Protein A, Protein G, and Protein 原理及应用

AnalysisandPurificationofAntibodyFragmentsUsingProteinA,ProteinG,andProteinL

RemkoGriepandJohnMcDougall

24.1Introduction

Today,monoclonalantibodies(mAbs)formthelargestcategoryofbiopharmaceu-ticalsinclinicaltrials,andtheirnumberisexpandingrapidly(DataMonitor2007a,b).Theantibodiesorfunctionalantibodyfragmentsarebeingproducednotonlyinartificialproductionsystemssuchasmammaliancells,yeast,bacteria,andplantcellsbutalsointransgenicanimalssuchasgoats,sheep,andcows.Regardlessoftheproductionmethod,thequalitycontroldemandisthesameforallofthem.Hostcellproteins,cellculturemediaadditives,DNA,andendotoxinshavetoberemovedfromthemAbpreparationtoallowtheproteinstobesafelyappliedforhumantherapy.Moreover,antibodyaggregates,clippedandlowmolecularweightspecies,shouldalsoberemoved.

Severalproteinswithaninherentaffinityforimmunoglobulins(Ig)havebeenisolatedfromvariousbacteria.Thesemoleculesincludeprotein-A,derivedfrom

¨quist1966);protein-G,derivedfromaStaphylococcusaureus(ForsgrenandSjo

¨rkandKronvall1984);andfinallyprotein-L,derivedgroup-CStreptococcus(Bjo

˚kerstro¨mandBjo¨rk19;Housdenetal.2003,fromPeptostreptococcusmagnus(A

2004).Theyallcontainrepetitive55–76aminoacidresidues(Fig.24.1)thatmediatetheactualIgbinding(Kasternetal.1992).Therecombinantprotein-Lcanbeproducedatayieldofupto3g/Linpilot-scalestudies.Ityieldsahighlypure,stable,andactiveprotein-Lfractionafterpurification,whichisbindingefficientlytomostofthehumanantibodiesoftheKappaisotype(Fig.24.2).

Protein-GbindsnotonlytotheFc-regionbutalsototheCH1-domainofthehumanIgG1-isotype.Therefore,ithasabroaderapplicationcomparedtoprotein-A.Someacademicgroupshavealsoreportedtheuseofgeneticallyfusedprotein-LG(Kihlbergetal.1996;Harrisonetal.2008)orprotein-AG(Eliassonetal.1988;

R.Griep(*)andJ.McDougall

´en21,Oslo3490,NorwayAffitechAS,Gaustadalle

e-mail:r.griep@affitech.com

¨bel(eds.),AntibodyEngineeringVol.2,R.KontermannandS.Du

DOI10.1007/978-3-2-01147-4_24,#Springer-VerlagBerlinHeidelberg2010

301

302

ASPB1B2B3B4B5SR.GriepandJ.McDougall

C1C2WM174246318390471524578Fig.24.1Structureoftheprotein-Lmoleculecomprising719aminoacids.Thenumbers,indicat-ingtheaminoacidsofthebeginningofeachdomain,arelistedbelowtheboxes.Includedarethesignalpeptide(SP),thesignalpeptidecleavagesiteisindicatedbythearrow,theNH2-terminal(A),therepeatedunitswithIg-bindingactivity(B1–B5),thespacerregion(S),therepeats(C),thewallspanningdomain(W),andthetransmembraneregion(M).Therecombinantprotein-LconsistsoffourIg-bindingdomains(B1–B4),whichcanbindtotheKapparegionwithoutinterferingwiththeantigen-bindingsiteoftheimmunoglobulin

Bergmann-Leitneretal.2008)andprotein-LA(Svenssonetal.1998)formonoclo-nalantibodypurification.Theyindeedobtainedbroaderfunctionalligandsbecausethebindingcharacteristicsofbothparentalproteinsweremaintained.Theabilityofprotein-A,-G,or-Ltomaintaintheirfunctionality,onconjugationwithfluoro-chromes,enzymes(Fig.24.3a),orgoldparticles,makesthemhighlyvaluablesecondaryreagentsforthedetectionofprimaryantibodiesinELISA,immunohis-tochemistry,flowcytometry,andelectronmicroscopy.

Protein-AmainlybindstotheFc-regionoftheIgGfromseveralhumanisotypes(Table24.1)butonlytoasinglevariableregionoftheheavy-chainfamily(Starovasniketal.1999).Incontrast,protein-LbindstomostofthehumanKappalight-chainsofthekI,kIII,andkIVfamilies.Thesecomprise55–60%ofallIgA,IgE,andIgMantibodiesinthehumanserum(Solomon1976)andcanthusbeusedtopurifyallmonoclonalantibodiesofthoseKappasub-types(Nilsonetal.1992)orfragmentsderivedthereof.This,withouttheneedtogeneticallyengineeraffinity-tagsontotheproteinofinterest(Devauxetal.2001;Dasetal.2005;Cossinsetal.2007).ThekantibodiesdescribedinFig24.3bwereoriginallyderivedfromalargehumanunbiasedantibodyphagelibrary(Løsetetal.2005)andsixoutofthetenkantibodiesstronglyreactwithprotein-L(Fig.24.3b).Theseauthorsalsodemon-stratedthatpreselectionofthisparticularphage-libraryforthebindingtoprotein-Lcanbeofuse.Ityieldsphage-antibodieswithimprovedfunctionality,aseachphageisactuallyassayedforitsabilitytoexpressatleastonefunctionalscFvonitssurfacepriortoitsselectionagainstanantigen.Analternativeapproachistobuildahighlydiverselibrary,onthebasisofcertainwell-expressingandprotein-LbindingKappalight-chaingenes(Holtetal.2008).Moreover,protein-Lhasaclearadvantageoverprotein-Aandprotein-G,asitdoesnotbindtobovineIgGortobovineserumalbumin.Thismightbeofmajorimportancewhenoneisforcedtousebovineserumasadditivetothecellculturemediumtopreventcertaintypesofmammaliancellsfromdying.Thusfar,protein-Lhasnotbeenavailablefortheindustrial-scale

6971924AnalysisandPurificationofAntibodyFragments303

a

3.53.02.52.01.51.00.501

2

3

4

mg/mLHD148HD147HD146HD149HD150b

kDa75503725201510

Time after induction

c

1

Protein-L

234

+ IgG

-+ -

Fig.24.2(a)Apilot-scaleproductionsystemhasbeensetupforproductionofrecombinant

protein-LinE.coli.TheDNAsequenceencodingtheB1-4domainshasbeenclonedintoapJB-vector(Slettaetal.2004)andtherecombinantprotein-Lwasexpressedintracellularinhigh-cell-density-cultivationasshownhereforfiveseparatecases.Theproducedprotein-Lwasextractedfromthecytoplasm,purified,andanalyzedbySDS-PAGE.(b)SDS-PAGEanalysisofthepurifiedprotein-L(c)CNBRactivated-sepharosebeadswereconjugatedwithout(À)andwithpolyclonalhumanIgG(þ)andincubatedwithprotein-Lpreparationswhichwerestoredfor1month,eitherat4󰀃C(lane1and2)orat37󰀃C(lane3and4).Subsequently,theobtainedsupernatantswereanalyzedbySDS-PAGEforthepresenceofunboundprotein-L.Ascanbeobservedfromthispicture,themajorityoftheprotein-LisspecificallybindingtothepolyclonalIgG,evenafterstoragefor1monthat37󰀃C

purification,butrecently,adevelopmenttowardintroductionintothebulkmarkethasbeeninitiated.

Aprerequisitefor(cost)-efficientindustrial-scalepurificationofMAbsisthattheligandslikeprotein-A,protein-L,andprotein-Gcanbecoupledefficientlytosolidmatriceslikecontrolledporeglass(Millipore)andtoagarosewithvaryingdegreesofcross-linking(GEHealthcare).Thesematerialsarerigidandcanbeoperatedathighflowvelocities.Highlyporousmaterialsexertalow-pressuredrop,alowmasstransferresistance,andahighdynamiccapacity(LeVanetal.1997).Unfortunately,thesefeaturesarenonexclusivetoacertainextent.Ahighlyporousmediumcouldhavealowequilibriumcapacitybecauseofalimitedsurfaceareaandsimulta-neouslyhavegoodmasstransfercharacteristicsbutbadflowpropertiesasaresult

304R.GriepandJ.McDougall

a

OD4051.61.20.80.40012345672-fold serial dilutions101112b

3.02.5

OD4052.01.51.00.50IgG-1IgG-5G-4IgG-8G-7b-1IgG-6IgGIgGFv-sccontFaIgIgrol-2-31Applied antibody

Fig.24.3(a)Qualitycontroloftheproducedrecombinantprotein-LwiththeaidofanELISA.AmaxisorbELISA-plate,coatedwithhumanIgG,waspreincubatedwithdifferentconcentrationsofunconjugatedrecombinantprotein-L(rProtein-LTM,#101Actigen)priortoincubationwithaprotein-L/HRPconjugate(rProtein-LTMHRP,#301Actigen).Afterwashing,chromogenicsub-stratewasaddedandtheabsorbanceoftheindividualwellswasmeasuredatOD405nm.Thesignalshowsclearinhibitionbytheunconjugatedprotein-L(b)AmaxisorbELISA-plate,coatedwithdifferenthumanIgG(k)antibodies,ahumanFab(k)orwithascFv(k)fragment(allat0.1mg/well)wasincubatedwithaprotein-L/HRPconjugate.Afterwashing,chromogenicsubstratewasaddedandtheabsorbanceoftheindividualwellswasmeasuredatOD405nm

ofitssoftness.Incontrast,aresinwithahighequilibriumcapacitymighthaveincreasedmasstransferresistance.

Asthecostsofresinsarehigh,theligandsshouldmaintaintheirselectivityandhavegoodchemicalstabilitiesoveralongperiodoftime.Cleaninginplaceprocedures(CIP)withrepeatedalkalineexposurescanbedetrimentalforligandslikeprotein-Aandprotein-G.TofacilitateCIP,someoftheligands,suchasMabSelect(GEHealthcare)oraprotein-AanalogZ(F30A)(Linhultetal.2004),couldbeoptimizedandarenowavailableasanimprovedalkalineresistantalter-nativeforprotein-A.Also,forprotein-G,animprovedmutantwasengineered¨lichetal.2002),whileaccordingtotheresultsofEnever(Eneveretal.2005),(Gu

higheraffinityvariantscanalsobeexpectedforprotein-L.

BecauseoftheacidicelutionandthehighconcentrationofMabsonthecolumn,aggregatesareeasilyformed(Shuklaetal.2007).Inaddition,leachingandcleavageoftheligandisobservedforprotein-A(Carter-Franklinetal.2007)andprotein-G.Asaconsequence,bothaggregatesandleachedligandhavetoberemovedfromthe

24AnalysisandPurificationofAntibodyFragments305

Table24.1Bindingofimmunoglobulinisotypesandsomeoftheirsmallerderivativestoprotein-A,protein-G,protein-L,protein-AGprotein-LG,andprotein-LA,onthebasisofdatathatwereobtainedfromPierce;GEhealthcare;Bonifacino,andDell’Angelica1998;Hoberetal.2007;

ˆteauetal.1993,andSvenssonetal.1998.(?:unknown,À¼noKihlbergetal.1996;deCha

binding,Ƽverylowbinding,þ¼lowbinding,þþ¼goodbinding,þþþ¼highbinding,VH3andKk=bindingonlytothesespecifichumanheavy-andlight-chainfamilies)SpeciesSubclassProt-Prot-Prot-LProt-Prot-Prot-LA

AGAGLG

HumanIgG1þþþþþþþþ(k)þþþþþþþ

IgG2þþþþþþþþ(k)þþþþþþþIgG3–þþþþþ(k)þþþþþþIgG4þþþþþþþþ(k)þþþþþþþ

þþ(k)VH3þþþþþþ(k)IgEVH3–

(k)(VH3)

þþ(k)VH3þþþþþþ(k)IgAVH3–

(k)(VH3)

þþ(k)VH3þþþþþþ(k)IgMVH3–

(k)(VH3)

HumanLamdda-LC––––––

þþ(k)VH3þþþþþþ(k)AntibodyKappa-LCVH3–

(k)(VH3)fragments

VH3þþþþþþ(k)IgG1-FabVH3þþþþþ(k)

(k)(VH3)

þþ(k)VH3þþþþþþ(k)FvVH3–

(k)(VH3)

þþ(k)VH3þþþþþþ(k)scFvVH3–

(k)(VH3)

þþ(k-LC)VH3þþþþþþ(k)singledomainVH3–

(k)(VH3)

MouseIgG1þþ35%oftotalIgGþþþþþ

IgG2aþþþinmouseseraþþþþþþIgG2bþþþþþþþþþIgG3þþþþþþþ

GuineapigIgG1þþþþ<10%oftotalIgG?þþþþ

IgG2þþþþ?þþþþ

BovineIgGþþþþ–þþþþþþþCatIgGþþþþ?þþþþþþChickenIgYÆþ>50%oftotalIgGÆþ>50%of

totalIgG

DogIgGþþþþþ–þþþþþþþDonkeyIgG–þþ??þþ?HamsterIgGþþþþþþþþþþþþþþHorseIgGþþþþþ–þþþþþþþGoatIgGþþþ??þþþþþMonkeyIgGþþþþþþ?þþþ??PigIgGþþþþþþ50%oftotalIgGþþþþþþþþRabbitNoþþþþþþ–þþþþþþþþ

distinction

RatIgGþþþ35%oftotalIgGþþþþþþSheepIgGþþþ?þþþþþþ

306R.GriepandJ.McDougall

antibodypreparationbeforeitcanbeapplied.Therefore,IgGpurificationwithprotein-A,-L,or-Gisusuallyonlythefirststepandisusuallyfollowedbyaseriesofmultiplepolishingsteps.Acombinationofanionexchangechromatographyinflowthroughmodeandcationexchangechromatographyremoveshostcellproteins,DNA,endotoxins,leachedprotein,andaggregatesefficiently(Tugcuetal.2007).Despitethewidevarietywithintheappliedmonoclonalantibodies,suchas,chimeric,humanized,andfullyhumanIgGsofvariousisotypes,ageneralpurifica-tionstrategyisdesirable.Todate,severalcomparativestudiesareavailableintheliterature(Fuglistaller19;Fahrneretal.1999;Godfreyetal.1993;Hahnetal.2003,2006;Ghoseetal.2007;Swinnenetal.2007andKatohetal.2007),butnewmatricesareavailabletobeintroducedontheflourishingantibodymarket(Boietal.2008).Inaddition,atotalmatrixfreepurificationmethodhasbeendescribed(Kimetal.2005),whichisonthebasisofareversibletemperaturetriggeredprecipitationofantibodieswiththeaidofprotein-L,orprotein-LGfusedtoelastin-likeproteins.

Thebasicprotocolsforprotein-A,protein-L,andprotein-Gchromatographyarerelativelystraightforward.BindtheimmonoglobulinsataneutalpHandeluteatanacidicpH.SaltionsevenpromotebindingofIgGtoprotein-A.OftenastationaryphaseisemployedforthepurificationofmultiplemonoclonalantibodiesandalthoughtheFcregionisthesame,stilldifferentbindingandelutionparametersmighthavetobeestablishedfordifferentvariableregions(Ghoseetal.2005,2007).Asdemonstration,methodsaredescribedforthepurificationofpolyclonalhumanIgG/kfromserumIgG,ascFv(k)andaIgG1derivedCH1/lFabfragmentfromanE.coliextractusingprotein-Landprotein-G,respectively.DespitethedescribeddifferencesintheliteraturebetweenuniquehumanIgGmolecules,thepurificationmethodologydescribedbelowwillyieldpure,homogeneous,andhighlyactiveantibodypreparationsforalmostanyantibodywithoutanymajorchangestotheseprotocols.

24.2

PurificationofHumanIgG/kAntibodyFragmentswithProtein-L

FortheisolationofapolyclonalIgGfractionfromahumanserumorofanscFvfragmentfrombacterialperiplasmicpreparation,protein-Lisknowntobeanexcellentligand(Fig.24.4).TheisolatedIgGandscFvhaveahighpurityandthepurificationmethod,asdescribedbelow,iseasytouse.

24.2.1Materials

–Protein-Lagaroseslurry(rProtein-LTM–agarose,#201,Actigen)in50%ethanol;maximumbindingcapacityis10mgIgGpermLbeads–Humanserum

24AnalysisandPurificationofAntibodyFragments307

abcFig.24.4Representativeexamplesoftheversatileapplicationofprotein-L.(a)PurificationofPolyclonalantibodiesfromhumanserum.Thepooledfractionsareindicatedwiththedoublearrow;thesolidlinesindicatetheopticaldensityat280nm,whereasthedottedlinesreflectthepH.(b)Separationofaprotein-ApurifiedhumanIgGpreparationinaKappaandLambdafractionviaprotein-L.(c)PurificationofaKappascFvfromabacterialextractonaprotein-Acolumn

308R.GriepandJ.McDougall

––––––PBS

Elutionbuffer(0,1MGlycin-HCl,pH2,5)Neutralizingbuffer(1MTris-HCL,pH9,0)Polystyrenecolumns,2mL(Pierce,#29920)20%EthanolDeionisedwater

24.2.2Method

1.Setupa2mLcolumnandloadwith0.5mLProteinL-agarose(thus1mLasin50%volumewithethanol/PBS).

2.Waituntilthegelissettledandwashwith5mLPBS.3.Load5mLIgG-solution.

4.CollecttheIgGflowthroughfraction.5.Washwith10mLPBS.6.Collectthewashfraction.

7.Add350mL1MTris-HCl,pH9.0tothetubesinthefractioncollectorpriortoelutiontoimmediatelyneutralizethesampleuponelution.8.Elutewith5mLelutionbuffer.9.Collecttheeluate.

10.Washthecolumnwith5columnvolumesofdeionisedwater.

11.Washthecolumnwith5columnvolumesof20%ethanol,andstoreitat

4󰀃C.

12.Dilutetheeluate,flowthrough,wash,andelutedfraction1:10withPBS.13.Determinetheabsorbanceat280nm.

14.AnalyzethepurityofthesamplebySDS-PAGE.

24.3

PurificationofaMonoclonalHumanIgGFabFragmentwithProtein-G

TheisolationofrecombinantFabfragmentsfrombacterialextractsrequiresamoredemandingpurificationprocedurebecausetheheavy-andlight-chainfrag-mentsarenotproducedinequalamounts.Ingeneral,thelight-chainisproducedathigherlevelsandsecretedasacontaminatinglight-chaindimer.Therefore,theisolationprocedurehastoconsistoftwosubsequentsteps.Thefirststepisisolationofallthelight-chainsviaahis-tag,whichislocatedontheC-terminus.Thisisfollowedbyanaffinitypurificationoftheheavy-chainfragmentviaprotein-G,whichbindstotheCH1-regionofhumanIgG1.Asaconsequence,

24AnalysisandPurificationofAntibodyFragments309

alllight-chaindimerswillberemovedduringtheproceduredescribedbelow,whichiseasytouseandwillyieldhighqualityFabfragments(Fig.24.5).

24.3.1Step1:Ni-IMACPurificationofaFabFragment

24.3.1.1––––––

Materials

¨ktaTMPurifierA

¨ktacolumnHisTrapTMFF,1mL(GEHealthcare)A

20%EthanolDeionisedwater

0.8mm,0.45mmand0,20mmfilters

2MImidazole,pH7.0(PreferablyfromFluka,soldbySigma-Aldrich,ultra-pure,cat.no56749,whichhasnointerferingabsorbanceat280nm)

–Buffer-A:IMACloadingbuffer(20mMsodiumphosphate,500mMNaCl,pH7.4)

–Buffer-B:IMACElutionbuffer(20mMsodiumphosphate,150mMNaCl,500mMImidazole,10%glycerol,pH7.4)–DialyzedperiplasmicE.coliextracts

24.3.1.2Method

1.Filterallthebuffersthrougha0.20mmfilter.2.Preferablyprecoolthebuffersat4󰀃C.

3.Filterthepooledanddialyzedperiplasmicfractionsthrough0.8and0.45mmfiltersbeforeloadingitontotheIMACcolumn.

4.Add500mLofthe2Mimidazolestockper100mLofthefilteredperiplasmicfractiontoobtainafinalconcentrationof10mM.

5.Equilibratethecolumnwith5columnvolumesofbuffer-A.6.Loadthesampleonthecolumn.

7.Washthecolumnwith20mMimidazoleuntiltheunboundproteinshavebeenwashedoutofthecolumn(5columnvolumes)andtheOD280signalhasreturnedtothebaseline.8.Elutewith100%Buffer-B.

9.Washthecolumnwith5columnvolumesofdeionisedwater.

10.Washthecolumnwith5columnvolumesof20%ethanol,andstoreitat

4󰀃C.

11.Optional:analyzetheisolatedfractionsbySDS-PAGEbeforepooling.

12.Avoidfreezingsampleswithimidazole,asithasbeenobservedthatthiscan

severelydecreasetheactivityofthepurifiedantibodyfragments.

310R.GriepandJ.McDougall

a

b

c

Fig.24.5Representativeexampleoftheprotein-GpurificationofamonoclonalhumanIgG1/lFabfragmentfromtheeluentofanickel-NTAcolumn.(a)TheFabfragmentswereisolatedfromabacterialextractthroughtheinteractionoftheHis-tagofthelight-chainwithnickel-NTAbeads.

24AnalysisandPurificationofAntibodyFragments311

24.3.2Step2:Protein-GPurificationofaFabFragment

24.3.2.1––––––––

Materials

¨ktaTMPurifierA

Nickel-NTAprepurifiedFabfragments

HiTrapTM_ProteinG_HP_1mLFF,(GEHEALTHCARE)20%EthanolDeionisedwater1MTris-HCl,pH9,0

Loadingbuffer:20mMsodiumphosphatewith500mMNaCl,pH7.4Elutionbuffer:0,1MGlycine-HCl,pH2.5

24.3.2.2Method

1.Poolthefractions,preferablyobtainedfromaFabpreparation,whichwereprepurifiedonanickel-NTAcolumn.

¨ktaPurifierTMaswellasthe10mLsample2.WashthegeneralsystemoftheA

loopwithLoadingbuffer.

3.Add300mLTris-HCl,pH9.0tothetubesinthefractioncollectorpriortoelutiontoimmediatelyneutralizethesamplesuponelution.4.Loadthedialyzedsampleontothecolumn.

5.Washthecolumnwithminimal5columnvolumesofloadingbufferuntiltheunboundproteinshavebeenwashedoutofthecolumnandtheOD280signalhasreturnedtothebaseline.

6.ElutethecapturedFabfragmentsviaelutionwith100%oftheelutionbuffer.7.Washthecolumnwith5columnvolumesofdeionisedwater.

8.Washthecolumnwith5columnvolumesof20%ethanol,andstoreitat4󰀃C.9.ToobtainFabfragmentsofthehighestqualityanSDS-PAGEanalysiscanbeperformedbeforedecidingwhichofthefractionsshouldbepooled.

10.DialyzeagainstPBScontaining5%glycerol,preferablyataFabconcentration

below1mg/mL;thisistopreventprecipitation.

312R.GriepandJ.McDougall

11.DeterminetheproteinconcentrationwithaspectrophotometeratOD280.

12.Storethesamplesat4󰀃C(1day)oratÀ20󰀃Cforlongerperiodsoftime,but

storageatÀ80󰀃CisrecommendedtoguaranteelonglastingqualityofthepurifiedFabfragments.

24.4TroubleShooting

ItmightbevaluabletomonitorthebindingefficiencyforeachspecificantibodywithtechniquessuchasELISA,SDS-PAGE,andWesternblotting.Optimizationofthebindingpropertiesof,forinstance,rProteinLcanresultinatenfoldhigheryieldforaparticularantibody.Similaroptimizationshavebeenreportedforprotein-Gandprotein-Awiththeapplicationofsaltssuchassodiumchlorideandsodiumsulfate,whichfavorincreasesinhydrophobicinteractions.Inaddition,thepHoftheloadingbuffercanbeincreasedfromneutraltomorebasic(pH9)tomaximizetheyield.Inaddition,theconcentrationofthefeedstockshouldbealteredforeachantibodyduringtheoptimizationprocesstogainmaximumbindingandelutioncharacteristics.Incaseofproblemswithserumderivedimpurities,protein-Lperformsspecificallyinthepresenceofalargebackground(uptotenfold)ofbovineimmunoglobulins.Thisisparticularlyvaluablewhenisolatingantibodiesfromculturemediacontainingbovineserumorfromthemilkoftransgenicanimals.

24.5ConcludingRemarks

Beforepurifyinganantibody,regardlessthesource,considerationshouldbegiventothefinaluseoftheproduct.Formanyapplications,bothmonoclonalandpolyclonalantibodiesmaybeusedinanimpureform.However,forconjugationtofluorochromesorenzymes,simpleligand-basedpurificationissufficient,butforcell-basedassays,ahigherlevelofpurificationisanabsoluterequirement.Inaddition,italldependsonthenatureoftheantibodyfragmentcombinedwiththemethodusedforitsproductionwhetherproteinA,proteinG,proteinLorevenacombinationoftheseshouldbeusedtoobtainoptimalresults.Whichevermethodischosen,careshouldbetakennottoexposetheantibodiesforanextendedtimetoeitherstrongacidicorbasicconditions.Thiscanbeavoidedbyaddinganeutraliz-ingbufferinthecollectiontubespriortotheelutionstep.Inaddition,bufferconditionswithapHaroundtheisoelectricpointmightfavorprecipitation.Ageneralformulationbuffer(10mMNa-citrate/pH6containing:300mMsucrose,0.9%NaCl,50mMglycine,3.5mMmethionine,and0.05%polysorbate-80)canberecommended,whichpreventsprecipitation,aggregation,andoxidationofthepurifiedantibodyfragments.Finally,antibodypurificationcanbeperformedwith

24AnalysisandPurificationofAntibodyFragments313

fancyequipment,butthisisnotatallanabsoluterequirementtoobtainexcellentresults.Simplegravityflowalwaysworks,eveninthecaseofpowerfailure.

References

˚kerstro¨mB,Bjo¨rkL(19)ProteinL:animmunoglobulinlightchainbindingbacterialprotein.A

JBiolChem2:19740–19746

Bergmann-LeitnerES,MeaseRM,DuncanEH,KhanF,WaitumbiJ,AngovE(2008)Evaluationofimmunoglobulinpurificationmethodsandtheirimpactonqualityandyieldofantigen-specificantibodies.MalarJ7:129–139

¨rkL,KronvallG(1984)PurificationandsomepropertiesofStreptococcalproteinGanovelBjo

IgG-bindingreagent.JImmunol133:969–974

BoiC,DimartinoS,SartiGC(2008)PerformanceofanewproteinAaffinitymembranefortheprimaryrecoveryofantibodies.BiotechProg24:0–7

BonifacinoJS,Dell’AngelicaEC(1998)Immunoprecipitation.CurrProtocCellBiolChapter7:7.2.1–7.2.21

Carter-FranklinJN,VictaC,McDonaldP,FahrnerR(2007)FragmentsofproteinAelutedduringproteinAchromatography.JChromatogA1163:105–111

CossinsAJ,HarrisonS,PopplewellAG,GoreMG(2007)RecombinantproductionofaVLsingledomainantibodyinEscherichiacoliandanalysisofitsinteractionwithpeptostreptococcalproteinL.ProteinExprPurif51:253–259

DasD,AllenTM,SureshMR(2005)Comparativeevaluationoftwopurificationmethodsofanti-CD19-c-myc-His6-CysscFv.ProteinExprPurif39:199–208

DataMonitor(2007)MonoclonalAntibodiesReportMarketModel–Detailedanalysisofthemonoclonalantibodysegment,encompassingmarketdynamics,keytherapyareas,technologyandtargettypesthroughto2012,evaluatingthestrategiescompaniesareusingtocapitalizeonthislucrativemarket.ReferenceCode:IMHC0090,June2007

DataMonitor(2007)MonoclonalAntibodiesReportPart1.ReferenceCode:DMHC2291,June2007

ˆteauM,NilsonBH,ErntellM,MyhreE,MagnussonCG,Akerstro¨mB,Bjo¨rckL(1993)DeCha

OntheinteractionbetweenproteinLandimmunoglobulinsofvariousmammalianspecies.ScandJImmunol37:339–405

DevauxC,MoreauE,GoyffonM,RochatH,BillialdP(2001)Constructionandfunctionalevaluationofasingle-chainantibodyfragmentthatneutralizestoxinAahIfromthevenomofthescorpionAndroctonusaustralishector.EurJBiochem268:694–702

´nM(1988)¨sM,GussB,LindbergM,UhleEliassonM,OlssonA,PalmcrantzE,WibergK,Ingana

ChimericIgG-bindingreceptorsengineeredfromstaphylococcalproteinAandstreptococcalproteinG.JBiolChem263:4323

EneverC,TomlinsonIA,LundJ,LevensM,HolligerP(2005)Engineeringhighaffinitysuper-antigensbyphagedisplay.JMolBiol347:107–120

FahrnerRL,WhitneyDH,VanderlaanM,BlankGS(1999)PerformancecomparisonofproteinAaffinity-chromatographysorbentsforpurifyingrecombinantmonoclonalantibodies.Biotech-nolApplBiochem30:121–128

¨quistJ(1966)ProteinAfromstaphylococcusAureusIPseudoimmunereactionForsgrenA,Sjo

withhumangamma-globulin.JImmunol97:822–827

FuglistallerP(19)ComparisonofimmunoglobulinbindingcapacitiesandligandleakageusingeightdifferentproteinAaffinitychromatographymatrices.JImmunolMethods124:171–177GhoseS,AllenM,HubbardB,BrooksC,CramerSM(2005)AntibodyvariableregioninteractionswithproteinA:Implicationsforthedevelopmentofgenericpurificationprocess.BiotechnolBioeng92:665–673

314R.GriepandJ.McDougall

GhoseS,HubbardB,CramerSM(2007)BindingcapacitydifferencesforantibodiesandFc-FusionproteinsonproteinAchromatographicmaterials.BiotechnolBioeng96:768–779GodfreyMA,KwasowskyP,CliftR,MarksV(1993)Assessmentofthesuitabilityofcommer-ciallyavailableSpAaffinitysolidphasesforthepurificationofmurinemonoclonalantibodiesatprocessscale.JImmunolMethods160:97–105

˚lS,HoberS(2002)EngineeringstreptococcalproteinGforincreased¨lichS,LinhultM,StaGu

alkalinestability.ProtEng15:835–842

HahnR,SchlegelR,JungbauerA(2003)ComparisonofproteinAaffinitysorbents.JChromatogB790:35–51

HahnR,ShimaharaK,SteindlF,JungbauerA(2006)ComparisonofproteinAaffinitysorbentsIIILifetimestudy.JChromatogA1102:224–231

HarrisonSL,HousdenNG,BottomLeySP,CossinsAJ,GoreMG(2008)GenerationofaminimalhybridIg-receptorformedbetweensingledomainsfromproteinsLandG.ProteinExprpurif58:12–22

HoberS,NordK,LinhultM(2007)ProteinAchromatoghraphyforantibodypurification.JChromatogB848:40–47

HoltLJ,BasranA,JonesK,ChorltonJ,JespersLS,BrewisND,TomlinsonIM(2008)Anti-serumalbumindomainantibodiesforextendingthehalf-livesofshortliveddrugs.ProtEngDesSel21:283–288

HousdenNG,HarrisonS,RobertsSE,BeckinghamJA,GrailleM,SturaE,GoreMG(2003)Immunoglobulin-bindingdomains:ProteinLfrompeptostreptococcusmagnus.BiochemSocTrans31:716–718

HousdenNG,HarrisonS,HousdenHR,ThomasKA,BeckinghamJA,RobertsSE,BottomleySP,GrailleM,SturaE,GoreMG(2004)ObservationandcharacterizationoftheinteractionbetweenasingleimmunoglobulinbindingdomainofproteinLandtwoequivalentsofhumanklightchains.JBiolChem279:9370–9378

¨bringU,Bjo¨rkL(1992)StructureofpeptostreptococcalproteinLandidentificationKasternK,Sjo

ofarepeatedimmunoglobulinlightchain-bindingdomain.JBioChem267:12820–12825KatohS,ImadaM,TakedaN,KatsudaT,MiyaharaH,InoueM,NakamuraS(2007)Optimizationofsilica-basedmediaforantibodypurificationbyproteinAaffinitychromatography.JChromatogA1161:36–40

¨holmAG,Bjo¨rkL,sjo¨bringU(1996)CharacterizationofthebindingpropertiesofKihlbergB,Sjo

proteinLG,animmunoglobulinbindinghybridprotein.EurJBiochem240:556–563

KimJY,MulchandaniA,ChenW(2005)Temperature-triggeredpurificationofantibodies.BiotechnolBioeng90:373–379

LeVanMD,CartaG,YonCM(1997)AdsorptionandionexchangeIn:GreenDW(ed),Perry’sChemicalengineersHandbook,7thedn.McGraw-Hill,NewYork,Chapter16

¨slundT,SimonA,KarlssonM,Sjo¨bergA,NordK,Hober(2004)¨lichS,GraLinhultM,Gu

ImprovingthetoleranceofaproteinAanaloguetorepeatedalkalinephosphataseexposuresusingabypassmutagenesisapproach.Proteins:structure,function,andbioinformatics55:407–416

˚,LøbersliI,KavlieK,StacyJE,BorgenT,KausmallyL,HvattumE,SimonsenB,LøsetGA

BefringHovdaM,BrekkeOH(2005)Construction,evaluationandrefinementofalargehumanantibodyphagelibrarybasedontheIgDandIgMvariablegenerepertoire.JImmunolMethods299:47–62

˚kerstro¨rkL,A¨mB(1992)ProteinLfrompeptostreptococcusNilsonBHK,SolomonA,Bjo

magnusbindstotheklightchainvariabledomain.JBiolChem267:2234–2239

ShuklaAA,GuptaP,HanX(2007)ProteinaggregationkineticsduringproteinAchromatography,acasestudyforanFcfusionprotein.JChromatogA1171:22–28

˚gS,AuneR,EllingsenTE,VallaS,BrautasetTSlettaH,NedalA,AuneTE,HellebustH,Hakva

(2004)Broad-host-rangeplasmidpJB658canbeusedforindustrial-levelproductionofasecretedhost-toxicsingle-chainantibodyfragmentinEscherichiacoli.ApplEnvironMicro-biol70:7033–9039

24AnalysisandPurificationofAntibodyFragments315

SolomonA(1976)Bence-Jonesproteinsandlightchainsofimmunoglobulins.NEnglJMed294:17–23

StarovasnikMA,O’ConnellMP,FairbrotherWJ,KelleyRF(1999)AntibodyvariableregionbindingbystaphylococcalproteinA:ThermodynamicanalysisandlocationoftheFvbindingsiteonE-domain.ProtSci8:1423–1431

¨bringU(1998)ProteinLA,anovelhybridproteinwithuniqueSvenssonH,HoogenboomHR,Sjo

single-chainFvantibodyandFabbindingproperties.EurJBiochem258:0–6

SwinnenK,KrulA,VanGoidsenhovenI,VanTicheltN,RoosenA,VanHoudtK(2007)PerformancecomparisonofproteinAaffinityresinsforthepurificationofmonoclonalanti-bodies.JChromatogB848:97–107

¨klenKE(2007)MaximizingproductivityofchromatographystepsforTugcuN,RoushDJ,Go

purificationofmonoclonalantibodies.BiotechnolBioeng99:599–613