Ion-exchange chromatography for the characterization ofbiopharmaceuticals Journal of Pharmaceutical and Biomedical Analysis

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ContentslistsavailableatScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

jou rn al h om e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / j p b a

Review

Ion-exchange chromatography for the characterization of biopharmaceuticals

Szabolcs Fekete

^a,∗

, Alain Beck

^b

, Jean-Luc Veuthey

^a

, Davy Guillarme

^a

aSchoolofPharmaceuticalSciences,UniversityofGeneva,UniversityofLausanne,Boulevardd’Yvoy20,1211Geneva4,Switzerland

bCenterofImmunologyPierreFabre,5AvenueNapoléonIII,BP60497,74160Saint-Julien-en-Genevois,France¹

a r t i c l e i n f o

Articlehistory:

Received3December2014

Receivedinrevisedform18February2015 Accepted19February2015

Availableonline26February2015

Keywords:

Ion-exchange Salt-gradient pH-gradient Monoclonalantibody Methoddevelopment

a b s t r a c t

Ion-exchangechromatography(IEX)isahistoricaltechniquewidelyusedforthedetailedcharacter- izationoftherapeuticproteinsandcanbeconsideredasareferenceandpowerfultechniqueforthe qualitativeandquantitativeevaluationofchargeheterogeneity.Thegoalofthisreviewistoprovidean overviewoftheoreticalandpracticalaspectsofmodernIEXappliedforthecharacterizationoftherapeutic proteinsincludingmonoclonalantibodies(Mabs)andantibodydrugconjugates(ADCs).Thesectionon methoddevelopmentdescribeshowtoselectasuitablestationaryphasechemistryanddimensions, themobilephaseconditions(pH,natureandconcentrationofsalt),aswellasthetemperatureand ﬂowrate,consideringproteinsisoelectricpoint(pI).Inaddition,bothsalt-gradientandpH-gradient approacheswerecriticallyreviewedandbeneﬁtsaswellaslimitationsofthesetwostrategieswerepro- vided.Finally,severalapplications,mostlyfrompharmaceuticalindustries,illustratethepotentialofIEX forthecharacterizationofchargevariantsofvarioustypesofbiopharmaceuticalproducts.

Contents

1. Introduction... 44

2. TheoreticalaspectsofIEX... 44

2.1. Salt-gradientbasedseparations... 44

2.2. pH-gradientbasedseparations... 45

3. MethoddevelopmentinIEXchromatography... 46

3.1. Theimpactofstationaryphase... 46

3.2. Theimpactofmobilephasecomposition... 46

3.3. TheimpactofsamplepI... 47

3.4. Theimpactoftemperature... 48

3.5. Optimizationprocedure... 48

4. ApplicationofIEXforproteinseparations... 49

4.1. Characterizationofchargevariantsoftherapeuticproteins... 49

4.2. SeparationofmAbvariants... 49

4.3. Analysisofantibody–drugconjugates... 51

5. PerspectivesinIEX ... 51

5.1. Decreasingtheparticlesize... 51

5.2. CapillaryIEX ... 52

5.3. Monolithiccolumns... 52

5.4. Twodimensionalseparations... 53

6. Conclusion... 53

References... 53

∗Correspondingauthor.Tel.:+41223796334;fax:+41223796808.

E-mailaddress:szabolcs.fekete@unige.ch(S.Fekete).

1 www.cipf.com.

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1. Introduction

Proteins and monoclonal antibodies (mAbs) are an emerg- ing class of therapeutic agents currently being developed by manypharmaceuticalcompanies[1].Duetotheincreasingnum- berofapprovedtherapeuticproteinsinthepharmaceuticalarea and the number of biosimilars (or follow-on-biologics) poten- tiallyenteringthemarket,theneedforanalyticaltechniquesfor theirdetailedcharacterizationhasincreased.Severalcharacteris- ticsofprotein-basedtherapycontributetoitssuccessbyimproving therisk–benefitratio.Thesecharacteristicsincludeimprovedtol- erance, good efficacy, highspecificity, and limited side effects.

However,theintrinsic micro-heterogeneity isof majorconcern withbiomoleculesandshouldbecriticallyevaluatedbecausedif- ferencesinimpuritiesand/ordegradationproductscouldleadto healthimplications[2].Furthermore,producingbiosimilarsismore challenging than manufacturing generic small molecule based pharmaceuticals[3].

In general, the identity, heterogeneity, impurity content, and activity of each new batch of therapeutic proteins has tobe thoroughly investigated before release. This examination is achieved using a wide range of analytical methods, including ion-exchange chromatography (IEX), reversed-phase liquid chromatography(RPLC),hydrophobicinteractionchromatography (HIC),sizeexclusionchromatography(SEC),sodiumdodecylsulfate polyacrylamidegelelectrophoresis(SDS-PAGE),capillaryisoelec- tricfocusing(cIEF),capillary zoneelectrophoresis(CZE),circular dichroism(CD),Fouriertransforminfraredspectroscopy(FT-IR), ﬂuorescencespectrophotometry(FL),andmassspectrometry(MS).

Thegoalofthismulti-methodstrategyistodemonstratethesim- ilaritybetweenproductionbatchesbypreciselycharacterizingthe primary,secondary,andtertiarystructureoftheproteins[4,5].

IEXisahistoricalandnon-denaturingtechniquewidelyused forthecharacterizationofchargevariantsoftherapeuticproteins andisconsideredasareferencetechniqueforthequalitativeand quantitativeevaluationofchargeheterogeneityoftherapeuticpro- teins[1].ThehistoryandcontinuousevolutionofIEXwasreviewed byLucy[6].AmongthedifferentIEXmodes,cation-exchangechromatography(CEX)isthemostwidelyusedforproteinpuriﬁcation andcharacterization[7].CEXisconsideredasthegoldstandard forchargesensitiveanalysis,butmethodparameters,suchascol- umntype,mobilephasepH,andsaltconcentrationgradient,often needtobeoptimizedforeachindividualprotein[8].IEXseparates chargevariantsbydifferentialinteractionsonachargedsupport.

Thenumberofpossiblechargevariantsincreaseswiththemolec- ularweightoftheanalyzedsample.Inaddition,changesincharge maybeadditiveorsubtractive,dependingonanymodiﬁcations.

Thus,IEXproﬁlesbecomemorecomplex,andtheoverallresolution ofindividualvariantsmaybelost[1].Thispropertyisparticularly apparentforlargebiomolecules.Therefore,notonlytheintactbut alsothereducedordigestedforms(limitedproteolysisorpeptide mapping)oftherapeuticproteinsarecommonlycharacterizedby IEX.

Inthisreview,wefocusonthepossibilitiesofIEXchromatog- raphyforthecharacterizationoftherapeuticproteins.Moreover, theaimof this reviewis todetail thetheoretical and practical aspectsofmodernIEX.Last,methoddevelopmentapproachesand applicationsarealsoreviewedandexplained.

2. TheoreticalaspectsofIEX 2.1. Salt-gradientbasedseparations

IEXseparatesproteinsbasedondifferencesinthesurfacecharge ofthemolecules,withseparationbeingdictatedbytheprotein

interactionswiththestationaryphase[9].Asaclassicalmodeof IEX,alinearsalt-gradientisregularlyappliedfortheelution.Several modelsforchromatographicretentionofion-exchangeadsorbents havebeenproposedinthepastyears[10].Theretentionmodels canbedividedintostoichiometricand non-stoichiometricmodels.Stoichiometricmodelsdescribethemulti-facetedbindingof theproteinmoleculestothestationaryphaseasastoichiometric exchangeofmobilephaseproteinandboundcounter-ions[11].

Thisstoichiometricdisplacementmodel(SDM)predictsthatthe retentionofaproteinunderisocratic,linearconditionsisrelatedto counter-ionconcentration.Thismodelwasextendedtodescribe protein retentionunder linear gradient elutionconditions (LGE model)[12],aswellasundernon-linearproteinadsorptioncon- ditions(stericmassaction(SMA)model)[13,14]forisocraticand gradientelutionmode. Anotherextension ofthestoichiometric modelfortheion-exchangeadsorptionwhichaccountsforcharge regulationwasdevelopedrecently[15,16].

Even if stoichiometricmodels are capable of describing the behaviorofion-exchangechromatographicsystems,theyassume thattheindividualchargesontheproteinmoleculesinteractwith discrete charges onthe ion-exchangesurface. In reality, retention throughion-exchange is more complexand primarily due totheinteractionoftheelectricalﬁeldsoftheproteinmolecules and the chromatographic surface [11]. Therefore, several non- stoichiometricmodelsfordescribingproteinretentionasafunction of the salt concentration in the mobile phase have also been proposed [17–20]. Quantitative structure–property relationship (QSPR)modelshavebeenderivedforproteinretentionmodeling inIEXbymeansofdifferentnumericalapproachesthatattempt tocorrelateretentiontofunctionsofdescriptorsderivedfromthe three-dimensionalstructureoftheproteins[21–23].Morerecently, theoriesusedincolloidandsurfacechemistrytodescribeelectro- staticandotherinteractions havealsobeenappliedtodescribe retentionpropertiesofproteinsinIEX[24–28].

TheworkofSnyderandco-workersshowedthatIEXsystems follow non-linear solvent strength (LSS) type retention mecha- nism[29,30].Consequently,solute-speciﬁccorrectionfactorsare requiredtouseLSSmodelforretentionpredictions,therebylim- itingtheapplicabilityoftheLSSmodel.Thenon-linearityofLSS modelwasassessed bycomparingtheelutiondatatothestoi- chiometricdisplacementmodel(SDM)commonlyusedinIEX.The retentionfactor(k)canbewritteninthefollowingwayaccording totheSDMmodel:

log k=logK−zlogC (1)

whereKisthedistributionconstant,zisassociatedwiththepro- teinnetchargeornumberofbindingsites(effectivecharge)andC isthesaltconcentration(thatdeterminestheionicstrength).This modelisprobablythemostacceptedoneandisusefulfromaprac- ticalpointofview.Thenon-linearityofEq.(1)ismostpronounced forsmallvaluesofz[30].Ifz>6(whichisveryoftenthecaseof therapeuticproteins),anLSStypemodelmayprovidereliabledata forretentionfactor(retentiontime)[31].Fig.1Ashowsexperimen- tallyobservedlogkversusCplotforz=1,whileFig.1Bshowssome calculatedlogk–Cplotsforvariouszvalues.

Proteinsareelutedinorderofincreasingbindingcharge(corre- latesmoreorlesswiththeisoelectricpoint(pI))andequilibrium constant.Theretentionoflargeproteinsinsalt-gradientmodeis stronglydependentonthesaltconcentration(gradientsteepness orgradienttime)–duetotherelativelyhighzvalue–andasmall changecouldleadtosigniﬁcantshiftinretention.Therefore,iso- craticconditionsareimpractical,andgradientelutionispreferred inreal-lifeproteinsseparations.Forlinearsalt-gradientinIEX,the

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Fig.1. logk–Cplots:isocraticnon-LSSion-exchangeretentioncomparedwithLSSmodelwithz=1and1≤k≤10(A)andsometheoreticallogk–Cplotsassumingseveralz values(B).

Fig.1AwasadaptedfromRef[30],withpermission.

saltconcentrationvarieswithtimeduringthegradient,therefore Eq.(1)canberewrittenas:

log k=logK−zlog

C₀+C tg

(2) whereC₀isthesaltconcentrationatthebeginningofthegradient (initialmobilephasecomposition)andCisthechangeinthesalt concentrationduringthegradient.

InanalogywithRPLC,thefollowinggeneralequationcanbe writtenforsalt-gradientbasedIEXseparationsinthegradientelu- tionmode[31]:

k∗= tg

1.15[t₀|z|log(C_f/C₀)] (3)

wherek*isthemedianvalueofkduringgradientelutionwhenthe bandhasreachedthecolumnmid-point,t₀isthecolumndeadtime andC_fistheconcentrationofthecounter-ionattheendofthegradi- entprogram.PleasenotethatbothRPLCandIEXseparationsvary withgradientconditions inasimilarfashion.However,because of thedifferencesin thedependence of k onthemobile phase compositionCinIEX(log–logrelationship)versusRPLC(log–linear relationship),theLSSmodelistheoreticallynotapplicableforIEX.

Nevertheless,asshowninFig.1,thehigherthez,thelowerthe deviationfromnon-linearityis.Itwascurrentlyshown,thatLSS approachcanbeappliedforlargeproteins(mAbs)possessingan importantnumberofcharges[32].

2.2. pH-gradientbasedseparations

Ion-exchange chromatofocusing represents a useful alterna- tivetolinearsalt-gradientelutionIEX,inparticularforseparating proteinisoformswithminordifferences intheisoelectric point (pI).Chromatofocusingisperformedonanion-exchangecolumn employingapHgradientthatcanbegeneratedinternallywithin thecolumn[33,34]orbyexternalmixingofahigh-pHandalow-pH bufferusingagradientpumpsystem[35–37].Highlylinear,con- trollable,andwide-rangepHgradientscanbegenerated[37–40].

Thenumberofapplicationsreportedattheanalyticalscaleis large,butthenumberofpublicationsdealingwiththemathemat- icalmodelingoflinearpHgradientelutioninIEXisratherlimited [9].TodescribetheelutionbehaviorofproteinsinlinearpHgra- dientIEX,apHdependenceparameterhastobeincorporatedinto theion-exchangemodel.

InpH-gradientmode,theproteinsnetchargeismodiﬁedduring thepHgradient,due toprotonation–deprotonationofthefunc- tionalgroups.InCEX,theproteinisexpectedtoeluteat,orclose toitspI.Accordingtotheory,whenapplyingpH-gradientelution

modeandlowionicstrengthmobilephase,thechromatographic bandsshouldbefocusedinnarrowerpeaksenablinghigherreso- lutioncomparedtoapH-gradientperformedathighionicstrength.

ThewidthofaproteinpeakalongalinearpH-gradientexpressed inpHunitscanbewrittenasfollows[33,34,40]:

(pH)²≈D(dpH/dV)

ϕ(dZ/dpH) (4)

whereDisthediffusioncoefﬁcientoftheanalyte,dpH/dVisthegra- dientslope,ϕistheDonnanpotentialanddZ/dpHisthechangein proteinnetchargealongthepHgradient.SincetheDonnanpoten- tialdependsontheionicstrength,apeakfocusingeffectisexpected atlowerionic strength.Inagreementwiththisexpectation, pH gradientsatlowionicstrengthshowedbetterresolutionformAb variantscomparedtopHgradientsperformedathighionicstrength [40].

TheappliedpHrangeclearlydeterminestheproteinsthatcan possiblybeeluted.Retentiontimesandpeakwidthsdependonthe gradientsteepness,asbotharefunctionofdZ/dpH.

Theeffectofgradientsteepness(gradienttime)ontheretention oflargeproteins(intactmAbsandtheirvariants)wasrecentlystud- iedandshowedanLSS-likelinearbehavior[41].Thedependence ofapparentretentionfactor(kapp)ongradientsteepness(time)in pH-gradientbasedIEXseparationisshowninFig.2.

InpH-gradientIEXmode,theuseofamixtureofaminebuffer- ingspeciesinthehigh-pHrangeandamixtureofweakacidsin thelow-pHrangeisquitecommon[38,39,42].Insuchasystem,

Fig.2.Thedependenceofapparentretentionfactor(kapp)ongradientsteepness (time)inpHgradientbasedIEXseparation.TheappliedpHrangewasfrompH=5.6 topH=10.2at0.6mL/minﬂow-rateona100mm×4.6mmCEXcolumn.

AdaptedfromRef[40],withpermission.

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maintaininglinearityofthepHgradientslopemaybesomewhat difﬁcult.It wasshownthatanappropriatemixtureofTrisbase, piperazineandimidazoleprovidesalinearpHgradientfrompH 6–9.5[8].Triethylamineanddiethylaminebasedbuffersystems alsoofferedlinearpHgradientinthepHrangeof7.5–10.0[40].

Formassspectrometric(MS)detection,5mMammoniumhydrox- idein20%methanolyieldedareasonablepHgradientinalimited pHrange(between9.5and10.5)[40].Zhangetal.[43]applieda salt-mediatedimprovedpHgradientthatwasusedinawidepH range(between5and10.5).Intheirstudy,a0.25mM/minsodium- chloridegradientwasperformedtogetherwiththepHgradient.

OneofthebeneﬁtsofpH-gradientbasedIEXisthatthesaltcon- centrationcanbekeptlow,yieldinglessbufferinterferences(e.g.

on-lineoroff-linetwo-dimensionalLC).Inaddition,pH-gradient basedseparationusinga CEXcolumnwasfoundtobea multi- productchargesensitiveseparationmethodforlargetherapeutic proteins(mAbs)[43,44].

3. MethoddevelopmentinIEXchromatography

Positively charged molecules can be separated using CEX columns,typicallypackedwith3–10␮mparticlesandcontaining negativelychargedacidicfunctionalgroups.Thesecolumnsbind cationicspecies suchasprotonatedbasesthroughionicinterac- tion.Inanion-exchange(AEX)mode,thestationaryphasecarries positivelychargedbasicfunctionalgroupsthatarecapableofbind- inganions(e.g.ionizedcarboxylicacids).Themobilephaseusually containsabuffertomaintainstablepHandvaryingthesaltcon- centration(counter-ion)tocontroltheretentionofsampleions.

Thechargeofthecounter-ionhasthesamesignasthesampleions, thereforeitcanbeusedtocontroltheretentionofprotonatedbases inCEXorionizedacidsinAEX.

Thestrengthoftheinteractionisdeterminedbythenumber andlocationofthechargesontheanalyzedmoleculesandonthe functionalgroups.Byincreasingthesaltconcentration,thesamples withtheweakestionicinteractionsstarttoelutefromthecolumn ﬁrst.Moleculeshavingastrongerionicinteractionrequireahigher saltconcentrationandelutelaterinthegradient.

InthepH-gradientmode,theionicstrengthofthemobilephase iskeptlowandconstant,whilethepHisvariedthankstoalinear gradient.

3.1. Theimpactofstationaryphase

Regardingthestationaryphase,therearetwomainaspects:(1) thestrengthofinteractionandassociatedretention(strongorweak ion-exchanger)andthe(2)achievablepeakwidths(efﬁciency).

Bothcation and anionexchangerscanbeclassiﬁedaseither weakorstrongexchangers.Weakcationexchangersarecomprised ofaweakacidthatgraduallylosesitschargeasthepHdecreases (e.g.carboxymethylgroups),whilestrongcationexchangersare comprisedofastrongacidthatisabletosustainitschargeovera widepHrange(e.g.sulfopropylgroups).Ontheotherhand,strong anion exchangers contain quaternary amine functional groups, whileweakanionexchangerpossessesdiethylaminoethane(DEAE) groups.Stronganionexchangersremainunderionizedforminthe pHrangebelow12,whilestrongcationexchangersareionizedat pH>2.

Asaruleofthumb,itispreferredtobeginthemethoddevelop- mentwithastrongexchangertoenableworkingoverabroadpH range.Strongexchangersarealsousefulifthemaximumresolution occursatanextremepH.(However,silicabasedion-exchangerscan beoperatedonlyinarestrictedpHrange.Incontrast,polymeric ion-exchangerscanbeusedinawidepHrange.)

Inthecaseofproteins,thecationexchangemodeiswellsuited, butastronganionexchangercanbeappliedtobindtheproteins iftheirpIisbelowpH7.Weakexchangerscanonlybeusefulina secondinstance,iftheselectivityofstrongionexchangersisunsat- isfactory.However,itisimportanttokeepinmindthattheion exchangecapacityofweakionexchangersvarieswithpH.

CommerciallyavailableIEXcolumnsarebasedonsilicaorpoly- merparticles.Bothporousandnon-porousparticlesareavailable butforlargemoleculeswhichpossesslowdiffusivity,non-porous materialsareclearlypreferredtoavoidtheunwantedbandbroad- eningeffectsofthetransparticlemasstransferresistance(C-term of the van Deemter equation). Highly cross-linked non-porous poly(styrene–divinylbenzene) (PS/DVB) particles are most fre- quently used in protein separations due to their pH stability (2≤pH≤12).Thosematerialscannowwithstandpressuredrop ofuptoa500–600barandcanberoutinelyusedbeyond400bar.

Columnspackedwith10,5or3␮mnon-porousparticlesareoften used,butsub-2␮mmaterialsarealsoavailablesincerecently.On thosecolumnshighpeakcapacitycanbeattainedevenwithlarge biomolecules.Howeversomelimitationscanbeexpectedinterms ofloadingcapacityandretentionwhenapplyingthesenon-porous materials.Table1summarizesthemostpopularstate-of-the-art IEXcolumnsappliedfortheseparationofproteinchargevariants.

3.2. Theimpactofmobilephasecomposition

Inthesalt-gradientmode,themobilephasebufferpHmustbe betweenthepIofthechargedmolecule(e.g.therapeuticprotein) andthepKaofthechargedfunctionalgroupatthesurfaceofthe stationaryphase.InCEX,usingastrongcationexchangerwitha pKaof1.2,amoleculewithapI∼8(e.g.mAbs)maybeelutedwith amobilephasepHbufferof∼6.InAEX,amoleculewithapI∼6 mayberunwithamobilephasebufferatpH8whenthepKaofthe solidsupportisbeyond10.

IntheCEXmode,increasingthemobilephase bufferpHwill cause the molecule to become less protonated (less positively charged). Therefore,the proteinforms weaker ionicinteraction withthenegativelychargedstationaryphasegroups,whichresults inaretentiondecrease.Onthecontrary,decreasingthepHmani- festsinhigherretention.InAEXmode,–oppositely–decreasingthe mobilephasepHcausesthemoleculetobecomemoreprotonated (morepositivelyandlessnegativelycharged),thereforeadecrease inretentionisexpected.

ThemostoftenappliedpHrangeforproteinsIEXseparations isbetween5.5 and 7.0,however insomecaseslow pHaround 3.5isrequiredtoreachappropriateselectivityandretention.The most frequentlyused buffers for protein separations are 2-(N- morpholino)ethanesulfonicacid(MES),phosphateandcitrate.MES isusefulbetweenpH5.5and6.8(pKa∼6.15),phosphateisapplied forpHbetween6.7and7.6(pKa∼7.2),whilecitrateprovideshigh buffercapacityforpHbetween2.6and3.7(pKa∼3.1).Otheraddi- tivessuchasmalonicacid,aceticacidorformicacidhavealsobeen reportedforalimitednumberofapplications.Thebufferconcen- trationistypicallycomprisedbetween10and50mMandallowsa sufﬁcientbuffercapacity.

AfterselectingthemobilephasepHandbuffer,thesalt-gradient hastobeoptimized.Typicallysodium-orpotassium-chlorideare usedforproteinscharacterization,usingasaltgradientfrom0to 0.2–0.5M.Theproteinsamplesareinjectedontothecolumnunder conditionswhereitissufﬁcientlyretained.Then,agradientoflin- earlyincreasingsaltconcentrationisappliedtoelutethesample componentsfromthecolumn.Itisﬁnallyimportanttonoticethat thegradientsteepnesshasastrongimpactonretentionandselec- tivityandshouldthereforebesystematicallyoptimized.

InthepH-gradientmode,themaindifﬁcultyistoperformlinear androbustpHgradients.Theuseofamixtureofaminebuffering

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Table1

Propertiesofthemostpopularstate-of-the-artIEXcolumnsavailableforproteinseparations.

Columnname Chemistry Particle

size/macropore size

Max temperature (^◦C)

pHrange Maxpressure (bar)

Monoliths Proswift (Thermo)

SAX-1S Stronganionexchange

(quaternaryamine) Information notavailable

70 2–12

WAX-1S Weakanionexchange 70 (tertiaryamine)

60 WCX-1S Weakcationexchange

(carboxylicacid)

60 SCX-1S Strongcationexchange

(polymethacrylate)

60

Packed

TSKgel(Tosoh)

SCX Strongcationexchange (sulfonicacid)

5 45 2–14

SuperQ-5PW Strongcationexchange 50 (trimethylamino)

10 2–12

SP-STAT Strongcationexchange (sulfopropyl)

7,10 3–10

Q-STAT Stronganionexchange (quaternary ammonium)

7,10 3–10

BioMab(Agilent) Weakcationexchange (carboxylate)

1.7 3 5 10

80 2–12 270

410 550 680 Antibodix(Supelco,Sepax) Weakcationexchange

(carboxylate)

1.7 3 5 10

80 2–12 270

410 550 680 Protein-PakHi

ResIEX (Waters)

SP Strongcationexchange (sulfopropyl)

7

60

3–10 100

CM Weakcationexchange

(carboxymethyl)

7 100

Q Stronganionexchange

(quaternary ammonium)

5 150

MAbPacSCX-10(Thermo) Strongcationexchange (sulfonicacid)

3 5 10

60 2–12 480

480 200 Bio-Pro(YMC) QA

QA-F

Stronganionexchange (quaternary ammonium)

5 60 2–12 30

120 SP

SP-F

Strongcationexchange (sulfopropyl)

30 120 Poly(PolyLC) CATA Weakcationexchange

(polyasparticacid) 5 Ambient Information

notavailable

Information notavailable WAXLP Weakanionexchange

(polyethyleneimine)

speciesinthehigh-pHrangeandamixtureofweakacidsinthe low-pHrangeisquitecommon[38,39,42].Aspreviouslydiscussed, themostoftenusedbuffercomponentsareTrisbase,piperazine, imidazole,triethylamine, diethylamineand ammoniumhydrox- ide[8,40,43].Finally,a0.25mM/minsodium-chloridegradientwas successfullyperformedconcomitantlywithapH-gradientforthe characterizationofmAbspossessingisoelectricpoints(pI)between 6.2and9.4,tohighlighttheinterestofpHgradientseparationover saltgradients[43].

3.3. TheimpactofsamplepI

Thechargeofproteinsdependsonthenumberandtypeofion- izableaminoacidgroups.Lysine,arginineandhistidineresidues haveapositivelychargedsidechaingroupwhenionized,whereas glutamicacidand asparticacidresiduesare negativelycharged whenionized.EachionizablesidechaingroupshasitsownpK_a. Therefore,theoverallnumberofchargesonaparticularproteinat agivenpHdependsonthenumberandtypeofionizableamino acidgroups.ProteinstendtohavedifferentchargesatagivenpH and socan befractionatedonthebasisof theirnetand acces- siblecharges.EachproteinhasapIvalue,which correspondsto

thepHvaluewhereithasnonetcharge.Then,whenpHisequal topI,theproteinwillnotbindtotheion-exchangeresin.Below this pH value,the protein hasa net positive charge and binds toacationexchanger,whileabovethispH,ithasanetnegative chargeandbindstoananionexchanger.Inpractice,proteinsare stable and functionallyactive withina fairlynarrow pHrange, sothechoiceofion exchanger isoftendictatedby thepHsta- bilityofthedesiredprotein.IftheproteinisstableatpHvalues belowitspI,acationexchangershouldbeusedifitisstableat pHvaluesaboveitspI,thenananionexchangerphasehastobe chosen.

ThepIoftheproteinalsodeterminesthemobilephasepH.ThepI oftherapeuticproteinsdistributebetween3.6and11.0,andamong them,mAbspossesspIvaluesbetween6and10.Forsalt-gradient basedCEXmode,themobilephasepHshouldpreferablybeatleast 1–2unitsbelowthepIofthesample,tomaintainappropriatereten- tion.InAEXmode–oppositely–thepHhastobesetatleast1–2 unitsabovethepIoftheprotein.

InthepHgradientmode–performedonCEXcolumns–the startingpHshouldbebelow thepIof theless retainedprotein, whiletheﬁnalpHhastobesomewhathigherthanthepIofthe mostretainedprotein.

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3.4. Theimpactoftemperature

Theeffectoftemperatureonretentionfactor(k)isgenerally expressedinliquidchromatographywiththeGibbsfreeenergyor van’tHoffequation:

log k=−H RT +S

R +logˇ (5)

where H representstheenthalpy changeassociated withthe transfer of the solute between phases, S the corresponding entropychange,Rthemolargasconstant,Ttheabsolutetemper- atureandˇthephaseratioofthecolumn.Whenlog(k)isplotted against1/T,theenthalpyisgivenbytheslopeofthecurve.Withreg- ularcompounds,theseplotsgenerallyfollowalinearrelationship.

However,non-lineardependenceoflog(k)versus1/Toverawide rangeoftemperaturewasnoticedbydifferentauthorsusingsilica- basedaswellasnonsilica-basedstationaryphases[45].Theeffect oftemperatureontheretentionofpartiallyionizedcompounds whichmayexistintwoforms(i.e.molecularandionizedforms)can alsobedescribedwithEq.(5).However,bothenthalpyandentropy areexpectedtobedifferentforthetwoformsandasaresult,both HandScanvarywithtemperaturewhenbothformsarepresent toa signiﬁcantextent[45].Withlargebiomolecules, theeffect oftemperatureonretentionbecomesmorecomplex.Depending onthestabilityofthesecondarystructure,themoleculesunfold tovariousextents andhenceinteractwiththestationaryphase withvariousstrengths[46]. Duetothedifferentconformation- dependentresponses of proteinsat elevated temperatures, the changeinretentioncanbedifﬁculttoassess[47,48].InRPLCsepa- rationofproteins,temperatureisausefulparameterforadjusting selectivity.InIEXseparationsofproteins,theimpactoftempera- turewasfoundtobeespeciallyimportantforpeakcapacity(and thereforeforresolution),buthasa limitedimpactonselectivity [32,41].Itseemedthatinbothsalt-andpH-gradientbasedseparations,thetemperaturedoesnotmodifyseverelyselectivity,but impacttheachievablepeakcapacity.Therefore,insomecases,tem- peratureoptimizationcouldalsobeofimportanceduringtheIEX methoddevelopmentprocedure.

3.5. Optimizationprocedure

In contrastwithRPLC, the methoddevelopment inIEX was mostly basedon trialand erroror “one factor at time”(OFAT) approaches.However,therearesomeguidelinesavailablefromcol- umnproviders,whichexplainthebasicrulesformethodscreening (e.g.columnselection,bufferselection...).

Baietal.showedthedependenceofretentionandselectivityof IgGantibodiesonmobilephasepH,stationaryphasetypeandsalt- gradientsteepnessinCEXmode[49].Theystudiedtheeffectof thethreevariablesindependently,andfoundthatmobilephasepH wasthemostimportantparameterinCEXseparationsofproteins.

Ithadthebiggestimpactontheseparationandthereforeshould bedeterminedﬁrst[49].Itwasalsofoundthat(i)peakwidthof IgG-smostlydependsonthetypeofthestationaryphaseand(ii) resolutioncanbetunedbychangingthegradientsteepness.Fig.3 showstheimpactofsalt-gradientsteepnessontheseparationof IgGproteins.

Themobilephaselinearvelocityalsohasastronginﬂuenceon theseparationqualityoflargeproteins[50,51].Indeed,thelon- gitudinaldiffusionisnegligiblewithlargemolecules,whileband broadeningismostlydeterminedbythemasstransferresistance.

Therefore,lowﬂowrateisalwayspreferredforhighresolutionsep- arations,butacompromisehastobefoundbetweenresolutionand analysistime.

Fig.3. Theeffectofsalt-gradientslopeontheretention,selectivityandpeakwidth inCEXseparation.Sample:IgG1,mobilephase:40mMphosphatepH6.5,applying a0–0.4MNaClgradient.

Theinﬂuenceofsalttypecanalsobeimportant.Itseffecton theretentionofbovineserumalbuminwasreportedbyAl-Jibbouri [52].

Computerassistedmethoddevelopmentandoptimizationin RPLCproteinseparationsisquitecommon[53,54]andwasalso recently appliedin ion-exchange mode. Because of the system non-linearity,ﬁndingtheoptimumforprocessoptimizationischal- lenging[55].Thiemoetal.developedasoftwarecalledChromX fortheestimationofparameters,chromatogramsimulation,and processoptimization[55].ChromXprovidesnumericaltoolsfor solving varioustypes of chromatography models,includingthe modelcombinationofTransportDispersiveModel(TDM)andSMA.

SimilarlytoRPLCmethoddevelopment,anon-LSSand LSStype computerassistedmethoddevelopmentprocedurewasrecently reportedforbothsalt-andpH-gradientmodesinagreementwith QualitybyDesign(QbD)concept[32,41].

Forthesalt-gradientbasedproteinseparation,itseemedthat temperaturewasnotarelevantparameterfortuningselectivityand shouldbekeptat30^◦C,toachievehighresolvingpower(elevated peakcapacity) [32].Becausetherelationship betweenapparent retentionfactorsandgradienttime(slope)canbedescribedwitha linearfunction,onlytwoinitialgradientrunsofdifferentslopes are required for optimizing the salt gradient program. For pH dependence,asecondorderpolynomialmodel(i.e.basedonthree initialruns)ispreferredtodescribekversuspHdependence.When combiningtheexperimentsina designofexperiments(DoE),it appearedthatmethodoptimizationcanbeperformedrapidly,in anautomatedwaythankstoaHPLCmodelingsoftware,usingtwo gradienttimesandthreemobilephasepH(e.g.10and30mingra- dientona100mmlongstandardborecolumnatpH=5.6,6.0and 6.4).Suchaprocedurecanbeappliedroutinelyandthetimespent formethoddevelopmentwouldbeonlyaround9h.Therelative errorinretentiontimepredictionwaslowerthan1%,makingthis approach highlyaccurate[32]. Fig.4Ashows a generic DoEfor

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Fig.4. Designofexperiments(A)andresolutionmap(B)fortheoptimizationofsalt-gradientbasedCEXseparationofmAbs(tg–pHmodel).Column:YMCBioProSP-F (100mm×4.6mm).Mobilephase“A”10mMMES,“B”10mMMES+1MNaCl.Flowrate:0.6mL/min,gradient:0–20%B,temperature:30^◦C.Gradienttimes:tg1=10min, tg2=30min,pH1=5.6,pH2=6.0,pH3=6.4.Ontheresolutionmap,red-orangecolorsshowthehighestresolutionwhilethedark-blueareasindicatetheco-elutionofpeaks.

(Forinterpretationofthereferencestocolorinthisﬁgurelegend,thereaderisreferredtothewebversionofthisarticle.) AdaptedfromRef[31],withpermission.

themethoddevelopmentofsalt-gradientbasedCEXseparationof mAbs(possessingawiderangeofpIbetween6.7and9.1),while Fig.4Brepresentstheobtainedresolutionmapshowingthecriti- calresolutionofthepeakstobeseparatedasafunctionofmethod parameters.

InthepH-gradientmode,itwasfoundthattheretentionoflarge proteinscanbeaccuratelymodeledasafunctionofgradientsteep- nessand mobilephasetemperature[41].Becausetheretention modelswerealwayslinear,onlyfourinitialexperiments(2gradi- entstimesat2temperatures)wererequiredtomodelthebehavior inCEXpH-gradient.Then,only∼6hwererequiredtoﬁndoutthe optimalconditionsona100×4.6mmcolumn[41].

4. ApplicationofIEXforproteinseparations

Today,IEXismainlyappliedtoseparateproteinchargevariants andisoforms.IEXmaybeusefulatdifferentlevelsofproteinanaly- sis,including(i)theanalysisofintactproteins(topdownapproach), (ii)theanalysisofpartlydigestedlargeproteinfragments(mid- dledownapproach)and(iii)thecharacterizationoftrypticdigests (peptidemappingorbottomupapproach).

Inthecaseoftherapeuticproteinscharacterization,IEXismostly usedtoseparateC-terminallysinevariants/truncation,N-terminal glutamine-pyroglutamatevariants,deamidatedforms(asparagine formsasuccinimideintermediatethatresultsintwoproductsofits hydrolysis,eitheraspartateorisoaspartate),glycoformsespecially sialicacidvariants(usuallyattachedatterminalpositionsofgly- canmolecules).Inaddition,IEXalsoseparatestheproductsofthe PEGylationreactionaccordingtotheextentofconjugation,andpro- videstheseparationfortheisomericformsofPEGylatedproteins (whichdifferfromoneanotherbythelocationoftheconjugation sitewithinthepolypeptidechain)[55,56].

4.1. Characterizationofchargevariantsoftherapeuticproteins Peptide mapping is commonly used todemonstrate protein identity.Inthelastphasesofpharmaceuticaldevelopmentandin qualityassurance/control(QA/QC),peptidemappingoftheprotein drugsservesasaprimaryproteinQCmethod.Althoughreversed- phase separation is the typical choice for separating peptides, high-resolution ion-exchangechromatography is an alternative methodthatprovidesadditionalinformationandadifferentselec- tivity [57]. Asexample,a strong CEXcolumn wasused forthe peptidemappingofcytochromeCtrypticdigest[58]andImamura etal.appliedCEXforthepeptidemappingofhemoglobintoiden- tifyitschargevariants[59].IEXwaswidelyusedinthepastfor

peptidemapping,buttodayitislesspopularandismostlyreplaced byRPLC,duetotheinherentincompatibilityofIEXwithMSdetec- tion.

IEXisdeﬁnitelyoneofthemostpowerfultechniquestosepa- ratedeamidatedformsofnativeproteins.Deamidationisasource ofnonenzymaticproteindegradation,andshouldbestrictlymon- itored during the course of formulation development [60]. As example,CEXwassuccessfullyappliedtoseparatethedeamidated analogsofrecombinantgranulocytecolony-stimulatingfactor(G- CSF, ﬁlgrastim),asillustratedin Fig.5.Weitzhandler et al.[61]

presentedthebaselineseparationofcytochromeCvariants(bovine, horse,rabbit)and twodeamidation productsofribonucleaseA, suchastheAsp⁶⁷andisoAsp⁶⁷forms.Gotteetal.[62]appliedCEX fortheseparationofribonucleaseBdeamidatedforms.Abzalimov etal.[55]appliedthecombinationofIEXandtop-downtandemMS forthestructuralcharacterizationofprotein–polymerconjugates andtoassessheterogeneityofasmallPEGylatedproteinandmap- pingconjugationsites.Finally,Ganzleretal.showedtheseparation ofoxidizedanddeamidatedPEGylated-G-CSFusingCEX[63].

4.2. SeparationofmAbvariants

MAbsareaspeciﬁcclassoftherapeuticproteinsandgainedsig- niﬁcantinterestoverthepastfewyears.Then,adedicatedsection wasdevotedtotheirIEXbasedseparationandcharacterization.

Fig.5. Ion-exchangeHPLCofrhG-CSFandthedeamidatedanalogs.(A)Gln^11,20,67

→Gluanalog,Gln⁶⁷→Gluanalog,f-metrhG-CSF,Gln^11,20→Gluanalog,andmet rhG-CSF(fromleft);(B)Gln^12,21→Gluanalog;(C)Gln⁶⁷→Gluanalog;(D)Gln^11,20,67

→Gluanalog.

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ThemAbsareglycoproteinsthatbelongtotheimmunoglobulin (Ig)superfamily,whichcanbedividedintoﬁveisotypes:IgA,IgD, IgE,IgG,andIgM.BecauseonlyIgGsareproducedfortherapeutic purposesthroughgeneticengineering,thetermsrecombinantmAb andIgGareoftenusedinterchangeably.IgGsarelargetetrameric glycoproteinsmeasuringapproximately 150kDa that are struc- turallycomposedoffourpolypeptidechains:twoidenticalheavy chains(HC,∼50kDa)andtwoidenticallightchains(LC,∼25kDa) connectedthroughseveralinter-andintra-chaindisulﬁdebonds attheirhingeregion[64].Eachchainiscomposedofstructural domainsaccordingtotheirsizeandfunction,givingtheconstant, variable,andhypervariableregions.DifferencesbetweentheHC constantdomainsareresponsiblefortheIgGsub-classes(i.e.,IgG1, IgG2,IgG3,andIgG4).

Functionally,mAbs consist of two regions, thecrystallizable fraction (Fc) and the antigen-binding fraction (Fab) [65]. Fc (∼50kDa)iscomposedoftwo truncatedHCs andisresponsible fortheeffectorfunctions,suchascomplementfixationandrecep- torbinding.TheFcsequencealsohasaconservedN-glycosylation site,whichisgenerallyoccupiedbyabiantennaryoligosaccharide accountingforsignificanteffectsontheactivityandefficacyofthe IgGs[66].TheFabdomain(∼50kDa)iscomposedoftheLCandthe remainingportionoftheHC.Thisdomainisprimarilyinvolvedin antigenbinding[65].

There are several common modifications leading to antibody charge variants (or isoforms) on thepeptide chains(e.g., deamidation, C-terminal lysine truncation, N-terminal pyroglu- tamation, methionine oxidation, or glycosylation variants) and sizevariantsonthepeptide chains(e.g.,aggregationorincom- pleteformation of disulfidebridges). Thecombination of these micro-heterogeneity sources in the peptidechains significantly increasestheoverallmicro-heterogeneityofanentireIgG.There- fore,thecompletecharacterization ofanintactmAb isdifficult to achieve. In this context, various enzymes, such as pepsin, papain, Lys-C or IdeS are often used toobtain mAb fragments andfacilitatetheinvestigationofitsmicro-heterogeneity.Papain isprimarilyusedtocleaveIgGsintothree fragmentsattheHC hingeregiontocreateoneFcandtwoidenticalFabfragmentsof

∼50kDaeach,whilepepsinandIdeSgeneratesF(ab)₂fragments of∼100kDa.Thesetypesofdigestionarecalledlimitedproteolysis (LP).

Moorhouseetal.[56]wasamongthefirstonestodescribethe potentialofIEX formAbcharacterization. Papain-digestedmAb samplesweresuccessfullyseparatedandthecorrespondingfrag- ments were identified thanks to MS detection. The C-terminal lysine variability of the Fc and the N-terminal glutamine- pyroglutamatevariabilityoftheFabwereobserved.Arecentstudy demonstratedthesuitabilityofIEXforstudyingcomplexdegrada- tionprocessesinvolvingvariousIgG1molecules[67].Assignment ofcovalentdegradationstospecificregionsofthemAbswasfacil- itatedusingLys-CandpapaintogenerateFabandFcfragments.

Thismethodwasparticularlyusefulforcharacterizingproteinvari- ants formedin the presenceof salts under accelerated storage conditions.Theusefulnessofthisassaywasfurtherillustratedby characterizationof light-induced degradationsof mAbformula- tions.AnotherstudypresentedtheimportanceofIEXintheanalysis ofoxidizedmAbsamples[68].BothCEXandAEXapproacheswere usedandfoundsuitablefortheseparationofthemorebasicoxi- dizedvariantsoftheintactmAbs.Fig.6displaysmultipleacidicand basicisoformsobtainedinAEXmode,typicalofrecombinantmAb drugproducts.Vlasaketal.[69]combinedCEX,papaindigestion andapanelofMStechniquestoidentifyasparaginedeamidationin thelightchainregionofahumanizedIgG1mAb.Anotherstudyalso presentedandprovedtheimportanceofCEX(salt-gradientbased) inmAbcharacterizationandshowedtheseparationofdifferent isoforms[70].

Fig.6.CEXofXOMA3ABantibodies.ThethreeXOMA3ABreferenceantibodies(Ab- A,Ab-B,andAb-C)displaymultipleacidicandbasicisoformstypicalofrecombinant monoclonalantibodydrugproducts.Theforcedoxidizedsamplesofeachofthese antibodiesexhibitbroadpeakproﬁlesindicatingunderlyingstructuralheterogene- ity.

A pH-gradient based separation using CEX chromatography was evaluated in a recent study [8]. The method was shown toberobust formAbs and suitable for itsintended purposeof chargeheterogeneityanalysis.Simplemixturesofdeﬁnedbuffer componentswereusedtogenerate thepHgradientsthatsepa- ratedcloselyrelatedantibodyspecies.Validationcharacteristics, suchas precision,linearity and robustnesswere demonstrated.

Thestability-indicatingcapabilityofthemethodwasdetermined using thermally stressed antibody samples [8]. Another recent studyshowedtheapplicabilityofashallowpHgradientthrough CEX monolithic column and demonstrated relatively high resolution separation of mAb charge variants in three different biopharmaceuticals[40].Zhangetal.[43]presentedamultiprod- uctchargesensitiveseparation methodfor 16mAbs possessing pI-s between 6.2 and 9.4. This salt-mediated pH-gradient IEX method was demonstrated to be robust under various chro- matographicconditionsandcapableofassessingmanufacturing consistency and monitoring degradation of mAbs. As an illus- tration, Fig. 7 shows the charge heterogeneity proﬁle of 16 mAbs.

High-performance cation-exchange chromatofocusing meth- odsweredevelopedforthevariantsofneutraltoacidicantibodies, and base-line separation of a variety of antibody variants wasachieved [71]. Thehighresolution achieved indicated that the methods developed were useful alternatives to isoelectric focusingforcharacterizingthechargeheterogeneityofmAbsvari- ants.

ThefastandefﬁcientseparationofcetuximabFabandFcvari- ants were recently reported in both salt and pH-gradient CEX mode[32,41].Fourteen charge variantswere separatedin only 17min.

AsystematicstudycomparedthepossibilitiesofIEXmodesand showedthattheseapproachesarecost-andtimesavingalterna- tivetoclassicalproteinanalysismethods(e.g.gelelectrophoresis).

Theauthorspredictedthatinthenextstep,furtherbiologicals,e.g.

antibodies,willbeanalyzedandquantiﬁedmostlywithIEXand RPLCinthenativeaswellasinitsdenaturedform,respectively [72].AnotherstudyalsofoundbeneﬁtsofIEXcomparedtoelec- trophoreticmethodssuchasthepossibilityofbeingautomatedand betterquantitativeresults[73].

BesidestheseparationofmAbs’chargevariants,pH-gradient basedIEXchromatographycanalsobeappliedtoevaluatethepIof intactmAbs[74].

BesidesrecombinantmAbs,IEXseemstobeapromisingtech- niqueforthecharacterizationofotherantibody-relatedproducts suchasbispeciﬁcantibodies,recombinantpolyclonalantibodies (pAbs)andFc-fusionproteins[75–80].

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Fig.7.Thechargeheterogeneityproﬁlesof16mAbswithpIrangingfrom6.2to9.4obtainedwiththesalt-mediatedpH-gradientbasedIEXmethod.

4.3. Analysisofantibody–drugconjugates

Antibody–drug conjugates(ADCs) or immunoconjugates, are becoming another increasingly important class of therapeutic agents undergoing clinicalinvestigations for treatmentof vari- ouscancer[81].ADCsareproducedthroughthechemicallinkage ofapotentsmallmoleculecytotoxin(drug)toa mAbandhave morecomplexandheterogeneousstructuresthanthecorrespond- ingantibodies[82].ADCsareconstructedfromthreecomponents:

amAbthatisspeciﬁctoatumorantigen,ahighlypotentcyto- toxicagentandalinkerspeciesthatenablescovalentattachment ofthecytotoxintothemAbthrougheithertheproteinorthegly- can.Theprimarysitesusedforprotein-directedconjugationare theaminogroupsoflysineresiduesorthesulfhydrylgroupsofthe inter-chaincysteineresidues[82].Dependingonthecharacteristics ofthedrug,thelinkerandtheconjugationsite(i.e.,lysine,inter- chainsulfhydryl,carbohydrate),themethodscommonlyusedto characterizetheparentmAbmaynotbeapplicabletotheADCor maygivesigniﬁcantlydifferentinformation.

Attachmentof anunchargedlinker anddrug throughlysine residuesdecreasesthenetpositivechargebyoneforeachbound drug-linker.Inthiscase,separationbasedoncharge,suchasusing IEXoriso-electricfocusing(IEF),resultsinproﬁlesthatcharacterize thedrugload,ratherthanprovinginformationabouttheunderly- ingmAb[83].Despitetheutilityofthesemethods,thereareonlya fewpublishedreportsofcharge-basedassaysappliedtoADCs.

Forgemtuzumabozogamicin,theIEXproﬁleshowedthatmost ofthecalicheamicinwasonapproximatelyhalfof theantibody while45–65%oftheproductwasalowconjugatedfraction,essen- tiallyunconjugatedantibody[83,84].

Thiomabsareantibodieswithanengineeredunpairedcysteine residueoneachheavychainthatcanbeusedasintermediatesto generateADCs[85].Multiplechargevariantpeakswereobserved duringCEXanalysisofseveraldifferentthiomabs.Thischargehet- erogeneitywasduetocysteinylationand/orglutathionylationat

theengineered and unpairedcysteinesthrough disulﬁdebonds formedduringthecellcultureprocess[85].

Ina recentstudy,theeffectsof chemicalconjugationonthe electrostatic properties of Fc-conjugates were estimated [86].

Tominimizetheeffectsofpost-translational modiﬁcations(e.g., deamidation),asingleFcchargevariantwasisolatedpriortocon- jugationofaﬂuorescentprobe,tothesidechainsoflysineresidues.

TheresultingFc-conjugateswereassessedbyavarietyofanalyti- caltechniques,includingIEX,todeterminetheirchargeproperties [86].

5. PerspectivesinIEX 5.1. Decreasingtheparticlesize

TheUltra-HighPressureLiquidChromatography(UHPLC)tech- nologywasoriginallydevelopedforRPLCapplications,butitisnow alsoavailableforSEC[87]andIEXoperationsduetotheavailability of1.7and3␮mnon-porousparticles(PS/DVB)[88,89].

Applying1.7and3␮mparticlesmayopenanewlevelofper- formanceinIEX,butithastobekeptinmindthatonveryﬁne particles,theseparationqualityisimprovedatthecostofpressure (andtemperaturegradientsattributedtofrictionalheatingeffects).

Therefore,thereisariskofcreatingon-columndegradationwhen analyzingtemperatureorpressuresensitiveproteinsunderhigh pressure(i.e.,>300bar)conditions,asreportedforRPLCandSEC [90,91].Theotherdisadvantageofsub-2␮mIEXseparationsisthat currentlythereareonlyaverylimitednumberofcommercially availablestationaryphases.

Columnspackedwiththeseveryfineparticlesarestableupto 600barthatcouldbebeneficialforbothfastandhighresolution separations.Onshortcolumns,highthroughputseparationscan beachievedbyapplyinghighflowrates.Ontheotherhand,the separationpowercanbeimprovedbyincreasingthecolumnlength (e.g.couplingcolumnsinseries).Withrelativelylongcolumns(e.g.

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Fig.8. Separationofastandardproteinmixtureona0.32mmID×25cmstrong anionexchangercapillarycolumnusingpH-gradientmode.Theelutionorderofthe proteins:cytochromeC(1),myoglobinbasicband(2),myoglobinacidicband(3), conalbumin(4),and␤-lactoglobulinB(5)andA(6).

20–40cm)employedatlowﬂowrates,thepeakcapacitycanbe improvedatthecostofanalysistime.

5.2. CapillaryIEX

Toimprovethesensitivityofproteinvariantsdeterminationor handleverysmallamountsofsamples,theuseofcapillarycolumns inIEXappearstobeapromisingapproach.However,severalkey modificationstoacommerciallyavailableliquidchromatography systemarerequiredtoreducethesystemvolumeandassociated extra-columnbandbroadening,whichcouldbecriticalforcapil- laryIEXoperation.Untilnow,thenumberofapplicationsinthis fieldisratherlimited,buta0.32mmI.D.IEXcapillarycolumnwas successfullyappliedinpH-gradientmodeasthefirstdimensionin a2Dseparationofstandardproteinmixtures[92].Fig.8showsthe

chromatogramobservedwitha0.32mmID×25cmstronganion exchangercapillarycolumnusingpH-gradientmode.

5.3. Monolithiccolumns

Monolithic stationary phases are promising materials to improvechromatographicperformance[93,94].

A monolithic column can be deﬁned as a continuous solid matrix, porous in nature and containing interconnected ﬂow paths. Various types of inorganic (e.g. silica, zirconia, car- bon,titania)andorganic(e.g.polymethacrylate,polyacrylamide, poly(styrene-divinylbenzene...)monoliths can beprepared but onlypolymethacrylate,poly(styrene–divinylbenzene),andsilica- based monoliths are commercially available (mostly for RPLC separations).

Thelargeﬂow-throughchannelsandessentiallynonporoussur- facessupport fast masstransfer, especially for largemolecules (possessingslowdiffusion),resultinginhighresolutionorfastsep- arations.Thesechannelsalsoprovidehighpermeability,allowing theuseofhighlinearvelocities.

Monolithsaregenerallyclassifiedasorganicandsilica-based monoliths. The organic monoliths are usually applied for the separationsofbiomolecules,includingoligonucleotides,peptides, and intact proteinssuch as protein isoforms [95,96]. They are better accepted for protein separations than inorganic monoliths because of their biocompatibility. However, low surface areaandbinding capacity,swelling and shrinkagein somesol- vents, as well as deficiency in mechanical stability are their major drawbacks. Contrarily, silica-based monoliths are well adapted to the analysis of small molecules. They consist of a singlerodof silicawithtwo typesofpores:macropores,which enablelowflowresistance,andmesopores,whichensureenough surface area to reach high separation efficiency and loadabil- ity.

Currently,only5cmlongorganicmonolithscontainingstrong andweakexchangersareavailableineither4.6or1.0mmIDformat [97].

Fig.9. The2DproteinexpressionmapofE.colibacteriallysate.ThexaxisisinpIunitfrompH4.0to7.0(measuredbypH-gradientIEX)andtheyaxisdisplaysincreasing hydrophobicity(%B)(measuredbyRPLC).