ContinuousperfusionwithastableproducerHEK293celllineforscalinguplentiviralvectorproduction.MVCattaneo1ARodenbrock2S.Lanthier2,E.Burney2,AManceur21.ArtemisBiosystems,4HancockStreet,Cambridge,MA021392:HumanHealthTherapeutics,NationalResearchCouncilofCanadaAbstractLentiviralvectors(LV)representakeytoolforgeneandcelltherapyapplications.Theproductionofthesevectorsinsufficientquantitiesforclinicalapplicationsremainsabarrier,drivingthefieldtowardsthedevelopmentofcellsuspensionprocesseswhicharemoreamenabletolargescaleproduction.StableproducercelllinesderivedfromHEK293cellsgrowwellinsuspensionatcelldensitiesof10millioncells/mLthusofferingdirectscalability.Furthermore,theproductionofLVisinducedbytheadditionoftwosmallmolecules(cumateanddoxycycline)avoidingtheneedforplasmidsandtransfectionreagents.Lentiviralvectorscanthusbeproducedinthe10^7TU/mlinbioreactorsoperatedunderbatchor10^8TU/mlinperfusionmode.Theperfusionbioreactorwasrunat40%DO,37°CandpH7.1for4dayspostinduction.InallperfusionrunsharvestswerecollectedandtheLVV-containingsupernatantwaskeptoniceat-4degreescentigradeuntilclarification(oncedaily).ThisstudydemonstratedthatLVproductionusinganovelandscalableperfusionprocessincreasedviraltiters30-foldcomparedtobatchprocessingandreachedacumulativetotalyieldof6.25x10^11TUina3Lbioreactor.Thisincreaseinyieldistheresultofincreasedvolumetricoutputaswellasimprovedspecificproductivitywhenoperatinginperfusioncomparedtobatchmode.TheperfusionapproachpresentedhereiseasilyamenabletolargescaleproductionofLV.BackgroundandExperimentalOverviewA.StableProducerCellLinesforscalablemanufacturingofLentivirus.Lentiviralvectors(LV)areusedforgeneandcelltherapyapplicationsbecauseoftheiruniquecharacteristics(stablegeneintegrationintothehostgenomeofdividingandnon-dividingcells,andbroadtissuetropismviaVSV-Gpseudotyping)AninduciblesystemisrequiredinordertokeepatightcontrolontheproductionoftheproteinscomposingLV,whicharehighlycytotoxic.Cumatepreventsbindingofthecumaterepressor(CymR)tothecumateoperator(CuO)whileDoxycyclinepromotesbindingofthereversetetracyclinetransactivator(rtTA2s-M2)tothetetracyclinepromoter(TR5):Figure1.Thecumate-induciblesystemofclone92:

-TranscriptionofRevandtheenvelopeprotein(VSV-G)isunderthecontrolofthetetracyclineandcumateswitches-AdditionofdoxycyclineandcumateintheculturemediumisrequiredtoinducetheproductionofLV.

B.ScalableManufacturingofLVCurrentLVproductionmethodsarenotadequatebecause:(1)Theyarenotreadilyamenabletolargescaleproductionwhentransfectionisused.(2)Thetitersobtainedarelowwhenstableproducercelllinesareused(adherentand/orinserumcontaining-media).(3)LVarelabileandsensitivetochangesinenvironmentalfactors.Operatinginperfusionmodeinsteadofbatchmodemighthelpaddresstheseissues.

Figure2.VHU®Perfusionsystemsfordifferentmanufacturingscale.VHU1for0.5Lbioreactors,VHU2for5LbioreactorsandVHU3for50Lbioreactors.C.ScalableVHU®PerfusionSystemSeveralLVcellculturesweredesignedutilizingthescalableArtemisBiosystemsVHU®perfusionsystemasshowninFigure3.

Figure3.TFFBioreactorsetupusingArtemisBiosystemsVHU®perfusionsystemconnectedtoa3LApplikon®bioreactor.

D.UpstreamProcessDevelopmentTheupstreamprocessconsistsofaseedtrainusingourHEK293stableproducercelllinefollowedbycontinuousharvestingusingtheVHU®Perfusionsystem(Figure4).

Figure4.UpstreamProcessincludingtheHarveststepResultsTheVHU®Perfusionallowsacontinuousharvestofproducedviralparticlesandalsoalleviatesnutrientlimitationsleadingtohighercellspecificproductivity.ContinuousHarvestingofLVwithashorthalf-lifeof4hat37°CinthebioreactorincreasestheyieldoffunctionalLV.ForthePerfusionRunsweobtained:

• MaximumLVof>1x108tu/mL• MaximumVCDof8x106vc/mL• Negligiblecellsintheharvestline

Figure5:(A)LVproductionusingtheVHU®PerfusionBioreactor.Viablecelldensities(Xv),LVinbioreactor(GTAvessel),andLVinharvestline(GTAharvest).(B)Turbidityofcell(emptycircles).Turbidityofharvestline(orangecirclesandgreensquares).AsshowninTable1,theturbiditytheVHUperfusionfilterwasabletosignificantlylowertheturbidityofthecellculturefrom>500NTUsdowntolessthan16NTUs.Table1:TurbiditydataforBatchvs.Perfusion

TheVHU®PerfusionresultsinunprecedentedLVyieldcomparedtootherperfusiontechnologiesresultingina30-foldimprovementinfunctionalvectoryieldcomparedtobatchmodewasobservedasshowninFigure6.

Figure6.TotalLVyieldfroma3Lvesselbatchculture(2x1010TU/L-blackbar)comparedtoa3LvesselcontinuousharvestculturecoupledtotheVHUPerfusionsystem(6.25x1011TU/L–redbar).ConclusionsandFutureWork

• ContinuousharvestingofLVusingastableHEK293producercelllineusingaVHU®perfusionsystemresultedin30-foldincreaseinviraltiterscomparedtobatchcultures.

• Thecontinuousharveststreamcanbeconnecteddirectlytoacapturestepthusreducingtheequipmentfootprintandenablingautomationtospeeduptheprocess.

• TheLVcontinuousharvestingprocessisidealforscalingupstableproducercelllines.

WecouldenvisiongoingdirectlyfromtheharvestlineoftheVHUperfusionfiltertoanLVpurificationtrainconsistingofionexchangeandsizeexclusionchromatographycolumnsthusenablingintegratedcontinuousbioprocessing(ICB)asshowninfigure7.

Figure7.VHU®PerfusiontoenableContinuousManufacturingtoincreaseyield,reduceequipmentfootprintandaccelerateviralvectorproduction.