DRAFT

Achieving intensification and flexibility in virus purification with state-of-the-art chromatography tools

Authors:MochaoZhao,MelissaVandersluis

Abstract Vaccinesgreatlyreducedisease,disability,death,andinequityworldwide.TheyevenpermittedtotaleradicationofSmallPox.However,mostvaccinesavailabletodayarestillmanufacturedwiththeoriginalprocesstechnologiesincomplexandcapital-intensivefacilities.Thelabor-intensive,low-yieldandlowthroughputprocesses,andthelackofmanufacturingflexibility,diminishaffordabilityfordevelopingcountries,forcingmanufacturerstoaddresstheproblembysupplyingthesemarketswithsubsidizedproductsand/ornon-sustainableprices.Advancementsinvaccinesmanufacturingwouldbenefitbothglobalhealthandtheindustry.

Needs for better processes Viruses,widelyknownfortheirprophylacticuseasvaccinesinthebiotechnologyworld,arealsogainingpopularityintherapeuticfields.Viralvectorsarebeingusedasdeliveryvehiclesinmorethantwo-thirdsofgenetherapyclinicaltrials.Oncolyticvirotherapies,with2commerciallyavailableand48currentlyinclinicalpipelines,representthenextpotentialbreakthroughtherapeuticmodalitiesforcancertreatment.However,despiteoftheprovensuccessofviralvaccinesortherapeuticpromisesofemerginggeneandviraltherapies,therearestillmultiplebarrierstoextendingtheaccesstotheseproductsaroundtheworld.Thelackofbreakthroughsinmanufacturingtechnologyisoneofthem.Whilecurrentviralvaccinemanufacturingmethodsareinsomecaseseffectiveforsmall-scaleproduction,thesemethodsshareseveralcriticalshortcomings:theyaretooslow,toocomplicated,lackrobustness,andrequireexpensivespecializedfacilitiesandequipmentforlarge-scaleproduction.Thelackofmodern,efficientproductiontechnologyisrestrictingprogressonseveralfronts(bb).Duetohighcapitalanddevelopmentcostsandthelowproductprofitabilitycreatedbyhighmanufacturingcostsandlimitsonproductpricing,nichevaccineopportunitiesareseenasfinanciallyrisky,withlowprobabilityofbreakevenorprofitability.Promisingnewvaccinesdevelopedfromlargevirusesremainundevelopedduetothelackofviableprocessingcapabilitiestohandlethelargeviruses.Likewise,progressindevelopmentofoncolyticvirotherapiesislimitedbecauselargedosesofhighlypurifiedmaterialarerequired,butefficientmanufacturingprocessesarelacking(cc).Meanwhile,viralvectorshavebeenwidelyexploitedforgenedeliveryapproaches,furtherincreasingthedemandforlarge-scaleproductionofhighlypurifiedviralmaterial.Inallcases,progressisdirectlylimitedbygapsinmanufacturingtechnologies.

The Solution Resides in Innovation State-of-the-artvirusproductionprocessesandtechnologiesholdthepromiseofenablingproductionofnewvaccinesaswellasviralvectorplatformsforotherapplications,whilealsostreamliningandstrengtheningcurrentprocesses.Thesetechnologies,manyofwhicharebeingusedsuccessfullyinmodernbiotherapeuticsmanufacturing,canalsocreatedramaticcostandproductivityimprovementsforvirusmanufacturing(dd).

Anewgenerationofhigh-productivity,disposableprocessingtechnologiesgreatlysimplifyproductionarchitectureandreducefacilityfootprint.Inupstreamprocesses,stainlesssteelbioreactorsandmicro-carriersarebeingreplacedbymicrofibers(ex.iCELLis)thatcanachievecelldensityupto150millioncells/mL(ee,ff).Thenextgenerationfixed-bedcontinuousperfusionbioreactorsdevelopedbyUnivercellscanreachhighcelldensitywith50-70%volumereduction(gg).Withreducedbioreactorsize,mediausageaswellasfacilitydesignandcostcanbesignificantlyimproved.Indownstreamprocesses,advancementsinchromatographytechnologysuchasaffinitymediaspecificallyforviruscaptureandsingle-usehigh-productivitymembranechromatographyofferseveralperformanceandcostadvantagesoverthetraditionalpurificationtechniques.Withapowerfulcapturetool,theprocessarchitecturecanbesimplifiedwithfewerunitoperationsyetstillmaintaincriticalqualityattributes,leadingtosignificantcostsavingsaslessspace,utilities,andlabourarerequired.Single-use,high-throughputmembranecolumnsfurtherreducevalidation,cleaning,andstorage-relatedexpenseswhileimprovingproductivityviahighbindingcapacitiesandfastflowrates.Theeaseofscalabilityofchromatographycolumnsalsoenablessmoothproductiontransitionfromthelabbenchtothemanufacturingsite.Whencombined,newsingle-use,highproductivityprocesstechnologiescanovercomethemanufacturingconstraintsthathavelimitedmorewidespreadproliferationofvaccineuse,whilealsoopeningdoorstopromisingnewvaccinesandviral-basedtreatments.Replacingtraditionallarge,inflexibleandcomplexstainlesssteeloperationswithsmallfootprintfacilitiesenablesquickbatch-to-batchturnoverandrapidchangeoverbetweendifferentproducts.Thesmaller,morecompactprocessarchitectureallowsvirusestobeprocessedinsmallfacilitiesacrossmultiplegeographies,withthepromiseofeventuallyplacingproductionproximatetothepointofcare.Inaddition,simplified,smallfootprintprocessesenableflexibleprocessingwhichallowanewlevelofagilityforrespondingtochangesindemandandhandlingmulti-productmanufacturing.

Acornerstoneofthenewpurificationtechnologiesishighproductivitymembranechromatography,astate-of-the-arttechnologythatcombineshighbindingcapacityandrapidmasstransfertoimprovepurificationthroughput.High-productivitymembranechromatographywithcommonmodalities(ex.cationexchange,anionexchange,ormixed-mode)havebeenavailableandimplementedinmanybiotherapeuticsmanufacturingprocess.Insomecases,suchmembraneswithhighselectivitycandeliversufficientpuritylevelinasinglestep,makingsingle-steppurificationaviablesolution.Inaddition,affinitychromatographycanbecombinedwithmembranechromatographytoreplacemanypurificationunitsforhighlyintensifiedandproductiveprocesses.Threecasesstudiesofprocessintensificationusingstate-of-the-artchromatographytoolsarediscussedbelow.

Case Study 1: Newcastle Disease Virus single step purification using HD-Q Inthisexample,engineeredNewcastleDiseaseVirus(NDV)producedforoncolyticvirotherapyisamplifiedinembryonatedeggsandpurifiedfromtheallantoicfluid.Theconventionalcentrifugation-

basedpurificationmethodresultsinlowvirusrecovery(12%-35%)andonlypartialremovalofimpurities(upto6.3mghostcellproteinleftafterTFFandUC)(hh).

Thisconventionalpurificationschemeisslow,non-scalable,labor-intensiveandalsoleadstolowproductivity,non-optimalprocesseconomics,andhighfailureratesinamanufacturingenvironment.Consequently,themanufacturersofNDVforoncolytictherapywilllikelysufferfromthehighcostofgoodsandinabilitytoreliablymeetmarketdemand.Thisapproachforcesatradeoffbetweenproductpurityandproductrecovery.

Thenewsingle-stepmembranechromatographymethodforpurifyingengineeredNDVisfast,scalable,andachieveshighvirusrecovery(78.1%)with>99%purity(hh).

FigureYY:

Case Study 2: Influenza Single-Step purification using affinity membrane

Inthisexample,affinitymembraneswereemployedtopurifyinfluenzavaccinefromallantoicfluidharvest.

Theaffinitymembrane’shighproductivityenabledprocessintensification,resultinginsuccessfulpurificationinasinglestep:targetpurity,highcapacity,andhighrecoveryareallachieved(99.9%HCPclearance(3LRV),87%yield,and30-foldconcentration)(aa).Theprototypeaffinitymembranesachievedperformanceequaltoorexceedingcommercialaffinityresins.

Theaffinitymembrane’shighproductivityenables100Xsmallercolumns(comparedtocommercialresins)forsamebatchsize.Inthisexample,a500mLmembranecolumncanpurifyallantoicfluidharvestfrom1Millionembryonatedchickeneggsin1shift(8h,average104HAU/mL@5mL/egg).

FigureXX:Comparisonofinfluenzapurificationusingaffinityresinsandaffinitymembranes.Productivitycalculationsarebasedonahypotheticalloadof200columnvolumes(CV)plus20CVforadditionalequilibrationandcleaningstepsforeachproduct.

ImpurityClearance Productivity

Media BC(HAU/mL) Recovery Virusin

FTFlowrate(CV/min)

CycleTime(min)

Productivity(HAU/L.h)

AffinityResin#1 1x106 96% 2% 0.7 315 1.9E+08

AffinityResin#2 7x105 87% 4% 1 220 1.9E+08

AffinityResin#3 2x105 74% NA 2.9 76 1.6E+08

PrototypeH15 2x106 81% 10% 33 7 1.8E+10

PrototypeX40 2x106 95% 3.6% 33 7 1.8E+10

Case Study 3: Modelling a VLP-based vaccine process Avaccinemanufacturerre-engineereditstraditionalvaccinepurificationprocessforVLP-basedvaccines,enabledlargelybythepowerofaffinitymembraneperformance.Thenewmembrane-basedprocessaccomplishedthepurificationinthreeunitoperations,ascomparedtothirteenoperationsinthebaselineprocess.Thesimplifiedprocessresultedinmuchgreaterproductrevoery(lessproductloss),whichfurtherenableddownsizingoftherequiredupstreamoperations.Thisupstreamdownsizingenabledtheadoptionofsimpler,single-usebioreactors.Thenewprocessmodelsignificantlyreducedthecapitalinvestmentrequired,whilealsoimprovingcostperdose,processreliabilityandplantfootprint(CANWEQUANTIFYANYOFTHESECAPITALANDCOSTIMPROVEMENTS??).

(modellingslidesbelowmaybeeliminatedforfinalsubmission)

Conclusion Thehighproductivityandcost-efficiencyofthesingle-usechromatographymembraneprocessenablefavorableprocesseconomicscomparedtothelabor-andcapital-intensivereferenceprocesses.The

simplifiedarchitectureofthemembraneprocess,combinedwithpotentiallyfullycontinuousandclosed(aseptic)operation,shouldenablemanufacturinginatrulyflexible,lowcostandsmallfootprintfacility.Thehighthroughputmembranepurificationprocesscanimprovecurrentegg-basedvirusprocesses,andwillalsomarryperfectlywithimproved,high-throughputupstreamprocess(ex.adaptNDVstraintocellcultureandimplementintoascalablemanufacturingstrategyusingPalliCELLisreactor).

References (aa)R.Jacquemart,WorldVaccineCongress(Washington,DC,2017)

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