Thèse Raval Keyur

7/28/2019 Thse Raval Keyur

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Characterization and Application ofLarge Disposable ShakingBioreactors

VonderFakulttfrMaschinenwesender

Rheinisch-WestflischenTechnischenHochschuleAachenzurErlangungdesakademischenGradeseinesDoktorsder

IngenieurwissenschaftengenehmigteDissertation

vorgelegtvon

KeyurRavalaus

Rajkot,Indien

Berichter: UniversittsprofessorDr.-Ing.J.Bchs

UniversittsprofessorDr.-Ing.U.Renz

TagdermndlichenPrfung:14.April2008

DieseDissertationistaufdenInternetseitenderHochschulbibliothekon-

lineverfgbar.


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Acknowledgement

IamhighlyindebtedtomyparentsandmyGuruShreeJayantikakaforshapingupmy

lifefromchildhoodtodateandhelpingmetobuildupinternalstrength.

IfeelgreatpleasureinexpressingmygratitudetomyguideProfessorDr.-Ing.Jochen

Bchsforhisexpertguidanceandconstantencouragementthroughouttheperiodof

theprojectwork.Iamextremelyindebtedforhismotivation,professionalacumenand

precioustimethathedevotedforsuccessfulcompletionofmyprojectwork.Hewas

andstillremainsafatherfigureinmylife.

IexpressmyheartiestthankstomycolleaguesCyrilPeter,AndreasDaubandArnd

Knollwhowere always therenot only for technical brainstormingbut also at vitalmomentsofmylife.

Lastbutnotleast,Iamverythankfultomywife,Ritu;whoisalwaystheretoholdand

supportmeduringthemostpainfulmomentsofmylife.


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Kurzfassung

In dieser Forschungsarbeit wird die Anwendung eines geschttelten

Bioreaktorsystems imPilotmastabdargestellt. Diese sehreinfache, vielfltige und

allgemein verwendbare Technologie wurde mit zylinderfrmigen Einwegreaktoren

kombiniert,umsiezueineridealenWahlfrdieKultivierungvonPflanzen-,Tier-und

InsektenzellkulturenzurProduktionimPilotmastabzumachen.Diezylinderfrmigen

Reaktoren der Gre 2L, 20L und 50L wurden in Bezug auf wichtige

Betriebseigenschaften wieMischen, Leistungseintrag, Wrmebertragungsrate und

Sauerstofftransferrate eingehend charakterisiert. Die vollstndige Vermischung der

Flssigkeit wurde innerhalb weniger Sekunden bei einer Schttelfrequenz von 80

U/min erreicht. Die Leistungsaufnahme von Flssigkeiten, deren physikalische

Eigenschaften sichnicht drastischmit der Temperatur verndern, wurdedurchdie

Temperaturmethodegemessen.DieMethodewurdemodifiziert,umdienderungen

physikalischer Eigenschaften der Flssigkeiten mit der Temperatur zu

bercksichtigen, wie z.B. die Viskositt und die Dichte. Betriebsbedingungen, in

denen eine sehr schlechte Vermischung beobachtet werden konnte, wurden

identifiziertundderLeistungseintragdesReaktorsystemsdimensionslosbeschrieben.

Hohe Wrmeerzeugungsraten wurden in 20L- und 50L-Reaktoren, besonders frSchttelfrequenzenber230U/minbeobachtet.Experimentezeigteneinemaximale

ZunahmederFlssigkeitstemperatur frWasser und fr ein80%-Glycerol-Wasser-

Gemischvon16Kbzw.30Kbei300U/min.WhrendeinevollstndigeBelftungfr

langsam wachsende Tier- und Insektenzellkulturen nicht zwingend erforlich ist, ist

einevollstndigeBelftungmitUmgebungsluftjedochbesondersfrHochzelldichte-

Kultivierungen schnell wachsender Pflanzenzellkultursysteme wie z.B. Nicotiana

tabacum notwendig, um eine Temperaturbeanspruchung zu vermeiden. Die

Sauerstofftransferrate wurde durch die gut erforschte Sulfitoxidationsmethode

gemessen. Die maximalen Sauerstofftransferraten, die im 20L- und 50L-Reaktor

gemessen wurden, waren 0.032 mol/L/h bzw. 0.028 mol/L/h. Der

Stofftransferkoeffizient wurde mit der Energiedissipation korreliert. Die

MastabsvergrerungeinesProduktionsprozesses freintherapeutischesProtein,

basierend aufNicotiana tabacum Pflanzenzellsuspensionskultur, wurde erfolgreich

vom 250mL-Schttelkolben zum 50L Einwegbioreaktor durchgefhrt. Die

Mastabsvergrerungzum2L-EinwegbioreaktorfreinenProzesszurKultivierung

tierischerZellen,basierendaufhybridoma-cmycZellen,warebenfallserfolgreich.


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Abstract Application of a shaking bioreactor system at pilot-scale level is presented in this

researchwork.Thisverysimple,versatileandwidelyusedtechnologywascombined

with the cylindricaldisposable reactors tomake itan ideal choice for cultivationof

plant,animalandinsectcellculturesforpilot-scaleproduction.Cylindricalreactorsof

size2L,20Land50Lwerethoroughlycharacterizedintermsofimportantengineering

parameters such as mixing, power consumption, heat transfer rate and oxygen

transferrate.Completemixingof fluidwasachievedwithin few secondsatshaking

frequencies as low as 80 rpm. Power consumption for fluids whose physical

propertiesdonotvarydrasticallyovertemperaturewasmeasuredbythetemperature

method.Themethodwasextendedtoincorporatechangesinfluidphysicalpropertiessuch as viscosity, density etc. over temperature. Operating conditions where poor

mixingmightbeobservedwereidentifiedandanon-dimensionaldescriptionofpower

consumption is given for the reactor system. High rates of heat generation were

observedin20Land50Lreactorsespeciallyforshakingfrequencieshigherthan230

rpm.Experimentsrevealedmaximumof16Kand30Kincreaseinfluidtemperature

for water and a 80% glycerol/water mixture at 300 rpm, respectively. Although

thoroughventilationmay not bemandatory for slowgrowinganimal and insect cell

culture,athoroughventilationofthesurroundingatmosphereismandatory,especially

forhighcelldensitycultivationoffastgrowingplantcellculturesystemse.g.Nicotiana

tabacum suspension culture toavoidany temperaturestress.Oxygen transfer rate

wasmeasuredbyawellresearchedsulfiteoxidationmethod.Themaximumvalueof

oxygentransferratemeasuredin20Land50Lreactorswere0.032mol/L/hand0.028

mol/L/h,respectively.Masstransfercoefficientwascorrelatedwithrespecttoenergy

dissipation.Atherapeuticproteinproductionprocessbasedonrelativelylesshydro-

mechanical stress sensitive and one of the fastest growingN. tabacum plant cell

suspensionculturewassuccessfullyscaled-upfroma250mLshakeflaskcultureto

50Lcylindricaldisposableshakingbioreactor.Thecellgrowthandproteinproduction

wascomparabletothatobservedinotherbioreactorsystems.Ananimalcellculture

process based on hybridoma-cmyc cells was also scaled-up successfully to a 2L

cylindricaldisposableshakingbioreactor.


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I

Tableofcontents

1. Introduction and objectives .....................................................................................1

2. Literature review .......................................................................................................6

2.1. Shakingbioreactors .............................................................................................6

2.1.1. Mixingandpowerconsumption ....................................................................6

2.1.2. Masstransfercharacteristicsofshakingbioreactors ....................................7

2.1.3. Ventilationinshakingbioreactors .................................................................9

2.2. Applicationofdisposableshakingbioreactors .....................................................9

3. Theory ......................................................................................................................13

3.1. Conventionaltemperaturemethod.....................................................................13

3.2. Extendedtemperaturemethod...........................................................................13

3.3. Oxygentransferratemeasurement ...................................................................15

3.3.1. Thesulfitesystem.......................................................................................15

3.3.2. Onlineoxygentransferratemeasurement..................................................16

3.3.3. Calibrationoftheoxygensensor ................................................................17

3.3.4. Materialbalanceonoxygenatsteady-state(intherinsingphase) .............18

3.3.5. Materialbalanceinmeasuringphase .........................................................20

3.4. Ventilationinshakeflasks..................................................................................23

4. Materials and methods ...........................................................................................25

4.1. Hydrophilicshakeflasks ....................................................................................25

4.2. Hydrophobicshakeflasks ..................................................................................25

4.3. Mixingperformance ...........................................................................................25

4.4. Measurementofpowerconsumption .................................................................26

4.4.1. Torquemethod ...........................................................................................26

4.4.2. Conventionaltemperaturemethod..............................................................28

4.4.3. Extendedtemperaturemethod ...................................................................304.5. Determinationofoverallheattransfercoefficient(UA).......................................30

4.5.1. Characterizationwithoutlateralairflow ......................................................30

4.5.2. Characterizationwithlateralairflow ...........................................................30

4.5.3. Measurementofheattransferarea.............................................................32

4.6. Determinationofoxygentransferrate(OTR) .....................................................32

4.7. Determinationoftheventilationthroughaluminumfoilinshakeflasks ..............34

4.8. Biologicalexperiments.......................................................................................35 4.8.1. Maintenanceofplantcellsuspensioncultureinshakeflask.......................35


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II

4.8.2. CultivationofN.tabacumsuspensioncultureatlarge-scale ...................... 35

4.8.3. Hybridomacellculturecultivation...............................................................36

4.9. Analyticalmethods ............................................................................................ 38

4.9.1. Determinationoffreshweightanddryweightofplantcellculture .............. 38

4.9.2. Determinationofextracellularsugarconcentrationinplantcellculture......38 4.9.3. Determinationofphosphateconcentrationinplantcellculture .................. 38

4.9.4. DeterminationofHumanSerumAlbumin(HSA)producedbyplantcell

culturesofN.tabacum.............................................................................................. 38

5. Results and discussion..........................................................................................40

5.1. Effectofhydrophobicityonpowerconsumption ................................................ 40

5.2. Mixingperformanceandcriticalshakingfrequency ........................................... 42

5.3. Powerconsumptionindisposableshakingbioreactors ..................................... 435.3.1. Comparisonofthetemperaturemethodandthetorquemethod ................ 43

5.3.2. Extendedtemperaturemethod ................................................................... 46

5.4. Non-dimensionaldescriptionofpowerconsumption ......................................... 52

5.5. Heattransfercharacteristics..............................................................................58

5.5.1. Necessitytocharacterizethebioreactorsintermsofheattransfer ............ 58

5.5.2. CharacterizationofUAwithoutlateralairflow ............................................ 59

5.5.3. CharacterizationofUAwithlateralairflow................................................. 63

5.5.4. Estimationofoutsideheattransfercoefficient ............................................ 66

5.6. Masstransfercharacteristicsofdisposableshakingbioreactors ....................... 68

5.6.1. Oxygentransferrate................................................................................... 68

5.6.2. Correlationforvolumetricmasstransfercoefficient....................................70

5.7. Application......................................................................................................... 74

5.7.1. Scale-upofplantcellcultureprocess........................................................74

5.7.1.1. Ventilationinshakeflasks...................................................................75

5.7.1.2. CultivationofNtabacumcellcultureindifferentbioreactors...............78

5.7.2. Scale-upofanimalandinsectcellculture ..................................................81

6. Conclusion ..............................................................................................................83

7. References ..............................................................................................................84

Appendix A: Dimensionless numbers ......................................................................... 89

Appendix B: Symbols .................................................................................................... 90

Appendix C: Greek symbols ......................................................................................... 93

Appendix D: Proposed level o f hydro-mechanical st ress generation....................... 94


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Introductionandobjectives

1

1. IntroductionandobjectivesIn todays biopharmaceutical industry, a good technology portfolio, a strong

intellectual property position and access to capital do not guarantee success (1).

Flexibility,costeffectiveness,andtimetomarketarebecomingkeyissuesaswell(1-

4).Biopharmaceuticalcompaniesareallinaracetogettheirproductstomarketas

quicklyaspossiblesoastoattainthelargestpossiblemarketshare.Timelymarket

penetrationcanmakethedifferencebetweenablockbusterdrugandonethatbarely

makesaprofitablereturnonR&Dexpenditures(5).Thepotentiallossesinrevenue

resultingfromdelaysinproductapprovalcanbeconsiderable;itisoftenreportedthat

foramoderatelysuccessfuldrug(onewithannualsalesof$350million)eachdays

delaytomarketincursalossof$1million(6).Itisalsoknownthatbiopharmaceutical

productshavehigh failure rates(7).Therefore, decisionof future expansion ofany

product development process becomes bottleneck as this decisionmust bemade

quiteearlyduringproductdevelopmentstage.Suchdecisionsaredifficulttochange

laterduetoregulatoryconstraints(2,8).Hence,toachieveanacceptablereturnon

investment, biopharmaceutical companies focus on cutting down the cost of drug

development and improving the overall time-to-market. Therefore, companies are

moving rapidly towardsDisposableprocessingunits.Therewasatime inthenot-too-distantpastwhenall processing from laboratory scale toproduction scalewas

dedicated to glass, hard plastic and stainless steel components. Supporting such

equipmentsrequiredlabour,money,timeandeffort.Forexample,thebioprocessing

unitmustbeassembled,sterilizedandcleaned,whichrequiressupplies,labourand

downtime. Later, the equipments being used must be validated, maintained and

stored. With rigid, reusable components such as glass and stainless steel, cross-

contaminationbecomesanaddedrisk (9).Ontheotherhand,apart fromflexibility,

disposableunitshavethefollowingadvantages

Safety:Single-usebagsandcomponentseliminatetheriskofcross-contaminationEfficiency:Noneedofassembling,sterilizing,cleaningandvalidationSpace savings:EmptysystemscanbestoredinasmallspaceProductivity: With less down time and fewer time consuming duties the otherimportantissuessuchasresearching,developing,discoveringandproducingcanbemetquickly.Moreover,useofdisposableequipmentalsoallowsforquickchangeover


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Shakingbioreactors

2

between products, which is invaluable in the clinical phase of development, when

oftenmultiple productsare evaluated simultaneously. A key factordetermining the

speedtomarketofdisposables-basedprocessesisassociatedwiththedecisionof

whentobuildthemanufacturingfacility(9).Thesimplerconstructionofdisposables-

basedplantsimpliesthatshorterimplementationtimecanberealizedwhichallowsformoredetailedprocessoptimizationbeforemovingintoconstruction.Further,shorter

constructiontimemayallowforearlierentrytomarketandatalowerriskduetothe

smallerinvestmentinvolved(2,9).

Muchefforthasbeenmadeforeconomicevaluationofdisposables-basedprocessing

and compared with other traditional bioprocessing methods (9). Novais et al.

compared several important parameters such as capital investment, running cost,

utilities cost, net present value etc. for disposable option and conventional optionusingacasestudyoftherapeuticproteinproductionbyE.coli(9).Theinitialcapital

investmentforadisposablesoptionwassubstantiallyreducedto60%ofthatfora

conventional option. Utilities cost halved due to absence of operations like CIP

(Cleaning-In-Place) and SIP (Sterilization-In-Place). However, disposables-based

running costs increased by 70% of those of the conventional option. Despite the

higher value, the net present valueof the disposableplantwas positiveandwithin

25%ofthatfortheconventionalplant.Thenetpresentvaluewasidenticaltothatofa

conventionalplantwhenaninemonths reduction intimetomarketarisingfromthe

adoptionofadisposables-basedapproachwasincorporatedinthemodelofNovaiset

al.(9).However,authorsassumedthatthetwocultivationoptionshadthesameyield

of biomass and therapeutic protein production. The net present value was also

calculated for the disposablesoption when therewas a25% reduction inbiomass

yieldandproteinexpressionyieldencountered.Resultsrevealedthatwhilebiomass

yieldwas25%less,therewasonlyaslightdropinthenetpresentvalueto91%of

thatofthebasecasebuta25%lowerproteinexpressionlevelhadahighimpacton

thenetpresentvalue,decreasingto83%ofthebasecase.

Because of these tremendous advantages, the disposables-basedoption iswidely

acceptedintodaysbiopharmaceuticalindustries.

Althoughdisposablebioreactorswerestudiedmainlyinthelatenineties,itsveryfirst

concept and use was reported decades ago by Falch et al. (10). They cultivated

bacterialandfungalculturesin300mLshakingtetrahedronplasticbagswith50mLfillingvolume.Theauthorsreportedthat thecellgrowthwasnot limitedbecauseof


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3

hydrophobic nature of the bags and cells did not attached to the wall of plastic.

Becauseofthelimitationsoftheshakingmachinesavailable,theexperimentswere

carriedoutat130rpmshakingfrequencyand4.1cmshakingdiameter.However,it

tookthreedecadestoidentifythepotentialofthedisposables-basedprocessconcept

tilllatenineties.Thenumbersofdisposablebioreactorsavailablenowinthemarketare increasing in leaps and bounds. Some of the successful trade names are,

CELLine,WaveBioreactors,CellMakerLite2,XDRbioreactors,CellPharm,miniPerm

etc. However,most of the bioreactros have limited fluid handling capacity, except

Wavebioreactors,CellMakerLite2andXDRbioreactors.XDRandWavebioreactors

areavailable in thesize of 2L to 1000L, whereas thesize of theCellMaker Lite 2

ranges from 1L to 50L. In spite of numerous bioreactors available, the Wave

bioreactorsaremostsuccessfulinbiopharmaceuticalindustry.TheWavebioreactors

weredevelopedbySinghetal.(11).Singhetal.reportedsuccessfulscale-upofa

number ofprocesses baseduponplant,animal, insect cellcultureaswell as virus

cultures up to 100L in Wave bioreactors. Although other disposable bioreactors

exceptWavebioreactorswerealsoavailableinthemarketinlateninetiesbutallof

themhadproblemsinscale-up,therefore,couldnotbeusedforpilot-scaleproduction.

Table 1.1 compares costs associated for production of Secreted Associated

Phosphatase(SEAP) invariousdisposablebioreactor systemsaswell asstandard

stirredtankfermentors(12).Table1.1indicatesthattheproductioncostofSEAPis

minimum for Wave bioreactors. There are numerous papers available about

successfulcultivationofarangeofcelllinesinWavebioreactors.Inspiteoftheirease

ofhandlingtheWavebioreactorspossessfollowingmajordisadvantages,

Ill-defined operating conditions: Wave bioreactors are not defined in terms of veryimportant scale-up parameters such as mixing, power consumption and hydro-

mechanicalstressgeneration.Thereisareportofmeasurementofoxygentransfer

rateinWavebioreactorsbutitislimitedtoonlyafewoperatingconditionsandforonly

afewreactorsizes.Inbio-pharmaceuticalindustriesthelargescaleproduction(>1m3)

isstillperformed instandardstirred tankreactors.Therefore, itmaynot beeasyto

scale-up the process from Wave bioreactors to standard stirred tank fermentors

becauseoftheill-definedoperatingconditions.

Thin Wave bags prone to wear and tear: The Wave bags are relatively thin ascomparedtostandardcarboys,whichmaketheman idealchoiceasit savesmuch

space.Butthesebagsbecomemorepronetopunctures,whichmaycausesevere

accidentsespecially,whenworkingwithviruscultures.


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Shakingbioreactors

4

Cultivationsystem

CELLine1000

miniPerm-classic kit

Cell-Pharm-100(BR 130)

WaveCellbase20 SPS(1L***)


Standardstirred tank

(2L***)

SEAPactivity(U) 1156 1102.5 495 5160 10320 4120

Investmentcosts*

(SFr)15,000 16270 4095 30,000 30,000 73,320

Cultivationsystem

CELLine1000

miniPerm-classic kit

Cell-Pharm-100(BR 130)



Standardstirred tank

(2L***)

Costsperbatch

(SFr)250 272 69 500 500 1222

Runningcosts

(SFr)750 478 305 526 1052 877

Cultivationunit

costs(SFr)540 130 1150 295 590 0

Personalcosts

(SFr)1160 1160 1200 1160 2320 2000

Cost**per1000

unitsSEAP(SFr)2336 1850 5503 481 432 995

*Cultivationsystemandperipheralsystemse.g.CO2incubatoretc.**Calculationisbasedupon60experiments.Costsassociatedwithenergy,cellculturelaboratoryand

analyticalsystemsnotincluded.***Workingvolume

Table 1.1:ComparisonofproductioncostofSEAPinvariousdisposablebioreactorsystemsalongwithastandardstirredtankreactorsystem.CourtseyofEibletal.(12).

Costs: The Wave bioreactors are expensive because of their monopoly in thedisposables-basedbioprocessmarket.


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5

Incontrasttosuchanexpensivebioreactor,largeshakingbioreactorspresentedin

Figure1.1 giveapromising choice forcellculturesystems from laboratory scaleto

pilotscale.

Figure 1.1:A50L(totheleft)anda20L(totheright)largedisposableshakingbioreactormountedonacommerciallyavailableRC-6shakerfromKhnerAG,Switzerland.Theshakerhasafixedshakingdiameterof5cmandcanoperateatshakingfrequenciesfrom0to400rpm.

Inthisresearchwork,atypeofdisposablebioreactorisintroduced,whichisbasedon

theshakingtechnology(seeFigure1.1).Thesizesofthebioreactorsusedare2L,

20Land50L.Themainobjectivesoftheresearchworkare:

Characterization of mixing performance and identification of operating

conditionswherepoormixingisobserved. Characterization of disposable shaking bioreactors in terms of power

consumptionandheattransfer.

Investigation of mass transfer characteristics of large disposable shaking

bioreactors.

Applicationofthelargedisposableshakingbioreactorstoavailablecellculture

systems.


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Literaturereview

6

2. Literaturereview2.1. Shaking bioreactorsShakemixinghasbeenwidelyusedatsmallscalelevelinbiotechnologylaboratoriesandindustriesforthescreeningofvaluablemicro-organismsandinbasicbioprocess

development experiments. Their simple operation, easy handlingand low cost are

someofthemajoradvantages(13).

2.1.1. Mixing and power consumptionThemixingcharacteristicsofconicalshakeflaskswerepioneeredbySuminoetal.

(14, 15). They used a temperature method for the determination of powerconsumptioninsmallshakeflasksofsize250mL.Shakemixinginconicalshaking

flasksofsizeupto5LisextensivelystudiedbyBchsetal.(16-19).Bchsetal.used

differentshakingdiameters(1.25to7cm)anddifferentshakingfrequencies(100to

400rpm)withvaryingliquidviscositiesupto200mPas.Theauthorsalsousedfilling

volumes in the rangeof 5% to 20%of the total flaskvolume.Bchset al. derived

following dimensionless equation between modified Newton number (Ne) and

Reynoldsnumber(Re),

' -1 -0.6 -0.2Ne 70 Re + 25 Re +1.5 Re= 2.1

wherefollowingconditionofaxialFroudenumber(Fra)mustbesatisfied,

aFr 0.4> 2.2

where,3 4 1/3

'L

PNe

n d V=

,

2n dRe

= and

( )2

02

2a

n dFr

g

=

,

where,

Ne ModifiedNewtonnumber(-)

Re Reynoldsnumber(-)

Fra AxialFroudenumber(-)

P Powerconsumption(W)

Densityoffluid(kg/m3)


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Literaturereview

7

n Shakingfrequency(1/s)

d MaximumInsidediameterofthevessel(m)

VL Fillingvolume(m3)

Dynamicviscosityoffluid(Pas)

d0 Shakingdiameter(m)

However,differentgeometriesofconicalshakeflasksandcylindricalshakingvessels

may make it difficult to extrapolate the experimental results to large shaking

bioreactors. On the other hand, Kato et al. (20) reported mixing performance of

cylindricalshakingvesselsofsizefrom0.5Lupto12L,whereliquidheighttovessel

diameterratiowaskeptconstant.However,theoperatingconditionswerelimitedfor

all the characterizationexperiments.ThemeasurementsofKatoetal.werecarried

outincylindricalvesselsforshakingfrequenciesof100-200rpm,whiletheshaking

diameter was kept in the range of 1 to 4 cm. Kato et al. derived following

dimensionlessequationforpowerconsumptionincylindricalshakingvessels,

0

313

2-42

dNe 934 Fr Re

d

=

2.3

3 5

P Ne n d=

2.4

2

r

n dFr

g

= and

20n dRe

=

wherefollowingequationmustbesatisfied,

0.166 -0.176 0.135 Re Fr 0.135 Re' < < 2.5

where,

Ne Newtonnumber(-)

g Gravitationalacceleration(m2/s)

2.1.2. Mass transfer characteristics of shaking bioreactorsMass transfer characteristics of conical shaking bioreactors are studiedby various

authors. It is thoroughly investigated over awide rangeof operating conditionsby

Maieretal.(21).Followingtabledescribestheoperatingconditionsusedbyvarious

authors.


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Shakingbioreactors

8

Authors Flask size(ml)

Filling volume(% of flask vol.)

Shaking speed(rpm)

Shaking diameter(mm)

Haarde&Zehner(22) S/SB(250,1000) 5%-20% 50-330 Notmentioned

Henzler&Schedel(23) S(1000) 5%-20% 150-400 25,50

VanSuijdametal.(24) S/SB(500) 8%-40% 230,320 25

Veglioetal.(25) S(300) 33%-50% 150-250 32

Veljkovicetal.(26) S(300,500,1000) 5%-20% 0-400 20

Maieretal.(21) S(50-1000) 4%-16% 50-500 12.5-100

Table 2.1:Investigationofmasstransfercharacteristicinshakeflasksbyvariousauthors. S denotesconicalshakeflaskandSB denotesconicalshakeflaskwithbaffles.Maieretal.usedasulfiteoxidationmethodformeasurementofoxygentransferrate

in shaking bioreactors of a size up to 1L at different filling volumes and shaking

diameters.Theydevelopedatwosub-reactormodelforthegas-liquidmasstransfer

inshakingbioreactors.Accordingtothismodel,themasstransfercharacteristicsinshaking flasks can be divided into a film reactor and a stirred tank reactor. The

experimentallyfoundvolumetricmasstransfercoefficientwasthencomparedwiththe

modelandcanbedescribedasfollows,

0~-0.83 1.16 0.38 1.92

L Lk a V n d d 2.6

where,

kLa Volumetricmasstransfercoefficient(1/s)

VL Fillingvolume(mL)

n Shakingfrequency(1/min)

d0 Shakingdiameter(cm)

d Maximuminsidediameteroftheflask(cm)

Katoetal.investigatedtheoxygentransferratebyadynamicgassing-inmethodin

cylindrical shaking vessels of diameter 12 cm and 15 cm with a liquid height to

diameterratioof0.5,1,and1.5(27).Theshakingfrequenciesusedwereintherange


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Literaturereview

9

of100to200rpmandtheshakingdiameterwaskeptintherangeof1to4cm.Mass

transfer occurred mainly on the surface of the bulk liquid and, hence, it was

characterizedintermsofpowerconsumptionasfollows,

~ 0.4 -0.6 -0.25L Vk a P H d 2.7

where,

kLa Volumetricmasstransfercoefficient(1/s)

PV Powerconsumptionperunitvolume(W/m3)

H Heightofthefluidinsidethevessel(m)

d Maximuminsidediameterofthevessel(m)

2.1.3. Ventilation in shaking bioreactorsHenzler&Schedel(23)measuredthemasstransferresistanceofsterileshakeflask

closures. Based on their basic model, Mrotzek et al. (28) developed an extended

model for determinationofresistanceofsterile closures inshakeflasks ofdifferent

sizes. Mrotzek etal. used sterile plugsmade of cotton, paper, urethane foamand

fibreglassalongwithcapsmadeofaluminiumandsilicon.Theauthorsfoundthatthe

masstransferresistancewasmainlydependentonneckgeometry,flaskclosureand

itspackingdensity.

2.2. Application of disposable shaking bioreactorsInfact,Millardetal.(29)investigatedrecombinantproteinexpressionbasedon E.coli

culturesin2Lpolyethylenebeveragebottles.Thenotchesatthebottomofthebottle

servedasbaffles.Atlowfillingvolumes(0.25L)thecellgrowthandproteinexpression

wasalmostdoubleascomparedtothatfoundin2Lbaffledshakeflasks.Athighfillingvolumes(1L) thecellgrowthandproteinexpressionwassimilar tothat foundin2L

baffledshakeflasks.Millardetal.(29)currentlyemploythese2Lbeveragebottlesfor

proteinexpressionbecauseoftheireaseofhandlingandreductioninlabourandtime.

Mller et al. (30) also cultivated HEK-293 EBNA and CHO-DG44 cell lines in 1L

borosilicatesquareshakingbottles.Theyemployeddifferentfillingvolumesat2.5cm

shakingdiameter.Thelivecellcountwas2to3timeshigherthanthatobtainedin

normal2Lspinnerflasks.Mlleretal.(30)observedoptimalcellgrowthandviabilityat30-40%fillingvolume,130rpmand2.5cmshakingdiameter.Useofthesesimple

thoughhighlyefficientshakingbioreactorsisnotlimitedto2Lscaleonly.Liuetal.(31,


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Applicationofdisposableshakingbioreactors

10

32) already showed the potential of large cylindrical shaking bioreactors for the

cultivationofanimalandinsectcellculturesatpilotscale.

Liuetal.(31)werethefirsttoscale-upproductionprocessesbasedontheanimaland

insectcelllinesindisposablelargeshakingbioreactors.Theysuccessfullycultivated

hybridoma cells, CHO cells and insect cell linesSf-9 andH-5. Normal batch, fed-

batch,semi-continuousandcontinuousoperationmodeswereused.Liuetal.used

differentsizesofcylindricaldisposableshakingbioreactorsrangingfrom3Lto50L.In

all the cases the cell growth was better than that obtained by spinner flasks or

standard fermentors. Sf-9 and H-5 cells have a higher oxygen demand than

mammaliancells.Amaximumviablecelldensityof14x106cells/mLwasachievedin

20Lbioreactorwith4LfillingvolumeduringcultivationofSf-9cells(Figure2.1).Singh

etal.reportedamaximumcelldensityof4.75x106

cells/mLduringafad-bathprocessbased on baculovirus/Sf-9 cells in a 20L Wave bioreactor system with 1L Bio-

Whittaker X-press insect cell medium. Similarly an expression system containing

Baculovirus/H-5 was scaled-up successfully in 20L shaking bioreactor with a

maximumviablecelldensityof7x106cells/mL.Themaximumcelldensityofthesame

baculovirus/H-5systemattainedinspinnerflaskandstandard3Ljarfermentorwas

3x106cells/mLand2.5x106cells/mL,respectively.

Figure 2.1:Insectcells(Sf-9)culturedin20Lpolypropylenebioreactors.Culturevolume:7L.Shakingdiameter5cm.ThisphotoisacourtesyofDr.Liu(personalgift).


17/102

Literaturereview

11

Figure 2.2:Hybridomacellsculturedina50Lpolypropylenebioreactor:culturevolume36L.Shakingdiameter5cm.ThisphotoisacourtesyofDr.Liu(personalgift).

Liu et al. (31) also evaluated the IgG production usinghybridoma cells in shaking

bioreactorsrangingfrom3Lto50Lsizeusingasemi-continuousmode(Figure2.2).

Theexperimentswereconductedwithan11%exchangeoftheculturebrothperday

withfreshmedium.IgGproductionreached150mg/Lperdaywhilemaintaining2 x106

viablecells/mL.Amaximumof250mg/Lof IgGwasproducedinthesameprocess

afterterminationofthedailyexchangeofbrothwiththefreshmedium.Acontinuous

process for the IgG production was maintained for 70 days in a 50L shaking

disposablebioreactorwith14.5Lfillingvolume.

A cultivation of CHO cells in a 20L disposable bioreactor with 5L filling volume isshowninFigure2.3.CHOcellswerealsogrowninafed-batchmodeina50Lshaking

bioreactorwithamaximumviablecellcountof6 x106cells/mL.Themaximumviable

CHOcellcountobtainedinafed-batchprocessina20LWavebioreactorwas4.5x106

cells/mLat6Lfillingvolume(11).Shakingbioreactorsof20Land50Lscalewitha

workingvolumeof5-10Land30-35L,respectivelyareroutinelyemployedbyLiuetal.

(31)(atRocheDiscoveryTechnologiesDepartmentinNJ,USA)togrowsuspension

adaptedmammalian (e.g., CHO, HEK293), insect cells andHel A and B cells for

recombinantproteinexpressionandlivecellproductiontosupporthighthroughput

drugscreeningprogramsatpilot-scalelevel.


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Applicationofdisposableshakingbioreactors

12

Figure 2.3:CHOcellsculturedin20Lpolycarbonatebioreactors.Culturevolume5L.Shakingdiameter5cm.ThisphotoisacourtesyofDr.Liu(personalgift).

Inspiteofsuccessfulcultivationofdifferentcellcultures,Liuetal.(31)stressedthe

needoffurthercharacterizationofthesesimplethoughefficientshakingbioreactors.


19/102

Theory

13

3. Theory3.1. Conventional temperature methodTheconventionaltemperaturemethodwasdevelopedbySuminoetal.(14).Theheatbalanceforagivenshakingvesselcanbedefinedbythefollowingequation,

PTTUAdt

dTCm of

f

p = )( 3.1

where,

m Massofthefluid(kg)

cp Specificheatcapacity(J/kg/K)

Tf Fluidtemperature(K)

t Time(s)

UA Overallheattransfercoefficient(W/K)

A Heattransferarea(m2)

To Temperatureofthesurroundingatmosphere(K)

where,-mCp(dTf/dt),UA(Tf-To)andPdenotethecoolingrateoftheliquid,the

heatlosstothesurroundingsandtheheatgenerationratebecauseofdissipationof

power.Accordingtotheexistingtheorythepowerconsumptionaswellastheoverall

heat transfer coefficient remains constant over time at given operating conditions.

Therefore, these two parameters can beestimated from the data of the liquid and

roomtemperatureprofileovertime,asdescribedbySuminoetal.(14,15).

3.2. Extended temperature methodIntheextendedtemperaturemethodthepowerconsumptionorheatgenerationrate

isnottakenasaconstantvaluebuttreatedasavariablevalue,whichchangeswith

respecttothetemperature.Thedynamicbehaviourofthepowerconsumptioncanbe

incorporatedintheextendedmethodasfollows:

AccordingtoBchsetal.(17)powerconsumptionofshakeflaskscanbedefinedin

termsofthedimensionlessmodifiedNewtonnumber(Ne'),


20/102

Extendedtemperaturemethod

14

3 4 1/3'

L

PNe

n d V=

3.2

Ne'canbecalculatedasafunctionoftheReynoldsnumber(Re)asfollows,

0.20.61

ReReReNe

++= 5.12570' 3.3

where,

2n dRe

= 3.4

Equation3.2 and 3.4 show that ata given operating condition,witha fixedvessel

diameter,Reisafunctionoftheliquidphysicalproperties,i.e.viscosityanddensity.

However,inthetemperaturerangesencounteredinthetemperaturemethodforthe

measurementofthepowerconsumptionotherphysicalpropertiesexceptviscositydo

notvarytoagreatextent(variation


21/102

Theory

15

onlythesecondandthirdtermofequation3.3istakenintoaccount,hence,Ne'can

becorrelatedwithReas,

' -0.6 -0.2fit1 fit2Ne C Re +C Re= 3.8

Hence,foreachtemperatureprofileobtainedforagivenoperatingcondition,therearethreeparameterstobefittedasdescribedinequation3.1and3.8,namely, UA,Cfit1

andCfit2.Itshouldbenotedherethatintheconventionaltemperaturemethod,power

consumptionwasthefittingparameter,butnowtheyaretheparametersCfit1andCfit2,

which describes the hydrodynamic behaviour of the system. Henceforth, power

consumption will vary according to the relationship given in equation 3.5 with

temperature. However, the power consumption measured with the extended

temperature method can only be specified with respect to a given standardtemperature.Thistemperaturewaschosentobe30Cinthisthesis.

3.3. Oxygen transfer rate measurement3.3.1. The sulfite systemMass transfer characteristics of solutions having water-like viscosity are readily

measured by an aqueous chemical model system developed and well studied by

LinekandVacek(35).Thismodelsystemcomprisesofasodiumsulfitesolutioninaspecificbuffer.ThesulfiteionsareoxidizedintosulfateionsinpresenceofCo+2,Cu+2,

Fe+2orMg+2,actingascatalysts.Theoxygentransferredfromtheairtotheaqueous

solution characterizes the mass transfer capacity of a given reactor at a given

operatingcondition.Theirreversibleoxidationreactioncanbewrittenas,

22 23 2 4

1

2

CoSO O SO+ + 3.9

Themasstransferrate(transferofoxygenfromairtosolution)aswellasthereaction

rate defines the oxidation reaction regime. The reaction regime can bedefined as

dimensionlessHattanumber(Ha),

reaction rate=

mass transfer rateHa

Depending upon the valueofHa, the reaction regimes can bedefinedasdelayed

reaction regime (Ha


22/102

Oxygentransferratemeasurement

16

oxidation reaction must take place in the non-accelerated reaction regime to

determinethemaximumoxygentransferrate(36).

ThecompletionoftheoxidationreactioncanbeeasilymeasuredusingapHindicator

sincesulfateionsaremoreacidicthansulfiteions.Thereareanumberofparameters

which influence the oxidation mechanism of above mentioned reaction, such as,

concentration of buffer solution, pH of the solution, ionic strength, catalyst

concentration etc. All these parameters were very well studied and optimized for

shakingbioreactorsbyMaieretal.(21)andHermannetal.(36).Maieretal.studied

themasstransfercharacteristicsinconicalshakingbioreactorsatdifferentoperating

conditions using above mentioned optical sulfite oxidation method in the non

acceleratedreactionregime.Theauthorsalsomeasuredandcomparedtheprogress

of the oxidation reaction online using the Respiration Activity Monitoring System(RAMOS)developedbyAnderleiandBchs(37).

3.3.2. Online oxygen transfer rate measurement

time

Sensorsignal

21 21

1: rinsing phase

2: measuring phase

Um

m

U

time

Sensorsignal

21 21

1: rinsing phase

2: measuring phase

Um

m

U

Figure 3.1:Sensorsignalprofileobservedduringthemeasurementofonlineoxygentransferrate.Um=sensorsignalatmidpoint,m=slope,U=sensorsignalatsteady-state.

TheRAMOStechnologyisbasedontheprincipleoftherateofchangeoftheoxygen

partial pressure in a closed head space of a shake flask. An oxygen sensor is

mounted on the top of the shake flask whichmeasures the change in the partial

pressureofoxygen.Theshakeflask isaeratedforaspecificperiodoftime(rinsing


23/102

Theory

17

phase)andthenaerationisstoppedforaspecificperiodoftime(measuringphase).

Therateofchangeoftheoxygenpartialpressureismeasuredduringthemeasuring

phaseusingtheoxygensensor.Theoxygentransferrateisthendeterminedusinga

material balance on oxygen in the head space. Diagrammatically the process is

showninFigure3.1.AsshowninFigure3.1,duringtherinsingphase,theoxygenpartial pressure reaches a steady-state value which is followed by themeasuring

phase. The oxygen sensor measures the rate of decrease of the oxygen partial

pressurefromwhichtheoxygentransferrateiscalculated.Afteradefiniteperiodof

time the rinsingphasestarts again. The oxygen transfer rate calculationstepsare

explainedbelow.

3.3.3. Calibration of the oxygen sensorThereisa linearrelationshipbetweentheoxygenpartialpressureandthevoltageof

thesensor.Thisrelationshipcanbewrittenas,

2op a U b= + 3.10

where,

a Slopeofthecalibrationcurve(bar/V)

b Interceptonthey-axis(bar)

Atwopointcalibrationisperformedtoavoidanysensordrifts.Voltageofthesensoris

measured at 0% partial pressure of oxygen and the value is taken as the first

calibrationpoint.Airisthenpassedthroughthesystemandvoltageofthesensoris

measured till steady-state condition is reached.This value is takenas the second

pointofthecalibrationcurve.

Mathematically,

letU0andPo20bethevoltageandoxygenpartialpressureat0%oxygen,

letUandPo2bethevoltageandoxygenpartialpressureatsteady-state.

Then,fromequation3.10itcanbeconcludedthat

2 2

0

o odp p dU

dt U U dt

=

3.11


24/102


18

3.3.4. Material balance on oxygen at steady-state (in the rinsing phase)

VL

2

in

oy

inn&

2oy

outn&

VG2 2

out

o oy y=

2o

n

&

VL

2

in

oy

inn&

2oy

outn&

VG2 2

out

o oy y=

2o

n

&

Figure 3.2:Molarflowofoxygeninandoutofthesystemintherinsingphase.Here,VLrepresentsfillingvolumeandVGrepresentsheadspacevolumeintheshakeflask.

Since,thegasphaseiswellmixed,

2 2

out

o oy y= ,therefore,

molaroxygenbalanceatsteady-statecanbewrittenas,

2 2 2

in in out out

o o oy n y n n = +& & & 3.12

where,

2

in

oy Molefractionofoxygeninthegasphaseenteringthesystem(-)

inn& Molarflowrateofairenteringthesystem(mol/s)

2 2

out

o oy y= Molefractionofoxygeninthegasattheendoftherinsingphase(-)

outn& Molarflowrategoingoutofthesystem(mol/s)

2on& Molarflowrateofoxygentransferredfromthegastotheliquidphase(mol/s).

Materialbalancebasedonthetotalmolarflowratescanbewrittenas,

2

in out

on n n= & & & 3.13

Fromequation3.12and3.13itcanbewrittenas,

2 2

2

in in

o o

o in

o

n ny

n n

=

& &

& & 3.14

Molarvolumeofagascanbewrittenas,

wellmixedgasphase


25/102

Theory

19

N

m N

R TV

p

= 3.15

where,

mV Molarvolumeofgasatnormaltemperatureandpressure(L/mol)

R Idealgasconstant(8.31410-2barL/mol/K)

NT Normaltemperature(273.15K)

Np Normalpressure(1.013bar)

Oxygentransferratecanbedefinedas,

2o Ln OTR V

= & 3.16

where,

OTR Steady-stateoxygentransferrateattheendofrinsingphase(mol/L/s)

Accordingtoidealgaslaw,

pn V

R T

=

&& 3.17

where,

n& Molarflowrate(mol/s)

V& Flowrateofagas(L/s)

p Pressure(bar)

T Temperature(K)

Since,yi=pi/p,equation3.14canberearrangedas

2

2

in ino

m

Lo in

m

L

p VV OTR

p Vp p

VV OTR

V

=

&

& 3.18

where,

yi Molefractionofcomponenti(-)


26/102


20

pi Partialpressureofcomponenti(bar)

inV& Totalvolumetricflowrateenteringthesystem(L/s)

The unknown parameterOTR in equation 3.18 can be calculated from themass

balanceofoxygeninthemeasuringphase.

3.3.5. Material balance in measuring phase

VL

VG

2on&

2odn

dtA

VL

VG

2on&

2odn

dtA

Figure 3.3:Molarflowofoxygenduringmeasuringphase.Here,VLrepresentsfillingvolumeandVGrepresentsheadspacevolumeintheshakeflask.

Oxygen in the head space is transferred into liquid during the measuring phase,

therefore,

2

2

o

odn n

dt= & 3.19

Theoxygentransferrate(OTR)canbedefinedas,

2 21o o

L L

n dnOTR OTR

V V dt = =

&

3.20

Usingtheidealgaslawrelationship,aboveequationcanbewrittenas,

21 oG

L

dpVOTR

V R T dt =

3.21

where,

GV Volumeofgasintheheadspace(L)

Combiningequation3.11and3.21resultsin

21 oG

L

pV dUOTR

V R T U U dt

=

o 3.22


27/102

Theory

21

Inthemeasuringphaseoxygenpartialpressuredecreasesandhenceoxygentransfer

rate also decreases which may result in non-liner decrease in the voltage of the

sensor. However, the duration of the measuring phase is always very short and

hence, during this short duration, the decrease in oxygen partial pressure can be

takenasnegligible.Therefore, the decrease in the sensor signal can be takenaslinear.Hence,

dUm constant

dt= = 3.23

where,

m Slopeofthedecreasingvoltagesignaloftheoxygensensor(V/s)

Therefore,theoxygentransferrateusingtheslopemcanbecalculatedas,

21 oG

m

L

pVOTR m

V R T U U

=

o 3.24

where,

OTRmOxygentransferratecorrespondingtoslopem(mol/L/s)

However,thisoxygentransferratecorrespondstothemidpointofthesensorsignalUmandnotU.Theactualoxygentransferratecanbecalculatedasfollows.

Oxygentransferratecanbedefinedas,

( )2 2L o o

OTR k a c c= 3.25

where,

c

*

O2 Oxygenconcentrationatthegas-liquidinterface(mol/L)cO2 Oxygenconcentrationinthebulkliquid(mol/L)

The reaction regime of the sulphite system being used is in the non-accelerated

reactionregime,where0.03


28/102


22

where,

k1 First order reaction constant of sulfite oxidation (0.6625 1/s,accoding to

Hermannetal.(36))

Theoxygensolubilityintheliquidphaseisverylow,therefore,accordingtoHenrys

law,

2 2o oHe c p = 3.27

where,

He Henryslawconstantforoxygeninsulphitesystem(barL/mol)

Fromequation3.10,3.26and3.27,itcanbewrittenas,

( ) ( ) ( )2 2o o

OTR c p U 3.28

Hence,fromequation3.18,3.24and3.28,theoxygentransferratecanbecalculated

as

( )( )

2 4

2

in

G m

m

m L

A A m p V V V U U T ROTR

V V U U T R

=

o

o

&

3.29

Where,

( )in m GA V U U T R V V m p= + o& 3.30

Since 0U U


29/102

Theory

23

where,

OTRmax Maximumoxygentransferrate(mol/L/s)

3.4. Ventilation in shake flasksTheshakeflasksareclosedwithsterileclosuresinbiotechnologylaboratories.The

ventilationofair from the surroundingatmosphere into the headspace of the flask

dependsonthegeometryoftheneckoftheflaskandtypeofsterileclosure(28,38).

AccordingtoMrotzeketal.(28),theoxygentransferratebecauseoftheventilation

throughthesterileclosurecanbedefinedas,

( )2 2

1plug plug O out O

L abs

OTR k p p

V p

=

3.34

where,

OTRplug Oxygentransferratethroughtheflaskclosure(mol/L/s)

Kplug Gastransfercoefficient(mol/s)

VL Flaskfillingvolume(L)

pabs Absolutepressure(bar)po2out Partialpressureofoxygeninthesurroundingatmosphere(bar)

po2 Partialpressureofoxygenintheheadspaceoftheshakeflask(bar)

Theoxygentransferredthroughtheaerationofshakeflaskcanbegivenas,

( )2 2

inflow O out O

abs m

qOTR p p

p V=

3.35

where,

OTRflow Oxygentransferratebecauseofaerationofflask(mol/L/s)

Vm Molarvolumeofair(L/mol)

qin Specificaerationrateintheflask(L/L/s)

Equatingequation3.34and3.35,resultsinaaerationvaluewhichisequivalenttothe

ventilationthroughthesterileclosure,mathematically,


30/102

Ventilationinshakeflasks

24

min plug

L

Vq k

V= 3.36

Thegastransfercoefficientcanbeobtainedbytheneckgeometryoftheflaskandthe

diffusioncoefficientofoxygenthroughtheflaskclosure(28).Thevalueofgastransfer

coefficientand oxygen transfer rate through flaskclosure can becalculatedby the

combinationofthemodeldevelopedbyHenzlerandSchedel(23),Mrotzeketal.(28)

and unsteady-state model developed by Amoabediny et al. (39). From the gas

transfercoefficient,theaerationvaluescanbecalculatedbythemodeldevelopedby

Amoabedinyetal.(40).Detaileddescriptionofthesemodelsisbeyondthescopeof

thisthesis.Therefore,readersarerequestedtoreadthecitedreferencesforthorough

understandingoftheventilationinshakeflasks.


31/102

Materialsandmethods

25

4. MaterialsandmethodsForalltheexperiments2Lpolycarbonate(PC)bottlesand20Land50Lpolypropylene

(PP) bottles (Nalgene, USA) were used. A table-top shaker (LS-W, Kuehner AG,

Switzerland)wasusedtoimpartshakemixingin2Lbioreactorsandacommercially

available pilot scale shaker RC-6 (Kuehner AG, Switzerland) was used to impart

shakemixingin20Land50Lbioreactors.Alltheexperimentswereperformedat5cm

shaking diameter. All the chemicals were obtained from ROTH, Germany, unless

otherwisestated.

4.1. Hydrophilic shake flasksShakeflasksofsize5Lwerewashedwithdeionizedwater.Laterca.1Lofa65%(v/v)

nitricacidsolutionwaspouredintotheshakeflasks.Followingthis,theshakeflasks

wereheatedonaheatingplateinanexhaustchamberandthesolutionwasbrought

toboil.Theywerekeptunderthisconditionfor5min.Then,theshakeflaskswere

removedfromtheheatingplateandcooleddowntoroomtemperature.Subsequently

theseflaskswerethoroughlywashedwithdeionizedwateranddriedinadryingoven

at50Cfor24h.

4.2. Hydrophobic shake flasksTheinnersurfaceofthe5Lshakeflasksweremadehydrophilicasdescribedabove.

Thentheinnersurfaceoftheshakeflaskwasmadehydrophobicbysilanisation.A5%

(w/v) Dichlorodimethylsilan (Sigma) solution was prepared in toluene (Sigma) and

about1Lof itwaspoured intoeach flask.Subsequently, the flaskswerevigorously

shakenunderthehoodfor15mininsuchawaythattheliquidmadeauniformfilmall

overtheinnersurface.Theremainingsolutionwasdiscardedfromtheshakeflasks.The flasks were kept in an exhaust chamber at room temperature for 24 h to

evaporateremainingDichlorodimethylsilansolution.

4.3. Mixing performanceMixingperformanceofthedisposableshakingreactorswasmeasuredbyanelectrical

conductivity method (20). The vessel was filled with deionized water. At given

operating condition, the tracer, 0.5 mL of 1M sodium chloride, was addedinstantaneouslyintothevesselatsteadystate.AsshowninFigure4.1,theelectrical


32/102

Measurementofpowerconsumption

26

conductivityoftheliquidwasmeasuredbyaconductivitymeterafteradditionofthe

tracer.Themixingtimeisdefinedasthetimerequiredfor99%ofthetotalchangein

concentrationresultingfromtheadditionofthetracer.

3

M5

4

1

2

3

MM5

4

1

2

Figure 4.1:Experimentalsetupforelectricalconductivitymeasurementin20Land50Lvessel.1)Cylindricalvessel,2)Electrode,3)Electricalconductivitymeter,4)Shakingmachinemotor,5)Mechanicalsupport.

4.4. Measurement of power consumption4.4.1. Torque methodPowerconsumption in2Land 20L disposable reactorsmountedona shaker table

wasmeasuredbythemethoddevelopedbyBchsetal.(41).Themethodisbasedon

themeasurementofthetorquedevelopedbytheliquidwhichrotatesaroundtheaxis

ofthevessel.The torque isgeneratedontheaxisofthemotor.Mechanical friction

lossesandwindresistanceofthevesselarecompensatedbymeasuringthetorque


33/102

Materialsandmethods

27

generatedbyadeadweightwhichshouldbe thesameas thatoftherotatingliquid

andthevessel.

B

1

2

A

3

B

1

2

A

3

Figure 4.2:ExperimentalsetupforthedeterminationofthepowerconsumptionbythetorquemethodinA)2LcylindricalvesselsandB)20Lvessel.1)Shakertable,2)Torquesensor,3)Shakermotor.

Thepowerconsumptioncanbecalculatedbyfollowingequation,

( )

LK

21

L V

n2MM

V

P

= 4.1

where,

M1 Torquedevelopedbyrotatingliquid(Nm)

M2 Torquedevelopedbythedeadweight(Nm)ZK Numberofshakeflasksmountedontheshakertable(-)

Figure4.2AandBshowthearrangementsusedfortorquemeasurementfor2Land

20Lvessels,respectively.AsshowninFigure4.2A,four2LPCbottlesinsteadofone

were mounted on the shaker table to maximise the accuracy in the torque

measurements. The shaker was rotated by the external motor which had an

integrated torque sensor (ViskoPakt, HiTech-Zang, Germany). The control of this

motorwasautomatedbyaLabViewsoftware(NationalInstruments,Germany)which

also recorded the data from the torque sensor. Figure 4.2B shows the same


34/102

Measurementofpowerconsumption

28

arrangementfora20LPPvessel.Tokeepthetorqueproducedwithinthelimitsofthe

torque sensor (0 to 1 Nm), only one 20L vessel was used. Measurements were

performedatdifferentshakingfrequenciesaswellasdifferentfillingvolumes.

4.4.2. Conventional temperature methodPower consumption in disposable bioreactors was measured by the method

developedbySuminoetal.(14).Themethodisdescribedinsection3.1.Figure4.3A

andBshowsthearrangementsusedtomeasurepowerconsumptionfor2L,20Land

50Lvessels,respectively.Aninsulated2Lvesselwasused.Polyurethanefoamwas

used to make insulated vessel from anon-insulated vessel of the same size. The

insulation was done by Mr. H. Ptz at Institute frWerkstoffe der Elktrotechnik,

RWTHAachen.Thediameterandheightofthe2Lvesselwas12.5cmand20cmrespectively.Thicknessofthepolyurethanefoaminsulationwas3cm.Itwasproved

inpreliminaryexperiments that the insulationwas necessary toprevent rapid heat

losses tothe surrounding.Temperatureof the liquid inside the20Land50L vessel

wasmeasuredasshowninFigure4.3.

2

2

3

4

5

B

1

A5

2

2

3

4

5

B

1

A5

Figure 4.3:Measurementofpowerconsumptionusingthetemperaturemethodin(A)insulated2Lcylindricalshakingvessel,(B)insulated20Landnon-insulated20Land50Lcylindricalshakingvessel.1)Shakingtable,2)temperaturesensor,3)shakingmachinemotor,4)heatinsulation,5)digitalmultimeter


35/102

Materialsandmethods

29

Sincepowerconsumptionina20Land50Lvesselishigherthanthatobtainedin2L

vessel,insulationofthe20Land50Lvesselwasnotrequired.Theheight,diameter

andwallthicknessof20Lvesselwere40cm,27cmand4mm,respectively.The

height, diameter and thickness of 50L vessel were 50 cm, 38 cm, and 15 mm,

respectively.Theentiresurfaceofthe20Lcylindricalvesselexceptitsopeningwascoveredwith5cmthickpolyurethanefoamtomakeinsulatedvessel.Theinsulation

was done by Mr. H. Ptz at Institute fr Werkstoffe der Elktrotechnik, RWTH

Aachen.APT100(Conrad,Germany)temperaturesensorwasmountedatthecentre

ofthevesselusingathinstainlesssteelrodtomeasuretheliquidtemperature.The

roddidnotresultinsignificantadditionaldisturbanceoftheshakemixingbecauseof

itscentrallocation(42).Thetemperatureofthesurroundingairwassimultaneously

measured by another PT100 sensor. Both sensors were connected to digital

multimeters(GMC-instruments,Germany)torecordthetemperatureovertime.The

fluid filling volumes in the range of 20% to 75% were employed. The shaking

frequencywasvariedfrom100to300rpm.Thefluidatroomtemperaturewaspoured

intothenoninsulated20Land50Lvesselsandthenheatedtoca.5Chigherthanthe

desired initial temperatureby immersion heater (KarlRothGmbH,Germany). This

differencewasaccountedforinitialadjustmentssuchasclosingthevessellid,setting

updesiredshakingfrequencyetc.Whenusing2Linsulatedvessel,previouslyheated

fluidtoabout50Cwaspoured.However,for20Linsulatedvessel,fluidwaspoured

atroomtemperature.Thevesselwasclosedair-tightatthetopandoperatedatgiven

operatingconditions.Due tothepowerconsumption inducedbyshaking, the given

fluidwasheatedupin the20L insulatedvessel,whereasitwascooleddownin the

20L and 50L non-insulated vessels and 2L insulated vessel until a steady state

temperature difference between the fluid and surrounding air was reached. The

temperatureprofileofthefluidinnon-insulatedandinsulatedvesselsweremonitored

over time using above mentioned Pt100 temperature sensors. This data oftemperatureprofileoffluidbeingheateduporcoolingdownwasusedtodetermine

theparametersPandUAinequation3.1.Forthispurpose,amathematicalmodel

was developed in the differential equation solver software ModelMaker (Cherwell

Scientific, UK), fitting the simulation to the measured data by optimising the

parametersPandUA.


36/102

Determinationofoverallheattransfercoefficient(UA)

30

4.4.3. Extended temperature methodTheexperimentalsetupfortheextendedtemperaturemethodremainedthesameas

inthecaseoftheconventionalmethod.Theliquidandroomtemperatureprofileswere

measured in a similar way as described in the conventional temperaturemethod.

However,theanalysisof theexperimentaldatadifferedfromthatoftheconventional

method. A model was developed as described in section 3.2 in the differential

equationsolversoftwareModelMaker(CherwellScientific,UK),fittingthesimulation

tothemeasureddataforeachoperatingconditionbyoptimisingtheparametersUA,

Cfit1andCfit2.Consistencyofthemodelparameterswasvalidatedbyimplementingthe

samemodel in the gPROMS (PSELtd,UK) process simulationsoftware. Dynamic

parameter estimation was performed using all the experimental data sets

simultaneously.

4.5. Determination of overall heat transfer coefficient (UA)4.5.1. Characterization without lateral air flowAn 80% (w/w) glycerol-water mixture and water were used as fluids. Figure 4.3B

represents the experimental set up used for measurement of liquid and room

temperatureprofile. The procedure for determination ofUA remained the same as

describedinsection4.4.3.

4.5.2. Characterization with lateral air flowThe experimental set up used for determination of UA with lateral air flow is

representedinFigure4.4.Waterwasusedasafluidin20Land50Lnoninsulated

vesselwith50%and40%fillingvolumes,respectively.Theshakingfrequenciesfrom

150 to250 rpmwereemployed.Theexperimental procedure todetermine the fluid

temperature profile remained the same as described in section 4.4.2. However,producing andmeasuring the lateralair flowwas different. The lateralair flowwas

producedbytworevolvingfans(HV-181E,Honeywell,Germany)placedoppositeto

eachotheroneithersideoftheshaker.Thefanswereplacedinsuchawaythatthe

aircurrentproducedbythemwouldbreaktheswirloftheairproducedbecauseofthe

shakingmotionofthevesselandthusenhanceturbulenceinaircurrentaroundthe

vessel.Thedistanceofthefanwithrespecttotheaxisoftheshakingvesselisgiven

inFigure4.4B.Thedistanceof80cmwasthenearestfromtheshakingmachine.The


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Materialsandmethods

31

farthestdistanceofthefansfromthecentreofthevesselwasca.100cmbecauseof

thelackofthespaceintheshakingmachineroom.

Figure 4.4:Experimentalsetupfordeterminationofoverallheattransfercoefficientwithlateralairflow.1)shakertable,2)temperaturesensor,3)motor,4)digitalmultimeter,5)fan,6)anemometer

The air flow was controlled by a 4-stage fan speed regulator. The fans produced

maximumlocalairvelocityof9m/s.However,theairvelocitiesshowninthisthesis

areaverageairvelocities.Theaverageairvelocitywasmeasuredbyananemometer(Windmaster 2, Conrad, Germany) over the entire surface of the vessel. The


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Determinationofoxygentransferrate(OTR)

32

anemometercalculatedandshowedtheaverageairvelocityalongwiththemaximum

and minimum local air velocity. The fan position was tilted manually about the

horizontalaxiswheneverrequiredtominimizethedifferencebetweenthemaximum

andminimumlocalairvelocitiesandthusminimizecavitiesalongthesurfaceofthe

vessel.Themaximumaverageairvelocityof6.5m/swasencounteredusingtwofans.Thedeviationof0.2 to 0.5m/swasencounteredforaverageairvelocitiesupto4.5

m/s.Thisdeviationincreasedfrom0.8to1.5m/sforaverageairvelocitiesfrom4.5

to6.5m/s.ThemathematicalprocedureremainedthesamefordeterminationofUA

withoutlateralairflowanditsvaluewascalculatedasdescribedinsection4.4.3.

4.5.3. Measurement of heat transfer area

Forsimplicity,itwasassumedthattheshakingvesselisaperfectcylinder.Waterwasusedasafluid.BromothymolBluewasaddedtogivebluecolortowaterandenhance

visibilityofthesiquelformedinsidethevessel.Thedisposablebioreactorwasfilled

with different fillingvolumes in the rangeof25% to75%. The shaking frequencies

usedwereintherangeof100rpmto300rpm.Ameasuringscalewasmarkedfrom

thebottomtothetopofthebioreactorwithincrementsof1cm.Thebioreactorwas

operatedatagivenoperating condition.The liquid heightwasmeasuredby taking

photographofthebioreactorandcomparingtheliquidheighttothescalemarkedon

thebioreactor.Theheattransferareacomprisedofthesurfaceareatouchedbythe

rotatingfluidi.e.surfaceareaofthevesselbottomandthecylindricalsurfaceoverthe

vesselwall.Therefore,theheattransferareawascalculatedasthesummationofthe

surface area of a cylinder with height equivalent to liquid height and diameter

equivalent tovessel diameterand the surfaceareaofacirclewhosediameterwas

equivalenttovesseldiameter.

4.6. Determination of oxygen transfer rate (OTR )The oxygen transfer rate was measuredby sulfite oxidation method (36). Shaking

bioreactorsofsize20Land50Lwereemployedwithfillingvolumesintherangeof

25%to75%.Sincelargefillingvolumeswereemployed,the OTRwasdeterminedby

themethod based on theRAMOS technology (37) to save experimental time and

chemicals. Sodium sulfite (98% purity, Roth, Karlsruhe, Germany), cobalt sulfate

(Fluka, Neu-Ulm, Germany) and sodium phosphate buffer (Merck, Darmstadt,

Germany)wereusedwithoutfurtherpurification.Allexperimentswerecarriedoutwitha 0.5M sulfite solution including 10-7 M CoSO4, 0.012M phosphate buffer and a


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Materialsandmethods

33

2.410-5Mbromothymolblue(Merck,Darmstadt,Germany)atpH8(36).Beforeand

duringthepreparationofthesodiumsulfitesolution,thedeionizedwaterwasgassed

withnitrogentoavoidaprioroxidationofsulfite.Thesulfitesolutionwaspouredinto

thevessel.Figure4.5showstheexperimentalsetupforOTRmeasurement.

Figure 4.5:Experimentalsetupfordeterminationofonlineoxygentransferratemeasurementin20Land50Lvessels.1)vessel,2)airinlet,3)airoutlet,4)oxygensensor,5)digitalmultimeter,6)mechanicalsupport,7)motor.

The vessel was closed air tight and air waspassed in thehead space during the

rinsingphase.Afteradefiniteperiodoftime,themeasuringphasestarted.Theair

inletwasclosedfirstandthentheoutletvalvewasclosedtoavoidincreaseinthe

headspacepressure.Thedecreaseintheoxygenpartialpressureintheheadspace

wasmonitoredbyanoxygensensormountedontopofvesselasshownintheFigure

4.5.Afterthemeasuringphase,theairinletandoutletvalveswereopenedagainand

therinsingphasestarted.Theoperatingconditionwaschangedeitherbychanging

shaking frequency or by changing filling volume and the same rinsing phase and

measuringphaserepeatedagaintomeasuretheOTRatthisnewoperatingcondition.

Thesurroundingatmospherewasventilatedtoavoidanytemperatureincreaseoffluid


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Determinationoftheventilationthroughaluminumfoilinshakeflasks

34

inthevesselduetomixing;especiallyatshakingfrequenciesmorethan200rpm.The

changeinthepHofthesulfitesolutionwasobservedbyapHindicator,bromothymol

blue.TheoptimalpHforsulfiteoxidationmethodinnon-acceleratedreactionregime

is8.ThispHdecreasesto6.2attheendoftheoxidationreactionwhenallthesulfite

isconverted intomore acidic sulfate.The bromothymol bluesolutionchanges fromdarkblueatpH8toyellowatpH6.2.However,thereisatransitionphasewhenthe

sulfitesolutionhaspHofca.7andatthispHthesolutioncontainingbromothymol

blue isgreencolored.This conditionmay lead to non-accelerated reaction regime

because of high absorption rate of oxygen in the solution (36). Since, large filling

volumes inthe rangeof 25% to75%wereemployed, thisnon-accelerated reaction

regimemayprevailforlongertimeandgivehigherOTRvaluesascomparedtothat

measuredinnon-acceleratedreactionregime.Toavoidsuchconditions,whenever,

the sulfite solution in the disposable bioreactor changed to green color, it was

replacedbyafreshlymadesolution.

4.7. Determination of the ventilation through aluminum foil in shake flasksThepurposeofthedeterminationofventilation insmallshakeflaskswas tofindan

equivalentvalueofaerationforlargedisposableshakingbioreactors.Dr.Amoabediny

developedascale-upstrategyfromlaboratoryshakeflaskstostandardstirredtank

fermentorsbasedonaeration(40).Inthisstrategy,anequivalentvalueofaerationfor

standardstirredtankfermentorisobtainedfromtheventilationthroughtheshakeflask

closure.

ItisusualinInstituteofMolecularBiotechnologytousealuminumfoilasshakeflask

closures.Theplantcellculturesarecoveredwithaluminumfoilin250mLwideneck

shake flasks with 20% filling volume. Therefore, it was necessary to measure the

mass transfer resistance of aluminum foil for ventilation of shake flask. Dr.

Amoabediny developed a scale-up strategy to calculate the aeration for standard

stirred tank fermentorsusingthemass transferresistanceof the flaskclosure(40).

Thisaerationvalueisequivalenttotheventilationfoundinanormalventilatedshake

flaskcoveredwithaclosure.Themasstransferresistanceofaluminumfoilasaflask

closurewas determinedby two flaskmethodmentioned byMrotzeketal (28) and

Anderleietal (38).Waterwasusedasafluid inoneflaskandotherflaskwasfilled

withasaturated,aqueoussodiumchloridesaltsolution.Since,tightnessofaluminium

foilcoveredonflaskcanvaryfrompersontoperson,fourpersonswerechosenforthe

experiment.Eachpersoncoveredaluminumfoilonfourwideneckflasks(threeflasks


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Materialsandmethods

35

filledwithwaterandfourthflaskfilledwithsaturatedsodiumchloridesolution)ofsame

size (250mL)with same filling volume (60%) and incubatedat 25C.The shaking

frequencyandshakingdiameterwere180rpmand5cm,respectively.Weightofeach

flaskwasmeasuredatthestartofincubationandagainafter8daysattheendofthe

experiment. Decrease in flask weight indicated water loss at given operatingcondition.Thewaterevaporationratewascalculatedfromthewaterloss.Thiswater

evaporationratewasincorporatedinmathematicalmodeldevelopedbyAnderleietal.

to calculate the oxygen transfer coefficientmentioned in section 3.4 through flask

closure(38),whichwasfurtherusedinamodeldevelopedbyAmoabedinyetal.(39,

40)toobtainaerationvaluesequivalenttotheventilationinshakeflasksasdescribed

insection3.4.

4.8. Biological experiments4.8.1. Maintenance of plant cell suspension culture in shake flaskThesuspensionculturewassubculturedeverysevendaysinto250mLshakeflasks

with 20% filling volume of freshnutrientmedium containingMurashige and Skoog

(MS)medium(43)+Kinetin(Kn)(0.2mg/L)+2,4,dichlorophenoxyaceticacid(2,4

D) (0.2 mg/L). For sub culturing, 10% inoculum was used. The cultures were

incubatedonorbitalshaker(KhnerAG,Switzerland)at180rpmand5cmshakingdiameterat25C.ThepHofthemediawasadjustedto5.8beforeautoclaving.

4.8.2. Cultivation of N. tabacum suspension culture at large-scaleThecellsuspensioncultureof N.tabacumwasscaled-upina10L(7Lfillingvolume)

standardstirredtankfermentorandindisposableshakingbioreactorsofsize2L(1L

fillingvolume),20L(10Lfillingvolume)and50L(35Lfillingvolume).Theaerationwas

keptat0.1vvm.Preliminaryexperimentsat theInstituteofMolecularBiotechnology,

RWTHAachenrevealedthattheGamborgsB5mediumwasoptimalforcellgrowth

and human serum albumin production. Therefore, the cells were cultured in

GamborgsB5medium(44)+Kn(0.2mg/L)+2,4-D(0.2mg/L).ThepHandpO2of

the suspension culture was measured by the fiber-optic sensor spots whose

luminescence was measured by Fibox (PreSens GmBH, Regensburg, Germany).

Following is the diagram which shows experimental set up for plant cell culture

cultivation.


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Biologicalexperiments

36

Figure 4.6:Experimentalsetupforplantcellcultivationin20Land50Lbioreactor,5cmshakingdiameter.Thezoomedfigureshowstheoxygensensorgluedtoatransparentpolycarbonatedisk.Thediskisfittedintoapipe.Alightsourceisinsertedintothepipe.

Thesensorspotsweremountedonthetransparentpolycarbonatedisk.Thediskwas

fixedontoathinstainlessstillpipe.AlightsourcecomingfromFiboxwasinsertedinto

thepipeuptothepointwheresensorwasmountedondisk.

4.8.3. Hybridoma cell culture cultivation

Figure 4.7:Experimentalsetupforhybridomacellculturein2Lpolycarbonatebioreactorwith1Lfillingvolume,95rpmand5cmshakingdiameter.


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Materialsandmethods

37

Theexperimentalsetupforthecmyc-hybridomacellcultivationisshowninFigure4.7.

Thesensorwasgluedonthetransparentvesselwall.ThepHandpO2oftheculture

mediumwasmeasuredbyFibox(PreSensGmBH,Regensburg,Germany).Thecells

werecultivated ina 2LPCdisposableshakingbioreactorwith50% filingvolume.A

shakingdiameterof5cmandashakingfrequencyof95rpmwereemployed.Thecellculture was aerated with sterile air enriched with 5% CO2 when the pO2 level

decreasedbelow19%saturationorthepHdecreasedbelow7.Thecellcultureand

medium compositionwere taken from the institute of molecular biotechnology. All

media components were obtained from Sigma-Aldrich, USA. Typical medium

formulationwas,

RoswellParkMemorialInstitute(RPMI)mediumcontainingglutamine(0.3g/L)(45)

5%FetalCalfSerum(FCS)

-mercaptoethanol(1ml/L)

penicilin/steptomycine(100ug/ml)

Glucose(2g/L)

Cell-culturetestedglutaminesolution200mM(0.2g/L)

The above medium was inoculated with 1105

cells/mL at 37

C. The cells werecountedusingthehaemocytometer.Thehaemocytometerconsistedoftwochambers

eachofwhichwasdividedintonine1mm2.Acoverglasswassupported0.1mmover

thesesquares, thus total volume over eachcellwas 0.1mm3.A0.4%trypanblue

solution(SigmaAldrich,USA)wasusedasacolorindicatortodifferentiatebetween

liveanddeadcells.A0.2mLof0.4%trypanbluesolutionwasaddedin0.8mLof

balancedsalt solution (Sigma Aldrich,USA). This diluted trypan blue solutionwas

added(0.1mL)toawellmixed0.5mLsamplesolution.After5minutes,thesamplesolutionwasplacedonthetopofhaemocytometerwiththehelpofamicropipette.The

cellswerecountedunderamicroscope.Thedeadcellstookthetrypanbluestainand

wereblueincolorwhichdifferentiatedthemfromthelivecells.ThecellspermLwere

countedasfollows,

Cells/mL=averagecountpersquare104

The countwas repeated thrice tocheck reproducibility. The error should be in the

rangeof15%.


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Analyticalmethods

38

4.9. Analytical methods4.9.1. Determination of fresh weight and dry weight of plant cell cultureTheplantcellsuspensionwascentrifugedat3000rpmfor20min.Cellpelletswere

collected in pre-weighed aluminum trays. The difference in weight indicated fresh

weight(FW)ofthecells.Thefreshcellswerethendriedinanovenat60Cuntilfinal

weightbecameconstant.Thefinalweightwastakenasthedryweight(DW)ofcells.

4.9.2. Determination of extracellular sugar concentration in plant cell cultureTheplantcellsuspensionwascentrifugedat3000rpmfor20min.Residualsucrose,

glucose and fructose concentrations in the supernatantwere determined using an

HPLC system (Dionex with Chromeleon Software, 232 XL Sampling Injector

(Abimed/Gilson),UVD 170S (Dionex, Idstein,Germany), Shodex RI71 (Dionex), P

580Pump(Dionex),1mMsulphuricacid,flow0.6ml/min,organicacidresincolumn

(RP8,CC125/4sperisorb50-5C8,CS-Chromatographie,Langerwehe,Germany).

4.9.3. Determination of phosphate concentration in plant cell culturePhosphateconcentrationinthesupernatantwasestimatedusingacolorimetricassay

based on the formation of a blue colour complex with molybdate ions. Molybdate

reagent was prepared by mixing 2.6 g Ammonium Molybdate tetrahydrate

[(NH4)Mo7O24 4H2O)], 20 mL deionized water, 0.07 g potassium antimony oxide

tartrate hemi hydrate [K(SbO)C4H4O60.5H2O], 60 ml sulphuric acid and 100 mL

deionized water. 100 L of sample was taken in a 10 mL vial. To this 9 mL of

deionizedwaterwasadded,followedby200Lofascorbicacidsolution(0.1g/mL)

and400Lofthemolybdatereagent.Thevolumewasmadeupwithdeionizedwater

to 10mL.Theabsorbance wasmeasured at 680nm, 15minafter addition of the

molybdate reagent. Toobtain the standard curveof concentration vs. absorbance,differentconcentrationsofKH2PO4intherangeof0.061mg/Lto2.45mg/Lwereused

insteadofunknownsample.

4.9.4. Determination of Human Serum Albumin (HSA) produced by plant cell culturesof N. tabacum HSAwasdeterminedbyenzymelinkedimmunosorbentassay(ELISA).Theprotocol

wasdevelopedandstandardisedattheinstituteofmolecularbiotechnology,RWTH

Aachen. Goat-anti HSA antibody (stock solution 1mg/ml) (Bachem) was diluted

1:1000 in TBSbuffer (pH8, Tris-HCl, pH 8: 0.05M;NaCl: 0.138M; KCl: 2.7mM).


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Materialsandmethods

39

Eachwell ofa 96-wellmicrotiterplatewas coated with200 Lofabovementioned

goat-antiHSAsolution.Thesampleapplicationschemeisshownbelow

Figure 4.8:Afigureofthesampleapplicationona96wellmicrotiterplate.Here,Srepresentssample.Theplatewascoveredwithparafilmandkeptovernightat4 C.Afterincubationwith

antibody,theplatewasbroughttoroomtemperatureandwashedthricewithwashing

buffer(pH8;TBSBuffer;Tween-20:0.05%).Thiswasfollowedbyblockingwith200

Lofblockingbuffer(pH8;TBSBuffer;5%skimmedmilksolutionindoubledistilled

water) for 30min at room temperature. After blocking, microtiterplatewas washed

thricewithwashingbuffer(pH:8,TBSBuffer,Tween-20:0.05%).Thiswasfollowed

by the addition of HSA standard solution in the range of 5 to 100 ng/mL and the

dilutedsamples(incaseofsupernantant,inrangeofthe1:10,1:20and1:50;incase

ofcellextractinrangeof1:50,1:100:1:200)totheELISAplateaccordingtosample

applicationschemeshownintheFigure4.8.Theplatewasincubatedfor24hat4C.

After incubation, the plate was washed thrice with washing buffer. A 100 L of

1:20,000 diluted rabbit anti-HSA antibody conjugated with peroxidises (Rockland,

USA) wasplaced into each well and incubated for 1 h at room temperature. Afterincubation,theplatewaswashedwithwashingbufferasdescribedbefore.Thebound

anti-HSAantibodywasdetectedusing2-2-azinobis(3-ethylbenzothiazoline-6-sulfonic

acid (ABTS) substrate tablets dissolved in ABTS buffer (Boehringer Mannheim,

Germany).A100Lofthisbufferwasplacedineachplateandincubatedfor2h.The

microtiterplate was then placed in multichannel photometer and absorbance was

measuredat690nm.


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Resultsanddiscussion

40

5. Resultsanddiscussion5.1. Effect of hydrophobicity on power consumptionThedisposablecylindricalshakingbioreactorsaremadeofeitherPPorPCmaterialwhich is hydrophobic. In a previous study,Maier et al. concluded that theOTRis

reduced substantially in hydrophobic shake flasks (21). Therefore, the effect of

hydrophobicityonpowerconsumptionwasexamined.Thetorquemethodwasusedto

measurepowerconsumptionin5Lshakeflaskswith300mLfillingvolumeat2.5cm

shaking diameter. A very interesting phenomenon called as out-of-phase was

observedinshakeflasks(16,46).Inthiscondition,thefluidinsidetheshakeflaskwas

eithernotmixinguniformlyorjustrotatingalongthesurfaceoftheflaskwall.During

in-phase operating conditions, power consumption increased with increase in

shaking frequency. But in out-of-phase operating conditions, power consumption

suddenly decreased to a very low value with increase in shaking frequency.

Hydrophobicnature of the reactor surfacemay havechanged the onsetof out-of-

phase condition. Since, this phenomenon was distinguishable and can be readily

measuredbythetorquemethod;itwasusedascriteriontostudytheeffectoftheflask

surfaceonpowerconsumption.Water,Water+surfactant (TX-100)and a30mPas

PVPsolutionwasusedasa fluidforthispurpose.Theresultsofpowerconsumption

areshowninFigure5.1forA)water,B)water+surfactantandC)a30mPasviscous

solution.Powerconsumptionincreasedwithincreasingshakingfrequency.However,

atoneoperatingcondition,thepowerconsumptionstartedtodecreasewithincrease

in shaking frequency and out-of-phase operating condition was started. Some

shakingmachinesacceleratequicklytocometothedesiredshakingfrequencyand

thendeceleratetillsetpointisreached.Toincorporatethiseffect,thisexperimentwas

carriedoutwithincreasinganddecreasingshakingfrequencies.

ThedashedcurvesinFigure5.1representmeasurementofpowerconsumptionwith

increasing shaking frequency. The dotted lines represent measurement of power

consumptionwithdecreasingshakingfrequency.Whenwaterwasusedasafluid,the

out-of-phase condition started at ca. 101 rpm with increasing shaking frequency,

whilewithdecreasingshaking frequency, the in-phase condition startedatca. 90

rpm.Thesamedifferenceofca.10rpmwasalsoobservedforwater+TX-100solution.

Thishysteresisbetweenin-phaseandout-of-phasewasalsoobserved inrotatingcylindersandshakeflasks(16,47).


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41

Figure 5.1:Powerconsumptionvsshakingfrequencyfor(A)water,(B)water+TX-100,(C)30mPasviscoussolution.()&()hydrophobicshakeflask,()&()hydrophilicshakeflask.Dashedlineswithfilledsymbolsindicateincreasingshakingfrequencyanddottedlineswithopensymbolsindicatedecreaseinshakingfrequency.Theshakeflasksize,fillingvolumeandshakingdiameterwere,5L,300mLand5cm,respectively.

However,suchahysteresiswasnotobservedfortheinvestigatedviscoussolution.Moreover,thevalueofpowerconsumptionobservedwasalsosimilarforallsolutions


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Mixingperformanceandcriticalshakingfrequency

42

exceptwater.Whenshakingfrequencyincreased,amaximumdifferenceof50%in

thevalueofpowerconsumptionwasobservedinhydrophilicandhydrophobicshake

flask (water as fluid), however such a huge difference was not observed while

measuring power consumption with decreasing shaking frequency. This could be

because of the measuring error encountered at such a low value of powerconsumption.Thesefindingsindicatethatthehydrophobicitydoesinfluenceonsetof

out-of-phaseconditionsdependinguponthefluidphysicalproperty.However,more

investigationsarerequiredtoreachanyfinalconclusionandhence,forthepresent

work,itseffectisnottakenintoaccount.

5.2. Mixing performance and critical shaking frequency

Cell culture systemsare sensitive toconcentration,pHand temperaturegradients.These gradients may appear because of non-homogeneous or poor mixing.

Therefore, bioreactors used for animal/plant cell culturemust possessgoodmixing

characteristics and at the same time should not generate large hydro-mechanical

stress(11,31).Mixingperformanceofdisposableshakingreactorswasmeasuredby

electrical conductivity method as described in section 4.3. The mixing time was

definedasthetimerequiredfor99%ofthetotalchangeinconcentrationafteraddition

ofthetracer.

Figure 5.2:Mixingtimein20Land50Lvesselwith15Land35Lfillingvolumerespectivelyat5cmshakingdiameter.

AsFigure 5.2 depicts,for shaking frequencies larger than80rpm,mixingoccurred

withinafewsecondsafteradditionofthetracer.Theelectricalconductivitymeterhad

aminimum time interval of 5 seconds. Therefore, it was not possible to measure


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43

mixingtimeslessthan5seconds.Althoughmixingoccurredatfrequencieslowerthan

80 rpm, it is not recommended to operate under these conditions as there is no

regularliquidflowpatternobserved.Katoetal.(20)investigatedcylindricalshaking

vessels of different sizes for the determination of critical shaking frequency. They

definedthecriticalshakingfrequencyas theminimumshakingfrequencyrequiredtoachievecompletemixing.Katoetal.derivedfollowingempiricalcorrelationforcritical

shakingfrequency,

0

-0.46 -0.16 0.08

cn 1.137 d d = 5.1

Where,

nc criticalshakingfrequency(1/s)

d vesseldiameter(m)

d0 shakingdiameter(m)

fluidviscosity(Pas)

Basedontheaboveequation, the calculatedvalueofcriticalshaking frequency for

20Land50Lvessel,at5cmshakingdiameteris116rpmand102rpmrespectively.

However, the experimental findings indicate that the mixing occurs at shaking

frequenciesaslowas60rpm.Itshouldbenotedthat,Katoetal.investigatedshaking

vesselsofsizerangingfrom8.5to20.6cm,shakingdiameterintherangeof1cmto

4cmandshakingfrequenciesintherangeof75rpmto200rpm.Exceptforshaking

frequency,all theotherexperimental conditionsapplied inthisthesisareout ofthe

rangeoftheoperatingconditionsinvestigatedbyKatoetal.Thiscouldbethereason

for the deviation of the experimental values and predicted values of the critical

shakingfrequency.

5.3. Power consumption in disposable shaking bioreactors5.3.1. Comparison of the temperature method and the torque methodTodateitwasassumedthatthetemperaturemethodcanbeusedtomeasurepower

consumptionfor fluidshavingwater-likeviscositiesbut the accuracyof thismethod

wasunknownincomparisontoothervalidatedmethodslikethetorquemethod(18).

Therefore,bothtorqueandtemperaturemethodswereusedforthedeterminationof

powerconsumptionin2Land20Ldisposableshakingbioreactorstocheckthevalidity

of the temperature method. Figure 5.3 shows the values of specific power


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Powerconsumptionindisposableshakingbioreactors

44

consumptionobtainedusingthetorqueandthetemperaturemethodin2Ldisposable

shakingvessels.

Figure 5.3:Specificpowerconsumptionmeasurementin2Lcylindricalvesselatdifferentshakingfrequencies(A)usingthetemperaturemethodand(B)thetorquemethodwith()0.25L,({)0.5L,(U)0.75L,(V)1L,()1.5Lfillingvolumes.Shakingdiameter5cm.

As shown in Figure 5.3, the tendency and magnitude of the specific power

consumption was almost identical with both methods. The power consumption

increasedwithincreasingshakingfrequency.Thisgeneraltendencywasalsofoundin

other cylindrical shaking vessels where power consumption was measured by an

electricalmethod(20).Katoetal.useddifferentvesselsizesbutkeptaconstantratio


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45

ofliquidheighttovesseldiameter.Moreover,theshakingfrequenciesusedwerein

therangeof100to200rpmandshakingdiameterwaskeptintherangeof1cmto4

cm. In this work wide ranges of shaking frequency were used (100 to 350 rpm).

However,theshakingdiameterwaskeptconstantat5cm.IntheworkofBchsetal.

on shake flasks the same tendency and same order of magnitude of powerconsumption was found (18). As the filling volume increased, the specific power

consumptiondecreased due toa decrease of the surface (friction) area to volume

ratio.Herefillingvolumeswerechangedfrom12.5to75%ofthetotalvesselvolume.

Bchsetal.observedsimilartendencyinconicalshakeflasksbychangingthefilling

volumesfrom4to20%ofthetotalflaskvolume(18).

The specificpowerconsumptionin 20L shakingvesselswasalsomeasuredby the

temperaturemethod over awide range of filling volumes (5L to 15L) and shakingfrequencies(100to300rpm)(48).Sincetheappliedintegratedtorquesensorhasa

maximumlimitof1Nm,itwasnotpossibletomeasurethepowerconsumptionby

torquemethodforalltheoperatingconditions.Therefore,afillingvolumeof5Lwas

chosen.Powerconsumptionwasmeasuredforshakingfrequenciesfrom110rpmto

160rpm.Itwasnotpossibletomeasurethepowerconsumptionbeyond160rpm.

Figure 5.4:Aparityplotofvaluesofspecificpowerconsumptionmeasuredbythetorquemethodandthetemperaturemethodin2Land20Lcylindricalshakingbioreactorsatdifferentoperatingconditions.Waterwasusedasafluid,5cmshakingdiameter.Thefillingvolumesinthe2Lvesselwere()0.25L,({)0.5L,(U)0.75L,(V)1L,()1.5Landinthe20Lvessel(X)10L.


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Powerconsumptionindisposableshakingbioreactors

46

Theavailabledataofspecificpowerconsumptionfor2Land20Lvesselsareplotted

in a logarithmic parity plot in Figure 5.4. Almost all the data points lie within the

tolerance of 30%. This proves that the temperaturemethod is also a reasonably

accuratemethod.

5.3.2. Extended temperature methodTo date, the conventional temperature method was used to determine the power

consumption indisposableshakingbioreactors(48).Katoetal. used insulated and

non-insulated20Lshakingvessels.Inmostoftheexperimentsconductedwithnon-

insulatedvessel,theyusedinitialfluidtemperaturesofca.40C(48).

Figure 5.5:Comparisonofvaluesofpowerconsumptionduringheatingu

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