved Bioprocesses, Research Division Biochemical Engineering, Institute of Chemical Engineering, Vienna University of Technology, Vienna, Austria, [email protected]

Thegoalofbioprocessingistooptimizeprocessvariables,suchasproductquantityandqualityinareproducible,scal-ableandtransferablemanner.However,bioprocessesarehighlycomplex.Alargenumberofprocessparametersandrawmaterialattributesexist,whicharehighlyinteractiveandmayvaryfrombatchtobatch.Thoseinteractionsneedtobeunderstood,andsourceofvariancemustbeidentifiedandcontrolled.Whilepurelydatadrivencorrelations,suchaschemometricmodelsofspectroscopicdata,maybeemployedfortheunderstandinghowprocessparametersarerelatedtoprocessvariables,theycanhardlybedeployedoutsideofthecalibrationspace.Currently,mechanisticmodels,modelsbasedonmechanisticlinksandfirstprinciples,areinthefocusofdevelopment.Theyareperceivedtoallowtransferabilityandscalability,becausemechanisticscanbeextrapolated.Moreover,themodelsdeliveralargerangeofhardlymeasureablestatesandphysiologicalparameters.Forimplementationofmechanisticmodels,however,modelsneedtobesimplifiedandlinkedtoprocessparametersforrealtimeexecution.Forthis,hybridmodels,hencelinksbetweenDataDrivenandMechanisticModelsmaybeahelpfulsolution.Moreover,modelsneedtobedeployedinthecontrolcontext:Bioprocessesneedtobecontrolledontheonehandondifferentparameterssimultaneously(e.g.constantprecursorconcentrationandspecificgrowthrate)andontheotherhandmayhavedifferentobjectivefunc-tions(maximumproductivityandcorrectproductquality).Hencenovelsolutionsandcasestudiesformultipleinputandoutputcontrolsneedtobedeveloped,astheyalreadyexistinothermarketsegments.Thecurrentcontributionwantstodisplaycurrentsolutionsandcasestudiesofdevelopmentanddeploymentofhybridmodelsandmulti-parametriccontrolofbioprocesses.Thefollowingelementscanbecovered:

y Hybridmodelsolutions,combinationsofdatadrivenandmechanisticmodels.y Workflowshowmechanisticmodelscanbedevelopedfromdatadrivenapproachesandviceversa.y Discussionbetweenexplorative(DoE-based)andmodelbasedexperimentaldesigny Implementationofhybridmodelsinthereal-timecontexty ModelsasPATtool:Demonstrationsofcasesinwhichmodelswereasolutiontomeasurelessy Observersolutionsforreal-timeparameteroptimizationy Multiparametriccontrolandeventpredictiony LifeCyclemanagementofmodelsy Knowledgemanagementusinghybridmodels

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17:00 Industrial case study: Data science investigations to improve bioprocess scale-up (L07)Patrick Sagmeister1, Sandra Abad2, Christoph Herwig3

1Exputec GmbH, Vienna, Austria, [email protected], 2Boehringer Ingelheim RCV, Vienna, 3Vienna University of Technology

Scale-upofbioprocessesreferstothetransferofprocessesfromlaboratorytopilotandproductionscale.Thisisanessentialandcriticalstepforthecommercializationofanybiotechnologicalproduct.Thisstepcanbeaccompaniedbyunexpecteddeviationsimpactingonprocessperformanceandproductquality.Scale-dependentdeviationsaretypi-callyrecognizedverylateinthecommercializationofbiotechnologicalproducts,therebyexhibitingahigheconomicimpact,suchasfailedbatchesandreprocessingunderGMPregulations.Therefore,itisoftheutmostimportancetoachieveathoroughandscience-basedunderstandinginordertomakethescale-upofbioprocessesevenmorerobustandpredictable.Inthepresentedindustrialcasestudyisdemonstratedhowthescale-upofanindustrialE.colimanufacturingprocesswasimprovedbasedonadatascienceapproach,leveragingintegrateddatafromprocessdevelopment,pilotingandmanufacturing.Firstly,wepresenthowadatascienceframeworkwasputtopracticetoidentify-andunderstandscale-dependentchanges.Secondly,wediscusshowscale-upimprovementstrategies,includingthedefinitionofscale-downmodels,werederivedfromtheobtainedmechanisticinsights.Thirdly,wediscusshowasystematicanalysisofavailablebigdatafrommanufacturingdatabasescanbeleveragedtomakebioprocessscale-upmorepredictableandderiveclear-andinterpretabledesignguidelinesforthedesignandscale-upofbioprocesses.

17:15 Multivariate data- and knowledge-driven tools for robust and efficient cell culture process development (L08)Michael Sokolov, Alessandro Butte, Massimo MorbidelliETH Zurich, Zurich, Switzerland, [email protected]

Inrecentyears,theEuropeanMedicinesAgency(EMA)andtheUnitedStatesFoodandDrugAdministration(FDA)havestressedtheneedofgreaterprocessunderstandingandqualitycontrolintheareaofpharmaceuticaldevelopment,manufacturingandqualityassurance,allwhichissummarizedintheProcessAnalyticalTechnology(PAT)initiative.Inbioprocesses,andparticularlyincellcultures,extensivedataacquisitionandanalysisarelikelytoprovideversatileandsignificantreal-timeinformation.Similarly,theQualitybyDesign(QbD)initiativewaslaunchedinordertopromotetheneedforbuildingproductqualityintotheprocess.Inprocessdevelopment,thiscanbeachievedbyadeepprocessunderstandingcombinedwithabroadinvestigationoftheproductqualityinordertoconnectthosetwodomainsandensurerobustproductqualityinanefficientmanner.Allofthesegoalsgoalongwithverylargedatasets,whichusuallyfeatureahighdegreeofcorrelatedvariables.Thismotivatedtodevelopmultivariateapproaches,whichreducethosehigh-dimensionalproblemstothemajoruniquepropertiesoftheprocessaswellasthecorrespondingproduct.Thisworkpresentsseveralcasesstudies,wheredifferentmultivariatedata-andknowledge-driventoolsweresuccessfullyappliedatvari-ousstagesofcellcultureprocessscreeninganddevelopment.Inparticular,highthroughputscreeningforearlyprocessdevelopment,productqualitypredictionatmultiplescalesduringprocesscharacterization,processmonitoringbyRamanspectroscopyaswellashybridapproachesincorporatingadeterministicprocessmodelbackbonewillbediscussed.Allthepresentedresultsgowellbeyondthepos-sibilitiesofferedbycommercialtoolsandprovideanimportantbasisforriskreductionandefficiencyduringprocessdevelopment.

17:30 Implementation of Computational Fluid Dynamics (CFD) as a Quality by Design (QbD) tool for industrial process development (L09)Thomas Wucherpfennig1, Erik Hasenfus1, Johannes Wutz2, Ralf Takors2 und M. Berger1 1Biopharma Process Development, Biberach an der Riss, Germany, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany, [email protected], 2Institute of Biochemical Engineering, University of Stuttgart, Stuttgart, Germany

Thedemandforcomplextherapeuticproteinsandantibodiesfortreatmentofvariousdiseaseshasincreasedcon-tinuouslyinthelastdecades,promptingthecontinuousdevelopmentofnewprocesses.Forthedevelopmentofthesenovelprocesses,theQualitybydesign(QbD)approachhasgainedpopularitysincetheFDA´sPAT-publication.However,thispracticerequiresathoroughunderstandingofaproductandespeciallyofthemanufacturingprocessandequipment.Toolstofurthersuchanequipmentunderstandinge.g.forthesimulationofthecomplexturbulentflowsinstirredtankbioreactorshavebeenaroundforsometime.Theirapplicationinthebiomanufacturingindustry,however,hasremainedscarceduetothedemandofcomputationalpower,complexunderlyingmathematicsandthehighamountoftrainingneeded.Inthisstudyitisdemonstrated,howthesimulationofscale-upcriterialikeP/V,kLa,ormixingtimescanfacilitatearobustscale-uporbeusedinafacilityfitanalysistodeterminewhetheradevelopedprocessissuitedtotheproduc-tionequipmentatvarioussites.Furthermore,aspecificapplicationexampleisprovidedbyusingCFDmethodsfortheset-upofaprocessdesignspaceforanacceptablerangeofthevolumetricoxygenmasstransfercoefficient(kLa).Agitationandaerationaresomeofthefirstparameterswhicharetestedduringprocessdevelopment;basedontheseparametersandthefillingvolumeasinputparametersanDOEstudy(MODDE10,Umetrics,Sweden)wasconductedinanaerated2Lstirredbenchtopbioreactor.ForeachexperimentalsetupatwophaseEuler-LagrangeCFDmodelwasestablishedandakLavaluedeterminedinsilico(Fluent16.6,ANSYSInc.,Canonsburg,USA).AprocessdesignspacewasestablishedformaintainingarequiredkLavalueofa6.5±1h-1neededtosupporthighdensitycellculture.Atafillingvolumeof1.5Lbetween0.09and0.1LL-1min-1andagitationratesbetween197and300rpmaresufficienttomaintainakLavalueof6.5h-1withaprobabilityof95%inthestudiedbioreactorsystem.Allsimulatedvalueswerevalidatedexperimentallyandshowedagoodfit.Theconductedinsilicoap-proachcouldbeusedinfutureprocesscharacterizationstudiesandsavevaluableexperimentaltime.

Programme


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17:50 Scale down of industrial scale fermentors using Euler-Lagrange CFD: A penicillin case study (L10)Cees Haringa1, Wenjun Tang2, Jianye Xia2, Amit T. Deshmukh3, Matthias Reuss4, Joseph J. Heijnen5, Robert F. Mudde1, Henk J. Noorman3,5 1Chemical Engineering department, Delft University of Technology, Delft, The Netherlands, [email protected], 2East China University of Technol-ogy, Shanghai,China, 3DSM Biotechnology Center, Delft, The Netherlands, 4Stuttgart Research Center Systems Bioloogy (SRCSB), University of Stuttgart, 5Biotechnology department, Delft University of Technology, Delft, The Netherlands

Micro-organismslivinginsideindustrialscalefermentersmaybeexposedtorapidvariationsintheextracellularenvi-ronment,forexample,variationsintheobservedsubstrateconcentration.Boththemagnitudeandfrequencyofthesevariationsmayimpactthemetabolismoftheorganismandtherebytheindustrialviabilityoftheprocess.Toestimatetheirupfrontviascale-downsimulation,itisessentialtogaininsightinthemagnitudeandfrequencyatwhichorgan-ismsareexposedtothesevariations.BasedonpioneeringworkofLapinetal.andDelvigneetal.,weproposetheuseofanEuler-LagrangeCFDframeworktostudytheseaspectsonanindustrialscale.TheLagrangianpointofviewallowstoquantifytheobservedfermenta-tionenvironmentfromthepointofviewoftheorganism,andanalysetheobserved‘lifelines’toextractthefrequenciesandmagnitudeofenvironmentalvariations.Fromthisinformationitispossibletodeterminethedesignparametersforascaledownsimulator,asspecifiedbyNoorman,andset-upanindustriallyrelevantscale-downsimulation.Wepresentacasestudy,basedonthefermentationofPenicilliumchrysogenum,inwhichweshowcasetheproceduretoextracttherelevantinformationfromCFDsimulations,andtheconsecutivedesignofscale-downsimulatorsbasedonthisinformation.Withthis,weshowacomprehensivemethodologyforthedesignofquantitativelyrepresentativescale-downsimulatorsbasedonCFDdata,andhowthismethodologycanbeappliedinpracticalsituations.Thisisamulti-partyresearchproject,betweenDSM-SinochemPharmaceuticals,TUDelft,EastChinaUniversityofScienceandTechnologyandGuojia,subsidizedbyNWOandMoST.

[1]ALapinetal.ModelingthedynamicsofE.colipopulationsinthethree-dimensionalturbulentfieldofastirred-tankbioreactor,astructuredsegregatedapproach.ChemEngSci61(14):4784-4797,2006.[2]FDelvigneetal.Amethodologyforthedesignofscale-downbioreactorsbytheuseofmixingandcirculationstochasticmodels,BiochemEngJ28(3):256-268,2006.[3]HJNoorman,Anindustrialperspectiveonbioreactorscale-down:whatwecanlearnfromcombinedlarge-scalebioprocessandmodelfluid-studies.BiotechnolJ,6(8):934-943,2011

18:05 A modelling framework for bioreactor simulations connecting physical and biological heterogeneities (L11)Maxime Pigou1,2,3 and Jérôme Morchain1,2,3

1Laboratoire d’Ingénierie des Systèmes Biologiques et des Procédés, Toulouse, France, [email protected], 2INRA, Ingénierie des Systèmes Biologiques et des Procédés, Toulouse, France, 3CNRS, Toulouse, France

Largescalegradientsofsubstrateandoxygenareoftenobservedatthereactorscalebecauseofinsufficientmacromix-ingandheterogeneousgas-liquidtransferrates.ItappearsthatthemostpromisingapproachresidesinthecouplingofpopulationbalancemodelsalongwithCFDandmetabolicapproaches.Thisrequiresahighlevelofexpertiseinseparatescientificdomains.Moreovertheset-upofsuchhighresolutionmodelsisverytimeconsuming.Inordertoeasethetaskwehavedevelopedasimulationtool,namedADENON,thatcombineseachtypeofmodels(hydrodynamics,populationbalanceandmetabolic)inageneralframework.RatherthanusingafullyresolvedCFDmodel,hydrodynamicsdescriptionisbasedonacompartmentapproach.Apopulationbalancemodeldescribingadaptationdynamicsisformulatedintermsofuptakeratedistribution.Thepopulationbalanceapproachsolvestheproblematicofaccessingindividualhistory,atlowercostthanLagrangiantrackingmethods,whilehandlingtheissuethatriseswhentwopopulationswithdifferentpropertiesaremixed.Finallyametabolicmodelisinvokedtocalculatethefateofthesubstrateinsidethecell,bytakingintoaccountthecellstateandlocalconcentrations.Thespecificityhereliesinthefactthatthereisnooptimizationproceduretocomputethemetabolicfluxes.Altogether,thesemodellingchoicesallowforasimultaneouscalculationofallaspectsoftheproblematareasonablecomputationalcost.Severalexamplesofdynamicsimulationofmicrobialcultivationinunsteadycontinuous,multi-stageandlargescalebioreactorswillbepresentedandtheresultscomparedtoavailableexperimentaldata.

18:20 Investigation of scale-up effects on mammalian cell culture process performance and critical quality attributes (L12)Matthias Brunner1,2, Jens Fricke1,2, Christoph Herwig1,2

1Research Division Biochemical Engineering, Institute of Chemical Engineering, Vienna University of Technology, Vienna, Austria, [email protected], 2Christian Doppler Laboratory for Mechanistic and Physiological Methods for Improved Bioprocesses, Institute of Chemical Engineering, Vienna University of Technology, Vienna, Austria

Inlarge-scalemammalianprocessesheterogeneitiesofcriticalprocessparametersaremostlikelytooccur,duetodif-ferentgassingstrategiesandelevatedmixingtimescomparedtosmallscalefermentations.Understandingtheeffectoftheseinhomogeneitiesonmammaliancellcultivationsisanessentialrequirementforprocessscale-up.Furthermore,comprehensiveknowledgeoftherelationshipbetweentheprocessandtheproductscriticalqualityattributes(CQAs)isnecessarytosatisfyQualitybyDesign(QbD)guidelines.InthiscontextacomprehensiveDesignofExperiments(DoE)approachincludingvariousproductqualityanalyticswasperformedtoinvestigatetheinfluencesandtheinteractionsofscaleuprelevantparametersaspH,dissolvedoxygentension(pO2)anddissolvedcarbondioxidetension(pCO2)onchinesehamsterovary(CHO)cellbatchperformanceandmonoclonalantibody(mAb)quality.ThroughindependentcontrolofprocesspH,pO2andpCO2wecouldderivesingleprocessparametereffectsandinteractionsonCHOphysiologyaswellasonmAbchargeandglycosylationvari-ants.Finally,processpHwasrecognizedasthekey-parameter,exertingstrongeffectsonCHOmetabolismandmAbCQAs.Subsequently,wefurthermoreinvestigatedtheeffectofpermanentpH-shiftstoelevatedlevels,similartothosepresentinlarge-scaleduetobaseaddition(pH7.8and9.0).Therefore,onecompartmentbatchfermentationsincludingpH-shiftswereconductedandshort-termaswellaslong-termeffectsofexternalpH-shiftsonintracellularpHandoverallCHOmetabolismcouldbederived.

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Programme

Withinthiscontributionacomprehensivestudyregardinglarge-scaleheterogeneitiesonmammaliancellcultureprocessperformanceandcriticalqualityattributeswillbepresented.ThisincludesDoEresultsregardingtheeffectofcriticalscale-upparametersonprocessperformanceattributesandindustry-relevantCQAs.Furthermore,theeffectoflarge-scalepHinhomogeneitiesonintracellularpHandCHOphysiologywillbeevaluatedoutofonecompartmentexperiments.

18:30 Keynote lecture: Modelling populations of microorganisms – the Anaerobic Digestion Model No. 1 as an example (L13)Krist V. Gernaey1, Ulf Jeppsson2, Damien Batstone3 and Xavier Flores-Alsina1

1Department of Chemical and Biochemical Engineering, Technical University of Denmark (DTU), Lyngby, Denmark, [email protected], 2Division of Industrial Electrical Engineering and Automation, Department of Biomedical Engineering, Lund University, Lund, Sweden, 3Advanced Water Man-agement Centre, The University of Queensland, St Lucia, Brisbane, Queensland, Australia.

Manyindustrialscalefermentationprocessesrelyonpurecultures.However,thereareexceptions,forexampleayogurtproduction.Withthecurrentfocusontheproductionofbiofuelsandchemicalsfromrenewables,newproc-essesaredeveloped,andthereisalsoincreasedfocusonthepotentialbenefitsthatcanbeobtainedbyusingmixedculturesinsteadofpurecultures.Forprocessdesignandoperation,itisimportanttodevelopmathematicalmodelsthatcandescribeaprocessrelyingonsuchamixedculture.Fromamodellingpointofview,andtakingapurecultureasareference,thecomplexityofmodellingamixedcultureishigher,sincetheco-existenceofseveralpopulationsofmicroorganismsandtheirinteractionshavetobedescribed.Inthewastewatertreatmentfield,almostallprocessesrelyonmixedcultures,andmathematicalmodellinghasduringthepastdecadesplayedanimportantroleinbuildingupprocessknowledge,andindevelopmentofnoveloperationandcontrolstrategies.TheIWAAnaerobicDigestionModelNo.1(ADM1)istakenasanexamplehere,andstartingfromtheoriginalmodel,aseriesofrecentlypublishedexten-sionsoftheADM1arehighlightedtofunctionallyupgradetheADM1toallowforplant-widephosphorus(P)removalsimulation.ThecloseconnectionbetweentheP,sulfur(S)andiron(Fe)cyclesindeedrequiresasubstantial(andunavoidable)increaseinmodelcomplexityduetotheinvolvedthree-phasephysico-chemicalandbiologicaltransformations.

19:00 Keynote lecture: Need for speed: Tools for enhanced microbial bioprocess development (L14)Johannes Hemmerich, Joachim Koepff, Holger Morschett, Marco OldigesResearch Center Jülich, Institute of Bio- and Geosciences: IBG-1 Biotechnology, Jülich, Germany, [email protected]

Thebiotechnologicalproductionoffineandbulkchemicalsaswellasheterologousproteinproductionusingmicrobialsystemsisoneimportantfieldinthegrowingbioeconomy.OneoftheimportantkeystosuccessistheincreasingspeedofgeneticmanipulationspossibleforplatformorganismslikeEscherichiacoliorCorynebacteriumglutamicumandothers.Thisallowstoengineermicrobialstrainsinafastwayandeasilyprovidesstrainlibrariesharbouringlargebiologicalvariance.However,thecapabilityofdetailedphenotypingofsuchgenotypelibraryatwell-definedbioprocesslevelisordersofmagnitudeslower.Thus,itrepresentsasubstantialbottleneckinstrainengineeringaswellasbioprocessdevelopment.Especially,thisholdstrueforthechallengeofidentifyingthebestperformingstrainunderbioreactorprocesscondi-tionsinsteadofartificialscreeningconditions.Allthisdemandsforincreasedexperimentalthroughputinmicrobialphenotypingatwell-definedbioprocessconditions.Tofullyreleasethepotentialofrapidstrainandbioprocesscharac-terisationnotonlythecultivation,butalsoupstreamprocessing,analyticsanddatatreatmentneedtobeaccelerated,inordertoprovideavaluableintegratedtoolformicrobialstrainengineering,syntheticbiology,gene-functionrelationshipaswellasbioreactionengineeringandbioprocessdevelopment.ThisisillustratedtakingmicrobialapplicationexampleswithCorynebacteriumglutamicum,StreptomyceslividansandChlorellavulgarisascasestudies,showinghowdevelopmentsinminiaturizedcultivationtechnologycombinedwithsmartlabautomationanddataprocess-ingfacilitatesmicrobialphenotypingandbioprocessdevelopment.Suchelevatedthroughputinmicrobialcultivationtechnologyandbioprocessengineeringprovideacomprehensivedatabasisandpavesthewaytogenerateimprovedknowledgeaboutmicrobialsystemsintermsofmetabolism,regulationandapplication.

19:30 Poster session & Exhibition & Welcome Reception Foyer of the Institute for Architecture

21:30 End

ChairofBioprocessEngineering


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Metabolic adaptation and population dynamics Chair MarcoOldiges(ForschungszentrumJülich,Jülich,Germany),ChristianReitz(TUBerlin,Berlin,Germany)

9:00 Keynote Lecture: Impacts of large-scale gradients mirrored on metabolic and transcrip-tional regulation (L15)Michael Löffler, Joana Simen, Ralf Takors Institute of Biochemical Engineering (IBVT), University of Stuttgart, Stuttgart, Germany, [email protected]

IndustrialworkhorsessuchasEscherichiacoliareappliedinlargescalebioreactorsandaretherebyexposedtogradientsmirroringimpactsofhydrostaticpressure,insufficientmixingconditionsandspatiallysegregatedcellularactivities.Cellscirculatinginproductionvesselsareexposedtofrequentlychangingmicro-environmentalconditionsthattriggernotonlymetabolicbutalsotranscriptionalcellularresponses.Yet,cellularperformanceinlargescaleisrequestedtomeetexpectationsoflabscalestudiesinordertofulfileconomicdemands(Takors,2012).AtIBVTascale-downdeviceconsistingofastirredtankbioreactorandaplugflowreactorwasinstalledforsimulatinglargescalegradients.Incontrasttothecommonapproachscale-downexperimentswereperformedundersteady-stateconditions.Consequentlyanaccuratelydefinedreferencescenariowassetthatallowedthein-depthelucidationofshort-andlong-termmetabolicandtranscriptionalresponsesofE.coliontheexposedheterogeneities.Morethan600geneswerefoundtobeup-ordown-regulated(threshold1.5fold)duringthefirst110sinPFR.Distinctpatternsofshort-andlong-termadaptationstrategieswerededucedwithrespecttocarbon(C-)andnitrogen(N-)gradients.Theoccur-renceofstablesubpopulationswasobserved.Biologicalmotivatedcriteriaforscale-upwereformulated.TakorsR.2012.Scale-upofmicrobialprocesses:Impacts,toolsandopenquestions.J.Biotechnol.160(1-2):3-9.

9:30 Rapid feast-famine cycles for studying metabolic response and adaptation representa-tive for large-scale (L16)Liang Wu1, Lodewijk de Jonge2, Camilo Suárez Méndez2, Amit Deshmukh1, Sef Heijnen2, Walter van Gulik2, Aljoscha Wahl2, Henk Noorman1,2

1DSM Biotechnology Center, Delft, the Netherlands, [email protected], 2Department of Biotechnology, Delft University of Technology, the Netherlands

Inlarge-scalebioreactors,microorganismsfacecontinuouschangesintheirenvironment.Mostprominentareglu-cose,oxygenandshearratedynamics,butalsopH,temperatureandotherfactorscanplayarole.Inlab-scaleresearch,suchdynamicsconditionsareoftennotapplied,oratbestbasedonqualitativescale-downreasoning.Asaresult,theperformanceofcellsinlarge-scalecanbedisappointingcomparedtoexpectationsraisedfromlabtests.Metabolicmechanismsbehindsuchscale-uplossescanbemultiple,butrelativelylittleisquantitativelyknowntoday.Wewillreportonsomerecentresultsfromstudieswithrepetitiveinputcycles,appliedatlab-scale,whereweinvesti-gatedthemetabolicresponseandadaptationoffungiandyeast.Akeyobservationfromthesefeast-faminestudiesisahighturnoverofintracellularstoragecompounds,associatedwithATPfutilecycles.Alsootherregulatorymechanismsarebecomingmoreclearly,gainingvaluableinsightinscale-upperformanceandscale-downdesignrequirements.

9:50 Metabolic regulation of carbon metabolism: Key element for robustness to bioreactor inhomogeneities (L17)Michael Limberg, Tita Aryani, Mathias Joachim, Wolfgang Wiechert, Marco Oldiges Forschungszentrum Jülich / Institute of Bio- and Geosciences- IBG-1: Biotechnology, Jülich, Germany. [email protected]

Performancelossesduringthescale-upfromlaboratoryintoproductionscaleandtheassociatedincreaseofproduc-tioncosts,imperilsthecompetitivenessofsustainablebiotechnologicalproduction.Onereasonforthesedeficienciesisbasedonthedecreasingmixingqualityandtheresultingglucose,oxygenandpHgradientformationattheindus-trialscale.Butnotallorganismsseemtobenegativelyaffectedbysuchinhomogeneouscultivationenvironmentsinasimilarmanner.EspeciallytheindustrialplatformorganismCorynebacteriumglutamicumprovidesanastonishingdegreeofrobustnesstooscillationsthroughsuchgradients.Thisraisesthequestionwhichkeypathwaysareneces-saryforsuchanabilitytoadaptandhowtheyareregulated.Thispotentiallycanleadtonoveltargetsforthedesignofevenmorerobustproductionstrains.ThisstudypicksupthisquestionbygeneratingabroadrangeofoscillatingpH,oxygenandsubstratesupplycondi-tionsandinvestigatingtheirinfluenceona1,5-diaminopentaneproducingCorynebacteriumglutamicumstrain.Thereforeatwocompartmentscale-downdevicecomposedoftwoconnectedstirredtankreactors(STR-STR)wassetup.Thisdeviceprovidesthepossibilityofmonitoringandcontrollingtheinhomogeneouscultivationparametersineachreactorforinvestigatingthesoleinfluenceofoneparameterorthecombinationofseveralinhomogeneities.DuringthisstudyonecompartmentprovideswellaeratedandpHneutralcultivationconditions.Theadditionalcompartmentwasusedforthegenerationofabroadspectrumofadversegrowthenvironmentswhichmimicdifferentsectionsinlargescalebioreactors.Thesesectionsarecharacterizedbyvaryingoxygenavailability,changingpHandoscillatingsubstratesupplyconditions(excessandlimitation).Dependingontheresidencetime(upto3min)andthecombinationofdisturbingparametersinthiscompartmenttheintermediaryformationofsideproductslikeorganicacidsaswellasgrowthrateweresignificantlyinfluenced.ByinvestigatingtheintracellulartranscriptandproteomelevelsthecompartmentspecificactivationofdifferentstressresponsemechanismsaswellasregulatoryactivationandoverexpressionofNAD+regeneratingpathwaysunderO2-limitationwereobserved.Thismatcheswithoscillatingchangesofmetaboliteconcentrationlevelsintheglycolysisandpentosephosphatepathway.Thestudyisthusthefirststeptowardsaglobalunderstandingofbiologicalrobustnessandpredictingtheirlimits.

Thursday, 7 April 2016

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Programme

10:05 Fermentation process gradient effect on Corynembacterium glutamicum 1945 ΔACT3 PTUF-LDCCOPT producing cadaverine (L18)Williams Olughu1, Chris Rielly1, Chris Hewitt2 and Alvin Nienow3

1Loughborough University, UK, [email protected], 2Aston University, UK, 3University of Birmingham, UK

Typicallyonscale-upofabioprocess,itisuneconomicaltomaintainhighspecificpowerinputsandhencethemixingtimetendstoincreaseandthelocalmasstransfercoefficientsarereduced,withincreasingsizeofthefermenter.Anincreaseinthetimescalerequiredforthefermentersystemtohomogeniseincreasesthelikelihoodofthepresenceofphysicalandchemicalgradients,i.e.thesubstrate,dissolvedoxygenconcentrations,temperatureandpHbecomemorespatiallyinhomogeneousonalargerscale.Ascellscirculatewithinsuchalargereactor,theyrespondtothevariedenvironmentwithinthesystem,whichmayresultinadeclineintheirproductivityorachangeintheformationofmetabolites.Hereatwo-compartmentmodelconsistingofaplugflowreactor(PFR)andastirredtankreactor(STR)wasusedtoinvestigatehowC.glutamicum1945Δact3puf-ldccopt(modifiedtoproducecadaverine)respondstogradients(pH,dissolvedO2,nutrient)andvaryingmixingtimes,inanattempttomimicwhathappensinalarge-scaleindustrialfed-batchprocess.GlucoseandpHcontrollingagentswereintroducedtoeitherthePFRorSTRreactorstocreatechemicalgradientsofsubstrateand/orpHconcentrationsindifferentregionsoftheflow;thePFRsectionhadvolumesof10%and20%oftheSTRsection.Theresultsshowednodeclineinbiomassproductivityinallsimulationsstudied,butinthesimulationwhichwasclosesttomimickingalarge-scalefed-batchenvironment,a26%lossinfinalcadaverinetitrewasrecordedwhenthemeanresidenceinthePFR(PFRƮmean)wassetat60s.Afurtherincreaseincadaverinelossof36%wasobservedwhenthePFRƮmeanwasincreasedto120s.Thissuggeststhatasthecellsfacedanincreasinglystressfulenvironment,moreenergywasredirectedtoproducestabilisingaminoacidstomaintainhomoeostasis,resultinginalossofcadaverineproductivity.Forthefirsttime,adualstainingprotocolinflowcytometrywasdevelopedforC.glutamicumtoprobeviabilityattheindividualcelllevel.Theflowcytometryanalysesshowedthatcellviabilityimprovedinallcasesstudied,above95%attheendoftheprocess,whichsuggeststhatthecellsbecamebetteradaptedtotheirenvironmentasfermentationprogressed.SamplestakenfromthetopsectionofthePFRhadthehighestconcentrationoflactateandatdrycelldensitiesabove25g/LwastwicethetitreintheSTR.Thisindicatesthatmixedacidfermentationoccurredpredominantlyintheoxygen-depletedPFRsectionofthistwo-compartmentmodel.Comparingthisstudywithothersimilarworks(withE.coli,B.subtilisandS.cerevisiae)confirmsthatonscaleupofbiologicalsystems,thedegreeofresponseorlossofproductivityisorganismspecific.

10:20 Performance loss of Corynebacterium glutamicum cultivations under scale-down conditions (L19)Anja Lemoine1 , Michael Limberg2, Marco Oldiges2, Peter Neubauer1, Stefan Junne1

1Technische Universität Berlin, Department of Biotechnology, Chair of Bioprocess Engineering, Berlin, Germany, [email protected], 2Research Centre Jülich, Institute of Bio- and Geosciences, IBG-1: Biotechnology, Jülich, Germany

Scale-downreactorsarefrequentlyusedtostudytheimpactofoscillationsinthelaboratory-scale,astheyoccurinlarge-scalenutrientlimitedfed-batchprocesses.Corynebacteriumglutamicumisawidelyappliedhostorganismofindustrialrelevanceforlarge-scalelysineproduction.Recentexperimentsinmulti-compartmentreactorsindicatedrobustnessofC.glutamicumagainstenvironmentaloscillationsinoxygenandsubstrateavailability[1,2].However,incontrasttoindustrialapplication,mineralsaltmediaismostlyusedintheseexperiences.Inthisstudy,wecultivatedC.glutamicumwithcomplexmediabasedonmolasses,sucroseandcornsteepliquorinathreecompartmentreactor.Theset-upofthescale-downexperimentswerebasedoncomputationalfluidflowstudiesperformedforanindustrial-scalereactorwithfeedadditioninthebottomoftheliquidphase.Threedistinctzonesweremimicked:(i)substratestarvationandoxygenlimitation,(ii)substratelimitationandoxygenexcess,and(iii)substrateexcessandoxygenlimitation[3].Incontrasttopreviousstudies,weobservedceasedgrowthandareducedaccumulationofthemainproductcadaverineaftertenhoursoffeeding,whilethesubstrateuptakeofsucrosewasreducedby20%.Atthesametime,severalorganicacids,amongthemlactateandacetatewereaccumulated,bothwitharateof2mmolL-1h-1.Proteomeanalysisshowedhigherconcentrationlevelsofseveralenzymes,whichindicatesageneralstressresponsetotheinhomogeneousconditions.Insitudeterminationofthecellsizedistributionwithlaser-lightbackreflectionshowedagglomerationunderoscillatorycultivationcondi-tions.Thesefindingsindicatethatthemorphologicalandphysiologicalstatusofcellsarebothdrasticallyinfluencedbytheunfavorablecultivationconditionsastheyappearinthelateproductionphaseatanelevatedcelldensity.

[1]KaessFetal.ProcessinhomogeneityleadstorapidsideproductturnoverincultivationofCorynebacteriumglutamicum.MicrobCellFact,2014.13.[2]OldigesMetal.Scale-downstudyofoscillationsinoxygenandsubstratesupplyforCorynebacteriumglutamicum.NewBiotechnol,2014.31:S50-S50.[3]LemoineAetal.ResponseofCorynebacteriumglutamicumexposedtooscillationscultivationconditionsinatwo-andanovelthreecompartmentscaledownbioreactor.BiotechnolBioengin,2015.116(6):1220-1231.

10:35 Studies on the clavam biosynthetic pathway: a controling step for clavulanic acid production (L20)Howard Ramirez-Malule1, Albeiro Restrepo2, Wilson Cardona3, Stefan Junne4, Peter Neubauer4, Rigoberto Rios-Estepa1

1Grupo de Bioprocesos, Departamento de Ingeniería Química, Universidad de Antioquia UdeA, Medellín, Colombia, [email protected], 2Grupo de Química Física Teórica, Instituto de Química, Universidad de Antioquia UdeA, Medellín, Colombia, 3Grupo de Química de Plantas Colombianas, Instituto de Química, Universidad de Antioquia UdeA, Medellín, Colombia, 4Chair of Bioprocess Engineering, Department of Bio-technology, Technische Universität Berlin, Berlin, Germany.

Clavulanicacid(CA),producedbyStreptomycesclavuligerus(Sc),isasecondarymetabolitewithclinicalinterestduetoitspotentialtoinhibitserineβ-lactamases;nevertheless,lowyieldsarecommonlyobtainedduringcultivationproc-esses.ThesynthesisofCAismediatedbythesocalledclavambiosyntheticpathway,forwhichsomestepsstillremainunclear;thus,improvedknowledgeaboutitsintricacieswouldhelptoeventuallyidentifycontrollingsteps,specificallythestepsinvolvedintheconversionof(3S,5S)-clavaminicacidinto(3R,5R)-clavulanicacid.Inthiswork,achemostatcultivationofScwasconducted;acetateaccumulationoccurredwithinthetimeframeinwhichCAwasaccumulated.Althoughtheacetylgroupispresentinβ-lactamintermediatecompoundssuchasN-acetyl-glycyl-clavaminicacid(NAG-clavam),itisstillunknownhowitisincorporated.Here,weproposeareactionmechanism–basedonacomputa-tionalsimulationapproach–foracetateincorporationduringNAG-clavamformation,inCAbiosynthesis.


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10:50 Sponsor Talk: Single-use online capacitance biomass measurement supporting process scale-up from 10 L to 1000 L in cell culture application (L21)Jochen Scholz, Stuart Tindal, Sebastian RuhlSartorius Stedim Biotech GmbH, Göttingen, Germany, [email protected]

Onlinecapacitancemeasurementhasalreadybecomeawell-establishedPATtoolinbiopharmaceuticalapplicationsutilizingtraditionalmulti-usebioreactorandfermentationequipment.AccordingtoAspenReport2013andReportandSurveyofBiopharmaceuticalManufacturingCapacityandProduction(2015),over40%ofthebiomanufacturingrespondersusethistechnologyforbiomassprocessmonitoringorcontrol.AsthisPATtoolemergesinthemulti-useprocessworld,simultaneouslythereisarapiduptaketosingle-use(SU)cultivationequipmentinprocessdevelopmenttoproductionscale(2000L).Consequently,SUcapacitancesensorsforbiomassmonitoringhavebeendevelopedandpresentedtothecommunitythroughoutthepastyears.Recently,BioPAT®ViaMassSUcapacitancemeasurementbecamecommerciallyavailablewithsignificantuptakeinmarket.Capacityadaptationandbioprocesstransferaretwokeyrequirementsthatpresentsignificantchallenges.Duringthescale-upprocess,differenttypesofbioreactorsareused.Today,SUcultivationequipmentisavailablewithgoodscalabilityfromsmallprocessdevelopmenttoproductionscale.Here,alignedgeometricalaspectratiosandwellcharacterizedprocessparametersarethekeyforgoodcomparability.Inthisscale-upcontext,onlinebiomasscapacitancemeasurementcanplayakeyroletomonitorconsistentcellgrowthrateandcultivationperformance.ThistalkdemonstratestheprocessscalabilityofonlinecapacitancemeasurementinaCHOcellculturefrom10Lto1000L.Additionally,across-systemcomparabilityofconventionalstirredtankandrockingmotioncultivationbioreactorsisshown.Theinvestigationsconcludebyshowingacleargoldenbatchtrajectoryofcellgrowthandproductivityinallscalesandbothagitationsystems.Asverification,theonlinecapacitancemonitoringwascorroboratedbymultipleofflinereferencetechniquesduringthefrequentsamplingoftheprocess.Inconclusion,theSUcapacitancetechnologydisplaysthesamecapabilitiesasthemulti-usetechnologyandenablestheidenticalprocesscontrolpotentialatallscales/systems.


11:40 Keynote Lecture: Exploiting the heterogeneity of biotic and abiotic phase in order to increase bioprocess robustness: Toward new bioreactor design (L22)Frank DelvigneUniversity of Liege, Belgium, [email protected]

Cell-to-cellvariationrelatedtophenotypicdiversificationstrategiesisknowntoinducevariabilityandlackofpredict-abilityinbioprocesses.Actually,thesinglecelltoolboxcomprisesmodellingproceduresandadvancedexperimentaldevicesthatcouldbeusedtodecipherthisimportantphenomenoninprocess-relatedconditions.Modellingproceduresareactuallyfocusedonthestochasticityassociatedwiththeintrinsiccomponentofbiologicalnoise,theothercomponents,i.e.extrinsicandintracellulardiffusionareoftenomitted.However,inbioprocesscondi-tions,extrinsiccomponentofnoiseisofcriticalimportancesinceitisknownthatenvironmentalfluctuationsarelikelytooccurinlarge-scalebioreactors[4].Alternativemodellingproceduresarethusneededinordertoovercomeactuallimitations.Theactualsinglecellexperimentaltoolboxcompriseshighresolutionandhighfrequencyopticaldevices[5],butneedstobeadaptedforthemonitoringofrelevantcultivationdevices,suchasstirredbioreactor[1-3].Thesecondlimitationoftheactualtoolboxreliesonitsdependenceoffluorescentmarkers.Thisconstraintswillbeillustratedthroughtwobiosensingstrategies,andalternativeapproachescomprisingsub-populationculturingwillbeproposed.Alternatively,abetterunderstandingofphenotypicplasticityduringbioprocessescanleadtonewbioreactordesign.

[1]BaertJ.tal.2015.Phenotypicvariabilityinbioprocessingconditionscanbetrackedonthebasisofon-lineflowcytometryandfitstoascalinglaw.BiotechnolJ10,1316–1325.[2]BrognauxA.etal.2013.Alow-cost,multiplexable,automatedflowcytometryprocedureforthecharacterizationofmicrobialstressdynamicsinbioreactors.MicrobCellFact12.[3]DelvigneF.etal.2015.Dynamicsingle-cellanalysisofSaccharomycescerevisiaeunderprocessperturbation:comparisonofdifferentmethodsformonitoringtheintensityofpopulationhet-erogeneity.JChemTechnolBiotechnol90,314–323.[4]DelvigneF,GoffinP.2014.Microbialheterogeneityaffectsbioprocessrobustness:Dynamicsinglecellanalysiscontributetounderstandingmicrobialpopulations.BiotechnolJ9,61–72.[5]DelvigneF.etal.2014.Metabolicvariabilityinbioprocessing:implicationsofmicrobialphenotypicheterogeneity.TrendsBiotechnol32,608–616.

12:10 Multiparameter monitoring of gradients in the liquid phase of fermentation processes (L23)A. Bockisch1 , J. Biering2, S. Sachse3, W. Vonau3, S. Junne1, P. Neubauer1

1Technische Universität Berlin, Department of Biotechnology, Chair of Bioprocess Engineering, Berlin, Germany, [email protected], 2Versuchs- und Lehranstalt für Brauerei in Berlin e.V., Berlin, Germany, 3Kurt-Schwabe-Institut Meinsberg e.V., Meinsberg, Germany

Inlargescalefermentationlikeinthebrewingprocess,processconditionsasthelocalpowerinputandfluidflowmightbeuneven.Thiswouldleadtogradients.Sincetheavailablesensortechnologyisnotdesignedfortheconsiderationofsuchheterogeneities,theknowledgeaboutthemagnitudeofgradientsinbioprocessesisratherlow.Offlinesamplesoronlinedatafromdevicesinstalledatonesinglepositionofthetankarenotrepresentativeforthewholeliquidphase.Computationalfluiddynamicsisoftennotabletodescribegradientformation,asthisdependsonthecells’metabolicactivity,whichevenaltersduringaprocess.Inordertoimproveprocessmonitoringandtoidentifycriticalreactorzonesinafermentationprocess,mobilemulti-parametersensortoolshavebeendevelopedfortheinsituandonlinemonitoringofvariousprocessparameters(pH-value,dO2,dCO2,redoxpotential,conductivity,temperature,pressure)inindustrialbioreactors.Thetime-dependentandspatialdistributioncanbecorrelatedtodatafromofflinestudieslikemetaboliteconcentrationsandlipidcon-tentmeasuredwithHPLCandGCmethodsaswellasthecells’physiologicalstateinvestigatedbyflowcytometry.Whenthisdataisapplied,theimpactofgradientsonthecellularperformancecanbedescribed.

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Programme

12:25 Quantification of spatial heterogeneity in large bioreactors (L24)Anders Nørregaard1, Stuart M. Stocks2, Brian Madsen3 and Krist V. Gernaey1

1Department of Chemical and Biochemical Engineering, Technical University of Denmark, Lyngby, Denmark, [email protected], 2Novozymes A/S, Krogshoejvej 36, Bagsværd, Denmark, 3Novo Nordisk A/S, Hallas Allé, Kalundborg, Denmark

Substrategradientsareknowntoexistinproductionscalestirredtankbioreactorsduetoimperfectmixinganddiffer-encesinthemasstransferrateofoxygen[1,2].Accesstoexperimentalmixingandmasstransferdatafromlargescaleislimited,andtheresultsaredependentonthegeometryofthevesselsandimpellersinuse.Amethodformeasuringoxygengradientsinsituinaproductionscalebioreactorwithminimalalterationofthevesselisproposed.Themethodutilizesdifferenttypesoffiberopticaloxygensensorsthatareintroducedtothebioreactor.Theflexibilityofthesensorsmakesthemethodapplicableintheindustryandfacilitatesthecollectionofexperimentaldataatlargescalewithoutdisturbingtheworkflowattheproductionplant.Thesetuphasbeentestedina1m3pilotscalemixingvesselwithamodeloxygentransferexperiment.Furthermoreithasbeentestedinapilotscalebioreactorduringyeastfermentationinordertoinvestigatethedurabilityofthesensorsduringheatsterilizationcyclesaswellasfoulingfrombiologicalgrowth.Thesetupisabletoquantifydifferencesintheoxygendrivingforceinthemodelsystem,indicatingtheexist-enceofmasstransfergradients.

[1]LarssonGetal.1996.Substrategradientsinbioreactors:Originandconsequences.BioprocessEng14(6),281–289.[2]OosterhuisNM,KossenNW1984.Dissolvedoxygenconcentrationprofilesinaproduction-scalebioreactor.BiotechnolBioeng26,546–550.

12:40 Sponsor Talk: Connected in situ measurement system (L25)Yoann Gasteuil, Céline VinsonsmartINST 213 Rue de Gerland, Lyon, France, [email protected]

Theflowinamixingtanklikeabioreactordependsonseveralparametersasthereactorgeometry,thefluidproperties,theliquidvolume,theimpellergeometryandtherotationspeed.Numericalsimulationshelptooptimizetheproc-esses,buttheyusuallyprovideonlyacrudeapproximationoftherealdynamics.ThesmartINSTsystemallowstruemeasurementofthewayeachcontrolparameterimpactstheflowandthereforethemixingandinternalreactorprocess.ThesystemconsistsofthesmartCENTER,whichisthedatalogger,adataanalyzer,andcommunicationcenterwithsmartPARTs.ThesmartPARTsareembeddedsensors,whichfreelymovearoundthecoreofthefluidinordertomonitorsimultaneously,andinreal-time,theevolutionofproductsandprocesses.AsmartPARTinthereactorcharacterizestheflowandprovidesnewpossibilitiesforprocessoptimization,scale-upandscale-downstudiesandreactoroptimization.ThesmartPARTcharacterizestheflowthankstonewdatalikeagitationrateandhydrodynamicsignature.Theagitationraterepresentstheenergysuppliedinthefluid.ThisrateisobtainedinrealtimethankstoanaccelerometerembeddedinthesmartPART.Thehydrodynamicsignaturecharacterizesflow,providingdetailedunderstandingofit,arisingfromtheagitationratestatisticsconnectedtocontrolparametersettings.Itformsameasurablequantitativedescriptionoftheflow,allowing:

y Flowcomparisony Detectionofzonesofverystrongagitation,whichcandamagecellsy Detectionofdeadareas

Moreover,thewirelessmeasurementdevices,smartPARTs,measurealsoatthemostrelevantplace:atthecoreofthefluidwherethereactionistakingplace.ItcanmeasurepH,turbidity,conductivityandtemperature,withintheprocess.Thisuniquefeaturemakesthesystemextremelyreactiveandreliable.Havingtheabilityofin-linemeasurementsoftheseparametersmakestheinstrumentverysuitedforadvancedprocessdesignusingPATandQbDapproach.Such,itcanalsocontributetobettercontrolledprocesses,beingthebasisofreproducibleproductquality.Duringthepresentation,wewilldescribethetoolstocharacterizeaflowanddetailthebenefitsforprocessdescription,optimizationandcontrol.

12:50 Sponsor Talk: Optical Sensors for bioprocess development and control (L26)Gernot John PreSens Precision Sensing GmbH, Regensburg, Germany, [email protected]

Startingendofthe90schemical-opticalsensorsarenowwidelyusedinbioprocessdevelopmentandproduction.Thesamesensorisavailablefordifferentsizes.ExamplesandlatestdevelopmentsareshownforO2,pHandCO2sensors.

12:55 Lunchbreakwithpostersessionandexhibition


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Chair: KristGernaey(TechnicalUniversityofDenmarkDTU,Lyngby,Denmark),StefanJunne(TUBerlin,Berlin,Germany)

14:00 Keynote Lecture: Bioprocesses under magnification: Learning from single cells about large-scale processes (L27)Alexander Grünberger, Wolfgang Wiechert and Dietrich KohlheyerJülich research Center, Germany, [email protected]

Ourunderstandingoflarge-scalebioprocessesisstilldominatedbyanaveragecellapproach.However,asconfirmedinrecentyears,isogenicbacterialpopulationscanbephysiologicallyheterogeneous.Thispresentationwillgiveashortoverviewonmicrofluidicsingle-cellcultivation(MSCC)anditsapplicationinthefieldofbioprocessdevelop-ment.Microfluidicsingle-cellbioreactorsofferpreciselycontrolledexternalconditionsandallowsingle-cellanalysisatfullspatio-temporalresolution.OntheexampleofCorynebacteriumglutamicumIwilldemonstratehowpicoliterbioreactorscanbeusedtogetadeeperunderstandingofmicrobialbioprocesses.Finally,existingbottlenecksandthechallengeoftheintegrationofMSCCintotheexistingbioprocessdevelopmentworkflowwillbediscussed.

14:30 Development of a perfusion system in a microbioreactor using sedimentation as a scale down tool for ATF perfusion bioreactors (L28)Steffen Kreye, Rainer Stahn, Antje Danielczyk, Steffen GoletzGlycotope GmbH, Berlin, Germany, [email protected]

Withthebiotechindustrymovingtocontinuousbioprocessingandfocusingincreasinglyonproductquality,perfusionbioreactorsarebecomingmoreandmoreimportant.Whereasbatchorfed-batchbioreactorscanbesuccessfullyrep-resentedinasmallscalewithsufficientthroughputforearlystageprocessdevelopment,the1Lbenchtopbioreactorremainsthesmallestscaledownunitforperfusionculture.Here,wedemonstratethedevelopmentofasedimentationbasedperfusionsysteminthesingle-useambrsystemwithgoodcomparabilityto1LATFperfusionbioreactorswhencomparingcellgrowth,viabilityandproductquality,especiallyglycosylation,forGlycoExpressTM(GEX)andCHOcells.Thesystemisbettersuitedforclonescreening,mediaoptimizationandthepredictionofproductqualitycomparedtobatchorchemostatculture.Furthermore,datawillpresentedofGlycotope’sproprietaryGlycoExpressTMtechnologyshowingextremelyconsistentglycosylationofdifferentglycoproteinsbetweenbatches,batchsizes,reactorsizes,proc-esscontrolstrategies,DSPscalesandproductionsite.

14:45 Keynote Lecture: Issues that are relevant when scaling-up hMSC microcarrier production processes in stirred bioreactor (L29)Regine Eibl, Valentin Jossen, Dieter EiblZürich University of Applied Sciences, School of Life Sciences and Facility Management, Institute of Biotechnology, Switzerland, [email protected]

Thepotentialofhumanmesenchymalstemcells(hMSCs)asagentsforallogeneiccelltherapiesisundoubted.InordertoprovidetherequiredamountsofclinicalgradehMSCs,theavailabilityofefficientscale-upproceduresisstringentlyrequired.Promisingresultshavebeenachievedinstirredbioreactorsthatoperatewithmicrocarriersatbenchtop-andpilotscale.UsageofComputationalFluidDynamics(CFD)togetherwithbioengineeringdata(fluidflow,shearstress,microcarrierdistribution)hascontributedtoanincreaseinhMSCexpansionefficiency.However,formationoflargemicrocarrier-cell-aggregatesmayresultinmasstransferlimitationsandinhomogeneitiesintheculturebroth.Inourpresentationfocusingonmicrocarrier-basedstirredbioreactors,scale-upapproachesforhMScsfromadiposetis-sue(hADSCS)andbonemarrow(hBM-MSCS)arepresented.Inaddition,wediscussanownapproachforthe50Lscale.Cellnumbersbetween1*1010and3.6*1010cellswereachievedwithin7days,whilemaintainingstemcellpropertiesandqualities.Finally,thesuperiorityoftheNS1ucriterionandtheimportanceoftime-dependentmicrocarrier-cell-aggregatesizeforrapidandsuccessfulscale-upofhMSCexpansionsaredescribed.

15:15 Sponsor Talk: Evaluation of a stirred small scale single-use bioreactor for microbial applications (L30)Thomas Dreher1,Marco Leupold1, Ute Husemann1, Mwai Ngibuini2, Gerhard Greller1

1Sartorius Stedim Biotech GmbH, D-Göttingen, Germany, [email protected], 2Sartorius Stedim Biotech Ltd., Royston, UK

Forasuccessfulprocesstransfer,aswellasforbioprocessdevelopmentandoptimizationasuitablesmallscaleap-proachisessentialtoreducetimeandcosts.Tofurtherincreasetheefficiencyofthesetransferssingle-usetechnol-ogyisveryattractive.Unfortunately,smallscalesingle-usesystemsoftencannotmimictheconditionsofestablishedbioreactors,intermsofoxygentransferandheatremoval,especially,formicrobialapplications.Consequently,havingsuchamodelinsmallscaleisofspecialinterest.Basedmentionedlimitationsasmallscalesingle-usebioreactor,theambr®250modular(250mLmaximalworkingvol-ume),wasdevelopedandevaluated.Thedesigncriteriaandgeometricalratiosaresimilartoestablishedbioreactors.Duetointegratedpumps,processeswithhighautomationeffortscanberealized.Afurtherfeatureistheintegratedexhaustgasanalyzer,fulfillingallrequirementsforPAT.Tobenchmarktheambr®250modularanindustrialrelevantEscherichiacolihighcelldensityprocesswasperformed.Theachievedresultswerecomparedwithaconventionalglassbioreactor(Uni-Vessel®glass5L),wherethesameprocesswascarriedout.ItwaspossibletoreachapeakcelldensityofOD600=335(drycellweight=120g/L).Noprocessdeviationsoccurredduringthefermen-tationintermsofoxygentransferorheatremoval.Theresultsarecompletelycomparabletotheconventionalglassbioreactor.Overall,theambr®250modularisausefultoolforthetransfer,developmentandoptimizationofindustrialbioprocesses.Furthermore,processdevel-opmentcyclescanbeshortenedandthecostsarereducedincludingahighflexibilityduetotheadvantagesofthesingle‐usetechnology.

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15:25 Scalability of cultivations in the 2-dimensional rocking single-use bioreactor CELL-tainer (L31)Anna Maria Marbà-Ardébol, Peter Neubauer, Stefan Junne Technische Universität Berlin, Institute for Biotechnology, Chair of Bioprocess Engineering, Berlin, Germany, [email protected]

Whilescale-upofstirredSUBscanfollowtraditionalproceduresofconventionalSTRs,thescalingofrockingSUBsismoredemanding.However,theselastoneshavetheadvantagethattheyaremoreflexibleintermsoffillingvolumeduetotheabsenceofstirrers.Moreover,inthecaseofthe2-DrockingCELL-tainer,thepossibilitytoincreasethework-ingvolumeduringthecultivationdoesnotonlyreducecontaminationrisks,butalsoreducescellstressduetotransfersteps.Animportantparameterforthescalabilityofmicrobialaerobiccultivationisalsothegasmasstransfercoefficient(kLa),whichcanbemaintainedfromthemLtothe120Lscale.

15:40 Towards efficient microaerobic processes using engineered Escherichia coli strains (L32)Alvaro R. Lara1, Karim E. Jaén2

1Universidad Autónoma Metropolitana-Cuajimalpa (UAM), Mexico, [email protected], 2Posgrado en Ciencias Naturales e Ingeniería, Universidad Autónoma Metropolitana-Cuajimalpa Ciudad de México, Mexico

Operationalandeconomicconstraintsinlarge-scalebioreactorsoftenresultinlocalorglobalmicroaerobicconditions,whichleadtolessefficientbioprocesses.Escherichiacoli(E.coli)adaptstomicroaerobicitybyactivatingfermenta-tionpathwaysthataccumulateacidicby-products,indetrimentofgrowthrateandbiomassyield(YX/S).Thiswillbeillustratedwithastudyshowingtheeffectsofconstantandoscillatingdissolvedoxygen(DOT)conditionsontheproductionofplasmidDNA(pDNA).Constantmicroaerobicity(3%airsat.)affectedthemetabolism,butnotthepDNAproduction.Incontrast,heterogeneousDOTseverelyimpactedthequalityoftheproducedpDNA.Therefore,themetabolismofE.coliwasmodifiedtobettercopewithconstantmicroaerobicity.Forthatpurpose,genescodingforglobalregulatorslikeCreBandArcA,orforfermentativepathwayswereinactivated.Theperformanceofawild-type(W3110)andengineeredE.colistrainswasevaluatedinbatchculturesatconstantlowDOT.BycombiningthepartialeliminationoffermentationpathwaysandtheexpressionoftheVitreoscillahemoglobin(VHb),a32%decreaseoncar-bonwasteasby-products,24%increaseonYX/Sand13%increaseofgrowthratewereobtained.FluxbalanceanalysisofthebeststrainestimatedmajordifferencesinthefluxesthroughthepentosephosphatepathwayandtricarboxylicacidcycleasconsequenceofVHbpresence.Overall,ourresultsshowthatE.colicanbegeneticallymodifiedtoovercomesomeofthedisadvantagesofmicroaerobicgrowth,whichispotentiallyusefulforbetterbioreactorscale-upandoperation.


16:50 Keynote Lecture: Novel fluorescence-based online monitoring of cell cycle and growth rate for the control of mammalian cell culture (L33)Grischa Fuge, Yaeseong Hong, Uwe Jandt and An-Ping ZengInstitute of Bioprocess and Biosystems Engineering, TU Hamburg, Germany, [email protected]

Areliableandon-linemeasurementofcellgrowthrateishighlydesiredbutstilloutofreachforprocesscontrolinmostcases.Formammaliancellcultureanon-linemonitoringofthecellcycleisofparticularinterestsinceitdeterminesthecellgrowthrateandproductivity.Wehavedevelopedanon-linemonitoringtechniqueofcellcycledistributionofmammaliancellsinbioreactorwhichisbasedontwostablyexpressedFluorescentCellCycleIndicator(FUCCI)proteinsinthecells.AsdemonstratedwithtwoCHOcelllinesthistechniqueiswellsuitableforquantifyingchangesofcellcycleandgrowthratecausedbycultureconditionssuchasnutrientlimitationandthususefulforbioprocesscontrol.

17:20 Multiposition sampling, multiparameter monitoring and electro-optical tracking of microbial activity: New tools for better understanding biogas production (L34)Jörn Beheim-Schwarzbach*1,Erich Kielhorn2, Alexander Angersbach3, Emma Ritzi1, Manja Laqua2, Anna Vaupel2, Boris Habermann1, Peter Neubauer2, Stefan Junne2

1Institute of Agricultural and Urban Ecological Projects, Philippstraße 13,10115 Berlin, [email protected]; 2Depart-ment of Bioprocess Engineering, Technical University of Berlin, Berlin, Germany, [email protected]. 3EloSystems GbR, Berlin, Germany

Gradientsmightappearintheusuallylaminarmixedbiogasfermentation.Therefore,innova-tivesamplingandmonitoringtoolshavebeendevelopedtoachieveabetterunderstandingofgradientsintheliquidphaseinordertoidentifysuitableareasforsampling.Microsensorsforthedeterminationoftemperature,pH-value,redoxpotentialandCO2concentrationhavebeenoptimizedforlong-termuseintheculturebroth.Theyarecombinedtoamulti-parameterprobeandintegratedintoportablelancesystemsthatallowthemovementofsensorsinsidethereactorforthedetectionofgradientsinsitu.Sincetheestimationofthecells’physiologicalstateisdifficult,electro-opticalmonitoringofthepolarizabilityisappliedtogaininformationaboutcellactivity.Itisshownthatthemicrobialactiv-itycorrelateswellwithprocessparameterssuchasthemethaneformationrate.Thepresentedtoolsallowanimprovedmonitoringoftheliquidphaseandmicrobialactivityinindustrialplantsforonsiteprocessoptimi-zation.Theycontributetoabetterunderstandingandconcomitantlytoareductionofoperationalrisks,especiallyataflexiblefeedstockload,orwheneveraprocessrunsunstable.

Programme


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17:45 In-line photon density wave spectroscopy for bioprocess monitoring (L35)Roland Hass, Oliver ReichUniversity of Potsdam, Germany, [email protected]

Highlyconcentratedbioprocessescauseanalyticalchallengesforprocessspectroscopyduetothesignificantamountoflightscatteringgeneratedbythecultivatedbiomaterial.PhotonDensityWave(PDW)Spectroscopycanindepend-entlydetermineabsorptionandscatteringpropertiesofhighlyturbidliquidmaterials.Withthehelpoffiber-opticalprobesitcanbeusedasnovelprocessanalyticaltechnologyforbioprocessmonitoring.BesidesanintroductionintothePDWtechnology,exampleslikethemashingprocessduringbeerproduction,thecultivationofScenedesmusrubescensinnon-conventionalphotobioreactorsandenzymaticmilkcoagulationwillbediscussed.Itisfoundthatparameterslikebiomassgrowthandbiomassdecompositionorthecaseinconversionproc-essinfreshmilk(hydrolysis,aggregation,gelling)arewellmonitored.

[1]VargasRuizSetal.OpticalmonitoringofmilkfatphasetransitionwithinhomogenizedfreshmilkbyPhotonDensityWavespectroscopy,IntDairyJ26,120–126,2012.[2]HassRetal.IndustrialapplicationsofPhotonDensityWavespectroscopyforin-lineparticlesizing,ApplOptics52,1423–1431,2013.Alsoin:VirtualJournalforBiomedicalOptics8.p.1423–1431,2013.[3]HassRetal.OpticalmonitoringofchemicalprocessesinturbidbiogenicliquiddispersionsbyPhotonDensityWavespectroscopy,AnalBioanalChem407,2791–2802,2015.

18:00 Keynote lecture: Stirred, not shaken – Unsolved issues and new problems in scale-down for scale-up (L36)Alvin W. NienowEmeritus Professor of Biochemical Engineering, University of Birmingham, UK, [email protected]

Mostbioprocessesaredevelopedatthebenchscale(mLtoL)andthenscaled-uptostirredbioreactorsofmany10soreven100sofcubicmetresiftheproductoftheprocessofferscommercialpossibilities.Suchscale-uphasoftenbeenbasedonratherarbitraryrulessuchasimpellertipspeed.However,abetterapproachistoconsiderwhatwouldbethedesiredoperatingconditionsatlargescale,especiallytakingintoaccountreasonableoperatingcostsandthendevisesmallscaleexperimentsthatsimulatethelargescaleoperatingconditions-scale-downforscale-up.OneofthemajordifferencesassociatedwithlargescaleoperationistheincreasedmixingtimewhichismanifestedastemporalandspatialinhomogeneitieswithrespecttopH,nutrients,dO2,etc.;andmuchofthisconferenceisdevotedtodevisingsmallscaletestswhichcangiveaninsightintohowthesefluctuationsimpactoncelldensity,productyieldandquality.Otherimportantphysicalfeatureschangewithscale,suchasReynoldsnumberandsuperficialgasvelocity.Theformeroftheseleadstochangesinflowregimesothat,forexample,atverysmallscale(ambr15,whichiscurrentlyverypopularforcloneselectionfortherapeuticproteinproduction),theflowbecomestransitionalratherthanturbulent.Alternatively,withrheologically-complex,veryhighviscositybroths(asfoundwithmycelialfermentations),theflowmaybetransitionalorevenlaminaratthebenchscalebutturbulentornearlysoatthecommercial.Inadditiontotheaboveconsiderationofphysicalbioreactorscale,thereisalsoanimpactofthescaleofthebiologicalentity.Suchascaleisimportantwhenconsideringtheimpactoffluiddynamicstressonproc-essperformance.Acurrentexampleofthisscaleissueisthedifferencebetweenthecultureofanimalcells(~15-18μm)infreesuspensionandofstemcellsonmicrocarriers(~200μm).Thislecturewillgiveabroadbrushairingtoalltheseissues.

19:30 Conference Dinner

Restaurant Zillemarkt, Bleibtreustr. 48a, 10623 Berlin (Charlottenburg)(S Bahnhof Savignyplatz)


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Quality by design in bioprocess developmentChair: RalfTakors(UniversityofStuttgart,Stuttgart,Germany),

ErichKielhorn(TUBerlin,Berlin,Germany)

9:00 Keynote lecture: Next generation mAb production – Continuous manufacturing, PAT, and real time release (L37)Douglas Richardson, David Pollard, John P. HigginsMerck, New Jersey, USA, [email protected]

Recentsuccessinmonoclonalantibodybasedimmuno-oncologytherapeuticssuchasKeytrudahaveinitiatedamanufacturingrevolutionwithintheBiopharmaceuticalindustry.Preparationforawaveofimmuno-modulatoryrecep-tor-focusedclinicalcandidateshighlightstheneedforamoreflexiblemanufacturingandtestingnetwork.Tosupportthisrevolutiondevelopmenteffortsfocusingonsingle-usetechnology,continuousmanufacturing,processanalyticaltechnology,IT/MVDA,andrealtimereleasearebeingpursued.Thispresentationwilldescribetheinvestments,needs,andchallengesinnext-generationmAbproductionandtesting.

9:30 Keynote lecture: High-level protein production with Pichia: Case studies of development path from strain generation over bioreactor (L38)Roland WeisVTU Technology GmbH, Grambach, Austria, [email protected]

ProteinproductionprocessesusingPichiapastorisareincreasinglyrecognizedashighlyefficientbymanyindustries.Especiallyforbiopharmaceuticals,key-to-successistherapidandreliablegenerationofhigh-levelproductionstrainsmatchingproteinquantityandqualityattributes.Aseamlesstransferabilityoflab-scaleresultstomorecontrolledbio-reactorconditionsisofutmostimportance.Theapplicationofaversatilepromoterlibraryforfine-tunedexpressionofboth,thetargetgene(s)aswellasspecificorgeneralauxiliarygene(s)createsadiversityofexpressionstrainsthatcanonlybeanalyzedindepthwithasolidhighthroughputcultivationandscreeningsystem.VTU´sreliable96-wellscalecultivationandscreeningplatformdelivershigh-levelproductioncandidatestrainswhichperformequallywellunderbioreactorconditionssothattime-consumingandlaboriousintermediatestepslikeshakeflaskcultivationinordertoselectspecificstrainsareobsolete.ThecreationofnovelAOX1-promotervariantswithuniqueexpressioncharacteristicsinglycerol-orglucose-fedprocesses,fullydevoidofmethanolnowenablemethanol-freehigh-levelproteinproduction,aswell.Alargenumberofmodelproteinsweresubjectedtothishigh-throughputscreeningplatformfollowedbydirecttransferintobioreactors,oftenyieldingmultig/Llevelsofsecretedrecombinantprotein.Straingenerationandbioreactorproductionproceduresofsomeselectedexamplesincludinggrowthfactors,HSA-fusionproteinsaswellasenzymeswillbedisclosedindetail.

10:00 Keynote lecture: ESETEC 2.0, a new, secretory Escherichia coli strain for the production of complex recombinant proteins at industrial scale (L39)Guido SeidelWacker Biotech, Jena, Germany, [email protected]

SeveralyearsagoWACKERBIOTECHdevelopedauniqueE.colistrainwhichiscapabletosecretecomplexproteinsdi-rectlytothefermentationmedia.Wefocusedourresearchonthenextwaveofbiologicssocalledantibodyfragments(FAbs).OverthelasttwoyearsweredevelopedandengineeredourworkhorseandwehaveintroducedESETEC2.0asanoptimisedplatformfortheproductionofFAbs(mono-,bi-ormultispecific)andothercomplexproteinsassolubleproteinssecretedtothefermentationmedia.Thereforecelldisruptionand/orrefoldingstepsarenotnecessarytoproc-esstheproductofinterest.ESETEC2.0®combinedwithourfermentationprocessisabletoreachfermentationtitersinthe[g/l]-range.Thetalkwilldescribeournewengineeredandoptimisedexpressionsystem,theindustrialfermentationprocessandwewillgivesomeinformationinourintegratedantibodyfragmentpurificationscheme.Wehavedevelopedseveralintegratedprocessesbyusingcelllineselectionwithmediumthroughputscreening,alignedwithfermentationandpurificationbasedonstate-of-the-artanalytics.Ourprocessesarescalabletoindustriallevelstosupplyclinicalprogramswithlargeamountsofhighqualitydrugsubstances.

10:30 Different process strategies for the glutathione overproduction in Saccharomyces cerevisiae (L40)Martin Senz1, Eric Lorenz2, Maximilian Schmacht1

1Research and Teaching Institute for Brewing in Berlin (VLB), Department Bioprocess Engineering and Applied Microbiology, Berlin, Germany, [email protected] Berlin, Germany, 2 Technische Universität Berlin, Chair of Bioprocess Engineering, Berlin, Germany

Glutathione(GSH)playsakeyroleinprotectivemechanismsagainstreactiveoxygenspeciesineukaryoticandprokaryoticorganisms.Duetoitsanti-oxidativecharacteritiswidelyusedinpharmaceutical,cosmeticandfoodindustry.Forinstance,inbakeriesGSHenrichedyeastcellscanbeusedasdoughmodifierhavingtheadvantageofnoneedfordeclarationinthefinalproduct.TherearevariousstrategiesforthefermentationprocessofGSHproduction,themostcommonbeingfed-batchprocedurewiththeyeastSaccharomycescerevisiae.Foranefficientproductionprocess,factorslikemediumcostsandprocessrobustnessatvariousscales,aswellasachievinghighcelldensitywithhighintracellularGSHcontent,areessential.ThistalkprovidesacompilationofdifferentprocessoptimizationstrategiesfortheproductionofGSH-enrichedyeasts.

Programme

Friday, 8 April 2016


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Incontextofqualitybydesign,studiesaddressingthefermentationformat,feedingstrategy,mediumcomposition,precursorforGSHtransformationanditstypeofsupplementation,arepresented.Promisingprocessformatswerevariedconcerningtheappliedyeaststrainandprocessscale.Therefore,thehereshownresultsofferinsightsandprospectsforcurrentandfurtherprocessdevelopmentsinGSHproduction.


11:40 Keynote lecture: Parallel small-scale bioreactors with on-line monitoring to prove the consistency of medium ingredients and pre-cultures and to efficiently assess process characteristics (L41)Jochen BüchsLehrstuhl für Bioverfahrenstechnik, RWTH Aachen, Germany, [email protected]

ManufacturingofpharmaceuticalproductsstrictlyhastocomplywithGMP,PATandQbDrequirements.Therefore,robustandeasytousetoolsandmethodologiesareurgentlyneededtoassessthequalityofcomplexmediumingre-dientsandtoprovetheconsistencyofpre-cultures.Inaddition,moreprocessunderstandingisrequiredtopredicttheimpactofprocessdeviationsonproductquality.Theapplicationofparallelshakenbioreactorssystems(200µLto20mLscale)isintroducedwhichallowafastandefficientassessmentofbioprocesscharacteristics.Manydifferentstartingconditionscanbechosenandcriticalcomponentscanbepulsedintorunningculturestoinvestigatetheinfluenceofthesevariationsonproductquality.

12:10 Towards intensifying design of experiments: An industrial Escherichia coli feasibility study (L42)Moritz von Stosch1, Jan-Martijn Hamelink2, Rui Oliveira3

1Chemical Engineering and Advanced Materials, Newcastle University, UK - [email protected], 2GSK Vaccines, Laval, Canada, 3REQUIMTE/DQ, Faculty of Science and Technology, University Nova de Lisboa, Campus de Caparica, Caparica, Portugal

ThecombinationofDesignofExperiment(DoE)withmultivariatedataanalysisiscommonlyusedinupstreambioproc-essdevelopmenttoevaluatetheimpactofprocessparametersonthefinalproducttiterand,wherepossible,productquality.However,foranincreasingnumberofprocessparameters,i.e.:designfactors,thenumberofexperimentsincreasesexponentially,reachingnumbers,whichareinfeasibletoexecute.Thiscontributionintroducesanovelconcept,referredtoasintensifiedDesignofExperiments(iDoE),i.e.:intra-exper-imentvariationofthedesignfactors.TheiDoEwasexperimentallyimplementedforanE.coliprocessinindustryandtheresultingdatausedtodevelopadynamichybridmodel.ThemodelwasvalidatedandevaluatedagainstdatafromatraditionalstatisticalDesignofExperiments(DoE).Theresultsshowthatthemodel(whichwasdevelopedbasedonthedynamicexperiments)canaccuratelydescribe12experimentsofclassicalDoE.TheresultssuggestthatiDoEincombinationwithdynamicmodelscanbeusedtosignificantlyreducethenumberofexperimentsandtimelines.Inaddition,themethod-ologydeliversdetailedprocessunderstanding,thussupportingtheimplementationofQbDandPATinprocessdevelopment.

12:30 Robot based bioprocess development by automated on-line optimal re-design in parallel bioreactors (L43)Mariano Nicolas Cruz Bournazou1, Tilman Barz2, Florian Glauche1, Peter Neubauer1

1Chair of Bioprocess Engineering, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany, [email protected], 2Energy Department, AIT Austrian Institute of Technology GmbH, Vienna, Austria

Modernbio-industrydemandsafasterandmorerobustprocessdevelopment.Toachievethis,advancesinsensortechnology,miniaturization,parallelization,andautomationneedtobesupportedbypropercomputeraidedtoolswhichallowtheoptimaluseofallexistingresourcesincombination,canpredictthebehaviorofthesystemunderdif-ferentconditions,andcreateapaththatsupportsaconsistentflowofinformationbetweenallstagesandscalesduringdevelopment.Duetotheinherentcomplexityofbiologicalsystems,simpleadaptivemodelsandefficientautomaticexperimentalfacilitiesarerequiredtoallowafastandcheapfittingofthemodeltosmallvariations(e.g.betweensimilarstrains,dif-ferentoperatingconditions,changesinsubstratecomposition,etc.).Wepresentaframeworkfortheonlineoptimalexperimentalre-designofparallelnonlineardynamicprocessesandapplyittotheE.coliW3010fed-batchineightmini-bioreactorsinanautomaticliquidhandlingfacility.Bythis,an“intelligent”automaticfacilitythatcanfitsimplemodelstorealdatainminimaltimeisavailable.Thisworkshowsthatthere-computationoftheoptimalsetuponthefly,whiletheautomatizedexperimentisrunning,ispossible.Asaresultafiftyfoldloweraveragevariationcoefficientontheparameterestimates(4.83%insteadof235.86%)comparedtoasequentialOptimalExperimentalDesignprovesthestrengthsoftheapproach.

12:45 Plenary lecture: From advanced PAT to QbD-compliant quasi-continuous integrated pharmaceutical production (L44)Reiner LuttmannResearch Center of Bioprocess Engineering and Analytical Techniques, Hamburg University of Applied Sciences, Germany, [email protected]

NowadaysdreamsofQbD-compliantpharmaceuticalproductionarebasedonsmallfullcontinuousoperatedinte-gratedprocesseswithaglobalmonitoringofbiologicalvariablesandofcoursewithdisposabletechnology.Thesegoalsarefarawayfromtodayandmoreovercontradictory.Thiscontributiondescribesawaytorealizesomeofthesetaskswithstateoftheartequipmentandprocessstrategies,explainedataprocessdevelopmentofpotentialMalariavaccineproductionwithPichiapastoris.Afirstintensificationofprocessingisbasedonarecursiveexpressionofpharmaceuticalproteinsandcanbeachievedbyatwo-stageupstreamstrategy,includingarepeatedcellbreedingwithasubsequentproteinproductioninaparallelreactor.

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TheseprocessesweredevelopedinafullyautomatedmanneraccompaniedbyintensivePATapplications.Foradvancedon-linemonitor-ingofbiotechnologicalvariablesspectroscopicin-linemethodsusingNIR-,Raman-and2D-fluorescence-spectrometerandtheassociatedMVDAareappliedfordetectionofcelldensityandglycerolbutalsofortotalsecretedproteinsandcellinternalAOX.Moreoveranat-lineapplicationofIMACwithHPLCenablesthediscriminationofsecretedproteinsandthereforeatargetproteinevaluation.AQbD-compliantproductionofpharmaceuticalproteinsneedsariskanalysisandtheidentificationofCriticalProcessParameterswithappropriateDesign-andControl-Spaces.Thereforeafullyautomatedbioreactorplantwasdevelopedforoptimizationofexpressioncondi-tions.ThisplantimagesthesequentialproductionstrategyandfulfillstheprocessdevelopmentboundaryconditionsofICH-Q8.Thedevelopmentofaquasi-continuousoperatedproductionplantstartswiththeproceduralintegrationofallunitoperationsofanoverallprocess.Thisinturnresultsinatwo-stepupstreamandafive-stepdownstreamprocess,consistingofa10lcellbreedinganda30lproteinproductionreactor,acellclarificationviaaseparator,aretentionofcellulardebrisusingamicro-filtration,andaconcentrationofthesecretedproductbyultra-filtrationwithsubsequentbufferexchangethroughdia-filtration,followedbyafinalpurificationusingcolumnchromatography.Thethreemainoperationscellbreeding,proteinproductionandalldownstreamoperationshererunseriallyover3days,butalsoinparal-lelwithaone-day-offset.Withthisstrategy,aquasi-continuousprocessflowispossible.However,aQbD-compliantproductionprocessrequiresaknowledge-basedsupportandmonitoring.Thisincludes,forexample,anadditionaloptimizationofallindividualdownstreamstepsusingDoE,fromwhichtherespectivedesignspacesandcontrolspaceshavealsotobeidentified.Amonitoringofthereproducibilityoftheentirerepeatedprocessflowrequirestheuseofanon-lineevaluationalongwithaGoldenBatchapproach.ForthispurposetheplantisequippedwithcorrespondingsoftwaremodulessuchasSIPAT,SIMCAQ,andSIMCA-online,andalsowithMATLABforenhanceddataprocessingandin-silicoprocessdevelopment.

13:30 Concluding remarksPeter Neubauer Technische Universität Berlin, Institute for Biotechnology, Chair of Bioprocess Engineering – [email protected]

13:45 Endofsymposium

Programme


Pleasenote:

5th BioProScale SymposiumApril2018,Berlin


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Metabolic adaptation and population dynamics

Combination of scale-down approach with 13C-labeling experiments (P01)Pooth Viola1, Nöh K., Wiechert W., Oldiges M.2

1 Institute of Bio- and Geoscience, IBG-1: Biotechnology, [email protected], 2 Forschungszentrum Jülich GmbH, Jülich, Germany

Imperfectmixinginducesgradientformationinindustrialscalebioreactors.Thereisevidencethatmicroorganismsadapttheirmetabolismtosuchinhomogeneousconditions.Nevertheless,bioprocessesmaysufferfromreducedprofitabilityasmicrobialmetabolismanditsperformancearehighlydependentonenvi-ronmentalconditions.Thus,scale-downbioreactorsystemsareusedasapowerfultooltomimicgradientsduringearlylabscaleoptimization.Inordertofollowmetabolicchanges,13C-labelingexperimentsserveasbasisformetabolicfluxanalysis.Underdynamicprocessconditionsonlyveryshortperiodsofmetabolicsteady-state,be-ingapreconditionforacarbonlabelingexperiment,arepresent.So,forshorttransientbiologicalchangesanisotopicallynon-sta-tionarycarbonlabelingexperiment(INST-CLE),whereatransientlabelenrichmentintheintracellularmetabolitepoolscanbeob-served,isthepreferredexperimentalapproach(Nöhetal.2007).Currently,thereareno13Cmetabolomedatafromscale-downbioreactorexperimentsavailableformetabolicfluxanalysis.Espe-ciallyfastsamplingandfurthersampleprocessing,aswellastheanalysisofmetabolomedatafromdynamicscale-downcultiva-tionsremainchallenginganddemandforsinglebioreactorsetupwithperiodicoscillations.Atthispoint,westartedwiththebatchandfed-batchcultivationofEscherichiacoliK12inatwocompart-mentscale-downbioreactor(STR²,stirredtankreactor)underoscillatingoxygensupply.Duetotheoscillationbetweenaerobicandnon-aeratedbioreactorcompartmentsdifferentgrowthphenotypeswereobserved.Theadditionalsubstrateexcessorsubstratedepletionatfed-batchcultivationsrevealedimpactsonby-productmetabolism.TheresultsofthedynamicconditionsoftheSTR²willbecom-paredtoasingleSTRwithperiodicoxygenlimitationduringcultivation.TheconditionsoftheSTR²scale-downbioreactorwillbetransferredtothesingleSTRwithperiodicoscillations,becauseonlythattypeofbioreactorissuitableforINST-CLEsduetotheintegratedrapidsamplingdeviceandsubstratepulseunit(Nöhetal.2007).Afterthecompleteestablishmentoftheworkflow,thesystemistobeappliedundervariouslimitingconditionstarget-ingindustrialproductionstrains.Thereby,acloserlookintothemetabolicstateofthecellunderdynamiccultivationconditionsisfacilitated.

[1]Nöhetal.(2007).JBiotechnol129(2):249-267

Bacterial phase diagrams: Analyzing and predicting cell-to-cell heterogeneity of microbial gene expression (P02)Iska Steffens1, Alexander Grünberger1, Dennis Binder2, Thomas Drepper2, Wolf-gang Wiechert1, Dietrich Kohlheyer1

1 Institute of Bio- and Geosciences, IBG-1: Biotechnology, Systems Biotechno-logy, Forschungszentrum Jülich GmbH (Juelich Research Center), Germany, [email protected], 2 Institute of Molecular Enzyme Technology, Heinrich-Heine-University Düssel-dorf, Forschungszentrum Jülich GmbH (Juelich Research Center), Germany

Phenotypicheterogeneityrepresentsvaryingcellbehaviorofclonalpopulationsandisamajorissueinthecontextofindustrialbioprocessing.Populationheterogeneityisconsideredtoenhance

thefitnessofmicrobialpopulationsinnaturalenvironments,butappearstoberesponsibleforfailure,reducedcontrolandloweryieldinbacterialproductionprocesses.Accordingly,novelanalyticaltoolsandstrategiesneedtobedevelopedtopredictandcontrolbacterialpopulationheterogeneity[1].Inthiscontribution,weintroducetheconceptof“Bacterialphasediagrams”(BPDs).Similartochemicalphasediagrams,BPDscanbeusedtovisualizeandpredictthestate/behaviorofmicrobialgeneexpression(e.g.homogeneousorheteroge-neousgeneexpression)atcertainenvironmentalconditions.Inordertodeterminesingle-cellresponses,novelpicoliterbioreactorcultivationplatformswereused[2,3].AsaproofofprincipleT7-RNA-PolymerasebasedEYFPproductioninE.coliBL21(DE3)[4]wasinvestigated.Thismodelsystemgener-atesanon-gradualandpartiallyinhomogeneousinductionbehavioroverabacterialpopulation.Herewepresentthemanipulationofexpressionbehavior,bymeansofvaryinginductor(hereIPTG)andrepressorconcentrations(hereglucose).Especiallyforlowamountsofinducermolecules,aninhomogeneousall-or-nothingexpressionresponsecanbeobserved.Latestresults,potentialapplicationandongoingresearchdirectionswillbeshown.Ourresultsshowthattheconceptof“bacterialphasedia-grams”hasthepotentialtopredictbacterialbehaviouringene-expression.Thispavesthewayforanimprovedunder-standingofcell-to-cellheterogeneityandcouldleadtonovelstrategiesofcontrollingmicrobialbioprocesses.

[1]GrünbergerAetal.,2014,CurrOpinBiotechnol29:15-23.[2]GrünbergerAetal.,2012,LabonaChip,12(11):2060-2068.[3]GrünbergerAetal.,2015,CytometryA,DOI:10.1002/cyto.a.22779.[4]BinderDetal.,2014,IntegrativeBiol6:755-765.

Chemical evolution of a bacterial proteome (P03)Christian Schipp1,2, Robert Kaml2, Stefan Oehm2, Michael Georg Hösl2, Stefan Junne1, Peter Neubauer1, Nediljko Budisa2

1 Chair of bioprocess engineering, TU Berlin, Berlin, Germany, [email protected], 2 Biocatalysis, Department of chemistry, TU Berlin, Berlin, Germany

Theintroductionofalternativebiochemicalbuildingblockstothestandardizedchemistryofalivingcellremainsanimpor-tantmilestoneinsyntheticbiology.Changingthefundamen-talcomponentsoflivesuchasaminoacidsornucleicacidshasmostlydetrimentaleffectsonbiochemicalprocesses.Therefore,thesystemmustadapttowardsthenewsubstrateinanevolvingmannerrewiringitsgeneticbasistoovercomethestructurallyunfavorablereplacement.Duringalong-termevolutionexperiment(LTEE),atryptophanauxotrophE.colistrainwasforcedbyaserial-transferregimeinatryptophan(Trp)limitingenvironmenttoadapttowardsthenon-canonicalanalogL-beta-(thieno[3,2-b]pyrrolyl)-alanine([3,2]Tpa).Genomicandproteomicanalysisrevealeddeepchangesongeneticandtranslationallevel.SomemutationsobviouslyledtotheshutdownofE.coli’sstringentresponse,whichisawell-knownartifactofLTEE.Nevertheless,thereplacementofTrpto[3,2]Tpainmorethan20.000UGGcodonsyieldedinrobustgrowingdescendantsinminimalglucosemediumonlycontainingthesurrogate[3,2]Tpaasatryptophansource.Thisstudyillustratesanapproachfortheconstructionofsyntheticcellswithalternativebiochemicalbuildingblocksexploitingtheflexibilityofthegeneticcode.Furthermore,theexperimentwilldeepentheunderstandingtheevolvabilityoflivingcellsinrespecttonon-canonicalsubstrates.

Poster abstracts

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Let there be light – Quantitative analysis of phototrophic cyanobacteria in controlled microfluidic environments (P04)Christian Dusny, Kristin Lindstaedt, Andreas Schmid

Department Solar Materials, Helmholtz Centre for Environmental Re-search GmbH – UFZ, Leipzig, Germany, [email protected]

Recentbiotechnologyincreasinglyfocussesonharness-ingthephotosyntheticmachineryofcyanobacteriafortheproductionofenergycarriersandchemicalbuildingblocks[1].However,inordertofacilitateindustrialprocessesbasedonbioartificialphotosynthesis,thecyanobacterialcellasaliving,light-drivencatalystwithitscomplexmolecularmechanismsandcellularfunctionshastobeunderstoodindetailbymeansofsinglecellanalysis[2].Mostsurprisinglyanddespiteofthehugepotential,singlecellstudiesdealingwiththeanalysisofphototrophicmicroorganismsarescarceandstilllimitedtofeasibilitystudieswithoutproperphysi-ologicalcharacterization.Inourcontributionwedemonstratehowmicrofluidictechnologiescanbeappliedtostudysinglephototrophicorganismsandanswerquestionsconcerningtheirbiotechnologicalapplicationatthescaleofasinglecell.

[1]AngermayrSAetal.2009.Energybiotechnologywithcyanobacte-ria.CurrOpinBiotechnol20:257-263.

[2]OliverJW,AtsumiS.2014.Metabolicdesignforcyanobacterialchemicalsynthesis.PhotosynthRes120:249-261.

LEANPROT: Systems biology platform for the creation of lean-proteome Escherichia coli strains (P05)Sebastian Hans1, Nicolas Cruz Bournazou1, Egils Stalidzans2, Trygve Brautaset3, Klaus Bensch4, Raivo Vilu5, Peter Neubauer1

1 Technische Universität Berlin, Institute of Biotechnology, Chair of Bio-process Engineering, Berlin, Germany, 2 Latvia University of Agriculture, Faculty of Information Technology, Biosystems group, Jelgava, Latvia, 3 Norwegian University of Science and Technology, Department of Biotechnology, Trondheim, Norway, 4 biotechrabbit GmbH, Germany, 5 Competence Center of Food and Fermentation Technologies, Tallinn, Estonia

Therapiddevelopmentofgeneticengineeringand-omicstechnologiestogetherwithnewmetabolicmodelingtoolsallowsthecreationofrecombinantchassiswithsuperiorchar-acteristics.However,thecurrentstrainsstillcontainanumberofsynthesisandregulatorypathwayswhichhaveevolvedinnature,butwhicharenotneededinabioprocessandconsumeenergyandmetabolicresourcesandconsequentlylowerthefinalproductionyields.ThereforeintheLEANPROTERA-SysAPPprojectwecon-sidertodevelopleanproteomestrainsbasedonasystemsbiology-basedstrainengineeringapproach.Theproteomeisoptimizedbyiterativecyclesofmodelingandbiologicalexperimentswhichareperformedbyparallelconsidera-tionofdifferentparametersasDesignofExperiments(DoE)underwell-definedprocessrelatedenvironmentalconditions.Ourapproachincludestheuseofasinglecellmodel,whichincludesthesequestrationofmetabolitesandenergyintothedifferentmetabolicroutesunderconsiderationofthecellcycleandcellvolumedynamics(basedonamodelbyCooperHelmstetterDonachie).Webelievethatthisnewwayofap-proachingtheproblemofproteinproductiontogetherwithstateoftheartchemicalengineeringoptimizationprinciplescouldopenanewavenueforthecreationofsuperiorcellfactories.

Bioprocess modelling

Computational analysis of hydrodynamics and light distribution in photo-bioreactors for algae biomass production (P06)Varun Loomba, Gregor Huber, Eric von Lieres

Forschungszentrum Juelich, Jülich, Germany, [email protected]

Microalgaecanbedirectlyusedinhealthfoodorasbio-filtersforwastewatertreatment.Theyalsohavenumerouscommercialapplicationsincosmetics,aquacultureandchemicalindustryasasourceofhighlyvaluablemolecules,e.g.,polyunsaturatedfattyacids[1].Moreover,theyareincreasinglyrecognizedasapromis-ingsourceforbiodieselproduction[2].Torealizethefullpotentialofmicroalgae,optimaloperatingconditionsfortheircultivationinphoto-bioreactors(PBR)needtobeidentifiedinordertomaxi-mizeproductivity,lipidcontent,andefficiencyofphotosynthesis.ThemostimportantparametersaffectingPBRperformancearereactorshape,lightintensitydistribution,algaegrowthandothermetabolicproperties.ThepresentedstudyaimsatoptimizingtheseparametersusingComputationalFluidDynamics(CFD)simulationswiththeCOM-SOLMultiphysicssoftware.Specifically,flatpanelphoto-bioreac-torswithturbulentmixingduetoairspargingandone-sidedlight-ingarestudied.First,flowprofilesofbothliquidandgasphasesarecomputedusingtheEuler-Eulerapproachforanalyzingtheairsparginganddetectingpotentialdeadzones.Then,lightinten-sitydistributionsarecalculatedinsidedifferentPBRtypes,basedonabsorptionandlightscatteringbyalgaeandgasbubbles.Subsequently,thepathsofindividualalgaearetraced,andtheenvironmentalconditionstheyareexposedto,arerecordedovertime,inparticularaerationandlightintensity.Resultsoftheabovedescribedsimulationstageswillbepresentedanddiscussed.

[1]Spolaoreetal.Commercialapplicationsofmicroalgae,JBiosciBioeng101(2006):87-96.

[2]Bitogetal.Applicationofcomputationalfluiddynamicsformodelinganddesigningphotobioreactorsformicroalgaeproduction:Areview,ComputElectronAgr76(2011):131-147.

Nutrient distribution in perfusion based picoliter bioreactors (P07)Christoph Westerwalbesloh, Alexander Grünberger, Birgit Stute, Sophie Weber, Wolfgang Wiechert, Dietrich Kohlheyer and Eric von Lieres

Forschungszentrum Jülich GmbH, Jülich, Germany, [email protected]

Cell-to-cellheterogeneityhasbeenfoundtohavethepotentialtosignificantlyinfluencebiotechnologicalprocesses.Microfluidicde-vicesoffernewopportunitiesforresearchandoptimizationofmi-croorganismsonsingle-celllevel,whichcangivefurtherinsightsinunderlyingcausesandresultingeffectsofheterogeneity[1].Arecentlydevelopedmicrofluidicplatformallowscultivationofsev-eralhundredmicrocolonies,eachconsistingofuptoseveralhun-dredcells,inseparatecultivationchambersononedevice.Data,e.g.growthrates,canbegeneratedbyautomatedtime-lapsemicroscopywithspatiotemporalresolutiononsingle-celllevel[2].Especiallyperfusionbasedcultivationchambertypesareexpectedtooffergoodenvironmentalcontrolwithregardtothecultivationmedium,i.e.constantsubstrateandlowproductandbyproductconcentration.Duetolength-scalesinthemicrometerrangeandcorrespondingsmallvolumesofseveralpicolitersperreactionchamberitiscurrentlyimpossibletodirectlymeasurethosecon-centrationsandtheirgradientswithinthedevices.Thereforethemicrofluidicdeviceemployedin[2]wasmodeledandsimulatedusingCOMSOLMultiphysics.Theliquidvelocityfieldandthemasstransferwithinthesupplychannelsandcultivationchamberswerecalculatedtogaininsightinthespatialdistributionofsuppliednu-

Poster abstracts


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trientsandmetabolicproductssecretedbythecultivatedbacteria.Thegoalwastoidentifypotentialsubstratelimitationsorproductaccumulationswithinthecultivationdeviceandtheresultinginhomogeneityexperiencedbysinglecells.Themetabolicuptakeandproductionrates,colonysize,andgrowthmediumcomposi-tionwerevariedcoveringawiderangeofoperatingconditions[3].Thislaysthefoundationforfurtherstudiesandtheoptimizationofexistingpicoliterbioreactorsystems.

[1]AGrünbergeretal.Single-cellmicrofluidics:opportunityforbioprocessdevelopment.CurrOpinBiotechnol29,15–23,2014.

[2]AGrünbergeretal.Spatiotemporalmicrobialsingle-cellanalysisusingahigh-throughputmicrofluidicscultivationplatform.CytomPartA87(12),1101–1115,2015.

[3]CWesterwalbeslohetal.ModelingandCFDsimulationofnutrientdistributioninpicoliterbioreactorsforbacterialgrowthstudiesonsingle-celllevel.LabChip,15,4177–4186,2015.

Combination of modeling and environmental life cycle assessment approach for demand driven biogas production (P08)Funda Cansu Ertem, Juan A. Arzate, Peter Neubauer, Stefan Junne, M. Nicolas Cruz-Bournazou

Technische Universität Berlin, Institute for Biotechnology, Chair of Bioprocess Engineering, Berlin, Germany, [email protected]

Asuitablestrategytoeventhefluctuatingenergyprovisionbysomerenewableenergysourcesisaflexibleoperationofbiogasplants.Nevertheless,thisprocessneedstoremainstable,profit-ableandenvironmentallyfriendly.Inordertoidentifyreasonableandsuitableoperationpoints,processmodellingiscombinedwithanLCA.Inthisproof-of-conceptstudy,abiogasprocesswassimulated,whichflexiblygeneratesenergyfromtheco-digestionofmaize,grassandcattlemanure,whileemittingthelowestamountofgreenhousegasemissions.TheprogramwasstructuredinMatlabtosimulateanybiogasprocessbasedontheAMOCOmodelandcombinedwithequationsthatprovideclimatechange,acidificationandeutrophicationpotentialsofthewholeproduc-tionsystembasedonReCiPemidpointv.1.06methodology.Thesimulationwasoptimizedbasedonrealdataofbiogasplantsandexistingliteratureresearch.

Effect of light modeling on the prediction of microalgae growth (P09)Christopher McHardy, Cornelia Rauh

Technische Universität Berlin, Chair of Food Biotechnology and Food Process Engineering, Berlin, Germany, [email protected]

Thepredictionofgrowthofmicroalgaerequiresaccuratemod-elingoflightpropagationinphotobioreactors.AlthoughLambertsLawprovidesasimplewaytoapproximatelightattenuation,itdoesnotcaptureanyscatteringeffectsand,inconsequence,growthpredictionsdeviatefromexperimentalresults.Toreachhigherlevelofaccuracy,numericalsimulationoflightdistributioninphotobioreactorsmayserveasanalternative.Inthepresentcontribution,theeffectsoflightmodelingonpredictionsofmicro-algaegrowthareinvestigatedbynumericalsimulations.

Modelling dissolved oxygen and glucose gradients in pulse-based fed batch culture of Escherichia coli (P10)Emmanuel Anane, Christian Reitz, Franziska Vera Ebert, Mariano Nicolas Cruz, Stefan Junne, Peter Neubauer

Chair of Bioprocess Engineering, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany, [email protected]

Localadditionofsubstrateandair,coupledwithinsufficientmixinginindustrialscalefed-batchculturesresultintheoc-currenceofzonesthroughoutthebioreactor.E.colicellsthatareintermittentlyexposedtothesezonesreactmetabolicallybyproducingacetate[1],andphysiologicallybyaccumulat-ingnon-canonicalaminoacids(NCAA)inbothcellularandrecombinantproteins[2].Inthecurrentwork,wedevelopadynamicalmechanisticmodeltopredicttheresponseofanE.colistrainforrecombinantproteinproductiontoperturba-tionsinglucosefeedandoxygentransferinfed-batchculture.Themodelisvalidatedwithexperimentaldatafrompulse-basedfed-batchculturesofE.coli.Thesimulationsshowagoodagreementwiththeexperimentaldataandprovideaframeworkformodel-basedprocessmonitoringandoptimi-sationoffermentationstrategies.

[1]ESWernerssonetal.Substrategradientsinbioreactors:originandconsequences.14,1996.

[2]MBiermannetal.Traceelementassociatedreductionofnorleu-cineandnorvalineaccumulationduringoxygenlimitationinarecombinantEscherichiacolifermentation.Microb.CellFact.,vol.12,p.116,2013.

Bioprocess scale-up and scale-down

How to successfully scale up a microcarrier ba-sed expansion process of human mesenchymal stem cells from adipose tissue (P11)Valentin Jossen1, Cedric Schirmer1,2, Regine Eibl1, Matthias Kraume2, Ralf Pörtner3, Dieter Eib1

1 Zurich University of Applied Sciences, Institute of Chemistry and Biotechnology, Switzerland, [email protected], 2 Berlin University of Technology (TU Berlin), Institute of Process Engineering, Germany, 3 Hamburg University of Technology (TUHH), Institute of Bioprocess and Biosystems Engineering, Germany

Humanmesenchymalstemcellsisolatedfromadiposetissue(hASCs)areofincreasinginterestforclinicalapplications.Duetotheirgoodclinicalefficacyandtolerability,theycanbeusedforallogeneictherapies(e.g.acutemyocardialinfarc-tion).However,therequirednumberoftherapeuticallyactivehASCsforallogenicapplicationsisintheregionofatrillioncellsperyear.Therefore,alternativesto2-dimensionalplanarcultivationsystems,whicharetypicallyusedtopropagatethem,andsuitablescaleupstrategiesfortheexpansionprocedureareurgentlyrequired.Promisingalternativestotraditionalsystemsarestirredandwave-mixedsingle-usebioreactors.Inthesedynamicallymixedsystems,therequiredgrowthsurfacefortheadherentcellsisprovidedbymicrocar-riers(MCs)thataresuspendedinthebioreactor.However,thisraisesthequestionofhowaMC-basedexpansionprocessforhASCscanbescaledup.AsuccessfulscaleupapproachforaMC-basedproductionprocessispresentedforadiposederivedstromal/stemcellsinastirred(35L;pilotscale)andawave-mixed(2L;benchtopscale)single-usebioreactor.Todeterminetheoptimumme-dium-microcarrier-combination,extensivescreeningstudiesbasedontheexperimentaldesignwereperformedinspinnerflasks.Inaddition,experimentalandnumericalinvestigationswithaspinnerflask,aBIOSTATSTR50LandaBIOSTATCulti-BagRM2Lwerealsocarriedout.Thisallowedtheoptimum

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cultivationconditionstobepredicted,whilekeepingtheshearstresslevelslow.Thescaleupapproachresultedinpeakcelldensitiesof2.9x105hASCsmL-1(1.015x1010hASCs)intheBIOSTATSTR50Landin1.9x105hASCmL-1(2.85x108hASCs)intheBIOSTATCultiBagRM2L(proof-of-conceptculti-vation).Duringthecultivationthecellsmaintainedtheirstemcellpropertiesandsurfacemarkers.

Scalability of multiple strain co-fermentation (P12)Lene Dybdal Christiansen, David Kold

Chr. Hansen, Hørsholm, Denmark, [email protected]

Duringscale-upoffermentationprocesses,scaledependentchangescanhaveasignificantimpactontheprocess.Varia-tioninprocessparametersuchaspHandtemperature,whichcanbeeasilyinvestigatedinlaboratoryscale,mayshowtobeinsignificant,comparedtochangesinmediapreparationorprocesstime.Scale-upoffermentationprocessesweremultiplestrainsareco-fermentedcanbeespeciallychallenging,aseachstraincanhavedifferentsensibilitytoprocesschanges.Thismayleadtoachangedstrainbalanceandtheinteractionsbetweenthedifferentstrainsmaybeaffected.Identifyingthesignificantparameterstocontrolandinvestigatemaythereforprovecomplex.Inthisstudy,theeffectofchangesinthemediapreparationbetweenlaboratoryandproductionscalefermentationsofamultiplestrainlacticacidbacteriacultureisinvestigated.Furthermore,itisdemonstrated,howthescaleofthereactoritselfmayimpacttheculturecomposition,andwhichchal-lengescanarisewhentransferringmodelsfromlaboratoryscaletoanindustrialsetting.

Impact of dissolved oxygen tension on plasmid DNA production by Escherichia coli (P13)Karim Jaen1, Juan C. Sigala2, Roberto Olivares2, Alvaro R. Lara2

1 Universidad Autónoma Metropolitana-Cuajimalpa, Mexico City, Mexico, [email protected], Posgrado en Ciencias Naturales e Ingeniería; 2 Departamento de Procesos y Tecnología

DespitethegrowingpotentialofplasmidDNA(pDNA)asatherapeuticagent,littleisknownabouttheimpactoflarge-scaleconditionsonpDNAproduction.Thereareafewreportsontheeffectofdissolvedoxygentension(DOT)heterogenei-tiesonpDNAproduction,however,theresultsarenotclear.Inthepresentcontribution,weinvestigatedtheeffectofconstantaerobic(DOT=30%airsat.),microaerobic(DOT=3%)andoscillatoryoxygenavailabilityontheproductionofapDNAvaccinemodel(pVAX1)byE.coliW3110recA-in1Lbatchbioreactorcultures.DOToscillationswerecreatedbystirrerspeedshiftsfrom100to1200RPMevery10minthatcausedDOToscillationswithdecreasingamplitudefrom0-90%airsat.Oscillatoryconditionsdidnotaffectthespecificgrowthrate,butresultedinadecreaseofbiomassyieldonglucoseofca.24%,ahigheraccumulationoffermentationby-productsanda30%increaseoftherespiratoryquotient(RQ),comparedtoconstantmicroaerobicconditions.ThiscanbeattributedtothelowerO2uptakeundersuchconditions.DOToscillationscausedperiodicchangesoftherespira-toryactivity,asevidencedbyoff-gasanalyses.Interestingly,temperatureoscillationswerealsoobservedinresponsetoDOTchanges.pDNAyield,titerandproductivitywerenotsignificantlyinfluencedbythedifferentDOTconditions.Un-derconstantaerobicandmicroaerobicconditions,thepDNAsupercoiledfraction(SCF)remainedcloseto90%throughoutthecultures,andtheDNAfidelity(analyzedbyIlluminase-

quencingtechnology)wascloseto100%.Incontrast,underDOToscillations,SCFdroppedto82%andshowedmoreheterogeneityasrevealedbyagarosegelandchipmicrofluidicselectrophoresis.Overall,ourresultsshowthatdespitecellularbehaviorisstronglyinfluencedbytheDOT,pDNAproductionisaffectedmainlyonitsqualityattribute(SCF),ratherthaninproductivity,byDOThetero-geneities.

Increasing efficiency of baculovirus-based r-protein productions in orbitally shaken single-use bioreactors (P14)Ina Dittler1, Britta Badertscher1, Katharina Blaschczok1, Tim Bürgin2, Tibor Anderlei2, Peter Neubauer3, Dieter Eibl1 and Regine Eibl1

1 Zurich University of Applied Sciences, Wädenswil, Switzerland, [email protected], 2 Kühner AG, Birsfelden, Switzerland, 3 TU Berlin, Department of Biotechnology, Berlin, Germany

Insectcellsusedinconjunctionwiththebaculovirus-expressionvectorsystemprovideasuitableproductionsystemforthede-velopmentandmanufacturingofrecombinantproteinproductssuchasvirus-likeparticlevaccines.Inordertospeedupvac-cinedevelopmentandproduction,andtoreactmorequicklytopandemicscenarios,newstrategiesforefficientbaculovirus-basedproductiontechnologiesneedtobeinvestigated.Amongtheseistheimplementationofsingle-use(SU)bioreactors,whichhavebeenprovenascost-effective(48%lesscapitalcost),andready-to-usesystemswithoutthetime-intensivecleaningandsterilizationprocessesrequiredbystainlesssteelbioreactors.Furthertime-andcost-savingscanbeachievedbyusingthetiterlessinfected-cellpreservationandscale-up(TIPS)methodasanalternativetotheclassical2-phaseproductionprocess.TheTIPSmethodenablesalarge-scaleproteinexpressionin100Lbioreactorswithcryopre-served,baculovirus-infectedinsectcells(SpodopterafrugiperdaSf9)inonescale-upstep.Itsmajoradvantagesincludetheelimi-nationoftiteringvirusstocksupernatants,andthestablestorageofhighlyconcentratedrecombinantbaculovirusesenclosedintheinsectcells.Inthisstudy,acombinationoftheTIPSmethodwithSUbioreac-torswasinvestigatedwiththeaimofincreasingtheefficiencyofbaculovirus-basedproductionprocessesinorbitallyshakenbioreactorsfrommL-toL-scale.AftersuccessfulestablishmentoftheTIPSmethodbyinfectingSf9cellswiththerecombinantbacu-lovirusAutographacalifornicamulticapsidenucleopolyhedrovirus(AcMNPV),amodelprotein,thesecretedalkalinephosphatase(rSEAP),wasexpressedin125mLshakeflasks.Subsequently,theproductionprocesswastransferredto3LshakeflasksandtotheorbitallyshakenSUbioreactorSB10-X.TherSEAPactivitiesobtainedwerecomparableinallthreeorbitallyshakensystems(152–173UmL-1).Infact,theywereupto40%higherthanthosedescribedforstirredandwave-mixedSUbioreactorsusingtheclassical2-phaseproductionprocess.

Performance loss of Corynebacterium glutamicum cultivations under scale-down conditions (P15)Anja Lemoine1 , Michael Limberg2, Marco Oldiges2, Peter Neubauer1, Stefan Junne1

1 Technische Universität Berlin, Department of Biotechnology, Chair of Biopro-cess Engineering, D-13355 Berlin, Germany, [email protected], 2 Research Centre Jülich, Institute of Bio- and Geosciences, IBG-1: Biotechnology, 52425 Jülich

SeeL19

Poster abstracts


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Simulating large scale conditions in a scale-down bi-oreactor: Impacts on cell physiology of recombinant Escherichia coli (P16)Christian Reitz, Ping Lu, Stefan Junne, Peter Neubauer

Technische Universität Berlin, Department of Biotechnology, Chair of Biopro-cess Engineering, Berlin, Germany, [email protected]

Escherichiacoliisanimportantproductionstrainforindustrialandpharmaceuticalrecombinantproteinproductioninlarge-scalefed-batchbioprocesses.Limitedmixingcapacitiesleadtothedevelopmentofgradientsconcerningthenutrientandoxygenavailabilitywhenacriticallevelinscaleandcelldensityisreached.E.colirespondstohighsubstrateconcentrationsincombinationwithoxygenlimitationwithanincreasedproductionofmetabo-litesbasedonpyruvateviaoverflowmetabolismandmixed-acidfermentation.Furthermore,anincreasedfluxintothebranched-chainaminoacidpathwaycanbeseenleadingtoanincreasedproductionalsoincludingnon-canonicalaminoacidslikenorva-line[].Non-canonicalaminoacidscanbeincorporatedintopro-teins,e.g.norvalineasasubstituteforleucine.Also,methionineisknowntobeexchangedbynorleucine[].Theseexchangesmayhaveeffectsontheproductionandqualityofarecombinantpro-tein.Tobetterunderstandtheimpactofheterogeneousoscillatingconditionsonthephysiologicalstateofthecellsandqualityoftheproducedrecombinantprotein,experimentsinascale-downmulti-compartmentreactorsystembasedon[]areperformedandinadditioncomparedtoglucose-pulsed-basedstirredtankreactorcultivations.Metabolomeanalysisfromsamplesofcultivationsofarecom-binantE.coliK-12W3110strainoverexpressingaleucine-richrecombinantproteinshowsanaccumulationofmetabolitesderiv-ingfrompyruvate(acetateandlactate)aswellasanincreasedproductionofnon-canonicalbranched-chainaminoacidsunderscale-downconditions.Furthermore,weseeincreasedincorpo-rationofthenon-canonicalaminoacidsnorvaline,norleucineandβ-methyl-norleucinewhenoscillatingcultivationconditionsareapplied,influencingthequalityoftherecombinantproduct.Theseresultshaveimplicationsforthedesignofmolecularandengineeringapproachesfordevelopingrobustprocessesaimingtowardsahighqualityofthedesiredproteinproduct.

[1]JSoinietal.AccumulationofaminoacidsderivingfrompyruvateinEscherichiacoliW3110duringfed-batchcultivationinatwo-compart-mentscale-downbioreactor“,AdvBiosciBiotechnol2(5)2011:336–39.

[2]ZIRandhawaetal.IncorporationofNorleucineatMethioninePositionsinRecombinantHumanMacrophageColonyStimulatingFactor(M-CSF,4-153)ExpressedinEscherichiacoli:StructuralAnalysis“,Biochem33(14):4352–62.

[3]SJunneetal.ATwo-CompartmentBioreactorSystemMadeofCom-mercialPartsforBioprocessScale-downStudies:ImpactofOscillationsonBacillusSubtilisFed-BatchCultivations.BiotechnolJ6(8):1009–17.

Bioprocess development

Tools enabling Real-Time Raman identification of biofilm fouling (P17)Martin Kögler1, Bifeng Zhang2, Li Cui2, Yunjie Shi3, Marjo Yliperttula1, Timo Laaksonen1, Tapani Viitala1, Kaisong Zhang2

1 Division of Biopharmaceutics and Pharmacokinetics, University of Helsin-ki, Helsinki, Finland, [email protected], 2 Key Laboratory of Urban Pollutant Conversion, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, P. R. China, 3 DSXDSYS-R&D-Design and Manufacturing Company, Xiamen, P. R. China

Thisposterdescribesaproof-of-conceptforacompactreal-timesurface-enhancedRamanspectroscopy(SERS)-onlinesensingapproachfordetectionofbacteriaindrinkingwatermembranefiltration.Inthisstudywecreatedacustom-designedflow-cellthatmimicsacross-flowmembranefiltrationsystem.Thisenablesonetomeasurechangesinsurface-foulants,suchasBrevundi-

monasdimiuta(BD)bacteriaandadenine,underconditionsthataresimilartoconventionalmembranefiltrationsystems.FormeasurementsweusedacommonportableRaman-spectrometerwithalaboratoryRaman-probemountedintoaspecificallydesignedflow-cellwithanopticalwindowandthelaser-beamfocusedontospeciallydevelopedgoldnanoparticle(AuNP)SERS-sensingareaonfilter-membranes.Thisallowedreal-timedetectionoflowconcentrationsofsurface-foulantsimmediatelyafterinoculationintoanultra-purewaterreservoirunderpressure-drivenfiltrationcondi-tionswithalimitofdetection(LOD)ataround10-7M.Wecomparedtheseonlineresultswithstaticmeasurementsfromanoffline,sample-takingapproach,usingaconfocalRaman-laboratory-microscope.Thedevelopedgoldnanoparticle(AuNP)SERS-sensing-areaonthemembranesprovedtobestableoveralongperiodofsurfacefoulinginvestigationsandtosuppressthestronginterferingRaman-signaloriginatingfromthecompositionlayerofmostfiltrationmembranes.

Bioprocess optimization using culture transfer in multi-reactor system (P18)Sten Erm, Kaarel Adamberg, Raivo Vilu

Competence Center of Food and Fermentation Technologies, Tallinn, Estonia, [email protected]

Thephysiologyofproducingorganismisknowntoaffecttheoutcomeofproductionprocesses,thusphysiologymustbewellcontrolledinoptimizationstudies.Continuouscultivationfixesthephysiologicalstateoftheorganism,butistimeandmaterialconsuming.Wehavedevelopedasystemcombiningthehighthroughputofbatchcultivationswiththecontrolledenvironmentofcontinuousones.Inthissystemmicroorgan-ismsarecultivatedinonebioreactorfollowedbyculturedistributioninanetworkofreceiverbioreactorsandcontinu-ationofindependentsteadystateexperimentstherein-ef-fectivelythephysiologicalstateofthecultureismultiplied.Inadditiontostartingexperimentsfrompreciselyestablishedphysiologythenovelsystemisverysuitablefortheproduc-tionofrecombinantproteinsorotherre-searchandpro-ductionapplicationswhichentailphysiologydisruption,asgrowthandproductionunitsaredecoupled.Incurrentstudytheefficiencyanalysisofthesystemispresented,proofofconceptexperimentaldataofrecombinantproteinproduc-tionoptimizationisincluded.

Bio-Butanol production with Clostridium aceto-butylicum in a multi-stage continuous process (P19)Peter Götz, Katja Karstens, Sergej Trippel

Biotechnologie, Beuth University of Applied Sciences, Berlin, Germany, [email protected]

Finitenessoffossilhydrocarbonresourcesandenvironmentalpollutionresultingfromtheuseoftheseresourcesmakepro-ductionofrenewableenergysourcesnecessary.Wellknownproductsinthisareaareethanolandbutanol.Comparedtoethanol,butanolhashigherenergycontentandcanbeblendedwithgasoline.Inaddition,butanolactsasabulkrawmaterialinthechemicalindustry.TheOPTISOLVprojectconsortiumconsistsofindustrialandacademicpartnersfromItalyandGermany.Withintheframeworkoftheproject,newcontinuousprocessesforsolventproductionwithClostridiumacetobutylicumarebeingdeveloped.Experimentalinvestigationsupportedbymathematicalmodellingisusedtoevaluatefeasibleandproductivecontinuousbioreactorconfigurations.Whileinthewidelyusedbatchprocesses,thetwometabolicphasesofC.

26

acetobutylicum–acetogenesisandsolventogenesis–followeachotherintime,inamultistagecontinuousbioreactorsys-temtheyarespatiallyseparated.Achangeofmetabolicstatesthroughoutthemulti-stagebioreactorsystemallowstheircharacterization.Multistageprocessescanbeoperatedwithbothsuspendedcells(cellsaretravelingthroughreactorsandareexposedtoenvironmentalchanges)andimmobilizedcells(constantenvironmentofthecells).Inourgroup,wefocusonmodelingthedynamicsofmicro-bialmetabolicnetworkanditsregulationaswellasonthegenerationofdatafromcontinuouscultivationexperiments.Wealsoattemptoptimizingthebio-butanolproductionproc-essinacascadeofcontinuousstirredtankreactors(CCSTR)usingmodelpredictions.Severaloptimizationpossibilitiesareeligiblelikeanadding/removalofreactorstages,changeofresidencetimesinthestagesbyreactorvolumeadaptationandrecirculationorfeedingbetweenthestages.Currentlyoursystemachievesafinalbutanolconcentrationof11gBUTL-1withavolumetricproductionrateof1gBUT(Lh)-1.Mathematicalmodelingofthesuspendedcellssystemisstillachallenge.Theagent-basedmodel,developedinourgroup,includessimulationofchemicalconcentrationsineachbioreactorstageandbiologicalconversionratesofbiomassineachmetabolicstate.Agentsrepresentsubpopulationswithproperphysiologicalproperties.Movingtheseagentsaccordingtotherulesofresidencetimedistributions,thesesubpopulationsarechanginginsizeandaremovingtonewenvironments.Theperformanceofeachreactorstagewillthenbecalculatedfromthemeanmetabolicreactionsofthesubpopulations.

Improving mammalian cell culture process development by combining model-based simulations with DoE tools (P20)Johannes Möller1, Fabian Freiberger1, Regine Eibl2, Dieter Eibl2, Ralf Pörtner1

1 Institute of Bioprocess and Biosystems Engineering, Hamburg Univer-sity of Technology, Hamburg, Germany, [email protected], 2 Institute of Biotechnology, Biochemical Engineering and Cell Cultivation Technology, Zurich University of Applied Sciences, Wädenswil, Switzer-land

Bioprocessesinvolvingmammaliancellsaremostfrequentlybasedonoptimizedfeedingprofileswithinafed-batch.StatisticalDesignofExperiment(DoE)toolsarewidelyusedforthiskindofprocessoptimizationbecauseoftheirsimplestructureandeasyhandling.Limitationsoccurwithrespecttothehighnumberanddurationofexperimentsduringprocessdevelopment.Evenifhigh-throughputsystemscanperformthesenumbersofexperimentsinparallel,theheu-risticrestrictionofboundariesresultsinstepwiseiterationswithmultipleruns.Thisistime-consuming,cost-intensiveandfurthercomplicatesthepathfromprocessdevelopmenttoprocessestablishment.TheuseofDoEtoolsincombina-tionwithanappropriategrowthmodelmightbeavaluablemethodforevaluatingfed-batchstrategiesanddecreasingtheexperimentalspacerequiredforstatisticaloptimizationmethodsinsilicobeforeexperimentsarecarriedout.Toprovethisconcept,amodelwasusedtodescribethedynamicsofcellmetabolismofCHO-XM-111cellsinatwo-stepgrowthprocesswithmediumexchangefollowedbyafed-batch.Modelparameterswerefittedtoaverageddatafromthreeparallelshakeflaskcultivations,andmodelpredictionswereusedtodecreasetheexperimentalspaceandthenumberofcultivationparametersinsilico.Therefore,theconcentrationofL-glutamine(feed),constantfeedrate,pointintimeforthemediumexchange,andthestartoffeedingwereidentifiedascriticalprocessparameters.Atfirst,simulationswereappliedtominimizeexperimentalspace.Therefore,thepointintime

formediumexchangeandthestartingpointforfeedingweredeterminedfromabatchsimulationataminimalL-glutamineconcentrationof0.1mmolL-1.Secondly,toincreasethetotalcellnumberinafed-batch,theL-glutamineconcentrationandconstantfeedratewereoptimizedbysimulatedDoE.Intotal,twosimulatedanditerativeresponsesurfaceplotswereevaluated.Insteadofperformingmultipleexperimentstogeneratedataforresponsesurfaceplots,datagainedfromsimulationwasused.Finally,oneappropriatecombinationofcultivationparameterswastestedandcomparedwiththeinitialmodel.Themethodshownissuitableforthegenerationofdeeperproc-essunderstanding,e.g.thelinkageofdifferentprocessparameterstoqualityattributes.Furthermore,cultivationstrategiesformam-maliancelllinescanbecomparedandevaluated.Thisresultedinasignificantreductioninthenumberofexperimentsrequiredduringprocessdevelopmentandprocessestablishment.

Advanced pre-cultivation method for recombinant Escherichia coli cells (P21)Basant Ali1, Antje Neubauer2, Antti Vasala2 and Peter Neubauer1

1 Chair of Bioprocess Engineering, Technische Universität Berlin, Berlin, Germa-ny, [email protected], 2 BioSilta Oy, Oulu, Finland

E.coliculturesarewidelyusedforrecombinantproteinproduc-tion.Cultivationconditionsplayanimportantroleinfinalproteinyieldandcorrectproteinfolding.Thequalityofthestartercultureaffectstherecombinantproteinproductionprocess,howevertheredonotexistanystandardsforcontrolledpre-cultures.TheEnBasetechnology,basedonfed-batchprinciple,iswidelyappliedforenhancedrecombinantproteinproduction.Hereweinvesti-gate;whetherEnBasebasedEnPressomediacouldalsoprovidebenefitsforstartercultures.Thisstudy,attheexampleofaprocessforproductionofathermophilicnucleosidephosphorylaseinE.colishowsthatEnPressoBmediumbasedcultures,whichcanbedirectlyinoculatedfromafrozenstockculture,providearepro-ducibleandrobuststartercultureforproteinproductionproc-esses.

Enhanced plasmid DNA production by enzyme con-trolled glucose release and an engineered Escheri-chia coli (P22)Alvaro R. Lara1, Karim E. Jaén2

1 Universidad Autónoma Metropolitana-Cuajimalpa (UAM), México, [email protected], 2 Posgrado en Ciencias Naturales e Ingeniería, Universi-dad Autónoma Metropolitana-Cuajimalpa Ciudad de México, Mexiko

Toevaluatethecombinationofaculturemediumemployingglu-coamylase-mediatedglucosereleasefromagluco-polysaccharideandanE.colistrainengineeredinitsglucosetransportsystemforimprovingplasmidDNA(pDNA)production.TheproductionofpDNAwastestedusingE.coliDH5growninshake-flasksandtherecentlydevelopedVH33D(recAdeoR)-en-gineeredstrain,whichutilizesglucosemoreefficientlythanwildtypestrains.Threeglucoamylaseconcentrationsforreleasingglu-cosefromthepolysaccharidecarbonsourcewereused:1,2and3U/L.Bothstrainsreachedsimilarcelldensitiesrangingfrom5to8.8g/Lunderthedifferentconditions.ThehighestpDNAyieldsonbiomass(YpDNA/X)forbothstrainswereobtainedwhen3Uenzyme/Lwereused.Undertheseconditions,35±3mgofpDNAl-1wereproducedbyDH5aafter24hofculture.Underthesameconditions,theengineeredstrainproduced66±1mgpDNA/Lafter20h.pDNAsupercoiledfractionswerecloseto80%forbothstrains.ThepDNAconcentrationachievedbytheengineeredE.coliwas89%higherthanthatofDH5a.Thecombinationoftheengi-neeredstrainandenzyme-controlledglucosereleaseisanattrac-tivealternativeforpDNAproductioninshake-flasks.

Poster abstracts


27

Enzymatic production of nucleotide analogues for click chemistry-based applications (P23)Maryke Fehlau, Birthe Rausch, Anke Wagner, Peter Neubauer

BioNukleo UG and Department of Bioprocess Engineering, Technische Univer-sität Berlin, Berlin, Germany, [email protected]

Theuseofenzymesasefficientandsustainablebiocatalystsisespeciallyinterestinginthosefieldsofindustrialbiotechnologythatarecurrentlydominatedbychemicalproductionprocesses.Oneofthosefieldsistheareaofmolecularbiologywheremodi-fiednucleotidesareincreasinglyusedforlabelingofPCRproductsandtheproductionoffluorescentprobesforfluorescenceinsituhybridization(FISH)applications.Recentlytheapplicationofcop-percatalyzedclickchemistryisgainingmoreandmoreinterestforthoseapplicationsusing5-ethynyldUTP,C8-alkyne-dUTPorC8-alkyne-dCTPassubstrates.Inthepresentedstudyweareshowingtheenzymaticproductionofphosphorylatedbuildingblocksusing5-ethynyl-deoxyuridineasamodelcompound.Theheterologousexpressionofallinvolvedenzymeswasoptimized.Finally,thebiocatalystsweresuccessfullyproducedashis-orGST-taggedproteinsinLBorEn-Presso®1mediumandpurifiedbyaffinitychromatography.Theproductionof5-EthynyldUMPwasachievedbyusingdeoxy-nucleotidekinase2withconversionratesbetween50and100%.Theactivitiesofguanylatekinase(GUK),CMP-UMPkinase(CMPK)andapyruvatekinase(PK)werealreadyshownwithnaturalsub-strates.NDPsandNTPswereformedwithyieldsof30to70%.Theactivitiestowards5-EthynyldUMPremaintobeinvestigatedforthoseenzymes.

[1]UkkonenKetal.2011.High-yieldproductionofbiologicallyactiverecombinantproteininshakeflaskculturebycombinationofenzyme-basedglucosedeliveryandincreasedoxygentransfer.MicrobCellFact10,107.

[2]SerraIetal.2014.ImmobilizedDrosophilamelanogasterDeoxyribo-nucleosideKinase(DmdNK)asaHighPerformingBiocatalystfortheSynthesisofPurineArabinonucleotides.Adv.Synth.Catal.356,563-570.

Biological activity of halogenated cladribine and fludarabine derivatives (P24)Heba AbdElRahman1,2, Maryke Fehlau1,3, Anke Wagner1,3, Peter Neubauer1

1 Technische Universität Berlin, Berlin, Germany, 2 National Research Centre NRC, Cairo, Egypt, [email protected], 3 BioNukleo UG, Berlin, Germany

Yearsaftertheapprovalofcytarabineandedoxudineasanti-cancerandantiviralagents,respectively,in1969,nucleosidesanaloguesstillholdtheirplaceasanimportantclassofmedicinalcompounds.Thedevelopmentofnewmoleculesisfocusedontheimprovementofdrugpropertieswithregardto:longtermtoxicity,resistanceandbioavailability.Inthepresentproject,itistheobjectivetofindthebestconditionsfortheproductionofatleast25differentpurinenucleosideanaloguesusingthermophilicenzymesinthescaleof100mgwithpurity≥99%.Thestructuresareconfirmedusingdifferentspectroscopicanalyses(MS/NMR).Finallydifferentbiologicaltestswillbecarriedout.Duringtheinvitrobiologicaltests,it’sintendedtoanalyzethevirostaticactivityandtoperformcytotoxicassaysaccountingforbothsolidtumorsandhematologicmalignancies.

Reconstructing the biosynthetic machinery of Ruminococcin A, a lanthipeptide identified from a strictly anaerobic Gram positive bacteria, in Escherichia coli (P25)Ongey Elvis Legala1, Michel Fons2, Peter Neubauer1

1 Chair of Bioprocess Engineering, Department of Biotechnology, Tech-nische Universität Berlin, Berlin, Germany, [email protected], 2 ISM2/BiosCiences UMR CNRS 6263, Interactions Moléculaires Microbiote – Muqueuse Intestinale, Service 342, Faculté des Sciences et Techniques de St Jérôme, Université Paul Cézanne Aix-Marseille III, Marseille, France

RuminococcinA(RumA)isatrypsin-dependentlanthipetidethatisproducedbythestrictanaerobicGram-positivebacteriumRuminococcusgnavusE1,isolatedfromhumanintestinalmicrobiome.ThebiosynthesispathwayofRumAin-volves,amongstothers,themodifyingenzyme(RumM)whichcatalyticallyinstallsthreethioethercrosslinksandanα,β-unsaturatedamino,didehydrobutyrineintoitscorestructure.Theenhancedactivityofthepeptideagainstpathogenicclostridiafurthermakesitaplausibletargetforthetreat-mentofhumaninfectionsandapplicationsinlivestock.DuetothenatureofR.gnavusE1,standardcultivationstrategiesareobviouslynotoptimalforlarge-scaleproductionandsubsequentpharmaceuticalcommercializationofRumA.Inthiswork,weaimedatengineeringthebiosyntheticrouteofRumAinE.colitoenhancethedevelopmentofabioprocessschemeforaviableindustrialproduction.Weisolatedthegenesforthestructuralpeptide(rumA)andthemodifyingenzyme(rumM)fromR.gnavusE1andco-expressedtheminE.coliunderseparateregulatorymechanismsinordertobalanceexpressioninawaytoguaranteeaminimalloadforthecell.Wealsoinvestigatedthebehavioroftheengineeredstrainwithrespecttothetoxicityofthetargetproducttothehost.OurresultsshowthatactiveRumAisproducedinE.coliwhichpresentsaverygoodbasisforscale-upoptimization.Toourknowledge,thisisthefirsttimethatalanthipeptidefromastrictlyanaerobicGram-positivemicrobeisheterologouslyexpressedinaGram-negativehost.

Platform technologies for automated bioprocess development (P26)Florian Glauche, Peter Neubauer

Technische Universität Berlin, Chair of Bioprocess Engineering, Berlin, Germany, [email protected]

Bioprocessdevelopmentistimeandcostintensiveduetothelargedesignspacetobeinvestigated.Laboratoryautomationcombinedwithexperimentaldesignstrategiesoffernewwaystoincreaseexperimentalthroughput.However,oftenkeypa-rameterssuchareselectedfromscreeningexperimentswhichdonotresembletheconditionsatproductionscale.AtthechairofBioprocessEngineeringoftheTUBerlin,alaboratoryofthefutureconcepthasbeenestablishedwhichfeaturesautomatedhigh-throughputcultivationsystemscombinedwithfed-batchgrowthmedia,chemoopticalsensorsandamodulardatabasefordataevaluationandmodelling.Theplatformtechnologieswereappliedforofrecombinantpro-teinproductionprocessdevelopmentfrommicrobialhosts.

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Novel bioprocess types based on CHO lysate cell-free protein synthesis systems (P27)Lena Thoring1, 2, Doreen A. Wuestenhagen1 and Stefan Kubick1

1 Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses Potsdam-Golm (IZI-BB), Department of Cell-free and Cell-based Bioproduction, Potsdam, Germany, 2 Technical University of Berlin, Department of Biotechnology, Berlin, Germany

Nowadays,differenttypesofbioprocessesareavailablefortheproductionofpharmaceuticallyrelevanttargetpro-teins.Ingeneral,hostorganismsusedduringfermentationprocessescanbedividedintofourgroupsthatcompriseE.coli,yeast,insectcellsandmammaliancells.Theselectionofsuitableorganismsdependsontheproteinproductionstructureofthedesiredproteinproductprocessitself.Humanproteinsinparticularplayanessentialroleintherapeuticalapplications.Duetotheircomplexstructure,posttranslationalmodificationsandelaboratefoldingprocedures,thesetypesofproteinsneedspecialproductionrequirementsandsyn-thesismilieus.Thereforethemostfavorablesystemsfortheproductionoffunctionallyactivehumanproteinarebasedonculturedmammaliancells.Currently,ChineseHamsterOvary(CHO)cellsareoneofthemostimportantandwell-knownmammaliancelllinesusedforindustrialproduction.CHOcellfermentationprocesseswerealreadyimplementedinindustrialproductionduringthe1980s.Overthelastdecades,thesystemwascontinuouslydevelopedandprovidesvariouspossibilitiesthatcouldbeadaptedforspecificapplications.Besidestheconventionalinvivoproteinproduction,alterna-tivebioprocessmodes,socalledcell-freeproteinsynthesisplatformsweredeveloped.Cell-freeproteinsynthesissystemsenableproteinproductioninanopenmannerusingcelllysates.Thisprocedureprovidestheadvantagethatmilieuconditionscanbeeasilyadaptedtoeachtypeofprotein,byforexampleadjustingredoxconditionsoradditionofchaper-ons.Additionally,theproteinproductionprocessinthecell-freesystemisfastercomparetoinvivoprocesses.Cell-freesystemsharbortheentirefunctionallyactivetranslationalma-chinery,includingribosomes,translationfactorsandtRNAs.Prokaryoticcell-freesystemsenablehighproteinproductionrates,buttheyarelimitedinproteinfoldingandmodifica-tions.Forhumantargetproteinsandsocalled“difficult-to-ex-press”proteins,cell-freesystemsareavailablethatarebasedoneukaryoticlysates.Eukaryoticcell-freesystemsderivedfromSf21cellsandmammaliancelllinesharborendogenousmicrosomalstructuresduetoamildcelldisruptionandlysatepreparationprocedure.Endogenousmicrosomes,obtainedfromtheendoplasmaticreticulum,enabletoimplementpost-translationalmodificationsanddirectlyintegratemembraneproteinsintotheirappropriatelipidenvironment.Toclosethegapbetweenconventionallabscalecell-freepro-teinsynthesisandinvivoproteinproductionprocesses,wearedevelopingcell-freeproductionprocessesbasedonCHOcelllysates.CHOcell-freeproteinsynthesisisimplementedintworeactionsmodeswhichdistinguishthemselvesbyreac-tionconditions,proteinyieldandprocesscosts.The“simply-to-perform”batchmode,aonepotreaction,providesproteinyieldsupto50µg/ml.Synthesisofvariousproteinscouldbedemonstratedinbatchmodeincludingintegralmembraneproteins,e.g.GPCRs,ionchannelsandglycoproteinsaswellascytosolicproteins.Additionally,thepossibilitytoperformcell-freesynthesisinthepresenceofnon-canonicalaminoacidwasshownbyincorporationintotargetprotein.Inthismanner,newopportunitieswillbeprovidedthatenableforexamplespecificfluorescencelabelingofproteins.Besidesthementionedbatchmodeanovel,dialysisprocessmodeisdevelopedforcell-freeproteinsynthesis.Acompartmentali-zationoftheprocessreactionintoareactionchamberandanadditionalfeedingchamberleadstoaprolongedreaction

time,therebyincreasingthetotalproteinyieldbyremovinginhibi-torybyproductsanddeliveringenergycomponentsalongwiththediffusiongradient.Differentprocessoptimizationstrategiesforthedialysismodeenhanceproteinyieldsupto10timesyieldscomparedtobatchbasedsynthesis.Withregardtothepresentedresultsourtechnologyoffersanenormouspotentialintermsofanovelproductionprocessfor“difficulttoexpress”proteinsandtheirsubsequentfunctionalanalysis.

Enhancement of lipopeptide yield by integrated bioprocess development (P28)Julia Glazyrina1, P. Neubauer1, P. Götz2

1 Technical University of Berlin, Institute of Biotechnology, Department of Bioprocess Engineering, Berlin, Germany, [email protected]. 2 Beuth University of Applied Sciences Berlin, Bioprocess Engineering, Berlin, Germany

Lipopeptidearesurfaceactivecompoundsandcanbeusedasbio-surfactantsinmanyindustrialareasincludingallapplicationfieldsofchemicalsurfactantssuchasagriculture,foodproductionandcosmeticsandalsoinspecialfieldslikebioremediation.Moreover,theyhaveantibioticactivitythatallowstousethelipopeptidesasplantprotectionagents.Currentlyrelativelylowyieldandhigh-costdownstreamprocessingmaketheproductionprocessoflipopeptideeconomicallyinefficient.Inthisprojectabioprocessforproductionofthelipopeptidfengycin,startingfromstrainscreening,viaoptimizationofmediaandcultivationconditionstodownstreamprocessing,usinganin-situproductremoval(ISPR)strategybyfoamfractionation,wasdeveloped.Itwasshownthatusingacost-effectivecultivationmediumbasedonpotatofruitjuice(side–productfromstarchproduction),opti-mizedcultivationconditionssuchaslowtemperatureandoxygen-limitationandalsobyapplyingtheintegratedfoamfractionationsystem,afive-foldincreaseoffengycinyieldwasachieved.Attheendofthebioprocessdevelopment,concentrationoffengycininthecultivationmediaofmorethan1gL-1couldbeobtained.

Docosahexaenoic acid (DHA) production of new strain of Indonesian Thraustochytrid Aurantiochytrium sp. LR52 in single use TubeSpin 600 system (P29)Andri Hutari1,2, Wahyu Hidayati3, Apon Zaenal Mustopa4, Peter Neubauer1

1 Technische Universität Berlin, Department of Biotechnology, Chair of Biopro-cess Engineering, Berlin Germany, [email protected], 2 University of Muhammadiyah Prof. Dr. Hamka, Biology, Jakarta, Indonesia, 3 University of Muhammadiyah Prof. Dr. Hamka, Pharmacy, Jakarta, Indonesia, 4 Indonesian Institute of Science (LIPI), Research Center for Biotechnology, Bogor, Indonesia

ThraustochytridsAurantiochytriumsp.areuniquemarinemicro-organismintermofdocosahexaenoicacid(DHA)productionbe-causetheygrowfast,producehighcelldensity,highproportionsoflipidandDHAcontent.DHAhasalreadyprovenasimportantfattyacidforinfantdevelopment,maintenanceofnormalbrainfunction,visualacuity,andpreventionofneurologicaldisordersandinflammatorydiseasesinadult.TheapplicationofDHA-pro-ducingAurantiochytriumisrapidlyexpandingforfoodadditive.Inaddition,driedAurantiochytriumhasGRASstatusforuseasfeedtobroilerchickens,layinghens,andaquaculture.ThegenusAuran-tiochytriumsp.(before2007isSchizochytrium)belongingtotheLabyrinthulomycetesofthekingdomChromophyta(Straminipila).Aurantiochytriumsp.producedoverallDHAproductivitymorethantwicehigherthananotherestablishedspeciesfromdino-flagellateCrypthecodiniumcohniiwhereasthecultivationtimeofAurantiochytriumwasshorterthanthatofC.cohnii.Inaddition,AurantiochytriumalsoshowedalesserincidenceofunwantedmicrobialcontaminationthanoccurredinvariousCrypthecodin-iumcohniifermentations.ThishasattractedmanyresearchersandindustrytoscreennewstrainofAurantiochytriumandoptimize

Poster abstracts


29

thefermentationperformance.Recently,wehaveisolatedastrainofAurantiochytriumfromIndonesianmangrovehabitat,namelyLR52.CultivationofAurantiochytriumsp.LR52inTubeSpin600withlowartificialsaltwater(ASW)concentrationof3.6gL-1yieldeddriedbiomassandDHAcontentmorethan63and18gL-1,respectivelyin6days.TheDHAproductionrateofLR52isoneofthehighestvaluereportedforanyDHAproducingmicroorgan-ismsinshakeflaskscale.

Process analytical technologies

Spectroscopic bioprocess monitoring and control of CHO cell cultivations (P30)Jens Claßen, Shaobin Lu, Marko Sandor, Dörte Solle, Thomas Scheper

Leibniz Universität Hannover/Institut für Technische Chemie, Hannover, Ger-many, [email protected]

Processanalyticaltechnology(PAT),aninitiativeoftheAmericanFoodandDrugAdministration(FDA),providestoolsforholistic,knowledge-basedandriskavoidingcontrolofmanufacturingprocessesinchemicalandpharmaceuticalindustry.Onetoolisonlinemonitoringofcriticalprocessparameters(CPP)byspectro-scopicmethodscombinedwithmultivariatedataanalysis[1,2].Thisisparticularlyimportantforcost-intensivemanufacturingofbiopharmaceuticalstoenableanearlyprocessfaultdetectionandrealtimeproductrelease.Mammaliancellcultivation(CHO-K1cellline)in15lstirredtankreactorischosenasabenchmarkprocessformonitoringandcontrol.MonitoringofapoptosisiscarriedoutbyUV/Visspectros-copy.Bynon-invasiveMIRspectroscopysubstrateandmetaboliteconcentrations(glucoseandlactate)couldbemonitoredduringthecultivation.Theresultsofspectroscopicinlinemeasurementandconsequentialdevelopedpartialleastsquaresregressionmodels(PLS)areshownexemplarily.Basedontheonlineglucosepredictionanautomatedfeedstrategyisbuilt-ontopreventanearlycelldeathbysubstratelimitation.Theprocessstrategyischargedfrombatchtofed-batchprocessbythisapplicationandhighercellcountswererealized.[1]SandorMetal.Comparativestudyofnon-invasivemonitoringvia

infraredspectroscopyformammaliancellcultivations,JBiotechnol.,2013,168,636–645.

[2]BiechelePetal.Sensorsystemforbioprocessmonitoring,EngLifeSci,2015,00,1-20

Mathematical model of adherent vero cell growth and poliovirus production in animal component free medium (P31)Gerco van Eikenhorst, R. V. Ursache, Y. E. Thomassen, W. A. M. Bakker

Institute for Translational Vaccinology, Process Development, Bilthoven, the Netherlands, [email protected]

Sabin–IPV(orsIPV,inactivatedpoliovaccinebasedonattenuatedSabinstrains)isanticipatedtoreplacetheoralpoliovaccine(OPV)fortheendgameinpolioeradication[1,2].OptimizationofsIPVproductionwillleadtoabettereconomicallyfeasiblevaccine[3].TheaimofourstudywastodevelopadescriptivemathematicalmodelabletocapturedynamicsofadherentVerocellgrowthandpoliovirusinfectionkineticsinanimalcomponentfreemediumtofurthersupportsIPVprocessoptimizations.TobeabletomonitorVerocellgrowthclosely,theexperimentalbioreactorsetupincludedaFogalebiomassprobeandacellfreesamplingdeviceforonlinemetaboliteanalysesusingaNovaBioProfileFLEX.Onlinebiomassdatacorrespondedwellwiththeofflinemeasuredcellconcentrations,alsotheon-andofflinemainmetabolites(glucose,lactate,glutamine,glutamate,ammonia)werecomparable(Fig1).Thedevelopedmodelpredictsthecelldensity,metabolitesprofilesandviralyieldsintime.Theviralgrowthmodelimpliedthatthemultiplicityofinfection

(MOI)andthetimeofinfection(TOI)donotaffectmaximalpoliovirusyields.However,theydoaffecttheprocesstime,whichmaybereducedbyselectingalowTOIandahighMOI.Additionally,weobservedacorrelationbetweenviraltitersandD-antigen,ameasureforimmunogenicity,ofSabinpoliovirustype1.Thedevelopedmodelisadequateforfurtherstudiesofthecellmetabolismandinfectionkineticsandmaybeusedtoidentifycontrolstrategiestoincreaseviralproductivity.Increasedviralyieldscanpotentiallyreducethecostsofpoliovaccineswithcouldhavelargeimplicationsonpublichealth.Inaddition,thebiomassprobeandtheonlinemetaboliteana-lyzermaybeusedincombinationwiththemodeltomonitorthecellandvirusgrowthinaprocessanalyticalapplication(PAT).

Multiparameter monitoring of gradients in the liquid phase of fermentation processes (P32)A. Bockisch1, J. Biering2, S. Sachse3, W. Vonau3, S. Junne1, P. Neubauer1

1Technische Universität Berlin, Department of Biotechnology, Chair of Bioprocess Engineering, Berlin, Germany, [email protected], 2Versuchs- und Lehranstalt für Brauerei in Berlin e.V., Berlin, Germany, 3Kurt-Schwabe-Institut Meinsberg e.V., Meinsberg, Germany

SeeL23

Multiposition Sampling, Multiparameter Monitoring and Electrooptical Tracking of Microbial Activity: New Tools for better understanding biogas production (P33 )Jörn Beheim-Schwarzbach*1,Erich Kielhorn2, Alexander Angersbach3, Emma Ritzi1, Manja Laqua2, Anna Vaupel2, Boris Habermann1, Peter Neubauer2, Stefan Junne2

1Institute of Agricultural and Urban Ecological Projects, Berlin, Germany, [email protected], 2 Department of Biopro-cess Engineering, Technical University of Berlin, Berlin, Germany, [email protected], 3 EloSystems GbR, Berlin, Germany

SeeL34

Online-monitoring tools in batch and (semi-)perfusion cultivations for vaccine production (P34)Daniel Vazquez-Ramirez1, Alexander Nikolay1, Yvonne Genzel1, Udo Reichl1,2

1 Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstraße 1, 30106 Magdeburg, Germany, [email protected], 2 Otto-von-Guericke University Magdeburg, Chair of Bioprocess Engineering, Universitätsplatz 2, 30106 Magdeburg, Germany

Theincreasingavailabilityofonline-monitoringtoolsenablesabetterunderstandingandcontrolofanimalcellculture-basedprocesses.However,real-timeprocessmonitoringofcellconcentrations,nutrientsandotherparametersisstillbeinglimitedinlarge-scalevaccinemanufacturing.Neverthe-less,forfurtherintensificationofvaccineproduction,i.e.theestablishmentofhigh-cell-density(HCD)perfusionprocesses,online-monitoringisofcrucialimportance.Inthepresentwork,thesuitabilityofdevicesforonline-monitoringofpHanddissolvedoxygenconcentrationinshakeflasks,aswellasofglucose,lactateandcellconcentrationsin1Lbioreactorsforvirusproductionprocesseswastested.Highcellconcentrations(~50×106cells/mL)wereachievedeitherthroughmediumrenewalbyperiodiccentrifugation(semi-perfusion)in125and250mLshakeflasksorbyusingahollowfiber-based(ATForTFF)perfusionsystemfor1Lbiore-actors.Forshakeflaskcultivations,oxygenconcentrationandpHvalueweremonitoredduringcellproliferationandMVAviruspropagationinsuspensionAGE1.CR.pIXcellsusingthe

30

SFR®system(Presens).Inbioreactors,theconcentrationsofglucoseandlactateweremeasuredonlineduringperfusioncultivationofAGE1.CR.pIXandbatchcultivationofBHK-21cellsusingaBioPAT®Traceprobe(Sartorius).Additionally,viableandtotalcelldensitiesweremonitoredonlineusingtheIncyteandDencyteeprobes(Hamilton),respectively,duringthepropagationofMVAandinfluenzavirusA/PR/8/34(H1N1)inAGE1.CR.pIXcells,andyellowfevervirus(YFV17D)inadherentVeroandBHK-21cells.TheSFR®systemshowedthatpHvaluesof7.2to7.4anddis-solvedoxygenconcentrationsabove86%canbemaintainedwhilecultivatingAGE1.CR.pIXcellsupto45×106cells/mLinshakeflasksunderasemi-perfusionregime.Interestingly,areducedoxygenuptakerateofthecellscouldbeobservedwhendelayingthemediumrenewal.Thisseemedtocorrelatewiththebeginningoflactateconsumptionusuallyobservedinbatchprocesseswhenglucoseconcentrationsdropbelow~2.5g/Landlactateconcentrationsincreaseabove~2.0g/L.Duringthebioreactorcultivations,theBioPAT®TracereadoutscorrelatedwellwithofflineglucoseandlactatemeasurementsfromaBioProfile®100Plusandallowedperiodicadjustmentsofperfusionratestokeepoptimalglucoseconcentrations.However,particularlyduringBHK-21cultivations,otherkeymetabolitesratherthanglucosealsoseemedtoplayanim-portantroleforoptimalcellgrowth.Concerningthetotalandviablebiomass,online-measure-mentsobtainedwiththeDencyteeandIncyteprobesduringthecellgrowthphaseofAGE1.CR.pIX,BHK-21andVerocellscorrelatedfairlywellwithofflineanalysescarriedoutwithaVi-CELL®system(BeckmanCoulter).Asexpected,theonlinemonitoringofcellconcentrationwasproblematicforcultiva-tionsofBHK-21cellsathighercellconcentrationsandafterinfectionwithYFVduetochangesincellphysiologyaswellascellmorphology.Towardslate-stageinfectiontimepoints,measurementswerenotreliable.However,duringMVAviruspropagationinAGE1.CR.pIXcells,acorrelationwithofflineanalysiswasobservedevenatlate-stageinfectiontimes.Here,celllysisduringviruspropagationcouldbefollowedbythefrequencyandcapacitancereadoutsoftheIncyteprobe.Inaddition,itwasobservedthatstepwiseadditionoflargeamountsofbasetoadjustthepHvalue,andtheincreaseingasspargingcouldinterferewiththeestimationoftheviableandthetotalcellconcentrations,respectively.TheSFR®offersreliableandvaluableinformationofpHvaluesanddissolvedoxygenconcentrationsforcharacterizationofcelllinesinshakeflasks.AlthoughtheBioPAT®measurementscouldbeusedformanualperfusioncontrolbasedonglucoseconcentrations,additionalspecificnutrientrequirementsofeachcelllineshouldbetakenintoaccountandmonitoredifnecessary.OnlinedataobtainedfromtheIncyteandDen-cyteesensorscanbeusefultomonitorcultivations,andformanualandautomaticcontrolofperfusioncultures.TheIncytealsoenablesfrequencyandcapacitancemeasurementsthatmaysupportprocesscontrolduringvirusproduction.

A PAT tool for real-time determination of spore quality in filamentous fungi bioprocesses (P35)Daniela Ehgartner, Jens Fricke, Christoph Herwig

Institut für Verfahrenstechnik, Umwelttechnik und Techn. Biowissen-schaften, Vienna University of Technology, Vienna, Austria, [email protected]

Sporeinoculumqualityinfilamentousbioprocessesisacriticalparameterassociatedwithviablesporeconcentrationandsporegermination[1].Itinfluencespelletmorphologyand,consequently,processperformance[2].Anessentialstepbeforeinoculationisthedeterminationoftheviablesporeconcentration,inordertoapplyqualitycontrolanddecreasebatch-to-batchvariability.Thestate-of-the-artmethodto

investigatethisvariableistedious,associatedwithsignificantinherentbias,andnotapplicableinrealtime.Therefore,itisnotusableasaprocessanalyticaltechnology(PAT).PATaimstodesign,analyseandcontrolpharmaceuticalproductioninordertoensureproductquality[3].Monitoringofsporegerminationisnotonlynecessarytoinvesti-gatefurthersporequalityattributesbutcanalsobeusedasvalida-tionofthebeforehandmentionedmethod.Qualityattributesconnectedtosporegerminationarenotonlytheamountofger-minatingsporesbutalsohowlongsporesneedforgerminationandwhetherallsporesgerminateatthesametimeornot.Thoseelementsweresofarmonitoredusingimageanalysis[4],whichishamperedbycomplexmediumbackgroundoftenobservedinfilamentousbioprocesses[5].Themethodpresentedhereisbasedonthecombinationofvi-abilitystainingandlarge-particleflowcytometrywhichenablesmeasurementsinreal-timeandhenceaimstobeapplicableasPATtool.Itiscompatiblewiththecomplexmediumbackground,andallowsthequantificationofmetabolicallyactivespores.Further-more,sporeswellingasapreviousstepofsporegerminationandgerminationitselfmaybemonitored.Adistinctionofgerminatedsporesfromnotgerminatedsporeswasbasedonalogisticregres-sionusingmultiparametericdatafromforwardscatterandgreenfluorescencechannel.Inanindustrialbioprocesswithfilamentousfungi,agoodcorrela-tiontoCFUcountswasfound.Themorphologicalparametersassporeswellingandsporegerminationaswellasmetabolicactivitywerefollowedovertheinitialprocessphasewithclosetemporalresolution.Thevalidationofthemethodtomeasuresporegermi-nationshowedanerrorofsporeclassificationoflessthan5%.Differencesinsporegerminationforvarioussporeinoculaagesandsporeinoculumconcentrationsweremonitored.Notallsporesshowingmetabolicactivitygerminated.Theamountofsporesgerminatedwasfoundtobedependentonthequalityofthesporeinoculum.Thiscombinationofviabilitystainingandflowcytometryisapromisingtooltonotonlydeterminesporeinoculumqualitybe-forebatchinoculuationbutalsotoat-linemonitorsporeswellingandsporegerminationinordertoreactinreal-timeondeviationsandhencedecreasebatch-to-batchvariability.ThereforethemethodcouldbeapplicablefortheimplementationasPATtoolinfilamentousbioprocesses[6].

[1]J.Nielsen&P.Krabben,Biotechnol.Bioeng.1995,46(6),588-598.[2]K.G.Tucker&C.R.Thomas,Biotechnol.Techn.1994,8(3),153-156.[3]FoodandDrugAdministration,DHHS.2004,Rockville.[4]P.W.Coxetal.,Microbiol.1998,144(4),817-827.[5]A.E.Poschetal.,Microb.cellfact.2012,11(1),1-14.[6]D.Ehgartneretal.,Fung.Gen.Biol.2016,Manuscriptinpreparation.

Scalability of cultivations in the 2-dimensional rocking single-use bioreactor CELL-tainer (P36)Anna Maria Marbà-Ardébol, Peter Neubauer, Stefan Junne

Technische Universität Berlin, Institute for Biotechnology, Chair of Bioprocess Engineering, Berlin, Germany, [email protected]

SeeL31

Poster abstracts


31

Analysis of the single-cell size distribution as a pro-cess analytical tool in bioprocesses (P37)Anna Maria Marbà-Ardébol1, J. Emmerich2, S. Junne1, P. Neubauer1

1Technische Universität Berlin, Institute for Biotechnology, Chair of Bioprocess Engineering, Berlin, Germany, [email protected], 2 SOPAT GmbH, Berlin, Germany

Atlineandonlinemicroscopyisapromisingtechniquetogainvaluabledataaboutpopulationheterogeneityandevensubstrateconversionandproductsynthesis,asthesestepshaveanimpactonthecellsizeinvariousbioprocesses,e.g.cellulargrowth,intra-cellularlipidaccumulation,etc.Inthisstudy,real-timemonitoringofthesinglecellsizedistributionisinvestigatedforthequantifica-tionofintracellularaccumulationofthepolyunsaturatedfattyaciddocosahexaenoicacidintheheterotrophicmicroalgaeCrypte-codiniumcohnii.Thenovelphoto-opticalmicroscopyprobeofSOPATwasappliedinsituandtheperformancewascomparedtoatline3-dimensionalholographicinterferometricmicroscopy(Ovizio),whichprovidedalsodataaboutthesurfacestructure,andtraditionalflowcytometry.Itwasproventhatboth,theatlineandtheinsitumicroscopy,arewell-suitedforthepredictionofthefattyacidaccumulationincells,andthuscanbeappliedasaprocessanalyticaltool.FurtherstudiesinvestigatedtheimpactofoscillatingcultivationconditionsonthepopulationheterogeneityinSaccharomycescerevisiaecultures.Bothtoolsaresuitabletoreducetimesinprocessdevelopmentandoptimization.

Electrooptical monitoring of polarizability in Lactobacillus plantarum batch and fed-batch fermentations (P38)Klaus Pellicer Alborch1, Alexander Angersbach2, Peter Neubauer1, Stefan Junne1

1 Chair of Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, Berlin, Germany, [email protected], 2 EloSystems GbR, Berlin, Germany.

Thedegreeofpolarizabilityofcellsdependsparticularlyonthecells’physiologicalstate[1]andisrelatedtotheirviability:cellswithhighmetabolicactivityandintactenergyhouseholdpossessalargerpolarizability[2].Theanisotropyofpolarizability(AP)canbemonitoredonlineapplyingelectroopticalmethods[2]withthehelpofautomatedsamplepreparation.Inpreviousreports,weshowedthatthemonitoringoftheAPofrod-shapedbacteriainbatchcultivationsallowedthepredictionofmetabolicsynthesisrates[3,4].TheobjectiveofourresearchistheinvestigationofwhetherdifferentgrowthconditionsandvariablefeedratesinanaerobicL.plantarumATCC8014batchandfed-batchcultivationsinflu-encetheAP,aimingtoidentifysuitablecultivationconditions.Theeffectsofsubstratepulses,temperaturevariation,pHshiftsandpresenceofoxygenhavebeeninvestigatedconcerninginfluencesontheAP.Inallcases,thegrowthphasecouldbedividedinsev-eralstages,basedonAPspectraoffourfrequencies(200,400and900kHzand2.1MHz).Themonitoringofthisparameterenableadeeperinsightintotheactualphysiologicalstateofcellsinrealtimeandwithoutanystainingorfluorescentmarkers,asusedinflowcytometry.Finally,theautomatedmeasurementapproachallowsitsapplicationasaprocessanalyticaltool.

Simulation of biomass transport and deposition in expanded bed adsorption chromatography (P39) Vikas Yelemane, Martin Kangwa and Marcelo Fernández-Lahore

Downstream Bioprocessing Laboratory, Life science and Chemistry, Jacobs University, Bremen, Germany, [email protected]

Integrativeapproachesindownstreambioprocessplayanimportantroleindevelopingcosteffectivepurificationproc-essforbio-moleculeswithincreasedrecovery[1,2].Expandedbedadsorption(EBA)isoneofsuchintegratedunitopera-tionsinproteinpurificationwithestablishedhandsonappli-cationinresearchaswellasindustrialsectors.However,EBAhasbeenevidencedtosufferfromthebiomassinteractioninanunclarifiedfeedstock.Hence,understandingtherationalebehindthebiomassadsorbentinteractionformsthebasisofresearchwhileusingEBArelatedapproaches.Additionally,investigatingthepotentialstrategiestoreducethisinterac-tionformachallengingscientificquestionthathastobead-dressedwhichhasgainedcurrentresearchinterestinthisarea[3].PresentstudyconsistsofresultsobtainedfrombiomassdepositionexperimentsonexpandedbedusingYeastandCHOcellsasamodelorganismandStreamlineDEAEandFast-lineMabDirectProteinAasmodeladsorbents.Processwasmodelledinordertoextractquantitativeinformationsuchas,attachmentefficiency(α),single-collectorcontactefficiency(η),particledepositionratecoefficient(kdsec-1),attachmentrateka1[1/s]anddetachmentratekd1[1/s].Theseparam-etersarecomparedwithprocessmodificationbychangingmobilephasepropertiesoradsorbentsurfaceproperties.

[1]M.FernándezLahoreOetal.ExpandedBedChromatography,SurfaceEnergeticsofBiomassDeposition,in:EncyclopediaofIndustrialBiotechnology,JohnWiley&Sons,Inc.,2009.

[2]JThommesetal.Theinfluenceofcelladsorbentinteractionsonproteinadsorptioninfluidisedbeds,AbstrPapAmChemSoc216(1998)U246-U246.

[3]RVennapusaetal.Assessingadsorbentbiomassinteractionsduringexpandedbedadsorptionontoionexchangersutilizingsurfaceenergetics,JChromatogr.A,1181(2008)9-20.

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