Capto-Core-700在流感病毒去除卵清蛋白

Vaccine32(2014)3721–3724ContentslistsavailableatScienceDirectVaccinejournalhomepage:ficientchromatographicreductionofovalbuminforegg-basedinfluenzaviruspurificationHansBloma,∗,AnnaÅkerbloma,TheoneKonb,SabahShakerb,LeovanderPolb,MatsLundgrenaaBioProcessingR&D,GEHealthcareBio-SciencesAB,Björkgatan30,SE-75184Uppsala,SwedenbInstituteforTranslationalVaccinology,AntonievanLeeuwenhoeklaan9,Pb450,3720ALBilthoven,TheNetherlandsarticleinfoArticlehistory:Received23December2013Receivedinrevisedform27March2014Accepted14April2014Availableonline5May2014Keywords:InfluenzaOvalbuminAllantoicfluidUltracentrifugationCorebeadchromatographyabstractVaccinationisthemosteffectivepreventionstrategytoavoidinfluenzainfectionandforprotectionoflargepopulations.Thevastmajorityofinfluenzavaccinesarestillproducedwithallantoicfluidfromfertilizedchickeneggs.Thepresenceofovalbumin,whichcanconstituteover60%ofthetotalproteincontentinallantoicfluid,canresultinsevereallergies.Consequently,efficientreductionofovalbuminiscriticalduringeggbasedvaccinemanufacturing.HerewepresentCaptoCore700,anovelcorebeadchromatographicflowthroughmoderesinforremovalofovalbuminandcompareittosucrosezonalgradientultracentrifugation,whichistheindustrystandardforegg-basedvaccineproduction.Theresultsdemonstratethatcorebeadchromatographyisfullycomparabletozonalcentrifugationinremovingovalbumintomeetregulatoryrequirements.Furthermore,thescalabilityandtheshorterprocesstimesofthismethodhavethepotentialtosignificantlyimprovetheproductivityandeconomyforindustrialproductioncomparedtozonalcentrifugation.©2014ElsevierLtd.Allrightsreserved.1.IntroductionTheemergenceofnewInfluenzavirusstrains[1,2]causesseasonalinfluenzaepidemicsandmayresultinsevereworldwidepandemics.Thereisthusacriticalneedforefficientandeconomicalvaccineproductiontorespondtothisthreat.Thesupplyofvaccinestillmainlyrelyonusinginfluenzaviruspropagatedinfertilizedchickeneggsasasource,whichisthenprocessedwithpurificationprocessesthathaveremainedunchangedfordecades[3–5].Oneconcernforegg-basedvaccinesisthereductionofovalbumin,whichcancausesevereallergies[6].Industrialscaleprocessingofvirusiscommonlybasedonsucrosegradientzonalultracentrifu-gation(ZUC)combinedwithfiltrationtechniques.ThoughZUCprovidesbothefficientpurificationandconcentrationofinfluenzavirus,itistimeconsuming,requiresextensivemaintenance[7]andsuffersfromlimitedscalability.Consequently,improvedandmoreefficientandscalabletechnologiesforpurificationofegg-basedAbbreviations:HA,haemagglutinin;SRID,singleradialimmunodiffusion;ZUC,sucrosezonalgradientultracentrifugation;FT,flowthrough;kDa,kiloDalton;UF/DF,ultrafiltrationanddiafiltration;DBC,dynamicbindingcapacity;CV,columnvolume;CIP,cleaninginplace;GMP,goodmanufacturingpractice.∗Correspondingauthor.Tel.:[phone];fax:[phone].E-mailaddress:[e-mail](H.Blom).vaccineswouldbevaluable.Hence,variouschromatographicmethodsforpurificationofvirusparticleshavebeeninvestigated[5,8–10].Duetothesizeofthevirus,flowthrough(FT)modechromatography,wherevirionspassthroughthecolumnunre-tainedandsmallerimpuritiesareadsorbedtotheresin,maybeanattractivealternative.CaptoTMCore700isanewFTmoderesinengineeredforpurifi-cationofvirusesandotherlargebiomolecules.Itconsistsofbeadswithaninactiveshellsurfaceandanactivefunctionalizedmulti-modalcorewithoctylamineligands.Thebeadporeshaveanapproximateexclusionlimitof700kDa(Fig.1).Thisallowssmallermoleculestoaccessthecorewheretheycanbeefficientlyadsorbed,whilelargerentitieswillpassintheFT.ThepresentedworkaimtoevaluateCaptoCore700asanalternativetoZUCforproductionofinfluenzavaccineandisacollaborationbetweenGEHealthcareandtheInstituteforTranslationalVacci-nology(Intravacc).2.Materialsandmethods2.1.VirusproductionInfluenzastrainH3N2A/Uruguaywaspreparedfromeggallan-toicfluidproducedatIntravacc.Theeggswereincubatedat35◦C©2014ElsevierLtd.Allrightsreserved.3722H.Blometal./Vaccine32(2014)3721–3724Fig.1.Schematiccross-sectionalviewofaCaptoCore700particlewithanaveragediameterof75m.Smallproteinimpurities(green)canentertheinterioroftheresinparticleandbindtotheligand.Largermolecularentitiesabovetheapprox-imateexclusionlimitof700kDa(pink),suchasvirusparticlesarehinderedfromenteringtheresinporesandwillpassthroughthecolumnunretained.(Forinter-pretationofthereferencestocolorintext,thereaderisreferredtothewebversionofthearticle.)andtheallantoicfluidharvested72hpostinfection.Clarificationwasperformedbycontinuousdiskstackcentrifugationfollowedbyfiltrationusingtwoparallel10in.2.0mULTATMprimecapsulefilters.Theclarifiedharvestwasdividedintwoportions,ofwhich70LwasusedforZUC.Another10Lintendedforchromatographywassubjectedtoadditionalfiltrationusinga1.5in.0.6mULTAGFtoprotectthecolumnfrompossibleaggregates.2.2.DynamicproteinbindingcapacityThreeduplicaterunswereperformedtoevaluatethedynamicbindingcapacity(DBC)usingthreedifferentTricornTM5/50CaptoCore700columns(columnvolume(CV)approximately1mL)andanÄKTATMexplorer10system.Equilibrationwasperformedwith20mMTr