Metabolic engineering of CHO cells to alter lactate metabolism during fed-batch cultures

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Journal of Biotechnology, 217, pp. 122-131, 2015-11-18

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Metabolic engineering of CHO cells to alter lactate metabolism during

fed-batch cultures

Toussaint, Cécile; Henry, Olivier; Durocher, Yves

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ContentslistsavailableatScienceDirect

Journal

of

Biotechnology

jo u r n al h om ep ag e :w w w . e l s e v i e r . c o m / l o c a t e / j b i o t e c

Metabolic

engineering

of

CHO

cells

to

alter

lactate

metabolism

during

fed-batch

cultures

Cécile

Toussaint

a,b

_,

_Olivier

_Henry

c

_,

_Yves

_Durocher

a,b,∗

a_Département_de_Biochimie_et_médecine_{moléculaire,}_Université_de_Montréal,_Montréal,_Québec_H3C_3J7,_Canada

b_Life_Sciences,_NRC_Human_Health_Therapeutics_Portfolio,_Building_{Montreal-Royalmount,}_National_Research_Council_Canada,_Montréal,_Québec_H4P_2R2,

Canada

c_Département_de_Génie_Chimique,_École_{Polytechnique}_de_Montréal,_C.P._6079,_Succ._{Centre-ville,}_Montréal,_Québec_H3C_3A7,_Canada

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received13August2015

Receivedinrevisedform3November2015 Accepted13November2015

Availableonline18November2015 Keywords: CHOcells Pyruvatecarboxylase Metabolicengineering Fed-batch Monoclonalantibody

a

b

s

t

r

a

c

t

Recombinantyeastpyruvatecarboxylase(PYC2)expressionwaspreviouslyshowntobeaneffective metabolicengineeringstrategyforreducinglactateformationinanumberofrelevantmammaliancell lines,but,inthecaseofCHOcells,didnotconsistentlyleadtosignificantimprovementintermsofcell growth,producttiterandenergymetabolismefficiency.Inthepresentstudy,wereportonthe establish-mentofaPYC2-expressingCHOcelllineproducingamonoclonalantibodyanddisplayingasignificantly alteredlactatemetabolismcomparedtoitsparentalline.AllclonesexhibitingstrongPYC2expression wereshowntoexperienceasignificantandsystematicmetabolicshifttowardlactateconsumption,as wellasaprolongedexponentialgrowthphaseleadingtoanincreasedmaximumcellconcentrationand volumetricproducttiter.Ofsalientinterest,PYC2-expressingCHOcellswereshowntomaintainahighly efficientmetabolisminfed-batchcultures,evenwhenexposedtohighglucoselevels,therebyalleviating theneedofcontrollingnutrientatlowlevelsandthepotentialnegativeimpactofsuchstrategyon prod-uctglycosylation.Inbioreactoroperatedinfed-batchmode,thehighermaximumcelldensityachieved withthePYC2cloneledtoanetgain(20%)infinalvolumetricproductivity.

1. Introduction

Whethertosupportnewtherapeuticantibodydevelopmentor

torespondtomarketneeds,theabilitytocost-efficiently

gener-atehighyield and highqualityof biotherapeuticsis critical for

thebiopharmaceuticalindustry, owingtothe greatpressure to

lowerthecost and shorten theperiod betweendrugdiscovery

andproductmanufacturing.Thedevelopmentand

implementa-tionofhigh-yieldfed-batchstrategieshascontributedgreatlyat

increasingtheproductivityofmammaliancellcultures(Bibilaand

Robinson,1995; Wlaschin and Hu,2006; Wurm,2004).In

fed-batchprocesses, theprolongedculturetime is often associated

withsignificantlactateandammoniaaccumulationintheculture

mediumovertime,which canhave detrimentalimpacts oncell

growthandproductquality.Continuouscelllines,likeCHOcells,

showaderegulatedglucosemetabolismassociatedwithhigh

lac-∗ Correspondingauthorat:LifeSciences,NRCHumanHealthTherapeutics Port-folio,BuildingMontreal-Royalmount,NationalResearchCouncilCanada,Montréal, QuébecH4P2R2,Canada.Fax:+15144967251.

E-mailaddresses:[email protected](C.Toussaint),

[email protected](O.Henry),[email protected](Y.Durocher).

tateproductionthatcancausemediumacidificationorundesired

osmolalitychangesduetoalkaliadditionforculturepHcontrol

(Cruzetal.,2000;LaoandToth,1997;Omasaetal.,1991;Ozturk etal.,1992).Ammoniaiswell-knowntoaltertheglycosylation

pat-ternsofmonoclonalantibodiesandotherglycoproteins(Andersen

andGoochee,1995;Borysetal.,1994;ChenandHarcum,2006; Gawlitzeketal.,2000;Yang andButler,2002).Appropriate

gly-cosylationofmonoclonalantibodiesandrecombinantproteinsis

necessarytoensuretheirbiologicalfunctionandtheiruseas

ther-apeutics.Thus, maintaining consistentand comparable product

qualityinhighcelldensityculturesremainsacriticalchallenge.

Severalwastereductionstrategieshavebeenproposedusing

eithermetabolicorprocessengineeringapproaches.Thisincludes

the use of nutrients replacement strategies, for example the

substitution of glucose withgalactose (Altamirano et al., 2006,

2000, 2004) or pyruvate (Genzel et al., 2005) or substituting

glutamine with asparagine (Kurano et al., 1990) or glutamate

(Altamiranoetal.,2000;HasselandButler,1990).Whileeffective

atreducing wastemetaboliteformation,thesesubstitutionsare

oftenassociatedwithaconcomitantreductionincellulargrowth.

Reducedlactateandammoniaproductionduringfed-batchcanbe

achievedbymaintainingglucoseandglutamineatverylowlevels

http://dx.doi.org/10.1016/j.jbiotec.2015.11.010

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(Omasaetal.,1992;Zhouetal.,1995).Underlownutrient availabil-ity,thecellmetabolismisshiftedtowardamoreefficientmetabolic statecharacterizedbyalowermolarratiooflactate/glucose(Chee FurngWongetal.,2005;MarangaandGoochee,2006).However,

thisrequirestheconstantmonitoringofnutrientlevelsandfeed

rateadjustmentssincenon-limitingconcentrationsmustbe

main-tainedatalltimeduringtheculture.Evenshortperiodofglucose

starvationwereshown toalter theglycosylationoftheproduct

(Xieetal.,1997).Moreover,ithasbeendemonstratedthatlow

glu-coseconcentrations(<15mM)leadtoreducedsiteoccupancyand

decreasedgalactosylationandsialylationofantibodies(Liuetal.,

2014).Adynamicfed-batchstrategytomaintainlowglutamine

(0.1–0.3mM)andglucose(0.35–0.70mM)concentrationswasalso

showntodecreasecomplex-typeglycanformationandsialicacid

content(CheeFurngWongetal.,2005).Theseresultsemphasize

theneedtooptimizebothproducttiterandqualityconcurrently.

Different metabolic engineering approaches have also been

investigatedtogeneratecelllineswithimprovedmetabolic

char-acteristics.Theamplificationoftheglutaminesynthetasegenein

CHOandmyelomacellsallowedgrowthinglutamine-freemedium,

therebysignificantlyreducingammoniaformation(Paredesetal.,

1999;Zhangetal.,2006).Partialdisruptionofthegeneencoding

lactatedehydrogenaseAbythemeansofhomologous

recombi-nationinhybridomacells(Chenetal.,2001)orsiRNAinCHOcells (Jeongetal.,2006;KimandLee,2007a)wasshowntogreatlylower

theproductionoflactate,aswellasglucoseconsumption.Studies

byChenetal.(2001)andKimandLee(2007a)onthedown regula-tionofLDHAledtoincreasedfinalproductconcentrations,although

cellgrowthremainedunaffected.Incontrast,Jeongetal.(2006)

observedanincreasedcellgrowthrate,butnosignificantimpact

ontPAproductionunlessdownregulationofLDHwascombined

withtheoverexpressionofglycerol-3-phosphatedehydrogenase.

Arecentstudydemonstratedthatthesimultaneous

downregula-tionofLDHandpyruvatedehydrogenasekinasecansignificantly

reducelactateaccumulationandincreasethevolumetricantibody

production,withoutimpairingcellgrowth(Zhouetal.,2011).The

expressionofanti-apoptoticgeneswasalsofoundtosignificantly

alter thelactatemetabolismof CHOcells,inducing ametabolic

shifttowardlactateconsumptionandincreasingthefinalantibody

titerby40%(Doraietal.,2009).Anotherapproachconsistedinthe

expressionofthecytosolicyeastpyruvate carboxylase2 (PYC2)

geneinBHK(Iranietal.,1999)andHEK293cells(Eliasetal.,2003; HenryandDurocher,2011;Valléeetal.,2013).Inallcases,lactate

formationwassignificantlydecreasedand,dependingonthecell

typeand prevailingcultureconditions,translatedinto

improve-mentofeitherproductivityorcellgrowth,butnotboth.Inthecase

ofCHOcells,twostrategieswereexplored.First,theinsertionof

thehumanpyruvatecarboxylase(hPC)geneinDG44cells,which

ledtoaslightdecreaseinlactateproduction(KimandLee,2007b),

asthisenzymeisexpressedinthemitochondriaandistherefore

notcompetingdirectlywithLDHforpyruvateconversion.Second,

onestudyreportedontheexpressionofPYC2inCHOcells,which

allowedtodecreasethespecificlactateproduction(Fogolínetal.,

2004).However,themolarratiooflactate/glucoseremainedhigh

(∼1.5)andrelativelyunchangedcomparedtotheuntransformed

cells(∼1.7),indicatingthatthereductioninlactateaccumulation

wasmostlydue toadecreaseinglucoseuptakerate.Moreover,

whileprolongedcellviabilitywasobserved,thiswasattheexpense

ofthemaximumcelldensity,whichexhibiteda2-foldreduction

inbatchculture.Recently,PYC2overexpressioninCHOcellswas

showntoprolongculturelongevityandreducethelactate/glucose

ratioby25%,butattheexpenseofa50%reductioninthecell

spe-cificantibodyproductionrateandanoveralldecreaseinantibody

titer(WilkensandGerdtzen,2015).

Inthe presentwork,we showtheestablishmentof a

PYC2-expressing CHO cell line with a greatly improved metabolic

efficiencythattranslatedintoreducedlactateaccumulation

with-out impairing the cells’ specific productivity. All the

PYC2-expressing clones that were generated exhibited a prolonged

exponentialgrowthphaseleadingtoincreasesinmaximumcell

concentrationandvolumetricproductivity.Wealsodemonstrate

thatthePYC2-expressingcellscanmaintaintheirimproved

phe-notypeduringfed-batchculturesemployingconcentratednutrient

solutions.Infed-batchbioreactorcultures,thisresultedinfurther

significantincreasesintermsofmaximumcelldensityandfinal

producttiter.

2. Materialsandmethods

2.1. Stablecelllinedevelopment

TheCHO-EG2clone1A7stablyexpressingachimeric

human-llamaheavychainmonoclonalantibody(EG2)againstepidermal

growthfactorreceptor(EGFR)(Belletal.,2010)wasestablished

fromtheCHO-DUXB11cellline(Agrawaletal.,2012).The

CHO-EG2-PYC2clonewasobtainedbytransfectionoftheCHO-EG2cell

linewiththepTT18vectorencodingtheyeastpyruvate

carboxy-lase2gene.Aftertransfection,hygromicinBwasaddedtothecell

cultureatafinalconcentrationof900␮g/mL.Aftertwoweeksof

selection, thepool ofhygromycin-resistant cells wasclonedby

limiting dilution in96-well plate.After2weeks, colonies were

expandedandscreenedfortheirPYC2expressionlevelby

west-ernblot.SincethePYC2enzymeisnaturallybiotinylated,itallows

itsdetectionusingHRP-conjugatedstreptavidin(Sigma).Briefly,

cellspelletswereincubatedonice20mininalysisbuffer

contain-ing50mMHEPESpH7.4,150mMNaCl,1%Thesit® _(Roche),_0.5%

NaDeoxycholate,1×complete-proteaseinhibitorcocktail(Roche).

Cell lysates were centrifuged for 20min at 12,000×g at 4◦_C.

Supernatantswereincubated5minat90◦_C_in_SDS_sample_buffer

containing5mMdithiothreitol.Sampleswereelectrophoresedon

anSDSpolyacrylamidegelelectrophoresis(PAGE)andtransferred

onto a nitrocellulose membrane (Schleicher & Schuell).

Mem-branes wereprobed withHRP-conjugatedstreptavidin (Sigma).

Ananti-GAPDHrabbit polyclonalantibody (Sigma)wasutilized

for detection of the GAPDH to control total protein load. As

a secondary antibody, an anti-rabbit polyclonal antibody

HRP-conjugated(sigma)wasemployed.Theblotsweredevelopedusing

achemiluminescencekit (BoehringerMannheim, Germany)and

visualizedwiththeKodakimagersystem440CF.

2.2. Fed-batchcultureinshakeflasksforcloneevaluation

ElevenPYC2-positiveclonesand4PYC2-negativescloneswere

cultivatedin250mLshake-flasks.After4daysofcultivation,

glu-cosewasaddeddailyinordertomaintainaconcentrationof20mM.

Lactateformationwasmeasureddailyforphenotypicscreeningto

confirmthatthechangeinlactatemetabolismwastheresultsof

PYC2overexpressionratherthanclonaleffect.

2.3. PYC2cytosolicexpression

Threecloneswereselectedaccordingtotheirstableexpression

ofPYC2.InordertoconfirmcytosolicPYC2expression,thecytosols

ofthesecloneswereisolatedusingmitochondriaisolationkitfor

culturedcells(ThermoScientific-cat#89874).TheirPYC2

expres-sion level was assessed by westernblot using HRP-conjugated

streptavidin(Sigma)asdescribedbefore.

2.4. Mediumandcultureconditions

PowerCHO2chemicallydefinedmedium(Lonza)supplemented

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124 C.Toussaintetal./JournalofBiotechnology217(2016)122–131

clumpingagent(Gibco)wasusedforstablecelllinedevelopment

andbatchshake-flasksexperiments.Cultureswereinoculatedat

acellconcentrationof0.2×106_cells/mL_and_were_grown_at₃₇◦_C

and5%CO2underconstantagitation(120rpm).

2.5. pHmonitoringin6-wellplates

Cellcultureswereinoculatedatacellconcentrationof0.2×106

cells/mLandgrownat37◦_C_and_5%_CO

2underconstantagitation

(120rpm)in6-wellplateswithintegratedpHsensors(HydroDish®_,

PreSens-PrecisionSensingGmbH,Germany).

2.6. Fed-batchcultivation

For fed-batch cultivation in shake flask or bioreactor, cells

wereseededatatargetcellconcentrationof0.2×106_cells/mL_in

BalanCD(Irvine)chemicallydefinedmediumsupplementedwith

8mMglutamine,0.1%Kolliphor®_and_2%_(v/v)_anti_clumping_agent

(Gibco).Samplesweretakendailytomeasurethecelldensityand

nutrient/metaboliteconcentrations.Oncetheanalysescompleted,

additionofCHOCDEfficientFeedTM_B_(Gibco)_supplemented_with

40mMofglutaminewasperformedonadailybasis.Thedaily

feed-ingvolumeiscalculatedaccordingtothecellspecificconsumption

ofglucose:

FeedVolume=

R×IVCDpredicted

−

Ct×V−Ctarget×V

Cfeed

whereRdenotes thespecificnutrientconsumptionrate.Ct and

Ctargetarethenutrientconcentrationandthedesirednutrient

con-centration,respectively. Vdenotesthevolumeofthevesseland

Cfeedrepresentsthenutrientconcentrationinthefeed.

2.7. Bioreactor

Bioreactorexperimentswereperformedin2Lstirredbioreactor

(Labfors4cell,InforsHT,Switzerland)operatedata0.6Lstarting

volume.Dissolvedoxygenwasmaintainedat50%ofairsaturation

bysurfaceaerationusingagasmixtureofoxygen/nitrogen/airwith

agasflowsetat100mL/min.Whennecessary,pureoxygenwas

spargedat2mL/minwithanon/offcontroller.Thetemperaturewas

maintainedat37◦_C_with_a_double-wall_{water-jacketed.}_The_stirring

speedstartedat80rpmandwasgraduallyincreasedduringculture

toreducecellclumpingandoptimizegastransfer,untilamaximum

of120rpm.CulturepHwascontrolledat7.0byasurfacegassingof

CO2or1Msodiumhydroxideaddition.Additionoffeedingmedium

wasoperatedwithadigitaltubingpumpdeliveringthedesiredfeed

volumeataflowrateof280␮L/min.

2.8. Analyticalmethods

2.8.1. Cellcountandviability,glucose,lactate,ammonium,

glutaminequantification

Cellnumberandviabilitywereassessedbytrypanblue

exclu-sionandcountingonahemocytometer.Sampleswerecentrifuged

andthesupernatantswerefrozenat−80◦Cforsubsequentanalysis.

Theglucose,lactateandglutamineconcentrationsinsupernatants

weremeasuredwithYSI2700Selectbiochemistryanalyzer

(Yel-lowSpring,USA).Ammoniaconcentrationwasmeasuredbythe

VITROS350(OrthoclinicalDiagnostics,USA).

2.8.2. Antibodyquantification

Antibodyquantification in supernatants was determined by

highperformanceliquidchromatography (WATERSCorporation,

Milford,MA)using aprotein Acartridge(POROS® _A20_column,

2.1mmD×30mmH,104␮L,Invitrogen,GrandIsland,NY).Samples

Fig.1.GenerationofastablecelllineexpressingPYC2.(A)Exampleofwestern blotanalysisofCHOcellstransfectedwiththePYC2gene.PYC2andPCexpression levelsaredetectedusingHRP-conjugatedstreptavidin.Lane1:parentalcellline (untransfected),lanes:2,4,7,24:PYC2positiveclones.Lanes:3,5,6–23,25:PYC2 negativeclones.(B)Westernblotofthe11PYC2positivecloneschosenforbatch cultivation.(C)Westernblotofthe3PYC2clonesexhibitingoverexpressionofPYC2 after2monthinculture(D)WesternblotanalysisofPYC2expressioninthecytosolic fraction.Lanes1–3:PYC2positiveclones.Lane4:parentalcellline(untransfected).

werefilteredbycentrifugationat8000–11,000×gfor3minusing

NANOSEPMFGHP0.45␮mcentrifugaldevices(PALLLifeSciences)

priortobeinginjectedonthecolumnataflowrateof2mL/min

(PBS)andelutionwasperformedusing0.15MNaCl,pH2.0.UV

detectionwasdoneat280nm.

3. Results

3.1. GenerationofCHOclonesoverexpressingPYC2

ThepTT18vectorencoding theyeastpyruvatecarboxylase2

gene wastransfectedin CHOcells stablyexpressinga chimeric

human-llamaheavychainmonoclonalantibody(EG2).129

indi-vidualclones were assessed for theirlevel of PYC2 expression

bywesternblot(Fig.1A).For theparentalcellline(Lane1),the

observedsignalcorrespondstothepresenceoftheendogenous

pyruvatecarboxylase(PC)naturallyexpressedinthe

mitochon-driaofcellsandwhichbearsthesameapparentmolecularweight.

PYC2-expressingclones(e.g.,lanes2,4,7and24inFig.1A)all

dis-playedastrongersignalreflectingthecombinedexpressionofPC

andPYC2.ClonestransfectedwiththepTT18vectorbutshowinga

signalsimilartotheparentalcells(e.g.,lanes3,5,6,8–23and25in

Fig.1A)wereconsideredasnegative.Finally,11clonesthat

exhib-itedsignificantPYC2expressionwereselectedandusedforfurther

analysis(Fig.1B). Giventhat PYC2 is expectedtobe expressed

inthecytosol(incontrasttoendogenous PCthatislocalizedin

themitochondria),asubcellularfractionationwasperformedon

threePYC2-positiveclones(clones1,7and11fromFig.1C)that

wereselectedonthebasisoftheirstablePYC2expressionforover

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Fig.2.(A)Lactateand(B)viablecellconcentrationprofilesforPYC2clones com-paredtoPYC2negativeclonesandtheparentalcelllinecultivatedinshake-flask cultures.

revealedasignalcorrespondingtoPYC2thatwasindeedspecificto

thetransfectedclonesandconfirmeditsproperexpressioninthe

cytosol.Asexpected,GAPDHexpressionwasobservedinallthe

cytosolicfractions.

3.2. PhenotypicevaluationofPYC2clones

TorapidlyassesstheimpactofPYC2expressiononcellgrowth

andmetabolism,11PYC2-positivecloneswerecultivatedinshake

flasksoperatedinfed-batchmode,wherebyglucosewasaddedon

adailybasistomaintainatargetconcentrationof20mM.

Main-tainingglucoseinexcesswasusedtocreateconditionsfavoringan

overflowmetabolism(i.e.,lactateproduction)andtoavoid

limita-tionsthatcouldalterlactatemetabolismasreportedinprevious

studies(Altamiranoetal.,2006;Martínezetal.,2013).Toruleout potentialeffectsattributabletoclonalvariation,thekineticsofcell

growthandlactateproductionofthe11PYC2-positivecloneswere

evaluatedandcomparedtothatoftheparentalcelllineandto4

PYC2-negativeclonesthatwererandomlychosentoserveas

neg-ativecontrols.AsshowninFig.2Aandinstrikingcontrastwith

thenegativeclonesandtheparentalcells,allthePYC2-expressing

clonesexhibitedthesamealteredmetabolismcharacterizedbya

significantshifttowardslactateconsumptionaroundday6.

Note-worthy,cellgrowthwasnotnegativelyimpactedandmostofthe

positiveclonesevenreachedslightlyhighermaximumviablecell

concentrations(Fig.2B).

Severalstudieshavepreviouslyshownthattheculturemedium

composition can strongly influence the metabolism of a given

cellline,includingitspatternoflactateproduction/consumption

(Altamirano et al., 2006;Luo etal., 2012; Maet al.,2009; Yuk etal.,2014;Zagarietal.,2013a).Inordertoconfirmthe

consis-tencyof themetabolicshiftdisplayed bythePYC2clones, cells

Fig.3. (A)Finallactateand(B)ammoniaconcentrationsforPYC2andparental celllinescultivatedin4differentmediainshake-flaskcultures.Errorbarsdepict standarddeviationsofthemeasurements.ND:mediumnottestedforclones1and 2.

Fig.4.EvolutionofpHfortheparentalcelllineand3PYC2positiveclonescultivated in6wellplatesequippedwithpHsensors(HydroDish®_)._The₃_pH_peaks_indicated

byarrowscorrespondtosamplingofthecultures.

werecultivatedin5commonlyusedcommercialmedia.Asshown

inFig.3A,thefinallactateconcentrationsvarieddependingonthe

culturemedium,butwerealwaysconsistentlylowerinthecase

ofthePYC2-expressingclonescomparedtotheparentalcells.It

shouldbeemphasizedthatthedifferencesobservedwerenotthe

resultofsignificantchangesincelldensities,asthecalculatedcell

specificlactateproductionrateswerealsoconsistentlylowerfor

PYC2clonescomparedtotheparentalcellline(datanotshown).

Ofsalientinterest,significantreductionsinfinalammonia

concen-trations(upto50%)werealsoobservedforPYC2clonesinthreeof

thefivecommercialmediaformulationtested(Fig.3B).

Lactateaccumulationandtheconsequentmediumacidification

inuncontrolledflaskculturescanbedetrimentaltothecellsand/or

theproductquality.WethereforeinvestigatedtheimpactofPYC2

expressionontheevolutionofpH.Tothatend,cellswerecultivated

inbatchmodeina6-wellplatesandpHwasmonitoredusinga

sensordishreader(SensorDish®_)._The_recorded_pH_signals_are

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Fig.5. (A)Celldensity,(B)cellviability,(C)glucose,(D)lactate,(E)glutamineand(F)ammoniaconcentrationprofilesduringbatchandfed-batchcultivationsofparental andPYC2-expressingcellsinshake-flaskcultures.Errorbarsdepictstandarddeviationsofthemeasurements.

mediumissignificantlyreducedinculturesperformedwithPYC2

clones.

3.3. Batchandfedbatchcultivationsinshakeflasks

Inordertoassess thepotentialof PYC2clones forfed-batch

applications,cellswerecultivatedinshakeflasksunderbatchand

fed-batchmodesusingtheBalanCDmedium,whichyieldedthe

highestcelldensityinshakeflaskbatchcultures.Clone#11(Fig.1)

wasusedin allthesubsequentexperiments inshake flaskand

bioreactorcultures.Theresultingcelldensityprofilesareshown

inFig.5AforboththeparentalandPYC2-expressingcells.When

cultivatedin batch mode, thegrowthcurves differed markedly

betweenbothcelllines.ThePYC2celllinereachedamaximum

cell concentration of about 1×107_cells/mL, _a _nearly _two-fold

increasecomparedwiththeparentalcells.Themaximumcell

spe-cificgrowthrateswerealsofairlydifferentforthetwocelllines

(0.033and0.023h−1_for_the_PYC2_and_the_parental_clones,

respec-tively)andtheexponentialphaseofthePYC2clonewasextended

bymorethan24h.Uponreachingtheirmaximumcelldensityon

day6,thePYC2cellsexhibitedasharpdecreaseinviablecell

con-centrationthereafter(Fig.5B).Thissuddendropincellviabilitywas

mostlikelyduetoakeynutrientdepletiongiventhehighcell

den-sityreachedinthesebatchcultures.Whengrowninfed-batch,the

PYC2cellshadasimilarcellspecificgrowthrate(0.032h−1_),_but

thefeedingstrategyallowedtomaintainhighcelldensitiesover

aprolongedperiodoftime,asthecellviabilitydecreasedbelow

85%onlyafterday7.Incontrast,thesamefeedingregimehadonly

marginalimpactonthegrowthandviabilityoftheparentalcells

(Fig.5A&B)comparedwiththebatchmode.

Batchcultureswerecharacterizedbyanearlytotaldepletionof

glucosebyday8forbothPYC2andparentalcells(Fig.5C),while

thefed-batchstrategyallowedtomaintainglucosearoundthe

tar-getconcentrationof35mM.Noteworthy,despitethoseelevated

glucoseconcentrations,thePYC2cloneexhibitedthesamealtered

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Table1

Cellspecificconsumption/productionratesinshakeflaskcultures.

Batch Fed-batch

Exponentialphase PYC2 Parental PYC2 Parental

qglucose(pmol/cell.d) 1.00±0.17 1.24±0.21 1.18±0.19 1.65±0.30 qglutamine(pmol/cell.d) 0.35±0.06 0.37±0.07 0.47±0.08 0.53±0.09 qlactate(pmol/cell.d) 0.46±0.06 0.98±0.18 0.54±0.09 1.18±0.21 qammonia(pmol/cell.d) 0.57±0.10 0.86±0.18 0.93±0.20 1.31±0.28 Lactate/glucoseratio 0.46 0.79 0.46 0.72 Ammonia/glutamineratio 1.63 2.32 1.98 2.47 Stationaryphase qglucose(pmol/cell.d) 0.69±0.10 1.19±0.21 1.42±0.24 1.73±0.29 qglutamine(pmol/cell.d) – – 0.27±0.05 0.31±0.05 qlactate(pmol/cell.d) −0.36±0.06 −0.05±0.01 −0.15±0.03 −0.03±0.01 qammonia(pmol/cell.d) – – 0.41±0.09 0.36±0.08 Lactate/glucoseratio −0.52 −0.04 −0.11 −0.02 Ammonia/glutamineratio – – 1.52 1.16

themaincellspecificconsumptionandproductionratesevaluated duringtheexponentialandthestationaryphasesispresentedin Table1.Inbatchandfed-batchcultures,onlyslightreductionin

theglucosespecificconsumption rates wereobservedfor PYC2

cells,but markeddifferenceswerenotedforlactate asthe

spe-cificproductionwasreducedby50%duringtheexponentialphase.

Thedifferenceswereevengreaterduringthestationaryphase,as

lactate wassignificantly consumedby PYC2cells, unlike inthe

parentalcell line(Table 1).Asevident fromthecalculated

lac-tate/glucoseratios,thePYC2cloneexhibitedamuchmoreefficient

glucoseutilization.

In batch cultures, glutamine depletion occurred rapidly (by

day4),buttheaveragecellspecificglutamineconsumptionrates

were not significantly different for the two cell lines (Fig. 5E

&Table1).Thesupplementationofglutamine infed-batch

cul-turesledtoanincreasedconsumptionrateandgreaterammonia

Fig.6.(A)Cellviability,(B)celldensity,(C)glucose,(D)lactate(E)glutamineand(F)ammoniaconcentrationprofilesfed-batchcultivationsofparentalandPYC2expressing clonesinbioreactorcultures.Errorbarsdepictstandarddeviationsofthemeasurements.

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128 C.Toussaintetal./JournalofBiotechnology217(2016)122–131 Table2

Cellspecificconsumption/productionratesinbioreactorcultures.

Exponentialphase Stationaryphase

PYC2 Parental PYC2 Parental

qglucose(pmol/cell.d) 2.46±0.42 2.49±0.44 1.57±0.27 2.06±0.33 qglutamine(pmol/cell.d) 0.75±0.12 1.18±0.20 0.44±0.08 0.56±0.09 qlactate(pmol/cell.d) 0.43±0.07 2.34±0.41 −0.17±0.03 0.24±0.04 qammonia(pmol/cell.d) 0.31±0.05 1.11±0.18 0.19±0.03 0.24±0.02 Lactate/glucoseratio 0.17 0.79 -0.11 0.11 Ammonia/glutamineratio 0.41 0.94 0.43 0.82

Fig.7.Comparison ofmaximum antibodytitersbetweenparental and PYC2-expressingcellscultivatedinshakeflasksandbioreactors.Errorbarsdepictstandard deviationsofthemeasurements.

accumulation,particularlyforthePYC2cloneduetothegreater celldensityandtheprolongedculture.Whilethespecific produc-tionswerequitesimilarduringtheexponentialphase,bothcell linesshowedadecreasedproductionofammoniapercellduring thestationaryphase (Table 1).Whetherthecultures were

per-formedin batchor fed-batch modes, the final product titer in

cultureswithPYC2-expressingcellswas35%greatercomparedto

theparentalcellline(Fig.7).Thisaugmentationcanbeattributedto

boththeincreasedcelldensityandtheprolongedculturelongevity

associated with PYC2 expression. The cell specific productivity

ofPYC2-expressingcellswascomparableinbatchandfed-batch

modes(1.91±0.40and2.02±0.44pg/cellday,respectively),while

thatoftheparentalcloneincreasedfrom1.69±0.35pg/cellday

inbatchto2.69±0.54pg/celldayinfed-batch.Comparedtothe

parentalcellline,theslightlylowercellspecificproductivityofthe

PYC2cloneislargelycompensatedbytheincreasedcellgrowth.

3.4. Fed-batchcultivationinbioreactor

Thesharp andsudden viabilitydecrease witnessedin shake

flasks suggested that these uninstrumented culture conditions

couldnot supportthehighcell densitiesreachedbyPYC2cells

(around1×107_cells/mL)._Cells _were_thus _cultivated_under

fed-batchmodeusingfullycontrolledbioreactorssoastoprovidebetter

regulatedconditionsintermsofpHandoxygen.Thefeeding

strat-egyconsistedinthedailyadditionofaconcentratedsolution(CHO

CDEfficientFeedB)supplementedwith40mMofglutamine.The

resultingcelldensityprofilesareshowninFig.6A.ThePYC2cellline

reachedamaximumcellconcentrationofabout1.3×107_cells/mL,

amorethan2-foldincreasecomparedwiththeparentalcells.The

maximumcellspecificgrowthrateswerefairlysimilarforthetwo

celllines(0.023and0.024h−1_for_the_PYC2_and_the_parental_clones,

respectively),buttheexponentialgrowthphasewasextendedby3

daysinthecaseofthePYC2clone.Uponreachingtheirmaximumin

concentrationbetweendays9and12(13×106_cells/mL),_there_was

asharpdecreaseinviablecelldensity,similartothetrendobserved insmall-scalecultures(Fig.6B).Sincethedissolvedoxygen

set-pointwassuccessfullymaintainedataround50%throughoutthe

culture,thiswaslikelyduetosomeothernutrientlimitation.

Aswasobservedinshakeflaskcultures,theexpressionofthe

PYC2geneledtoasignificantalterationofthecellmetabolism,with

lactatebeingconsumedafter7daysofcultivationdespiteelevated

glucoseconcentration(Fig.6C&D).Thegreaternutrient

utiliza-tionefficiencyofthePYC2celllinewasevenobservedduringthe

exponentialphase,asthecellspecificlactateproductionratewas

reducedby80%foracomparableglucoseuptakerate(Table2).PYC2

expressiondidnotsignificantlyimpacttheglutaminemetabolism,

and,asaresult,theculturesachievedsimilarfinallevelsof ammo-nia(Fig.6E&F),althoughthespecificammoniaproductionrateof thePYC2clonewaslower(Table2).

Consistentwithsmall-scalefed-batchresults,theaveragecell

specificantibodyproductionrateswasslightlylowerforthePYC2

clone(2.21±0.45and2.86±0.58pg/celldayforPYC2andparental

cells),butthefinalproducttiterwasaugmentedby28%duetothe

highercelldensityachieved(Fig.7).Ofinterest,themaximum

vol-umetrictiterwasreached4daysearlierincultureswiththePYC2

cells.Whilethefed-batchstrategybroughtonlyaslightantibody

volumetricproductivityimprovementinshakeflasks,culturesin

bioreactorimprovedbyaround30–40%thevolumetricproduction

ofPYC2andparentalcelllines,respectively.

4. Discussion

TheaimofthisworkwastogenerateaPYC2-expressingCHO

celllinewithanimprovedmetabolicefficiencyanddemonstrateits

potentialforthedevelopmentofafed-batchprocessforantibody

production.Inanymetabolicengineeringstrategy,clonalvariations

shouldbeconsideredwhentryingtoassesstherealimpactofgene

overexpression.Clone-basedstudiesoftenfailtoestablisha

rela-tionshipbetweentransgeneexpressionandthedesiredtrait.Inthe

specificcaseoflactatereductionstrategies,avarietyofotherfactors

maycausedifferencesinmetabolicphenotypes,suchaschangesin

themediumcomposition(Kishishitaetal.,2015;Luoetal.,2012;

Zagarietal.,2013a).Inourstudy,wehaveinitiallycomparedthe

kineticsofgrowthandmetaboliteformationof11PYC2-positive,

4PYC2-negativeandtheparentalclones.Thefactthatall11

PYC2-positiveclonesexhibitedcomparablephenotypeswhentestedin

differentmediaformulationprovidedstrongevidencethatthe

dif-ferencesobservedwereduetoPYC2expressionandnottoclonal

variations.

ThePYC2expressingCHOcellsthatweregenerateddisplayed

a much more efficient metabolism characterized by a lower

lactate/glucose ratio, especially in fed-batch cultures using an

enrichednutrientsolution.Moreimportantly,theslightlylower

cellspecificantibodyproductionofthePYC2cloneislargely

com-pensated by the increase in maximum cell density, translating

(9)

maintainedtheir distinctive phenotype, even when exposed to highglucoselevelsduringfed-batchcultures.Inaddition,thecell

specificproductivityisnotsignificantlyalteredbythemetabolic

modification,as was previously observedwhen the same gene

wasoverexpressedinHEK293cellsproducinginterferoninbatch

(HenryandDurocher,2011)orfed-batch(Valléeetal.,2013)

cul-tures.Incontrastwithourresults,anapparenttwo-foldincrease

inspecificproductivitywasobservedinPYC2transfectedBHK-21

cellsproducingrecombinantEPO,althoughthesecellswere

culti-vatedinperfusionmodeunderlimitingglucoseconditions(Irani

etal.,2002).However,nopositiveimpactswereobservedinterms

ofmaximumcelldensityandcellspecificgrowthrate.Similarly,

atwo-foldincreaseof specifichGM-CSF productivitywasnoted

forCHOcellsexpressingPYC2cultivatedinbatchandfollowinga

temperatureswitch(Fogolínetal.,2004).Thecellspecificgrowth

rateandmaximumcelldensitywerereduced,butculturelongevity

wasextendedleadingtoariseinfinalproducttiter.Inanearlier

study,HEK293expressingPYC2showednosignificantincreasein

SEAPtiterin spiteofincrease celllongevityand reducedwaste

production(Eliasetal.,2003).Similarly,adecreaseinthespecific

antibodyproductivitywasfoundforCHOcellsexpressingPYC2,

eventhoughthecellgrowthrateandglucose/lactateratiowere

improved(Wilkensand Gerdtzen, 2015).Taken together,these

resultsindicatethattheeffectsofPYC2expressionaredependent

onthecelltypeandthecultureconditions.Inthepresentstudy,the

greatestimpactofPYC2expressioninCHOcellswasonthe

max-imumcelldensityattainedduringthecultures.Resultsfromour

shakeflaskandbioreactorculturesinfed-batchmodeshowedthat

theincreaseincumulativeviablecellconcentration,combinedwith

themaintenanceofantibodyspecificproductivity,resultedintoa

netincreaseinthefinalvolumetrictiter.

Inthetransitionfromsmall-scaletobioreactor,themetabolism

andgrowthofthePYC2cloneremainedmostly similar,butthe

producttiterincreasedsubstantially.Thistiterimprovementcan

likelybeattributed,atleastinpart,totightlyregulatedconditions inthebioreactor.InthecaseofPYC2cells,regardlessofthe

cul-turemode(batchorfed-batch)orculturedevice(shakeflaskor

bioreactor),thelactateshiftseemedtooccursystematically24h

beforereachingthemaximalcelldensity,asthecellsenteredthe

decelerationphase.FortheparentalCHOcellline,thenet

accumu-lationoflactateceasedwhencellgrowthstopped.Ineitherofthe

twocelllines,theseresultssuggestalinkbetweenacertainlactate

level,growthratereductionandlactateproductionshutdown.In

thecaseofcellsexpressingPYC2,theenzymecouldhaveanadditive

effectleadingtoanetlactateconsumptionduetothecompetition

forpyruvatebetweenthisenzymeandlactatedehydrogenase(Irani

etal.,1999).Lactateconsumptionhasbeenreportedtooccurunder

lowglucoseconcentrations (Altamirano etal.,2004;Cruzetal.,

2000;Ozturketal.,1992)orundernon-limitinglevels(Luoetal., 2012;Maetal.,2009;Mulukutlaetal.,2012;Ozturketal.,1997; Pascoeetal.,2007).Theco-consumptionoflactateandglucosewas identifiedasahighlydesirabletraitforindustrialcelllines,sinceit istypicallyassociatedwithincreasedculturelongevityandgreater

productyields(Leetal.,2012).Thisphenomenonandits

under-lyingphysiologicalmechanismsarestillnotfullyunderstoodand,

consequently,notyetwell-controlled.Recently,somestudieshave

suggestedthatthecopperlevelintheculturemediumcouldbea

triggeroflactateconsumption(Luoetal.,2012;Yuketal.,2014), butotherfactorshavealsobeenassociatedtothismetabolicshift,

includinganincreaseinoxygenconsumption,areductionof

gly-colysisandglutaminolysis,aswellascellgrowthslowdown(Luo

etal.,2012;Maetal.,2009;Mulukutlaetal.,2012;Pascoeetal., 2007).

StudiesonBHK-21expressingPYC2havereportedincreasesin

oxygenuptakeandintracellularATPpool,pointingtoanenhanced

TCAactivityresultingfromthisgeneticmodification(Iranietal.,

1999).Morerecently,13_C _metabolic_flux_analysis _conducted_on

HEK293-PYC2expressingcellsconfirmedanincreaseinthe

pyru-vate fluxtooxaloacetate(Henryand Durocher,2011),although

thisintermediatemetabolitecansubsequentlyhavemultiplefates.

First,inthecytosol,thelattercanbeaccumulatedorcouldbe

con-vertedintomalatethroughcytosolicmalatedehydrogenase(MDH)

withtheconcomitantrelease of NAD+_._Then_the_malate_can _be

recycledintopyruvateviacytosolicmalicenzymeorenterthe

mito-chondriaviathemalate/aspartateshuttletobecompletelyoxidized

intheTCA.ApreviousstudyonCHOcellsexpressingmitochondrial

MDHIIreportedenhancedglycolysis,reducedlactatesecretionand

elevatedintracellularATP/NADHlevelsalongwithantibodytiter

improvement(Chongetal.,2010).Thesefindingssuggesta

criti-calroleoftheredoxbalanceintheregulationoftheglycolyticflux

andtheproportionofpyruvateenteringtheTCA.Thecapacityof

theLDHreactiontoregenerateNAD+_allows_to_maintain_high

gly-colyticrates,decreasingtheavailability ofcytosolicpyruvateas

asubstratefortheTCAcycle.Themalate-aspartateshuttleplays

a pivotalrole inmaintainingtheredoxbalancebyallowingthe

regenerationofNAD+_in_the_cytosol._In_CHO_cells,_{overexpression}

ofaralar1,anaspartate/glutamatecarrierandcriticalcomponentof

themalate-aspartateshuttle,wasshowntopromoteametabolic

switchtolactateconsumptionwithoutanyreductionofthe

gly-colyticrate(Zagarietal.,2013b).ForPYC2overexpression,itcan

bespeculatedthatthereductionintheLDHfluxislikely

compen-satedbyanincreaseinthemalatedehydrogenasefluxtomaintain

theredoxbalance.

Thedistinctive phenotypeof PYC2-expressingCHO cellscan

provideadditionalbenefitsinthecontextofthedevelopmentof

acellculturemanufacturingprocess.First,itiswellknownthat

glycosylationpattern ofglycoproteinscanbenegatively altered

byelevatedwasteproductaccumulation(AndersenandGoochee,

1995;Gawlitzeketal.,2000;YangandButler,2002).Since

signif-icantlactate/ammoniaaccumulationislikelytooccurinstandard

highcelldensityfed-batchcultures,thelowerwastemetabolite

for-mationobtainedinPYC2cellscouldbeexpectedtoenhancefinal

productqualityandstabilityintheculturemedium,butfurther

workisrequiredtoassesstheimpactofPYC2overexpressionon

productqualityattributes.Furthermore,this phenotypicchange

eliminates theneedtocontrol glutamine/glucoseat low levels,

strategieswhichmakeprocessoperationmorecomplexandalso

have adverse effects ontheglycosylationprofileof theprotein

of interest (Liu et al., 2014).Second, it is generally recognized

thatsmall-scalestudiesperformedinshakeflasksduringprocess

developmentmaynottranslatewelltobioreactorduringscale-up,

particularlyduetolactateaccumulation.GiventhatthepHchanges

aredampedwithPYC2-expressingcells, thisrepresentsanother

significantadvantagetostreamlinetheoptimizationofculture

con-ditions.

5. Conclusion

WehavedemonstratedthatPYC2expressioninCHOcellscan

efficientlyaltertheirmetabolismandinduceadesiredshifttowards

lactateconsumptionthatwasshowntooccurconsistentlyinall

culturemodesandcommercialmediatested. Giventhemarked

reductioninwastemetaboliteformation,theresultspresentedhere

demonstratethatPYC2cellsarewellsuitableforfed-batchprocess

employingconcentratedfeedsolutions.Indeed,asimplefeeding

strategybasedonglucoseconsumptionwasabletosubstantially

increase culturegrowthand longevityand lead toa significant

improvementofthevolumetrictiter.Sincethecellspecific

pro-ductivityofPYC2cloneswasmarginallyreducedcomparedtothe

parentalcells,this enhancementwasattributabletothe

(10)

potentiallybeachievedbyscreeningkeyculturesupplementsor

processconditionsleadingtoincreasedculturelongevityand/or

stimulatingthecellspecificproductivity(e.g.mild hypothermia

conditions).

Acknowledgements

Theauthorswould like tothankthe following contributors:

LouisBissonforperformingproteinAanalysisandProf.Mario

Joli-coeur(CanadianResearchChaironthedevelopmentofmetabolic

engineeringtools)forgrantingaccesstothebioreactorsemployed

inthisstudy.

ThisworkwassupportedinpartbytheNaturalSciencesand

EngineeringResearchCouncilofCanada(NSERC)MabNetStrategic

Network.ThisisNRCpublication#NRC-HHT53305.

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