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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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Introductionandobjectives
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***)
WaveCellbase20 SPS(2L***)
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)
WaveCellbase20 SPS(1L***)
WaveCellbase20 SPS(2L***)
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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Introductionandobjectives
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).
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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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Figure 2.3:CHOcellsculturedin20Lpolycarbonatebioreactors.Culturevolume5L.Shakingdiameter5cm.ThisphotoisacourtesyofDr.Liu(personalgift).
Inspiteofsuccessfulcultivationofdifferentcellcultures,Liuetal.(31)stressedthe
needoffurthercharacterizationofthesesimplethoughefficientshakingbioreactors.
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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'),
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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
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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
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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
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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
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Oxygentransferratemeasurement
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
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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(-)
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Oxygentransferratemeasurement
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
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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
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Oxygentransferratemeasurement
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
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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,
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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.
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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
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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
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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
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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
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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.
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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
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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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Resultsanddiscussion
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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Resultsanddiscussion
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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Resultsanddiscussion
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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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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