Impact of aeration strategies on fed-batch cell culture kinetics in a single-use 24-well miniature

12 Pages·2014·2.81 MB·English

BiochemicalEngineeringJournal82 (2014) 105–116

ContentslistsavailableatScienceDirect

Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

RegularArticle

Impact of aeration strategies on fed-batch cell culture kinetics in a
(cid:2)
single-use 24-well miniature bioreactor

J.P.J.Bettsa,S.R.C.Warrb,G.B.Finkab,M.Udenb,M.Towna,J.M.Jandaa,
F.Ba ganza, G.J.Ly ea,∗

aTheAdvancedCentreforBiochemicalEngineering,DepartmentofBiochemicalEngineering,UniversityCollegeLondon,TorringtonPlace,LondonWC1E

7JE,UK
bBio PharmR&D,BioPharmProcessResearch,GlaxoSmithKlineR&D,StevenageSG12NY,UK

a r t i c l e i n f o a b s t r a c t

Articlehistory: Theneedtobringnewbiopharmaceuticalproductstomarketmorequicklyandtoreducefinalman-
Receiv ed10June2013 ufac turing c osts is driv ing early stage, sm all scale bi oproces s dev elopmen t. Th is work d escri bes a
R10ecNeoivveedm inb e rrev2i0s1e 3d form comprehen sive e ng ineering chara cterisa tion of a nov el, single-us e 24-well paral lel m iniatur e bioreact or
system.Cellcultureperformanceisalsoinvestigated,withparticularfocusontheaerationstrategies
Accepted11November2013
adoptedatthissmallscale(7mL)eitherbyheadspacespargingaloneorbydirectgasspargingintothe
Available online 18 November 2013
culturemedium.
KMeiynwiaotrudrse:shakenbioreactor 4–A5p3p ha− r1enfotr vhoelaudmspetarciec aoexryagtieonn manads sd itrreacnts gfears cspoearffigicniegn rte s(pkLeac)t ivveallyu.e Ts hrea nhgigehde rb ektLwa eveanlu e3s– w22it hh− d1iraencdt
EACnHngtOiibn coeedellry ic np ugrlo tcudhruaecrta icotnerisation gstihamas kesispn a(gtr mgfri)ne oqgvu ceeorn racr eyrla a(n5teg0de0 –od8fi r0ce0ocn trlpdymi twi)oi. ntDhsi trwheecetr i eng acgsree nsapesraear liglniyn gigna sa t–lhsloieq rsuaeindrvg ieend t1 et–ro1f ar3ce sida aul ncaedre tdame pcveraerl auusenesid tb vwyo ialtu hfma icnetc.o rMre aoixsf iiunnpgg
to19fold.

TheimpactofaerationstrategiesoncellculturekineticsofamodelCHOcelllinewasalsodeter-

mined.Culturesperformedwithheadspaceaerationaloneshowedthehighestviablecelldensity(VCD)
(15.2× 106cells mL−1),viab ilityr etent ionan dantibod ytitre (1.58g L−1) .Thesew eregr eate rthani ncon-
venti on alshake flaskcu lturesd uetothei mpr ovedcont rolo fthe (cid:2) 24bi oreact orsys tem.In allca se sthe
miniature biore actor managed go od con trolofpro cesspa ram ete rssu chaspH6 .95±0. 4,t em perat ure
T◦C37±0. 4andDO% 57±32.Cu lture sperfor me dwithd irectgasspa rging sh owe da2 5– 45% reductionin

VCD(dependingontheaerationstrategyused)andasimilarreductioninantibodytitre.Overallthiswork

showsthesuccessfulapplicationoftheminiaturebioreactorsystemforindustriallyrelevantfed-batch

culturesandhighlightstheimpactofthedispersedgasphaseoncellcultureperformanceatthesmall

scale.
© 2013 The Authors. Published by Elsevier B.V. All rights reserved.
1. Introduction Fromanengineeringperspective,theabilitytoconstructcommon

manufacturingplatforms[3,4]forarangeofsuchantibodyprod-

Biopharmaceuticalsareattractingsignificantcommercialinter- uctshasbeenakeyfactordrivingindustrialinterestinantibody

est because of enhanced success rates in clinical trials (30%) basedtherapeutics.

comparedtoconventionalpharmaceuticals(21.5%)[1].Todate,the Increaseddemandforantibodyproductshaspromptedindustry

most successful biopharmaceuticals have been monoclonal anti- to increase production capacity by construction of bulk manu-

bodies(mAbs)anditisestimatedthatafurther30%ofnewdrugs facturing facilities and to improve cell culture processes to raise

licensedinthenextdecadewillbebasedonantibodymolecules[2]. product titres [3]. However, product uptake is hindered by high

sellingprices.Sikora[5]reportspricesofupto£70,000perpatient

peryearintheUKforsomemonoclonalantibodytherapies.Accord-

ingly,thereispressurefromgovernmentsandhealthauthorities
(cid:2) This is an open-access article distributed under the terms of the Creative todec rease bi opharmac eutic alsellingpric esw hichin creasesthe

CommonsAttributionLicense,whichpermitsunrestricteduse,distributionand industrialincentiveforcost-effectivemanufactureoftheseprod-
reproducti on in any medium , provi ded the original au thor and source are ucts [6]. T o meet s uch pressures an d yet remain e conom ically
credited.
∗ viable,companiesmustfindwaystoaccelerateR&Dprogrammes
CE-omrraeislpaodnddreinssg: agu.ltyheo@ru. Tcle.al.c: .+u4k4( G02.J.0L 7y6e7).9 7942; fax: [phone]. andinc reasebiopr ocess effic iency.

1369-703X/$–seefrontmatter© 2013 The Authors. Published by Elsevier B.V. All rights reserved.

http://dx.doi.org/10.1016/j.bej.2013.11.010
106 J.P.J.Bettsetal./BiochemicalEngineeringJournal82 (2014) 105–116

systems have recently been commercialised. These include the
Nomenclature (cid:2)24bio reacto rsystem from Pall[14,15]andt heam brTM sys tem

from TAP Biosystems (Royston, UK). Both have the ability to
a area,mm2
independently measure and control pH, DO and temperature in
CD-CHO chemicallydefinedCHOcelllinemedia
individualwellsinparallelcellcultureexperiments.
CHO Chinese hamster ovary derived cell line The(cid:2)2 4bior ea ctorsyst em usesam icrotitreplateformatwith
dChPfr−/− ndiohrymdarloifsoelda tdeisrseodluvcetda soexydgeefinc iceonntcceenlltrliantieon, % system leve l control o f shaki ng fr e quency an d ind ividual well
controloftemperature,pHandDO[14].Thisworkdescribesacom-
df inner diameter of shaken vessel, m prehens iv eengineering an alys iso fthe (cid:2)24 biore actorsyst e mfor
do orbitalshakingdiameter,m
fed-batchsuspensioncultureofamodelChineseHamsterOvary
dw microwelldiameter,m
(CHO), mAb expressing cell line. In particular we show how the
DO dissolvedoxygen(%)
differentaerationstrategiesavailable(surfaceaerationorwitha
IhVLC dinistepglarcaeldo lfiqvuiiadb hleeigcehltl, mcomncentration, ×106 cells dispersed gas phas e) impact o n fluid mi xing, gas –liquid m ass tran s-
mL−1da y−1 fer and ultimately cell culture performance in terms of both cell
kLa volumetricoxygenmasstransfercoefficient,h−1 growth and antibody productivity.

kLaapp apparent volumetric oxygen mass transfer coeffi-
cient,h−1 2. Materials and methods

mAb monoclonal antibody 2.1. Descriptionof(cid:4)24bioreactorplatform
MTX methotrexate
N shakingfrequency,s−1 The(cid:2)24bioreactorplatform(MicroReactorTechnologies,Pall,
OD opticald ensity PortW ashin gton,USA) hasprevi ouslybeendesc ribedbyIsett etal.
P power input,W [15] andCheneta l.[14] .Bri efly,the(cid:2)2 4con sistsofasi ng lesha ki ng
P BS phosph atebu fferedsaline base plat e,wit ha n adjus tablesh aki ngfr equency fro m 0to8 00rpm
PER C non-direct gassparg edplatedesignforusewiththe atafi xed2 .5mm o rbitaldiam eter.Th ecellcult urec as se ttec om-
(cid:2)24biorea ctor pr is es a p re-s teril ised 24 deep wel l mi crot itre plat e, availa ble in
Ph phas enumber(asdefinedinEq.3),dimensionless PERCo r REGdesignsa ssh own inSu pplementa ryFig. 1.Eachw ell
PPG media Pro prietary pro duction gr owt hm edia hasa wo rking volume (w v)of3– 7m L.Individualw ellsa re equip ped
Qp ce llspecificpro ductivity,p gcell−1 day−1 with twother mistors that co rres pon dtoequiv alent tem perature
REG dire ctgassp argedplated esig nforusewiththe(cid:2)24 moni torin gandheatin gele mentsont he (cid:2)24basep late.Inaddi-
biorea ctor tion,eachw ella lsohasfl uorescen tp Han dDO sens ingpa tch esto
RO reverseosmosis allow opti calm onit orin gviaLED’sa nd dete ctor sonthe basepla te.
SRW standar droundwellmicrotitreplate Durin gshakin gavacuum isa pplied to sealthece llc ultu reca ssette
STR stirredta nkbior eact or totheb aseplat e. Individu al gasinje cti onp orts from theba seplate
t time,s fee din to0 .2(cid:2)m spargeme mbr aneswith inea chof the well s.For
t mt mass transfertime,kLa−1,s theP ERC plat ede signsp argedgaspa ssesup acen tra lsp argetu be,
tm mixin gtime,s whi chis highe rthant heliqui dsu rface,a nd th erefore passes into
V liquidv olum e,m3 thehe ad spaceo fthe wel ltopro videsur face aerationo nly.Fo rthe
V CD viable celldens ity,×106cellsmL−1 REG plate desig n, gas issp arg eddirec tlyinto thebase ofea chw ell
v/v concen trat ion,volu mebyvol ume crea tinga dispers edg as phase. Uptoth reed iffe rentg as esca nbe
VVM gas volume fl ow per un it of liquid volume per usedata n yonetime ;fo rmam mal ian cellc ulturepu rpose sthe se
min ute willt yp icall yinc ludea nox ygensource forD Ocontr olandac arbon
wv workingvolume diox ideconta iningga sf orpHco ntrol.

Greekletters
(cid:2) density,kgm−3 2.2. Engineering characterisation of the (cid:4)24 bioreactor
(cid:3) probere spo nsetime,s
P 2.2.1. Oxygenmasstransfercoefficient,kLadetermination

Thek avaluesweredeterminedexperimentallyusingthestatic
L

gassingoutmethodasdescribedbyvan’tRiet[16].Allexperiments
Biopharmaceuticals are ultimately manufactured at large scale; wereca rried outat3 7◦ C.Thefluo res cent DOs ensor of anindividual
however here there is limited opportunity to perform process wellf roma(cid:2) 24c as sette( PER CorREGdes ign )wasp re -ca libratedto
development and optimisation. Consequently most companies 100% oxyg e nsat uration inaira t3 7◦C .Thesys tem wasoperated in
havevalidatedscale-downmodelsoftheirpilotandmanufacturing
‘constantflow’mode,withoxygencontrolturnedontoenableDO
scalebioreactors[7].Theseusuallytaketheformof0.5–10Lscale
loggingbutoperatedsuchthattherewasnoactiveoxygensparg-
stirredtankbioreactors(STR)[8,9]andareusedforcellculturepro-
ing.Thepurgelinewasusedtospargetestgasesatdefinedflow
cessdevelopment.Nevertheless,thereisaneedformoreefficient,
high throughputan dminiaturised biore ac t orsth atc anbe usedear- rates. The test fluid was then sparged with N2until the DO reached
zero. The required bioreactor operating conditions (shaking fre-
lierduringprocessdevelopmenttofurtherreducetimeandcosts.
quency,aerationrate,etc.)werethensetandtheairspargeduntil
Thefundamentalchallengeliesincreatingabioreactormodelthat
accu ratelyrecrea testheen gine er ingenvir o nmentexp erience dat the probe reading reached approximately 100%. The kLa was deter-
minedtakingintoaccounttheproberesponsetimeusingEq.(1)
mediumandlargescalesinordertoyieldprocessrelevantdataat
[17]:
small scale. (cid:2) (cid:3) (cid:4) (cid:5) (cid:6)(cid:7)
Anumberofgroupshavepreviouslydemonstratedthepoten- 1 −t −t
tmiailc rt oow peelrlfpolram te ss u[1s0p–e1n3s i]o.Inn cou rldteurreto oofv mer acmommeatlhiaenl accekllos f icno nshtraokleonf Cp= tmt− (cid:3)p tmtexp tmt − (cid:3)pexp (cid:3)p (1)

keyprocessparameters(e.g.pH,dissolvedoxygen(DO))inconven- whereC isthenormaliseddissolvedoxygenconcentrationmea-
P
tion alplate formatsan umb ero fautomat edmini ature bi oreactor suredb ythe pro beattimet,t mtis1/kLa and(cid:3)Pi stheproberesp onse
J.P.J.Bettsetal./BiochemicalEngineeringJournal82 (2014) 105–116 107

time.Theresponsetimeoftheopticalprobe,18s,wascalculated forcellculture.Pluronic-F68(Sigma–Aldrich,P1300)wasaddedto
byde term iningthe time req uir edforth eDOr ead in gto dropfrom RO wat erat0.5 gL−1todeter minetheeffecto fthisc omp onento n

100%tobelow33%byspargingtheprobedirectly,firstlywithair bubblesizeanddistribution.
andth en rapid lysw itc hingtoN .Si ncethe (cid:2)24sof tware conta ins For both the PERCandREGplatedesignsimageanalysisofthe
2

a proprietary algorithm for averaging DO readings over time all wellsatvaryingshakingfrequencieswasperformedusingImageJ

kLavaluesreportedherearerepresentedasapparent,kLaapp,val- todeterminethedisplacedliquidheight(hL)andthustheareaof

ues,whichwillslightlyunderpredictthetruek avalues.Allk a thegas–liquidsurface(a).Thedisplacedliquidheightvalueswere
L L

experimentswereperformedintriplicate. determinedforbothplatedesignsata7mLfillvolumeassuming
anovalgeom etr y.Th egass parging me c ha nis mi nthe(cid:2)2 4bioreac-

2.2.2. Mixingtimedetermination tordelivers‘pulses’ofgas,openingvalvesfor22msanappropriate

Liquidphasemixingtimesweremeasuredexperimentallyusing numberoftimestoequaltheprogrammedgasflowrate.Assuch,

theiodinedecolourisationmethodasdescribedbyBujalskietal. discrete gas pulses were analysed and compared to determine

[18].Thebrown5mMiodinesolution(Sigma–Aldrich,Gillingham, reproducibilityofpulseswithvariationofgasbubblesizeandnum-

UK,CatNo.35089)turnscolourlessuponadditionofanequimo- ber.Fromthesevaluestherelativegas–liquidsurfaceareaperpulse

larsodiumthiosulphatesolution(1.8M)(Sigma–Aldrich,CatNo. wasdetermined.Fromthepulsebeginningandendingframenum-

S8503).Experimentswereperformedfromeitherastaticstartwith ber,andknownframerate,theaveragegasbubbleresidencetime

sodiumthiosulphateaddeddirectlytothebaseofthewellorwitha wasdetermined.Themeasurementofthebubblediameteranddis-

dynamicstartwheresodiumthiosulphateadditionwasmadedown placedliquidheightassumedthatallthebubblesweresphericalin

thesideofthewellatafixedposition2cmabovetheliquidsur- ordertocalculatethesurfacearea.Experimentswereperformedat

face.MixingtimeswerequantifiedasdescribedinSection2.3from leastintriplicate.

analysisofvideoimagesasdescribedpreviously[19].Allmixing

timeexperimentswereperformedintriplicate. 2.4. Cellculture

2.2.3. Determinationofevaporationlevels 2.4.1. Celllineandsubculture
Sp ecific evaporat ion levels from individual wells were deter- A mode lCH OD G44dihydrofolatereductasedeficient(dhfr−/−)
minedbase donmeasur edcha nges intheconc entrat ionof ablue cell l ine exp ress ing a w hole IgG1 m Ab produc t was kin dly pro-
dye(Su perC ook ,Leeds,UK ;atani nit ialc oncentrationo f0 .0 02%, vide d by GSK (Stev en age, UK ). Ce lls we re routi nely passage d in
v/v) over t ime. C ontrol expe ri me nts sho wed that the m easured aprop rie tarys ubculturem ediu msup pleme ntedwith methotre x-
incre asein optic aldensit y(OD)at630 nmwasd irect lypr oportional a te(MTX)(Ha nnaPharma ceuticalS upplyCompan y,Del aware,USA,
tothere du ctioni nliquid volu me .To de term inethe specificwell Cat No.55 390-033 -10)atafinalco ncentr ationof50 nM.Cellsw ere
ev apor ationrate ,a PERC platewa sfil ledwithdy es tocksolu tion rep eate dly subcultured b y dilu tion at 3–4 da y int erva ls us ing a
(7mL fill vo lume ) a nd se t in t he (cid:2) 24 bio react or s ystem at 37◦C seedingden sityof6×10 5c ellsmL−1 .C ultur esw eremaint ained in
for 9d ays .Standar dcu ltur ec ondi tions weresimu latedby u singa disposab levent ed c ap shakefl asksin aGalax ySin cubator(Wo lf
sha ki ngfre quencyof 650rpm andacon stant purgegasfl ow rateo f Laboratorie s,York ,UK )at37 ◦Cand 5 % CO ,on a norbitals haker
10mLm in−1.TheO D was dete rmi ne dforeac hwell byt rans ferrin g (CertomatMO II,Sa rtor ius Stedi m,A ub agn2e, Fra nce )atas haking
10 0(cid:2)L of sa mpl e to a s tandard mi cro titre plate a nd measur- frequency of15 0 rpmwith a25mm orbitaldi ameter.
ing the ab sorbanc e in a plate rea der (Safire , Teca n, Mä nnedorf,
Swi tzer land).Measu rem e ntsw ereblan kedaga inst10 0(cid:2)Lreverse 2.4.2. Fed-batchcellculture
osmosis (RO) water. Values prese nted her e are b ased on tripli- Fo r fed-bat ch experiments, cells were seeded at
catemea surem ents. Thefold evaporati onpe rwe llcoul dth enbe 8×105 cellsmL−1 into a PPG m edia (G ibco, In vitrogen, Cat
calcu latedusingEq. (2): N o. 041-962 14V)supple me nted withMT X,atafi nalconcentra tion
(cid:8) (cid:9)
A −A of 50nM, and an additional proprietary amino acid solution. A
foldevaporation= (cid:8) 630,final 630,blank(cid:9) (2) sta nd ard GSK fed -batch cult ure protoco l was f ollow ed using a
A −A
630,initial 630,blank single 5% (v/v) proprietary feed solution added on day 7.
Shakeflaskfed-batchexperimentsutiliseddisposable,vented

2.3. Visualisationofliquidphasehydrodynamicsandgas–liquid cap 125mL or 250mL Erlenmeyer shake flasks (Corning Life

dispersion Sciences, Amsterdam, Netherlands) with working volumes of

20–60mLand100–140mL,respectively.ForPERCandREGplate
Ahighspeedcamerawasusedtostudytheliquidphasehydro- culture sth ete mperatur ese tpointofall well swas 37◦ C,wi than
dyna mics , the m otion a nd d eform at ion of the gas–l iquid surface environm en ttemperature set point of 35 ◦C.In addi tion,e achw ell

andforbubblesizeandsizedistributionmeasurements.Thecamera used a pH set point of 6.95 and a DO set point of 57%. All wells

usedwasaDVRFastcam(Photron,California,USA).Theresolution were sealed with a cap which has a central gas permeable filter
was set t o 640× 480 pix els for all experime nts. Fo r m ixing time and a check valve t o lim it eva pora tio n from the culture. Fo r cell

experimentsthecamerawassettorecordat125framespersec- culture with the PERC plate design enough stock inoculum was

ondwithashutterspeedof1/framerate.Forthebubblesizeand made to fill an entire plate using a 7mL fill volume per well, in

distributionexperimentsandthesurfacedeformationexperiments order to minimise well to well variability. Similarly, for cell cul-

thecamerawassetat500framespersecondwitha1/1000sshut- tureusingtheREGplatedesigntherequiredvolumeofmediawith

ter speed and twice normal gain. Image analysis was performed AntifoamCemulsion(0.003%,v/v)(Sigma–Aldrich,CatNo.A8011)
usi ngIma geJso ftwar e(http:/ /rsbw eb.nih. gov/ij/).F orth eseexperi- wasaliqu ot edperwel l,thepla tewa splacedintothe (cid:2)24 so thatthe

mentsindividualwellsfromPERCandREGcassetteswerecutfrom desiredcontrolsetpointscouldbereachedbeforeinoculationfrom
a casse tte to aid visua lisatio n an d su bseq uent ima ge an alys is. A oneinoc ulumsu sp ension .The(cid:2) 24 bioreact orallow sfor‘active ’gas

small Perspex box was constructed around each individual well flowtocontrolpHandDOsetpoints.Thereisalsotheabilitytouse

andthiswasfilledwithglycerolsoastopreventimagedistortion. a‘constant’gasflowmodewhichspargesapurgeorbackground

AchemicallydefinedCHOcellmedia(CD-CHO)(Gibco,Invitrogen, gasatadesiredflowrateaswellashavinggasesfor‘active’con-

Paisley, UK, Cat No. 10743-029) was used to provide a compari- trol.PERCplateswereoperatedusinga‘constantflow’modeonly

sontotheproprietaryproductiongrowthmedia(PPGmedia)used whereasREGplatecultureswereoperatedeitherinthe‘constant
108 J.P.J.Bettsetal./BiochemicalEngineeringJournal82 (2014) 105–116

Fig.1. Iodinedecolourisationmixingtimeexperimentsfor(a)PERCplateand(b)REGplatedesigns.Imagesshowminimumtimerequiredtoachievecompletedecolourisation
in ea ch case fr om a dynamic s tart. Ex perim ental condit ion s: 5 mM iodine sol utio n; 7 mL fi ll volum e; 800 r pm sh aking freq uenc y; do2.5 m m ; 25◦C.
flow’or‘activeflow’modes.The(cid:2)24operatingconditionsforthese (AppliedBiosystems,California,USA,CatNo.2-1001-00)onanAgi-

differentculturesareshowninSupplementaryTable1. lent1100seriesHPLCusingChemstationversionA.0901software
The s haking f requ encies fo r the (cid:2)24 fed- batch experiments (Agi lentT echnol ogies, Edinb urgh,UK).Th eanalyt icalprot ocolwas

wereselectedinordertoprovideamatchedmixingtime[20].The 0.4minutesofa0.05Msodiumphosphate,0.5Msodiumchloride

cassetteswereweighedatregularstagestodeterminethedilution buffer(pH7.0)mobilephasetoloadsamplesfollowedby3.1minof

factorsrequiredforthestockbasefeed(1Msodiumbicarbonate, a0.05Msodiumphosphate,0.5Msodiumchloridebuffer(pH2.0)

1Msodiumhydrogencarbonate)additions.Largevolumeinocu- mobilephasetoelutesamplesandfinally2.5minoftheoriginal
lu m addition swerema deusingaR aininAutoR epE( MettlerT oledo, loading buffer to regen erateth ema trix;u sing a1 mL min −1 flow

Greifensee,Switzerland).Plateagitationwasprovidedbyanorbital ratethroughout.

microplateshaker(MS3Digital,IKA,Staufen,Germany;agitation
ratesetequ ivalent tobio reactor oper atingfre quency)in between 3. Resultsanddiscussion

individualwellsampling.Allexperimentswerecarriedoutwith24
3.1. Engineeringcharacterisationofthe(cid:4)24bioreactor
parallelreplicates.

3.1.1. Liquidphasehydrodynamicsandmixingtimes

2.5. Analyticaltechniques Initialexperimentsaimedtocharacterisethefluidflowinindi-
vidualwe llsfromeach ofthe tw oplatedesig nsa ndto quan tif ythe

Viable cell density (VCD) and cell viability were determined liquidphasemixingtimes.AsshowninFig.1,foralltheshakingfre-
using the Try pan Blu e excl usio n m ethod us ing a Vi-Cell XR quenc iesstu diedorb italsh ak ingind uc edd ef orm at ion oftheliq uid
(Beck man Coulter, High Wycombe, UK).Sam plesw er edilute das surfacew hichthe nmove dinano rbitalm otionaround th ew allsof
appropriat e with TrypL ETM Expres s (Gi bco, Invi troge n, Cat N o. thewel l.Visua llyth erewa sn od ifferen ceinthe nature oft heflu id
12605)andi ncuba tedat37◦Cfor10m inbefor eanalysisto brea kup flow betw eenthe twop late des igns.
anycell clum ps.Mean ce llsize me as urem entsw erealso ta kenfro m B üchsetal .[2 2]di scove redaphenomenonspecifictoshaken
the Vi-C ellwhic hemp loys opt icalanalysisfor them eas ureme ntof systems d ep ict ed b y the phase n umber, Ph. T he ‘in-p has e’ flow
cell diamet er. regimed escribes con ditio nswh erethem ajor ityo ftheliquid cir-
M etabolite analysis was performed using either a BioProfile culates aroundth eedgeoft hesha ken vessel,sy nc hron isedw ith
Analyzer (Nov a Biomed ical, Massachuse tts, U SA) or a 2700 Mul- theorb italmoti ono fthe sha king platfor m.‘Out ofphase’fluid flow
tiparame ter Bio analytical Sy stem (YSI, Ohio , USA ). I n both cases occ urswhe nonlya s mal lportion oftheflu idcir cu latesa round the
samples we re diluted wit h phosp hate buffer ed sa line as a ppro- wallso fthew ellw it hthe majority of the fluid remaining station ary
priatefo ranal ysis.The sesy stemsutilis eimmobi lisede nzy mesin inthe ce ntre oft heve ssel [22].The Ph ase num bercanbe calculated
biosen sor patchesw hiche nablem easurem entofspecifi cdissolv ed ac cord ingto Eq .(3 ):
(cid:10) (cid:11) (cid:12) (cid:13) (cid:14) (cid:15)(cid:16)
metabolitelevelsinsolution[21]. (cid:3) (cid:4) 2
ityPursoidnugctP rtoittreei nw aAs dHePtLeCr.mTi nheedc boalusemdn onu saendtibwoadsy abiPnOdiRnOgS a®ffi2n0- Ph =ddof 1 + 3log10 (cid:2)(24(cid:5)(cid:5)N)df2 1 − 1 −(cid:5)4 Vd1f/3 2 (3)
mi cronPr oteinAI DC artridg e(20 (cid:2)m,2.1 mmi. d.×3 0 mm,104(cid:2)L)
J.P.J.Bettsetal./BiochemicalEngineeringJournal82 (2014) 105–116 109

Table1
Measu redliquidphasemixingtimesforPERCandREGplatedesignsfromeitherastationaryordynamicstart.Experimentsperformedat25◦Coverarangeofshaking

frequencies and fill volumes with do2.5 mm. Errors represent one standard deviation about the mean (n = 3).
Mixingtime(s) Shakingfrequency(rpm)

500 650 800

REG PERC REG PERC REG PERC

Staticstart 3 11±1 14±4 4.3±0.6 4.3±0.6 3.3±0.6 3.0±0.1
5 31 ± 2 190 ± 3 4 5.0 ± 0.1 5.0 ± 0.1 4.3 ± 0.6 3.7 ± 0.6
Fillvolume(mL) 7 21 0± 78 3700 ± 1000 6.0 ± 0.1 6.7 ± 0.6 4.0 ± 0.1 4.0 ± 0.1
Dynamicstart 5 ND ND 1.4± 0 .2 1.0± 0 .2 0.8± 0 .10 0.8± 0 .18
7 13 ±1 ND 2.0 ± 0.1 2.0 ± 0.2 1.5 ± 0.18 1.3 ± 0.16

ND:notdetermined.

where dfis the inner diameter of the shaken vessel, dois the shaker 3.1.2. kLaappdetermination and gas–liquid interfacial area
diameter, N is the shaking frequency and V is the liquid volume ApparentkLavalues(kLaapp)forboththePERCandREGplate

[22]. For Ph>1.26 the shaken fluid will be ‘in-phase’ while for designsareshowninFig.2.Itisimportanttounderstandhowthese

Ph<1.26thefluidwillbe‘outofphase’[22].Previouslywehave valuesvarywithbioreactoroperatingconditionsastheywillinflu-

appliedthiscorrelationtocalculatePhforstandard24-roundwell enceoxygentransfer[8].Overall,increasingshakingfrequencyor

plates(24SRW),atashakingdiameterof20mm,fillvolumesfrom increasinggasflowrateincreaseskLa.IngeneralkLaappvaluesinthe
800to 200 0(cid:2)La nd s hakingf requencie sf rom 120 to 300rpm [10]. REGplatea re highe ran dshowa stro ng erdepen dencyongas fl ow

Underallconditionstheflowoffluidwasfoundtobe‘in-phase’(Ph ratethaninthePERCplate.Thisisexpectedgiventhepresenceof
8.2–12 ).F orthe(cid:2)24 sys tem us edhe re,a ssumi ng th eliquidpr op- thed isper se dga spha se.For theR E Gplateat highs haki ngfreque n-

ertiesofwater,Eq.(3)predictsflowconditionstobe‘in-phase’for cieshowever,i.e.800rpm,kLaapp valuesseemtodecrease,rather

fill volumes between 3 and 7mL and shaking frequencies above thanincreasing.Thisislikelyduetofluidvortexing,thusdecreasing

100rpm.Thisisinagreementwithourvisualobservations. theheightofliquidabovethespargerandreducingthebubbleresi-

Liquid phase mixing times were subsequently determined dencetime.Pluronicisanon-ionicsurfactantthatstabilisesbubble

undernon-aeratedconditions.Mixingtimesweredeterminedfrom formationwhichwillthereforecausethebubblestoseparate,thus

a dynamic position, i.e. shaking platform is in operation, which increasingaandhencek a.However,thewater–pluronicsolution
L

is important with regards to bioreactor monitoring and control; values,showninFig.2,aremostlikelylowerthanvaluesinactual

and also from a stationary start which is unique to this biore- culture media due to the presence of additional salts. Increasing

actor system due to the fact that the plate is removed from salt concentration will decrease the mean bubble size [26], thus

the shaker platform for liquid handling operations and is there- increasinga,henceleadingtoincreasedk avalues.
L

fore important for initial set up, sampling, and nutrient or base Variousmethodshavebeenusedtoassessk avaluesinshaken
L

feeds. microwells. Doig et al. [7] used a dynamic gassing out method

Time courses of these experiments for each plate design are to measure k a values and compared these to calculated values
L

illustrated in Fig. 1. Mean values of the calculated mixing times fromthemasstransferlimitedgrowthrateofastrictaerobicmicro
aregivenin T able 1. Increas ingthe sh akin gfrequency andde creas- organ ism .For a24-wel lplate,fi llvolum eo f1 18 2(cid:2)L, orbital diam-

ingthefillvolumeisseentodecreasethemixingtime.Thisisin eterfrom3to8mmandshakingfrequenciesfrom200to900rpm,
line wi th r esults fo r conve nt ional 24 SRW plates [10] wher e tm kLa value s we re rep orte d betwe en 36 and 1 80h− 1. F urt herm ore

values ranged from 2 to 12,900s with fill volumes ranging from theseauthorsestablishedacorrelationinordertopredictk aval-
L
800to 2000(cid:2)L .Tiss ot et al.[23] re porte dt mvalues below30 sfor uesin microw ellsystems as afunction of fluidp ro perties, sha king

a 1.5L STR fitted with a single 45mm pitched blade impeller at frequencyandwellgeometry[7].Hermannetal.[27]utilisedthe

agitation rates between 80 and 150rpm and Nienow reports all sulphiteoxidationmethodtodeterminek avaluesfora96well
L

valuesbelow40sforarangeofdifferentSTRconfigurations[24]. plate design, with reported values ranging from approximately
Thus,t hetmv alu e sre po rtedin T able1are with intherangesp revi- 25–1 50h−1. F or m ammalian cell cu ltures, h owev er, the oxygen

ously reported for conventional microwell plates and at shaking demandislesssevere[8].Barrettetal.[10]reportedvaluesranging
freque ncies ab ove 500rpm, are of a simila r mag nitud e to those from1.1 to 29 h−1 for 24 SRWp lat es atsh akingfre quenci esfrom

seeninlaboratoryscalebioreactorsundercellcultureconditions. 120 to 300rpm. In comparison to stirred bioreactors Micheletti
Spec ific allywithth ePER Cplate,the centra lspa rgetube appearsto etal .[2 8]re ports av alueof61.2h −1 fora3.5 LSTRequip pedwitha

retardmixingatlowshakingfrequencies,however,athighershak- 70mmthree-bladesegmentimpelleratanagitationrateof150rpm

ingfrequenciesdifferencesbetweenthetwowelldesignsarenot

significant.
Inconventionalstirredtankreactorsthepresenceofadispersed Table2
gasph aseisknown todecr ease liquidph ase mixingti me s [8].Simi- Measu redliquidphasemixingtimesfortheREGplatedesignasafunctionofgas

ilasrslyee, tnheto primespernocvee omf ai xdiinspgearsseidn dgiacsa ptehdasien iTna tbhlee R2E.GIn pclraetaes dinegsiggnass fl2.o5w m rmat;e 7. E mxpL efirlilm veonlutaml ec;o n2d5i◦tCio. nEsr:r ostras trieopnraersye sntta rotn; es hsataknindga rfrde dqeuveinactyio 5n0 a0b ropumt ;t hdeo
mean(n=3).
flowrateleadstoanalmost20-folddecreaseinthemixingtime.

Theinfluenceofthedispersedgasphaseonliquidmixinginshaken Gasflowrate Normalisedgas Mixing
syst emsispar tic ula rlypronou nce dsinc eth ebub blesadd a naddi- (mL min− 1) flow rate (V VM) time (s)
tionalax ia lcomponen ttothefluid flow .In Table2, the gas flow 0 0.00 210±78
ratesa reals oshownast he volu metr icgas flo wper un itli quid vol- 0. 2 0.03 59 ± 18
2 0.29 31 ± 7
ume per minute, or VVM. This is a useful term to consider when 4 0.57 26 ± 5
scalingupcellculturesystems.Catapanoetal.[25]reportsthatfor 6 0.86 20 ± 5
mamm alia nce llcultu reinstirr edbiorea cto rs the VVMsh ould be 8 1.14 12 ± 2
lowerthan0 .1. 10 1.43 11 ± 2
110 J.P.J.Bettsetal./BiochemicalEngineeringJournal82 (2014) 105–116

F(F(cid:4)-ig6)..8 E2 ar. nroAdrp PbpPaaGrrse m nrete pdkrLieaas rveenasltpu oeencst edi vseettaleynr.md Eaxirnpdee ddr ieumvsieianntgitoa tnlh caeob snotdauittti ictoh gneas sm: s7ien magnL o (fiunlt =l m v3o)e.l tukhmLoade v; fa odluor e(2as.5 da mnetdme rb;m) 0 Pi.n1Ee–Rd1C 0 aa smn ddLe m(scc iraninb−d1e dgd ai)n sR flSEeoGcw tpi orlaantt ee2 s.d2;e .s1shi.ganksin wg iftrhe qRuOe nwcaietesr 5 c0o0n (t(cid:2)ai)n, i6n5g0 0 (.(cid:3)5 )g a Ln−d1 8Pl0u0r orpnmic
and0.1VVMgasflowrate;whilstTissotetal.[23]reportsavalueof frequenciestested.Anincreaseinshakingfrequencyfrom500to
4h− 1fo ra1.5 LS TRem ploy inga4 5mm pit ch edb ladeimp e lleran d 800rpm is s een to app roximat ely double the gas–liq uid t ransf er
a gasflow rat e of0 .3mLmin−1 a nd agit ationra tesup to150r pm. area ;lea din gto an approximatedo ublingi nkL aapp(Fig.2 aandb).
T here fore, kLaa pp valu es reportedhe rerangin gfrom 4 –22 h−1 and Thisi ndicates th att heincreasesi nkLaappan d henceoxyge nt rans fer
4–53h−1 f or media; an d from 4– 24h −1 and 4 –46h −1 fo r water arep rimarily due tot heincrea se inawhile thefl uidhyd rodyna-
with 0.5gL−1 Pluroni csol ution forthe PERCan dREG plated esigns mic shavelitt lein flu ence onk .A ss ee ninF ig.1 the platedesign
L

respectivelyarewithintherangesreportedforsimilarsystems.In alsohaslittleimpactonthedisplacedliquidheightandtheavailable

termsofcellculture,specificoxygenuptakerates(qO )rangefrom gas–liquidtransferareaatthesurface.
2
2.3×1 0− 17t o1.7×1 0−16mo loxyge ncell−1 s−1[ 29]an dsok Laapp When t he REG plat e i s ae rated, Fig. 3 shows the nature of

valuesofthismagnitudewouldappearadequatetosatisfytheoxy- the gas–liquid dispersion produced. Visually this varies greatly

gentransferdemandsofmostmammaliancellcultureprocesses. between water (coalescing) or the water–pluronic solution and

Inordertounderstandhowtheavailablegas–liquidinterfacial the two types of cell culture media (non-coalescing) studied. In

areaimpactsonthemeasuredkLaapp values,thedisplacedliquid all cases, there is no significant entrainment of the gas bubbles,

heightwasquantifiedbyanalysisofhighspeedcameraimages.For but an ‘imperfect bubbly’ [30] flow regime; bubble packing is

eitherplatedesign,thedisplacedliquidheightincreasesby17,23 observedresultingina‘foam’layerappearingatthesurface;this

and32mmatagitationfrequenciesof500,650and800rpmwith phenomenonislesslikelytooccurinatraditionalSTR.Thisfoam
gas– liq uidi nte rfacialar easof220,3 10 and 410m m2 ,res pecti vely. layerwasobs er vedt opers ist forup to 4 min;henc eant ifoam was

Subsequently, the gas–liquid transfer area at the surface of the usedduringallREGplatecellcultureexperiments.

liquidwascalculatedbasedonthesevalues.Asexpected,increas- Analysisofthenumberandsizedistributionofthedispersed

ingtheshakingfrequencyincreasesthedisplacedliquidheightfor gas bubbles produced is presented in Table 3. This indicates a

bothplatedesignsandthusthecalculatedliquidareaincontact complex series of interactions between media composition and

withthegasattheliquidsurface.Thisreinforcesthetrendsseen the resultant gas phase characteristics in the bioreactor, which

inkLaapp valuesindicatingthatincreasingtheshakingfrequency canbeattributedtothepresenceofelectrolytesinsolution[31].

increasestheavailablegas–liquidtransferareaandthuskLaapp.For Forexample,incontrasttowater,forthewater–pluronicsolution

thePERCplate,wherethereisnodispersedgasphase,therelation- oreithermedia,themeanbubblediameterdissmaller,therefore

shipbetweenaandkLaappisalmostlinearovertherangeofshaking there are an increased number of bubbles and surface area, a,
J.P.J.Bettsetal./BiochemicalEngineeringJournal82 (2014) 105–116 111

Fig.3. VisualisationofgasbubblenumberandsizedistributionintheREGplatedesignfor(a)water(b)waterwith0.5gL−1Pluronic-F68,(c)CD-CHOmediaand(d)PPG.
Exp eri mental condit ion s: 7 mL fill volume; 650 rpm shaking freq ue ncy ; do2 .5 mm ; 5 mL min −1 gas flo w r ate.
availableforoxygentransferwitheachgaspulse.Thebubblesize bubblesizehasadramaticimpactonenergydissipationasaresult

willalsobeaffectedbytheorificediameterandthegasflowrate; ofgasbubbledisengagementattheliquidsurface[34].Similarly,

inthiscasethehighgasflowratewilldominatethisrelationshipas there is a progression to a decrease in average bubble residence

observedbytherelativelysmallgasbubblesproduced[30].Thegas timeperpulse.Forthedifferentfluidstested,asresidencetimes
112 J.P.J.Bettsetal./BiochemicalEngineeringJournal82 (2014) 105–116

Table3
Analy sisofgasbubblesize,sizedistributionandvolumetricgasholdupintheREGplatedesignforROwater(with/without0.5gL−1PluronicF-68),PPGandCD-CHOmedia.
Experim en tal c onditio ns: 7 mL fi ll volume; d o2.5 mm; 0.5 m L m in−1g as flo w r ate; s hakin g frequ enc y 6 50 rpm . Values based on im age analysi s of hig h sp eed video im ages as
showninFig.3.Errorsrepresentonestandarddeviationaboutthemean(n=3).

Fluid Bubblesper Meanbubble a(mm2)per Bubbleresidence Gasholdupper

pulse diameter,d(mm) pulse time(s) pulse(%,v/v)

Water 5.0±0.1 4.8±0.2 370 0.21±0.06 4.4
Water +0.5gL−1 22 ± 2 2.4 ± 1.1 410 0.11 ± 0.04 2.4

PluronicF-68
CD-CHO 56±2 1.8±0.5 560 0.15±0.02 2.4
PPG 34 ± 4 2.9 ± 0.6 930 0.14 ± 0.02 6.5

change,thegas–liquidsurfaceareachangesinversely.Accordingly, 3.2. (cid:4)24cellculturekinetics

there is relatively little difference between the media in terms

ofthetotalgas–liquidsurfaceareaavailablepergaspulsewhen 3.2.1. Achievementofconsistentwell-to-wellperformance

averagedoutoverthepulseduration.Itisworthnotingthatthe AsshowninSupplementaryFig.2(a)initialparallelbatchcul-
gasbubbl edis tribu tion andd ynamicso bs er vedhe rewillb erat her tures (n=24) of theCHOdhfr−/ − ce lllin einPE RCplat essho wed

differentinanSTR,duetoimpeller-inducedbubblebreak-upand considerable well-to-well variation even though all wells were

theirsubsequentinteractionandcoalescence[8]. controlled at the same set points. By day 8 the maximum VCD
was10.5× 10 6cel lsmL− 1an dthere wa sam a ximu mdifferen ceof
3.1.3. Evaporationstudies 6.4× 106 ce llsmL−1 equivalen ttoan 88% difference infinalma x-

Duetotheextendedcultureperiodsrequiredforcellculture, imumandminimumVCDvalues.Subsequentexperimentsinves-

evaporation,particularlyfromsmallscalesystemswithlowwork- tigatedwaystoreducethisvariability.Initialproblemsidentified

ingvolumescanbeproblematicleadingtouncontrolledchangesin includedinconsistentcellgrowthinsomewells,eitherduetovari-

metabolitelevelsandcultureosmolality[12].Ithasbeenreported ationintheinoculumaddedorduetocellsettlingwhilstsampling.

thathighosmolalityconditionsincreasecellspecificproductivity Tocountertheseissuesamicroplateshakerwasusedtogently

[35]thereforeevaporationeffectsneedtobeminimisedtoover- agitate the culture cassette when removed to a biological safety

comethesenon-specificinfluencesoncultureperformance.Inthe cabinetforsampling.Inaddition,alargevolume,automatedelec-
(cid:2)24s ystem ,thecassette isposition ed suchth atcolumn1is c los- tronic p ipe tte was e mp loyed to in ocula te the w ells in par allel.

est to the fans that help control the environmental temperature Using the optimised methodology cell culture kinetics across all

and6isfurthestawaywhilstrowsAandDareattheoutsideof wellswerevirtuallyidenticalasshowninSupplementaryFig.2(b)
thep la te .Eachwe llont he(cid:2)24 plate h asac ap ,wi th apl asticche ck andth efina lVCDin creasedto 1 9.3×10 6c ellsmL−1.There wa sno

valveandsterilemembranebarriertohelpreduceevaporation.The apparentsystematicvariationinperformanceacrosstheplatewith

specificfoldevaporationvaluesover9daysofasimulatedbatch well-to-wellvariationinviablecellnumberreducedtolessthan
culture are s hown in Fig . 4. It c an be s een t ha t n ot only is there 2.6×106cell smL−1 at da y 11, equi valent to a 14% d iff eren ce in

verylittleculturevolumereductionbyevaporationoverthistime maximumandminimumVCDvalues.

(averageof0.6%perdayoverthewholeplate)butevaporationrates

are generally consistent across the plate and less than 10% (v/v) 3.2.2. Fed-batchcellculturekineticsinPERCplates

overall. As shown in Section 3.2.1 the PERC plate design provides a

homogeneouscultureenvironmentwithadequategas–liquidmass

transferforcellcultureoccurringsolelyviaheadspaceaeration.The

detailedkineticperformanceof24parallelfed-batchculturesofthe
CHOdhf r−/−cel llinegrowni n PPG media underopt imisedc on di-

tionsusingthePERCplateisshowninFig.5(a).Undertheconditions

usedthemeasuredtmduringshakingwas2.0s(Table1)whilethe
maxi mum kLaappw asapprox imately8 h−1 (Fi g .2(b)).

AsshowninFig.5(a,i)reproducibleperformanceisagainseen

across all 24 wells. In terms of cell growth the peak cell density
wasal mo st2 0.4×1 06 cellsm L− 1at day14a ndv iabili tyre mained

above60%forallwells.Theon-lineparameters(Fig.5(a,ii))demon-

stratethatthesystemwascapableofmaintainingallwellsattheir

set points and that the control was reproducible across the cul-
tur ecasset te.D Owa sm aintaine dat5 7±12%andp Hat6. 95± 0.3.

ThespikesseeninthepHtrace,forexampleatday7,corresponds

tomanualsodiumbicarbonatebasefeedingtoreadjustonlinepH.

Themetabolitedata(Fig.5(a,iii))andantibodytitre(Fig.5(a,iv))

was shown to be consistent across the wells reaching a peak of
appr oximat ely 1.6 gL−1atda y14.

3.2.3. Fed-batchcellculturekineticsinREGplates

IncontrasttothePERCplate,wellsintheREGplateareindi-

Fig.4. Variationofevaporationperwellacrossa(cid:2)24PERCcassetteduringatypical viduallyaeratedleadingtotheformationofadispersedgasphase
batc hc ultureper iod .Evaporatio nw asme asured a sdes cribe dinSect ion2.2. 3. Exper- (Fig.3).U singth eREGp lat efor matdiffer en tg asoperati ngs trate-
iment alcondi tions:I nitial0.002% (v/v )bluedye inR Owater; 37 ◦C;9da ysdu ration;
shaking frequency 6 50 rpm ; do2.5 mm ; purg e ga s fl ow rate o f 10 mL m in−1 . Average bgieelsie wveedret oinavfefesctitgcaetleldg.r Tohwet phraensdenacnet iobfo tdhye pdrisopdeurcsteidon ga[2s 9p]h.aInsea wtraas-
(lian)e rserperperseesnetns tt±he1 m0%eavnar wiaittiho nerarbooru btatrhse inmdeicaant.ing one standard deviation. Dashed ditionalb io reacto ra carrierg asis sparged throughthe cult ur e ina
J.P.J.Bettsetal./BiochemicalEngineeringJournal82 (2014) 105–116 113

Fig.5. Influenceofplatedesignon24parallelfed-batchculturekineticsofadhfr−/−celllinefor(a)PERCplate,(b)REGplatesoperatedin‘constantflow’mode,and(c)REG

platesoperatedin‘activeflow’mode:(i)VCDandviability(ii)onlinepHandDOvalues,(iii)glucoseandlactateconcentrationsand(iv)mAbtitre.Experimentalconditions:

do2.5 mm; shaking frequency 650 rpm for the PERC plate and 550 rpm for the REG plate design. Experimental set points as described in Supplementary Fig. 2 and feeding
performedasdescribedinSection2.4.2.

continuousmanner,andothercontrolgases,i.e.oxygenorcarbon andSupplementaryTable1,wherebyairisconstantlyspargedata

dioxide,areactivelyblendedinasappropriateinordertocontrol desiredflowrate,interspersedbytheappropriateactivesparging

thesystempHandDOsetpoints.Thisaerationstrategywasrepli- ofgasestocontrolpH(CO )andDO(40%O ).Inordertoevaluate
2 2
cate dinthe (cid:2)2 4‘co nsta ntfl ow’pr otoco lasrepor tedinSe ction 2.4.2 th eeffec to fgassp arg ingon the sys tem an‘a cti veflo w’ protocol
114 J.P.J.Bettsetal./BiochemicalEngineeringJournal82 (2014) 105–116

Table4
Derive dgrowthparameterscalculatedfromaveragecellculturedataforshakeflask,(cid:2)24bioreactorusingaPERCplate,andREGplatedesignsoperatedina‘constantflow’

or‘activeflow’moderespectively,asdescribedinSection3.2.3.

System MaximumIVC CumulativeIVC Instantaneous Maximumvolumetric Maximum Maximum
d(×ay1−016)cells mL−1 (d×ay1−016)cells m L−1 Qdapy(−p1g) cell−1 p(mrogdLu−c1tidvai tyy−1) rsapteeci(fidca yg−r1o)wth (ddoauyb)ling time

Shakeflask 125 261 13.6 111 0.37 11.0

PERC‘constantflow’ 128 253 14.4 113 0.34 7.7

REG‘constantflow’ 62 119 12.2 53 0.32 8.7

REG‘activeflow’ 87 173 12.1 71 0.33 5.8

wasalsoinvestigatedasdescribedinSection2.4.2andSupplemen- order to control the bioreactor set points at these now high cell

taryTable1.Inthiscasenopurgegaswasusedtoconstantlysparge densities.

thecultureandthereforeonly‘active’spargingofgassesoccurred Anotherissuethatcouldbeaffectingcellularmetabolismwith

inordertomaintainpHandDOsetpoints. regardtothedifferentgassingstrategiesisthatofCO toxicity.Itis
2

Theperformanceof24parallelfed-batchculturesoftheCHO widelybelievedthatCO buildupisprimarilyanissueinlargescale
2
dhfr−/− celllineinPP Gm ed iausing theREGp latewith eit her ‘con- bioreac torswhe reth eincrease dh yd rostaticp res surei nc rease sCO
2

stantflow’or‘activeflow’protocolsisshowninFig.5(b)and(c) solubility,andthelowVVMgasflowratesthatuseanenrichedoxy-

respectively. Under the operating conditions used the measured genairsupplyareunabletosufficientlystriptheCO andhencelead
2

tm values was approximately 7.0s (Table 1) and the kLaapp was tocellulartoxicity[8].However,itisbelievedthatthisisnotthe
around12 h−1 (Fig.2(d)).The mea n bubbl es izew as2 .9mmand ca useofth elower VCD andtitre se xh ibitedby the REG pl ate cul-

thegasphasehold-upwas6.5%(v/v)(Table3). turesin‘constantflow’gasmodebecauseofthesmallvolumeof

AsshowninFig.5(b,i)theREGplateexperimentsoperatedusing thebioreactorsystemandthatthe‘constantflow’gassingregime

the‘constantflow’protocolagainshowedreproduciblecultureper- wouldbestrippingmoreCO thanthatoftheREGplateinthe‘active
2
form ance.Th epeak cellden sityw asalmos t12.9×106c ellsmL− 1at flow’m od eandthu sthe buildup ofdi sso lve dCO tot ox icle velsis
2

day14andviabilityremainedabove70%forallwells.Gassingin unlikelytooccur.Thusthereducedgrowthkineticsunderthe‘con-

this case appeared to retard cell growth at the beginning of the stantflow’regimeismostlikelysolelyattributedtotheincreased

growth phase, but the cells maintained a higher percentage via- gasspargingfrequency.

bilityoverthistimewhencomparedtothePERCexperiments.DO
wasm aint aine dat5 7±32 %andpHa t6 .95 ±0.4( Fig.5(b,ii)).G lu- 3.2.4. Comparisonof(cid:4)24andshakeflaskculturekinetics
cose andlactate co nce nt rati onsw er ec onsis te nta cros sthe par allel In the previou s secti on c ompa rison of PER C and REG plate
1ex.1p5Ien grci Lmo−ne1 tnr(tFasis gt(.,F u5i g(sb.e ,5 oi(vfb)t,)h .ieii)‘a) catnivde aflnotwib’ opdryo tcoocnocle(nF itgra.5ti(ocn , i)p)e sahkoewd eadt dpkLeraismaigpap nr)si ,ls yhu oantwdtreeidrb utsthiemad ti ltdaoirf tfheorepeepn rrcaeetsisen ingnc eccoeonlfld tcih uteilotdun irsse p (peloresw refo dtrmmg a, asnapdchee a qwsueearitnee
a gre ater degr ee of var iability in gr owth pr ofile s. H ow ever, in theREGp lates.Here th ep erformanc e ofth etwopla ted esigns is
g eneral, t here is g oo d consisten cy across t he wells in the plat es, com pare dtoco nvent iona lshakeflask fed -ba tchc ulture salsoat a
and the effect o f bubb le damage on cell cult ure p erf orm ance is matchedm ix ingtime(∼6s )assh own inFig.6.
clea rlys hown. In thiscas ethecell gro wth follows apatternmu ch Assho wninF ig.6( a)th e cel lgrowt hk inet icsinthePERCplates
moreli kethat sh own fort heP ERC plate,w ithav is ibleexp onen- outpe rformt ho sem easu red int heshak eflasksy st ems duet obet-
tialgr owt hpha se,rea chin ga peak VCDof 15.5 × 106cel lsmL−1at tercontrolo fcult ureparam ete rsin the(cid:2) 24.T heobser ved hi gher
day 11. Ho wever, after this point ther e i s a m o re rapid decline ant ibodyti tre inthes hakeflask( Fig .6(c ))isi nfa ctnotsign ificant
in c ell v iability, le ading to a final averag e via bility below 60% at duetoth eove rla ppi ngerro rbar s.Th esha ke fla skt itre isbelieved
da y 14 . This can be exp lain e d due to the increased freque ncy in tob es imil artothatof theP ERCp late result sdue toth e factthat
acti veg assin g,at the highercel lden siti es,i nordertoc ontrolbior e- th evi abilityi slo wer an dth erefor einco mplete anti bod yfr agm ents
actor set poin ts. On line pa ram eters are m aintai ne d with DO at will berelea se d,thus ove rpredictin gtheshake flaskant ibodytitre
57±2 4% and pH at 6.95 ±0.2 (Fig. 5(c, ii)). Glucose and lacta te valu e. The REG plate resu lts are low er due to incr eased cel lular
con c entr ation sfo llow cell g row th,bu twi thin creasedg luco seutil- stress asa resul tofga sspargi ng, andhe ncel ow erVCDval uesand
isation and lac tate pr odu ction (Fi g. 5 (c, iii )). This is assume d to lower pr od ucttit re .
bebeca use thecell sareunderg oing agr eater degr ee ofenviro n- De rived gro wth parameters are calculated and presented in
me ntalstre ss,a ndar ethu sutilisingm o reenerg yforce llu larrepair Table4fort hefourc ulturecondi tion s.Theinteg ralvi ablecell(IV C)
[32,33] .Thep eak anti body concent ration was1 .40 gL−1 (Fi g.5(c, count sh ow sth eme asureo fviablecells inth ecultur eatag iven time
iv)). point. ThePE RC platehas th ehigh estm ax im umIVC, th u sillus trat-
Taken together these results highlight the significant impact ingth ecap acity forth iss yste mtosu pporttheh igh estn umberof
that gas s parging h as in small s cale cell c ultu re formats like the viab lec ells.Simi larl y,the PERCp lat ehasthe hig hestins tantaneo us
(cid:2)bl2e4s .i Ezen,e er.ggy. idnispsuipr eatwioa nt err atfeo rinacb reuabsbelse rdaipa imdleyt ewriothf 6d.e3c2remams inegn be urgby- ciselalb slpe etcoifiscu pprpoodrutcatihv iigtyh ( nQupm) vba elureo. fT cheisll sh,igahn ldigahltsso tb heact atuhsee syosfttehme
diss ipati oni s1 05W m−3w hic h increas esto108W m−3 fora bubble m onito rin gandco n trolca pabilitie s, thece llsal soex pressag rea ter
diameterof 1. 7mm[ 29].Incom parison,th e energy dissipat io ngen- amountofp rodu ct.The maximums peci ficgr owth ratere fle ctsthe
eratedby an im pell erwh ic hwillhavea na pproxim ate101W m−3 cellgrow th data,wi thth egrowthr atesint heREGp late lowert han
volume av er agefora typical anim alce llb ioreactor[29 ].The con- that oftheP ERC form at.T hisalso high lig hts thef actth atthe neg-
stantga ssingpro toc ol appear stosign ifica ntlyretard cellg row that ative im pa ctdue tothe cellu lard amagecau sed byt hebu bbl esin
theex ponent ialgrowt hphase. H owever,this appear sto conditio n theR EGcultu res ha sag reateri mpactth anthe pos itive impact of
the cellstothes tressas aresu ltofbubbl eda mage.Th is isseenin mon itor ingandc ontr o llingcul turecon ditio ns.
the activ eg assi ngstra teg y ;after ap proxima telyday 11w he nthe re Monitor inga ndcontrolo fcultur eparameters,pHandDO,inthe
isa signifi cantde creasein thev iabilityofthec ellp op ulation asa (cid:2)24asinconv enti onalbe nc handp roductionbi ore acto rs,m e ans
re su ltofasign ificantin cre ase inthefre qu enc yof gasspargin gi n that m ore representati ve dat a is obtained as opposed to other