Protein separation using membrane chromatography 2.docx

Protein separation using membrane chromatography 2.docx

《Protein separation using membrane chromatography 2.docx》由会员分享，可在线阅读，更多相关《Protein separation using membrane chromatography 2.docx（29页珍藏版）》请在冰点文库上搜索。

Proteinseparationusingmembranechromatography2

Proteinseparationusingmembranechromatography:

opportunitiesandchallenges

RajaGhosh

DepartmentofEngineeringScience,UniversityofOxford,ParksRoad,OxfordOX13PJ,UK

http:

//dx.doi.org/10.1016/S0021-9673（02）00057-2,HowtoCiteorLinkUsingDOI

Permissions&Reprints

Abstract

Someoftheproblemsassociatedwithpackedbedchromatographycanbeovercomebyusingsyntheticmacroporousandmicroporousmembranesaschromatographicmedia.Thispaperreviewsthecurrentstateofdevelopmentintheareaofmembranechromatographicseparationofproteins.Thetransportphenomenonofmembranechromatographyisbrieflydiscussedandworkdoneinthisareaisreviewed.Thevariousseparationchemistrieswhichhavebeenutilisedforproteinseparation,alongwithdifferentapplications,arealsoreviewed.Thetechnicalchallengesfacingmembranechromatographyarehighlightedandthescopeforfutureworkisdiscussed.

Keywords

Reviews;

Membranechromatography;

Stationaryphases,LC

Keywords

Proteins

1.Introduction

Chromatographyisbyfarthemostwidelyusedtechniqueforhigh-resolutionseparationandanalysisofproteins.Theseprocessesaretraditionallycarriedoutusingpackedbeds,whichhaveseveralmajorlimitations.Thepressuredropacrossapackedbedisgenerallyhighandtendstoincreaseduringaprocessduetothecombinedeffectsofbedconsolidation（causedbymediadeformation）,andcolumnblindingcausedbyaccumulatedcolloidalmaterial.Anothermajorlimitationwithconventionalchromatographicbioseparationprocesses,particularlythoseemployingsoftchromatographicmedia,isthedependenceonintra-particlediffusionforthetransportofsolutemoleculestotheirbindingsiteswithintheporesofsuchmedia（seeFig.1）.Thisincreasestheprocesstimesincetransportofmacromoleculesbydiffusionisslow,andparticularlysowhenitishindered.Consequently,therecoveryliquidvolume（neededforelution）alsoincreases.Channelling,i.e.theformationofflowpassagesduetocrackingofthepackedbed,isamajorproblem.Thisresultsinshort-circuitingofmaterialflow,leadingtopoorbedutilisation.Otherproblemsincluderadialandaxialdispersionlimitationsarisingfromtheuseofconventionalpolydispersemedia.Someofthesefactorsandthefactthatthetransportphenomenoniscomplicatedmakescale-upofpackedbedchromatographicprocessesdifficult.

Fig.1.Solutetransportinpackedbedchromatographyandmembranechromatography.

Figureoptions

Someofthelimitationsofpackedbedchromatographyhavebeenovercomebyusingnewlydevelopedmonodisperse,non-porous,rigidchromatographicmedia（e.g.Refs.[1]and[2]）.However,thesemediaaregenerallyexpensiveandthesolutebindingcapacityisgreatlyreducedsincebindingcannowonlytakeplaceontheexternalsurfaces.Alsowiththesematerials,theproblemofhigh-pressuredropstillpersists.

Aradicallydifferentapproachtoovercomethelimitationsassociatedwithpackedbedsistousesyntheticmicroporousormacroporousmembranesaschromatographicmedia（e.g.Refs.[3],[4],[5]and[6]）.Inmembranechromatographicprocessesthetransportofsolutestotheirbindingsitestakesplacepredominantlybyconvection（seeFig.1）,therebyreducingbothprocesstimeandrecoveryliquidvolume.Thebindingefficiencyisgenerallyindependentofthefeedflow-rateoverawiderangeandthereforeveryhighflow-ratesmaybeused.Thepressuredropisalsosignificantlylowerthanwithpackedbeds.Anothermajoradvantageofmembraneadsorbersistherelativeeaseofscale-upwhencomparedwithpackedbeds.However,thispotentialhasnotbeenfullyutilisedasyetinthebioprocessindustry.Membranechromatographyisparticularlysuitableforlargerproteins（i.e.Mr>250 000）.Suchproteinsrarelyenterporespresentinparticulatechromatographicmediaandonlybindontheexternallyavailablesurfaceareaofsuchmedia.Therefore,forlargerproteins,thesurfaceareaavailableforbindingissignificantlygreaterwithmembranes.Thebindingcapacityofmembraneadsorbersforsmallerproteinsisgenerallylowerthanwithconventionalgel-basedmedia,butsignificantlyhigherthanwithmonodisperse,non-porous,rigidmedia.

Membranechromatographicdevicesaregenerallyeasierandcheapertomass-produce.Thismakesitpossibletohavedisposablemembraneadsorbers.Thesedevicescanbeuseduntilthedesirableproperties（i.e.hydraulicpermeability,bindingcapacity,selectivityandresolvingpower）aremaintained.Oncetheyceasetofunctionproperlythesedevicescanbereplaced.Thistypeofflexibilityeliminatestherequirementforcleaningandequipmentrevalidation.

Differentseparationchemistriesareutilisedinmembranechromatographyofproteins.Somemembranesalreadyinuseforothertypesofmembraneprocesses（e.g.microfiltration）havebeenfoundtobesuitableaschromatographicmedia.However,inmostcasestheseavailablemembraneshavebeenmodifiedtomakethemmoresuitableforuseasmembraneadsorbers.Novelsyntheticmembraneshavealsobeendeveloped.Anotheralternativetopackedbedchromatography,whichhascertainsimilaritieswithmembranechromatography,isbasedontheuseofmonolithcolumns.Thesecolumnsarepreparedusingrod-shapedporousstructuresthroughwhichconvectiveflowofmobilephasecantakeplace.Themainadvantagesofmonolithcolumnsaresimilartothoseformembranechromatography.However,monolithsdifferfrommembranesintermsofmaterialofconstructionandmorphology.Whileamembranebydefinitionisabarrierinwhichthelateraldimensionfarexceedsthelongitudinaldimension,theconverseisprobablytruewithmonoliths.Monolithsareperhapsmoresimilartopackedbedchromatographiccolumnsthantomembranes.

Inthisreviewarticle,thecurrentstateofdevelopmentintheareaofmembranechromatographyisdiscussed.Publishedliteratureintheareaofmembranechromatographyofproteinsisreviewed（e.g.Refs.[3],[4],[5],[6],[7],[8],[9],[10],[11],[12],[13],[14],[15],[16],[17],[18],[19],[20],[21],[22],[23],[24],[25],[26],[27],[28],[29],[30],[31],[32],[33],[34],[35],[36],[37],[38],[39],[40],[41],[42],[43],[44],[45],[46],[47],[48],[49],[50],[51],[52],[53],[54],[55],[56],[57],[58],[59],[60],[61],[62],[63],[64],[65],[66],[67],[68],[69],[70],[71],[72],[73],[74],[75],[76],[77],[78],[79],[80],[81],[82],[83],[84],[85],[86],[87],[88],[89],[90],[91],[92],[93],[94],[95],[96],[97],[98],[99],[100],[101],[102],[103],[104],[105],[106],[107],[108]and[109]）.Potentiallimitationsofmembranechromatographyarealsohighlighted.Thewideracceptanceofthistechnologydependslargelyonfindingsolutionstotheselimitations.

2.Transportphenomenaofmembranechromatography

Theadvantageofmembranechromatographyliesinthepredominanceofconvectivematerialtransport.However,asevidentfromFig.1,diffusivetransportisnottotallyabsent.Thepredominanceofconvectionalonedoesnotnecessarilyguaranteeefficiency.Convectiveflowofinappropriatetypecanbeaseriousdisadvantage.Flowdistributionisamajorconcerninchromatographicandindeedmosttypesofseparationprocesses.Rationaldesignofthemembranechromatographicprocessandequipmentispossibleonlywhenthetransportphenomenainvolvedareproperlyunderstood.However,itmaybeworthmentioningthat,inmanychromatographicprocesses,particularlythoserelyingonaffinity-typeinteractions,thebindingkineticsmaybelimiting.Insuchprocesses,improvementintransportphenomenaisnotlikelytoresultinsignificantimprovementinprocessefficiency.

Generallyspeaking,threetypesofmembraneadsorbersareusedforproteinbioseparation:

flatsheet,hollowfibreandradialflow.Singleflatsheetsarerarelyused.Moreoften,stacksofseveralflatsheetsarehousedwithinmembranemodules.Inadditiontoprovidingmoreadsorbentvolume,theuseofmembranestackshascertainotherbenefitswhicharediscussedbelow.Ahollowfibremembranehasatubulargeometrywiththetubestypicallyrangingfrom0.25to2.5mmindiameter.Ahollowfibremembraneadsorberusuallyconsistsofabundleofseveralhundredfibrespottedtogetherwithinamoduleinashellandtubeheat-exchanger-typeconfiguration.Radialflowadsorbersarepreparedbyspirallywindingaflatsheetmembraneoveraporouscylindricalcore.Fig.2summarisestherelativereportedusageofthethreemajortypesofmembraneadsorbers（basedonthepapersreviewedinthisarticle）.Flatsheetmembranesarebyfarmostwidelyused.Hollowfibres,eventhoughadvantageousinothertypesofmembranebasedtechnologies（e.g.microfiltration,ultrafiltration,anddialysis）,areperhapsnotsowellsuitedformembranechromatography.Thereasonsforthisareexplainedinthenextparagraph.Mostofthereportsontheuseofhollowfibresarefromresearchgroupsactivelyengagedindevelopmentofhollowfibremembranesfordifferentuses.Theuseofradialflowdevicesisalsonotthatwidelyreportedinthepublishedliteratureeventhoughseveraladsorbersofthistypeareavailableonthemarket.Table1listssomeofthecommerciallyavailablemembraneadsorbers.Thefactthattherearerelativelyfewmanufacturersofmembraneadsorbersindicatesthenewnessofthetechnology.

Fig.2.Membraneadsorbertypes（geometry）.

Figureoptions

Table1.Commerciallyavailablemembraneadsorbers

Productname

Membranematerial/type

Configuration

Manufacturer

SartobindMA5,

Reinforcedstabilisedcellulose,

Flatsheet,ready

Sartorius

MA15andMA100

strongcationexchange（Stype）,

touseadsorbers

strong