protein aggregates Theory and practice of size exclusion chromatography for the analysisof Journal of Pharmaceutical and Biomedical Analysis

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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

Theory and practice of size exclusion chromatography for the analysis of protein aggregates

Szabolcs Fekete

, Alain Beck

, Jean-Luc Veuthey

, Davy Guillarme

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

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

a r t i c l e i n f o

Articlehistory:

Received12February2014 Receivedinrevisedform3April2014 Accepted8April2014

Availableonline19April2014

Keywords:

Sizeexclusionchromatography Therapeuticproteins Aggregates Biopharmaceuticals

a b s t r a c t

Sizeexclusionchromatography(SEC)isahistoricaltechniquewidelyemployedforthedetailedcharac- terizationoftherapeuticproteinsandcanbeconsideredasareferenceandpowerfultechniqueforthe qualitativeandquantitativeevaluationofaggregates.Themainadvantageofthisapproachisthemild mobilephaseconditionsthatpermitthecharacterizationofproteinswithminimalimpactontheconfor- mationalstructureandlocalenvironment.Despitethefactthatthechromatographicbehaviorandpeak shapearehardlypredictableinSEC,somegenericrulescanbeappliedforSECmethoddevelopment, whicharedescribedinthisreview.

Duringrecentyears,someimprovementswereintroducedtoconventionalSECthatwillalsobedis- cussed.OfthesenewSECcharacteristics,wediscuss(i)thecommercializationofshorterandnarrower columnspackedwithreducedparticlesizesallowinganimprovementintheresolutionandthroughput;

(ii)thepossibilityofcombiningSECwithvariousdetectors,includingrefractiveindex(RI),ultravio- let(UV),multi-anglelaserlightscattering(MALLS)andviscometer(IV),forextensivecharacterizationof proteinsamplesand(iii)thepossibilityofhyphenatingSECwithmassspectrometry(MS)detectorsusing anadaptedmobilephasecontainingasmallproportionoforganicmodifiersandion-pairingreagents.

©2014ElsevierB.V.Allrightsreserved.

Contents

1. Introduction... 162

2. TheoryofSECseparation... 162

3. Occurrenceofproteinaggregationinpharmaceuticalformulations... 163

4. SeparationofaggregatesinSEC... 164

4.1. Possibleinteractionswiththestationaryphase... 164

4.2. Mobilephasecomposition... 164

4.3. Columns ... 165

4.4. Methoddevelopment... 165

5. DetectionmodesinSEC... 166

6. PossiblepitfallsofmodernSEC... 166

7. ApplicationsofmodernSECtothecharacterizationofbiopharmaceuticals... 167

7.1. ApplicationofSECforthecharacterizationofmAbs... 168

7.2. ApplicationofSECforthecharacterizationofotherproteins... 169

8. SomefuturetrendsinSEC... 170

8.1. InterlacedandparallelinterlacedSEC ... 170

8.2. Sub-3␮mparticles,ultrahigh-pressureSEC... 170

8.3. CapillarySEC... 171

9. Conclusion... 171

References... 171

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

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

http://dx.doi.org/10.1016/j.jpba.2014.04.011 0731-7085/©2014ElsevierB.V.Allrightsreserved.

1. Introduction

Theclinicaluseoftherapeuticproteinshasenabledthetreat- mentofawiderangeoflife-threateningdiseases.Manyofthese diseases were considered incurable or untreatable only a few decadesago.Almosthalfofthenewdrugsrecentlyapprovedby theUnitedStatesFoodandDrugAdministration(FDA)arether- apeuticproteins,andthedrug-pipelinelandscapeisnowshifting evenmoretowardthisdrugclassofdrugs[1].Thereareseveral hundredpotentialbiopharmaceuticals thathavebeenevaluated inclinicaltrialsforthetreatmentofgeneticdiseases,cancerand infectiousdiseases[2–4].Becausethedevelopmentofbiopharma- ceuticalsandbiosimilarsiscomplex,regulatorybodiessuchasthe FDAandEuropeanMedicinesAgency(EMA)requirecomprehen- sivedrugsubstancecharacterization,lot-to-lotandbatch-to-batch comparisons,stability studies,impurity profiling, glycoprofiling andrelatedproteinandexcipientdetermination,suchaspolysor- bateorproteinaggregateelucidation[5,6].

Duetotheuniquephysicaland chemicalproperties ofther- apeuticproteins,theyarepronetoa numberofchangesduring theirpreparation,formulationorstorage[7].Thesechangesinclude severalpossiblemodifications,suchasoxidation,deamination,gly- cosylation, aggregation,misfolding,or adsorption [8–11]. These modificationscouldleadtothepotentiallossoftherapeuticefficacy orunwantedimmunereactions[7].Aggregatelevelsinprotein- baseddrugsareacriticalqualityattributeduetotheirpotential immunogenicity[12,13].Inrecentstudies,cytotoxiceffectshave alsobeenobservedwithseveralbiotherapeuticproteinsduetosig- nificantdenaturationorchemicalalterationsofthenativeprotein [8,9,14].Inthiscontext,thecharacterizationofproteinmodifica- tionsrequiresanumberofanalyticalmethodsbecauseonesingle techniquedoesnotallowfortheassessmentofalltherequired parameters[6].

Proteinaggregates are generallycharacterized usingsodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) [15],size-exclusion liquidchromatography (SEC-HPLC) [16–18], asymmetricalfieldflowfractionation(AF4)[19],fluorescencespec- troscopy[20],circulardichroism(CD)[21]orlight-scattering-based methods,suchasmulti-anglelaserlightscattering(MALLS)[22].In addition,nativemassspectrometryappearstobeanalternative, emerging methodtoidentifyand characterizesmall oligomeric proteinaggregates[23–25].

Among these techniques, SEC-HPLC is employed today as thestandard separationtechniqueforthequantificationofpro- teindimers,trimers andoligomers.The mainadvantageof this approachisthemildelutionconditionsthatallowforthecharacter- izationoftheproteinwithminimalimpactontheconformational structureandthelocalenvironment.Recently,somedevelopments have been made in SEC-HPLC via the introduction of columns packedwithsub-2␮mparticlesorthetandemandparallelinter- laced approaches that enable a new level of chromatographic performanceorthroughput[26,27].Conversely,severaldetection techniquesarenowavailable,ensuringimprovedsensitivity.The aimofthisstudyistoreviewthepossibilitiesandnewdevelop- mentsinSEC-HPLCappliedtotheanalysisofproteinaggregates.

2. TheoryofSECseparation

SECseparatesbiomoleculesaccordingtotheirhydrodynamic radius. The stationary phase consists of spherical porous par- ticles witha carefully controlled pore size, through which the biomoleculesdiffusebasedontheirmolecularsizedifferenceusing anaqueousbufferasthemobilephase.

Basically,SECisanentropicallycontrolledseparationprocess inwhich moleculesareseparatedonthebasisofmolecularsize

differences(filtering)ratherthanbytheirchemicalproperties[28].

In SEC, the partitioning is ideally driven by entropic processes withoutanyadsorption,thusthegenerallyusedGibbsfree-energy equationbecomes

ln K_D=−S₀

R (1)

whereK_Disthethermodynamicretentionfactor,S₀isthechange inthesystementropyand Risthegasconstant.However,it is importanttonotice thatin practice,temperaturecanindirectly impacttheelutiontimetoasmalldegreebyalteringtheconfor- mationsoftheproteinsandaffectingmobilephaseviscosityand solutediffusivity.Thethermodynamicretentionfactor inSECis differentfromotherchromatographicmodes.Here,thethermo- dynamicretentionfactoristhefractionoftheintraparticlepore volumethatisaccessibletotheanalyte[29].

Conversely,ananalogousretentionfactor,k*,wassuggestedby Engelhardtastheratiooftheprobabilitiesofthesamplestaying inthestagnantmobilephaseinsidetheporesandinthemoving mobilephasebetweentheparticles(interstitialvolume)[30].In SEC,thisk*valueislimited,anditsmaximumvalueisgivenbythe ratiooftheporevolume(Vp)andtheinterstitialvolume(Vz)ofa columnorsimilarlybytheporeporosity(εp)andtheinterstitial porosity(ε_z):

k^∗= V_elu−Vz

Vz

(2)

k^∗_max= Vp

V_z = εp

ε_z (3)

Therefore, the ratio of the pore porosity to the interstitial porosity determines theachievable retention range for a given separation.Usingcurrentlyavailablestate-of-the-artcolumns,this maximumretentionfactorisaroundk*∼1.6–1.8.Theseparame- ters–andtheassociatedselectivity–canbealteredbypacking acolumnmoredensely(ordecreasingparticlesize)orbyusing particleswithlargerporevolumes.

InSEC,thesize-basedseparationallowstheconstructionofa calibrationcurvebasedonasetofknownanalytes,whichcanbe usedtoestimatethemolecularweightofanunknownanalyte.Typ- icalcalibrationcurvesarebasedonproteinsorpolymersofknown molecularweights.ByplottinglogMvs.theretentionvolume,one typicallyobtainsathirdorderpolynomialwithalinearregionpro- vidingthehighestresolutionandmolecularweightaccuracy[29].

Thelargestproteins,whichareexcludedfromthepores,elutefirst.

Subsequentproteinseluteinorderofdecreasingsize.Asproteins shapescouldalsovary(e.g.,globular,rod-likeorflexiblechains), theirStokesradiidonotcorrelateexactlywithmolecularweight.

Anothersourceoferrorinthecalibrationcurveisthatnon-ideal adsorptionmayaltertheretentionvolume[31–33].

Theslopeofthelineinthelinearportionofthecalibrationcurve isameasureoftheselectivityofthestationaryphase,whichcanbe definedbytherelationship

log M=m·K_D+b (4)

wheremandbaretheslopeandinterceptoftheline,respectively.

Astheporesizedistributionoftheparticlebecomesnarrower,the slopebecomeslesssteeper,whichresultsinagreaterselectivityto discriminateanalytesofsimilarsize.AtypicalSECcalibrationcurve isshowninFig.1.

Similarlytootherseparationtechniques,themaininterestof modernSECistoimprovetheanalysisthroughputbyreducingthe analysistime.TheanalysistimeinSECismostlydeterminedbythe mobilephaseflowratewithagivencolumnconfigurationbecause alloftheanalytesofinterestareelutedbeforethetotalvoidtime (volume)ofthecolumn(thereisnopartitionandretention)[34].

ToshortentheseparationtimeinSEC,theratioofthecolumnvoid

Fig.1.TypicalcalibrationcurveinSEC.

(ReprintedfromRef[29]).

volumetotheflowrateneedstobedecreased.Reducingthecol- umnsizeandincreasingtheflowratearethestraightforwardways forperformingfastSECanalysis[34].Shorteningthecolumnlength hasbeenappliedinfastSECanalysistokeepthebackpressureat areasonable leveleven athighflowratewhen separatingsyn- theticpolymers[35,36].However,decreasingthecolumnlength proportionallyreducesthenumberoftheoreticalplates.

Popovici and Schoenmakers reported several considerations for fastseparations usingdifferent commercialSEC columnsat roomtemperature[36].Theeffectsofparticlesize,columnlength, andmobilephaseflowrateonSECseparationwerestudiedand reportedinthepast[36,37].Themaindifficultyinachievingboth high-speedandhigh-resolutionseparationinSECistheslowmass transferofthelargesolutesbetweentheinterstitialspaceandthe porespace[37].Toincreasethemasstransferrate,thetempera- turecouldbeavaluableparameter.Asthetemperatureincreases, themobilephaseviscositydecreases,andtheanalytesdiffusiv- ityimproves.Furthermore,thisapproachallowstheuseofhigher flowratesforgivencolumndimensions.Asanexample,Parketal.

showedsomevery fasthightemperature SEC separations,per- formed at 110^◦C [34]. Therefore, and according to theory,the analysistime in SECcanbeshortened byapplyingsmall parti- cles,shortcolumnsandelevatedcolumntemperatures.Engelhardt

proposedthefollowingsemi-empiricalrelationshiptodescribethe columnperformanceinSEC[30]:

H=3.5·dp+1.3(1+k^∗)D_M

u +0.6 k^∗ (1+k^∗)²

d²_p

D_Mu (5)

whereHistheplateheight,dpistheparticlediameter,DMisthe moleculardiffusioncoefficientanduis themobilephaselinear velocity.Basedonthismodel,somepredictionshavebeenmadeto estimatetheimpactoftheparticlesizeandmobilephasetemper- ature(DMdependsonthetemperature)ontheH–uplotasshown inFig.2.

Today,variousparticlesof3–20␮mareroutinelyappliedwith differentpore-sizes[28].Themostcommoncolumndimensionin analyticalSECis30cmcolumnlengthand4.6–8mminternaldiam- eter.Withthese“conventional”SECcolumns,theanalysistimeis generallybetween15and50min.

Whenhigh-resolution separation is required,the separation power(resolution)ofanSECcolumnincreasesindirectpropor- tiontothesquarerootofthecolumnlength;thus,theseparation ofcomplexsamplesrequireslongcolumnsthatcanbeobtainedby joiningmultiplecolumnsinaseries.

3. Occurrenceofproteinaggregationinpharmaceutical formulations

Thecontrolofproteinaggregationisalwaysaconcernduringthe purification,formulationandmanufacturingoftherapeuticprotein products[37–40].Thekineticsorratesofaggregateformationare ofprimaryinterestbecausetheydeterminetheaggregatelevels overagivenperiodoftimeindifferentphysicalenvironmentsand atdifferentproteinconcentrationsandtemperaturesexperienced byaproteinduringitsprocessing,shipping,orstorage.

Ataminimum,aggregatesrepresentaprocessrelatedimpurity and/ordegradationproductthatmustbecontrolledatrelatively lowlevelsofconcentrationthroughoutthemanufacturinganddur- ingtheproductstorage[41].Recentconcernshavealsobeenraised regardingthepotentialimmunogenicityofaggregates,particularly those that are composedof multiple folded or partially folded monomers,although theprecise mechanismsthat make a par- ticularaggregatesize,morphology,and/orstructuremoreorless immunogenicremainuncertain[42].Therefore,itisalsoofinter- esttodifferentiatebetweentheratesofformationforaggregates rangingfromsmalloligomers tosolublehighmolecular-weight (HMW)aggregatesandlarger,effectivelyinsolubleparticles.

Fig.2.Theoreticallyexpectedimpactoftheparticlesizeandmobilephasetemperatureoncolumnperformance.(Forthecalculations,a50kDaproteinwasassumed.).

Fig.3. Schematicrepresentationofsomeofthekeystepsinnon-nativeaggregation.

(ReprintedfromRef[48]).

Inthiscontext,thetermsolubledenotesspeciesthataremolec- ularly dispersed in solution, while insoluble is used to denote aggregatesthathavecoalescedintoamacro-ormicro-phasesep- arateddomainsorparticles.Ithasalsobeenshownrecentlythat proteinaggregatescanreversiblytransitionbetweensolubleand

“insoluble”or“condensed”states[43,44].Alternatively,onecan differentiateaggregatesbasedonsizemeasurementusingother analyticaltechniques[38,45].

Assumingaggregationisthefastestdegradationroute,product shelflifeandtheeffectiveratecoefficientofmonomerloss(k_obs) areequivalentquantitieswhenoneisconcernedwithrelatively lowconversionpercentagesofmonomertoaggregate(<approx.

10%)[46].Aggregationpathways,suchasthermalunfoldingand structuralperturbations,werereviewedacrossawiderangeofpro- teinsbyRobertsetal.[47].Fig.3showsaschematicviewofprotein aggregationbasedontheworkofLietal.[48].

Alltherapeuticproteinshaveaninherentpropensitytoaggre- gate.Thisprocesscanbeacceleratedbyvariousexternalfactors, suchastemperaturevariations,freezing–thawing,agitation,pres- enceofimpurities,formulationchanges,exposuretointerfacesor light,andlong-termstorage[38,40].Aggregatesmayvaryinmany properties,suchassize,typeofintermolecularbonds,reversibility, morphology,andhydrophobicity.Theymayalsocontainproteins withdifferentphysicalandchemicaldegradationstates.

The United States, European and Japanese Pharmacopeias (USP,PhEurand JP,respectively) areharmonized regardingthe requirementsfor visible and sub-visible particles in parenteral formulations.Forsub-visibleaggregates,therearetwomainSEC strategies:(1) observetheaggregation profileand quantifythe percentageofhigh-molecular-weightspecieselutedfromthecol- umnor (2) indirectly estimate the fractionof largeaggregates (typically>100nm)asthelossoftotalpeakarea[49].However, thereisnocleardefinitionforaggregateacceptancelimitsinbio- pharmaceuticalproducts.TheFDAandInternationalConferenceon Harmonization(ICH)consideraggregatestobeprocessorproduct relatedimpurities[50,51].Intheparticularcaseofabiosimilarity study,theFDAguidelinesrecommendthefollowing:“Ifcompara- tivephysicochemicalanalysisrevealscomparableproduct-related impuritiesatsimilarlevelsbetweenthetwoproducts(originator’s andbiosimilar),pharmacological/toxicologicalstudiestocharac- terizepotentialbiologicaleffectsofspecificimpuritiesmaynotbe necessary.”However,ifthemanufacturingprocessusedtoproduce theproposedbiosimilarproductintroducesdifferentimpuritiesor higherlevelsofimpuritiesthanthosepresentinthereferenceprod- uct,additionalpharmacological/toxicologicalorotherstudiesmay berequired[50].

Themainchallengeinanalyzingthedegraded/aggregatedpro- teinformulationsliesinthecomplex,dynamic,heterogeneousand transientnatureofdegraded/aggregatedspecies.Thenumberof chemicalandphysicaldegradationpathwaysisveryhighandmany ofthemareintrinsicallyrelated.

4. SeparationofaggregatesinSEC

4.1. Possibleinteractionswiththestationaryphase

Proteinstendtointeractwiththechargedsurfacesitesofthe stationaryphase[29,31–33,52–54].Non-bindinginteractionsare dominatedbytwotypesofinteractions:(1)electrostaticinterac- tionsand(2)hydrophobicinteractions[32].

Due to theionic interactions, protein adsorption can occur, inducingelutiontimeshifting,bandtailingorasymmetricalband elution.Thethree-dimensionalconformationoftheproteinscan alsobechanged [31,55].Electrostaticinteractionscanbeclassi- fiedintotwogroups.(1)Iftheproteinandthestationaryphase surfaceareidenticallycharged,“ion-exclusion”canoccurdueto theelectrostatic repulsion.In thisway,theproteinisprevented fromenteringtheporesoftheparticleand,therefore,elutesfaster (decreaseinelutiontime).(2)Iftheproteinandtheparticleare oppositelycharged,the adsorptionof proteinonthestationary phasesurfacemayresultinincreasedelutiontime.

Possible hydrophobic interactions between the protein and hydrophobicsitesofthestationaryphasecanalsoleadtoincreases inelutiontime.

ThelastgenerationofSECmaterialsismuchmoreinertthan the older versions, minimizing the possible electrostatic and hydrophobic interactions between the proteins and stationary phase surface. Thisbehavior couldbe attributedto theregular improvementofsilicamaterials(typeBsilicaorhybridsilica)and theuseofdiolbonding.Exceptthenatureofthestationaryphase bondingandtheinteractionsbetweentheproteinsandthesta- tionaryphasecanalsobeminimizedbyproperadjustmentofthe mobilephasecomposition.

4.2. Mobilephasecomposition

Toreduceelectrostaticinteractions,acommonapproachinSEC istoincreasetheionicstrengthorsaltconcentrationofthemobile phase.Inthis way,theunwantedsecondaryinteractions canbe reduced, andthepeak shapes canbeimprovedand quantifica- tioncanbecomemoreprecise.Therecoveryofaggregatescanalso beincreasedbytheadditionofsalts(e.g.,≥100mMsodiumchlo- ride)[54,56].Itwasdemonstratedthatbyvaryingthemobilephase ionicstrength,theretentiontimeofsomemonoclonalantibodies (mAbs)wasshifted,andpoorpeakshapeswereobservedatlow sodiumchlorideconcentration[54].Theaggregaterecoverywas alsoimprovedwhenincreasingthesaltconcentrationinthemobile phase[56].Inadditiontotheionicstrengthincrease,anothervalu- ableapproachconsistsofincreasingtheconcentrationofacounter ioninthemobilephase.Indeed,veryhighconcentrationsofthe sameionscanincreasethehydrophobicorion-exclusioneffects [57].TomitigatetheundesiredsecondaryionicinteractionsinSEC, itisalsorecommendedtousesomeadditivesinthemobilephase.

ArginineiscommonlyusedasanSECadditive[58,59].Theaddition ofargininereducesthepossiblesecondaryinteractions between theproteinand thestationaryphase asit canbindtothepro- tein,leadingtoanimprovementinproteinaggregatesquantitation andpeakshape[29,58,59].Asanexample,Fig.4showstheSEC chromatographicprofilesofrecombinanthumanbasicfibroblast growthfactor(bFGF)inthepresenceandabsenceofarginine.How- ever,arginineshowssignificantUVabsorbanceatlowwavelengths (below220nm)thereforecandrasticallydecreasesensitivity.

Someorganicmodifiers(e.g.,methanolandethanol)couldbe usedtoreducethestrengthofhydrophobicinteractionsduetotheir highereluentstrengthcomparedtowater[60].Therefore,thepeak shapescanbeimprovedinsomecasesbytheadditionof5–10%of anorganicmodifier.

Fig.4.SECchromatographicprofilesofrecombinanthumanbasicfibroblastgrowthfactor(bFGF)in0.2MNaCl(A)and0.2Marginine(B).Thereislittleresolutioninthe separationofbFGF(arrow)fromthesaltin(A),whilebaselineresolutionisobservedin(B).

(ReprintedfromRef[58]).

ThepH of themobile phase canalso affectthe interactions betweenproteinsand thestationaryphase. In anoptimalcase, thepHshouldbeclosetotheisoelectricpoint(pI)oftheprotein tolimit secondary interactions. Indeed, there is a risk for ion- exchangeinteractions when thepHis lowerthan thepI,while ion-exclusioneffectscanoccuratpHvaluesabovethepI.Thiseffect becomesespeciallyimportantwhenworkingatlowionicstrengths [33].

4.3. Columns

TherearemostlytwotypesofSECpackingmaterials:(1)sil- ica, with or without surface modification, and (2) cross-linked polymeric packings, which possess non-polar (hydrophobic), hydrophilic, or ionic character [28]. The most common silica packingsconsistofchemicallybonded1,2-propanediolfunctional groupsthatprovideahydrophilicsurface.Thisstationaryphase blocksorreactswithmanyoftheacidicsilanolgroupsandneutral- izingthesurface.ThereforeitmakesitidealforSECseparationof biopolymers[28].Baresilicaisalsoausefulpackingmaterialfor non-aqueouspolarornon-polarorganicmobilephases;however, itisnotrecommendedwithaqueousmobilephasesbecauseofthe presenceofactivesilanolsites.Thelatesttypeofsilica-relatedpack- ingisanethylene-bridgedhybridinorganic–organic(BEH)material thatiscurrentlyavailableatsizesof1.7␮m–thefirstsub-2␮mSEC packing–and2.5␮m[26,61].Comparedtoregularsilicapackings, BEHparticleshaveimprovedchemicalstabilityaswellasreduced silanolactivity[61].

There have been a number of different hydrophilic cross- linkedpackings developed for theSEC ofbiopolymers. Mostof thesepackingsareproprietaryhydroxylatedderivativesofcross- linked polymethacrylates [28]. Unusual polymeric packings for aqueousSECincludesulfonatedcross-linkedpolystyrene,polydi- vinylbenzenederivatizedwithglucoseoranion-exchangegroups,a polyamidepolymer,andhigh-performance,crossed-linkedagarose [28]. However, when using organic or polar SEC packings for aqueousSEC, one mustprevent theadsorption thatis possible if there are extensive hydrophobic regions on the packing or polymer.

For interested readers, the most important SEC column providersandproductswererecentlyreviewedinRef.[28].

4.4. Methoddevelopment

In any SEC method, a variety of parameters can be adjusted to improve the resolution between the native pro- tein and aggregates and make the method more robust and reliable.

First,anappropriateporesizehastobeselected.Theporesize selectiondependsonthesize(ormolecularweight)oftheprotein anditsaggregatestobeseparated.Moleculeswithadiameterlarger thanthelargestporesinthestationaryphaseareunabletoenter theporousvolumeoftheparticles.Therefore,theypassthrough thecolumnandelutefirstwiththeinterstitialvolume(Vz).Smaller moleculesentertheporeswithintheparticlesanddependingon theirapparentsizetheyelutelaterinorderofdecreasingsize.For proteincharacterization,typicalporesizesbetween150and500 ˚A areapplied.Forcommontherapeuticproteins(MW∼15–80kDa),a poresizeof150–200 ˚Aiswellsuited,whilea200–300-Åporesize isusuallyappliedfor mAbs(MW∼150kDa).Forverylargepro- teins(MW>200kDa,e.g.,PegloticaseorPEGylatedproteins),the 500–1000 ˚Aphasesofferthebestselectivity.

Resolutionbetweentheaggregates andnativeprotein peaks isenhanced whendecreasingparticlesize.Theachievableplate numbersareinverselyproportionaltotheparticlesize,whilethe resolutionisinverselyproportionaltothesquarerootofparticle size.Historically,5–10␮mparticlesarecommonlyusedinroutine SECapplications,butseveralprovidersoffernow3␮mparticlesor evensub-2␮mpackings.Byusingthesestate-of-the-artcolumns packedwithveryfineparticles,theresolutioncanbesignificantly improvedortheanalysistimecanbeshortenedbyapplyingshorter columns.The useofvery fineparticles inSEC hasshown clear advantagesinresolution[30,62].

BecauseSECisanentropicallycontrolledseparationwhereno retentionoccurs,largeporevolumes(highporosity)arerequiredto ensureappropriateselectivity.Generally,thislargeporevolumeis providedbylong-andwide-borecolumns.InroutineSECapplica- tions,a30cmcolumnlengthwithinternaldiameters(I.D.)of6,7.8, 8or10mmisgenerallyemployed.TheseSECcolumnsarereferred toasstandardborecolumns.Now,severalvendorsoffernarrow borecolumnswith4.6-mmI.D.and15cmlengthsthatarepacked withveryefficient,smallparticles.Byusingthesecolumns,similar separationpowercanbeattainedaswith5␮mparticlesin30cm standardborecolumns,buttheanalysistimecanbereducedbya factorof3–4[26].

Afterselectingtheappropriatecolumn,themobilephasecom- positionhas to be optimized. As mentioned in Section 4.2, an appropriatepHandsaltconcentrationarerequiredtoensurecor- rectpeakshapeandresolution.Generally,thepHshouldbeclose totheisoelectricpointoftheprotein,andtheproteinshouldbe solubleandstableinthebuffer.Itisgoodpracticetouseabuffered solutionwithanionicstrengthof50–200mM.Thisconditionhelps toreducenon-specificinteractionsbetweentheproteinandthe stationaryphase.Themostcommonlyusedbufferisphosphateat 10–200mMconcentrationandpHbetween6and7.2.Whenthe isoelectricpointoftheproteinisunknown,apHof6.5maybea goodstartingpoint.Toimprovethepeakshape,sodiumchloride, potassiumchlorideorsodiumsulfateisoftenaddedintothemobile phaseataconcentrationof10–250mM.However,whenworking atlowionicstrength,non-idealconditionscanimproveselectiv- ityinsomecases.Enhancementinselectivityandresolutionwas reportedformodelproteinsinthesorptivemodewithmobilephase pHbelowthepI[63].Insomecases,theadditionoforganicmod- ifiers,suchasmethanolorethanol,canalsohelptoimprovethe resolutionandpeakshapeduetoareductionofpossiblehydropho- bicinteractionsbetweenproteinsandthesurfaceofthestationary phase.Pleasenotethatbufferswithhighsaltconcentrationand organicmodifierscanbehazardousforthepumpandsystem.Salt precipitationcanblockthetubing,sealsorplungers.Thereforeitis suggestedtoflushthesaltsfromthesystemregularlywithwater orwithahighaqueous(e.g.5%acetonitrileormethanol)mobile phase.

Intermsofmobilephaseflowrate,itiscommontoworkat 0.2–1.0ml/minwithstandardborecolumns.Inmostcases,theres- olutioncanbeimprovedbydecreasingtheflowrate,albeitthe throughputisreduced.

As in other chromatographic modes, sample loading (both volume and mass) affects SEC chromatographic resolution and sensitivity[64,65].Inthecaseofcolumnoverloading,resolution betweenanalytescanbedecreased.Theidealvolumeloadscorre- spondtosamplevolumeslowerthan1–5%oftheaccessiblecolumn volume.Ifthesamplevolumeincreasesbeyondthiscut-offvalue, theresolutionmaybereduced,andpeaktailingmaybeobserved.

To conclude the discussion of SEC method development, it remainspartlyempiricalbecausechromatographicbehaviorand peakshapearehardlypredictable.Therefore,theoptimizationof anSECseparationissometimestime-consuming.

5. DetectionmodesinSEC

InSECseparationsofproteins,UVisstillthepredominantdetec- tionmode[66,67].UVwavelengthsof270,275and280nmgive specificresponseforaromaticaminoacids(tryptophan,tyrosine, phenylalanine)and arecommonlyusedtoassessproteinaggre- gation[68].AtlowerUVwavelengths(210,214or220nm),the amidepeptidebondhasarelativelyhighabsorbance.Therefore, theselowerUV wavelengthsofferenhanced sensitivitybutalso lowerspecificity.Finally,higherwavelengthsprovideagreaterlin- eardynamicrange.Dualwavelengthdetectionincludingbothlow andhighdetectionwavelengthscanbeusefulforpurityprofiling inSEC[22,69].Thelowerwavelengthprovidesthesensitivityfor thelowabundancespecies(aggregates),whilethehigherwave- lengthprovidesagreaterlinearrangeforthemajorspecies(native monomer).

Using a fluorescence detector,both sensitivity and selectiv- itycanbesignificantlyimproved[70–73].Fluorescencedetection enablesthemeasurementoflowlevelsofaggregates.Intrinsicpro- teinfluorescenceisgenerallyobservedatanexcitationwavelength of280nmandupto295nm.Theemissionismostlyduetoexci- tationoftryptophanresidues,andscarcelyattributedtotyrosine

andphenylalanineresidues[74].Themaximumemissionoccurs between300and370nm,dependingonthefolding,conforma- tionandmobilephaseproperties.Itisalsoimportanttonoticethat proteinscanbecovalentlylabeledwithvariousfluorophores,thus producingfluorescentproteinconjugates.Theemissionfromthese attachedtagsiscalledextrinsicfluorescence.Taggingaproteinwith fluorescentlabelsisanimportantandvaluabletoolforstudying structureandmicroenvironment[74].

Modern analytical SEC platforms are capable of more than just sampledetectionwitha singleconcentrationdetector.The combination of multiple detectors including refractive index (RI),ultraviolet(UV),light scattering(LS)–ormulti-angle light scattering (MALS), multi-angle laser light scattering (MALLS) – andviscometer(IV)allowsextensivecharacterizationofprotein samples[75].RIandUVbothallowaccurateconcentrationmea- surements.Lightscatteringdetectors allowthemeasurementof themolecularweightwithouttheneedforcolumncalibration[76].

Intrinsicviscosityisameasureofthemoleculardensityandenables structuralchangestobeassessed.Acombinationoflightscatter- ingandintrinsicviscosityallowsthedeterminationofthesizeof themolecules.Thissetofdetectioniscalledtetra-detection[75].A typicaltetra-detectoranalyticalSECsystemisshowninFig.5.

MALSdetectorsaregenerallymoresensitivetothehighMW aggregatesandenabletheirconfirmationandanalysis.

Mass spectrometry (MS) is another possibility for obtaining molecularweightinformation.However,therearechallengesto interfacingSECwithMSduetotheinherentincompatibilityofthe mobilephasescontaininghighconcentrationsofnon-volatilesalts [77].SECmobilephasesaretypicallynon-denaturingaqueoussolu- tionsclosetothephysiologicalpHrange.Suchmobilephasesmade withphosphate bufferlead toion suppressionand contamina- tionofthemassspectrometer.Ammoniumformateorammonium acetateatreasonable ionicstrengthscouldbeusedas analter- nativetophosphatebuffer,butphysiologicalpHconditionsmay bedifficulttoachievebecausethesebuffersareadaptedforacidic conditions(pKaof3.75and4.75forformicandaceticacid,respec- tively)[78].Toovercomethisdifficulty,SEC-MSmethodshavebeen developedusingdenaturingmobilephasescontainingorganicsol- ventsand ion-pairingreagentsinsteadof largeamounts ofnon volatilesalts[79–82].Acetonitrile andtrifluoroacetic acid(TFA) in themobilephase allowsfor successful SEC-MSexperiments.

Anotherpossiblemethodforavoidingtheproblemsencountered withthemobilephaseistouseoff-lineMSdetection.Asanexam- ple,Matrix-assistedLaserDesorptionIonization(MALDI)combined withTimeofFlight(TOF)analyzerhasbeenusedinthepastfor off-lineSEC[83].

Comprehensivetwo dimensionalLC (2D-LC)is mostlikely a good alternative to make SEC separations compatible with MS detectionandtoimprovetheseparationpower.UsingSECinthe firstdimensionandreversed-phaseLCmodeastheseconddimen- sionwould beidealbecausethis strategyprovidesa highpeak capacity,evenforfastgradientseparations,inadditiontostraight- forwardcompatibilitywithMS.

6. PossiblepitfallsofmodernSEC

Today,awidevarietyofporouspackingmaterialswithdifferent particlesizesareavailablefortheseparationandcharacterization ofmacromolecules[84].Theporevolumeandtheporesizedis- tributiondeterminethemolarmassrangethatcanbeevaluated, whereas theparticlesizedetermines thediffusionprocesses of proteinsthroughthecolumn[84].Smallerparticlesleadtonar- rowerpeaksduetobettercolumnefficiency.Intermsofefficiency andachievableanalysistime,porousmaterialswithasmallpar- ticlesizesseemtobethemostfavorablecolumnmaterialforSEC

Fig.5.Schematicviewofamultiple-detectorSECsystemequippedwithultravioletUV/PDA),lightscattering(LS),refractiveindex(RI)andviscometricdetectors.

(AdaptedfromRef[73]).

[26].However,withveryfineparticles(sub-2␮mparticles),the separationqualityisimprovedatthecostofpressure.Ithasbeen establishedinreversedphaseliquidchromatography(RPLC)that highpressure(i.e.,>500bar)mightcauseimportantfrictionalheat- ingeffectsinsidethecolumn,andthisheatingisalsolikelytrue forSEC[85].Therefore,temperaturesensitiveproteinsmightsuf- ferfromon-columnaggregationordenaturation[86].Insummary, reducingtheparticlesizetothesub-2␮mrangeincreasestherisk ofsheardegradation[87].

Ingeneral,SECseparationsareperformedatlowtomoderate pressures(typicallyP<100bar)usingconventional SECcolumns packedwith5–10␮mparticlesandoperatingatrelativelylowflow rates[28].Undertheseconditions,thethermaleffects(longitudi- nalorradialtemperaturegradients)insidethecolumnsaswellas theshearingforcesarenegligible.However,whenapplyingvery fineparticlesand/orhighflowrates,theseadverseeffects could becomeimportantandcouldhaveastrongimpactonseveralchro- matographicproperties,suchasretention,peakshape,efficiency orselectivity[88,89].Usingtherecentlyintroduced1.7␮mparti- clesinSEC(aqueousmobilephase,15cmcolumnlength),veryhigh pressures(upto500–600bar)canbegenerated.Therefore,thermal effectsandshearingforcesmightbecomecriticalfortemperature orpressuresensitiveproteins[86,87].Asexplainedpreviously,ele- vatedtemperaturesinSECcouldbeaninterestingtooltoincrease the efficiency and are often used in ultra high-pressure liquid chromatography(UHPLC)todecreasethemobilephaseviscosity.

Undersuchconditions,thegeneratedpressuredropislower,thus allowingworkathigher flow rates.However,elevatedtemper- aturesmightalsocausesomechangesinproteinstructure, and thereisanadditionalriskofon-columnproteindenaturationand aggregation.

Oneofourrecentstudiessystematicallyinvestigatedtheeffects ofpressureand temperatureontheobserved amountofaggre- gateswhen usingSECcolumnspackedwithsub-2␮mparticles [26].Fig.6Bshowstheeffectofthecolumnheadpressureonthe measuredamountofIgGaggregates.Asthepressureincreased,the amountofmeasuredaggregatesalsoincreased.Asimilartrendwas observedwhentheaggregateamountwasevaluatedasafunction

ofthemobilephasetemperature.AsshowninFig.6A,themeasured amountofaggregateschangedfrom∼4%to∼10%whenincreasing thetemperaturefrom30to60^◦C.

Toconclude,usingveryfineparticles(highpressure)andele- vated temperature in SEC can generate serious artifactsin the quantitationofproteinaggregates.Theinductionofproteinsubunit associationsandaggregationsathighpressureiswelldocumented [90–92].Inaddition,thepossiblechangesinaggregatesolubility caused by thevery highpressure cannot beignored. However, somecontradictory resultscan befoundin theliterature. Both increases and decreases in protein aggregate solubility caused by highpressure were reported [93,94]. The adverse effects of UHPLC-SECapproacharemostlikelystronglyproteindependent [26].

The other importantissue in SEC is the column lifetime. In biopharmaceuticalformulations,severalexcipientsandstabilizers (e.g.,polysorbates,salts,PEG)areaddedtominimizethepossible chemical andphysical modificationoftheproteinsduringstor- age.Unfortunately,theseadditivesdecreasethelong-termcolumn stability and resultin changes in elution,peakshape or recov- ery.Varioustime-consumingcleaningprotocolsaresuggestedby columnsupplierstorestoretheSECcolumns,butinmostcases, theyareineffective.Theuseofguardcolumnsisavaluablestrat- egytoimprovecolumnlifetime.However,theexpectedlifetime of SEC columns is clearly shorter than for reversed phase or ion-exchangecolumns when analyzing therapeuticprotein for- mulations. Typically, only a few hundred injections (e.g., 300) can beperformedwithoutsignificant lossin efficiencyor peak symmetry.

7. ApplicationsofmodernSECtothecharacterizationof biopharmaceuticals

Inthissection,someimportantSECapplicationswereselected fromthebiopharmaceuticalindustrytoshowthepotentialofthis analyticaltechnique.

Fig.6. Representativechromatogramsontheeffectofcolumntemperature(A)andpressure(B)ontheobservedamountofantibodyaggregates.

(ReprintedfromRef[26]).

7.1. ApplicationofSECforthecharacterizationofmAbs

Recently,Latypovetal.showedanimpressivehigh-throughput methodthatisabletodifferentiatecandidaterecombinanthuman monoclonalIgG1and IgG2antibodiesbasedontheirpropensity toformaggregateswhensubjectedtoagitation(vortexing)stress [95].IntactmAbswereseparatedfromsolubleandinsolubleaggre- gatesusingsizeexclusionchromatography,undernon-denaturing conditions,andtheindividualcomponentsofthemixturewere identifiedwithaUHPLC-TOF-MSplatform andquantifiedvs.an unstressedcontrol.Aninternalstandard wasaddedtothemix- tureafter stress, and used to correct for ionization differences betweensamples.TreatmentofthesampleswiththeIdeS(Fabrica- tor)enzymesignificantlyreducessamplecomplexity,andallowed foralargenumberofcandidatemoleculestobeassessedinasingle analysis.TheIdeSenzymegeneratedF(ab)₂(fragmentofantigen binding)andFc(fragmentcrystallizable)domainsoftheantibody.

AvolatileammoniumacetatesaltwasusedtogenerateanMScom- patiblemobilephase matchingthepHformulation. Themobile phasewas25mMammoniumacetateand5%acetonitrileinwater, adjustedtopH5.2. SeparationwasperformedusinganAcquity UPLCBEH200SEC1.7␮m×4.6mm×150mmcolumnatambient temperature.Amixtureof8intactmAbsatt=0h,1hand4hof agitationandthefragmentsgeneratedbydigestionwithIdeS,were analyzedatthesametimepoints.ThegrowthofHMWspeciesdid notaccountfortheoverallmonomerlossbecauseoftheformation ofinsolubleaggregates[95].Fig.7showsthecorrespondingSEC chromatogramsobtainedatdifferentagitationtimes.

Anotherstudy reported an online fluorescent dye detection methodsuitablefor SECand asymmetricalflow fieldflow frac- tionation (AF4)[96]. Thenoncovalent,extrinsic fluorescentdye (Bis-ANS)wasaddedtothemobilephaseorthesample,andthe fluorescenceemission at 488nm wasrecorded atan excitation wavelengthof385nm.BycombiningSECandAF4withonlinedye detection,itwaspossibletosimultaneouslydetectheat-induced aggregationandstructuralchangesofmonomericandaggregated IgG[96].

Wätzigetal.showeda15minSECseparationofIgG1antibody aggregatesusingaconventional30cmlongcolumnpackedwith 5␮mparticlesanddemonstratedtheprecisionandrepeatability ofmonomerandaggregatequantitation[97].However,byusing state-of-the-artcolumntechnologyandUHPLCinstrumentation,it wasdemonstratedthattheanalysistimecanbereducedtoless than3min[26].

AhighlysensitivecapillarySECmethodologywasdevelopedfor Fabaggregateanalysisinhumanvitreoushumor[98].Thecapil- larySECmethodenabledpicogramsensitivitywithanRSDofless than8%fortherelativepeakareaofHMWoftheFabfragments.

Fig.8showsrepresentativechromatogramsobtainedwithcapil- larySECbyinjectingantibodyFabfragments.Anotherstudyalso

Fig.7. UVchromatogramsat280nmofamixtureof(A)8intactmAbsatt=0h (black),1h(blue),and4h(red)ofagitation,and(B)thefragmentsgeneratedby digestionwithFabricator,atthesametimepoints.ThegrowthofHMWdidnot accountfortheoverallmonomerlossbecauseoftheformationofinsolubleaggre- gates.(Forinterpretationofthereferencestocolorinthisfigurelegend,thereader isreferredtothewebversionofthearticle.)

(ReprintedfromRef[93]).

Fig.8.Representativechromatogramsofco-mixedoxidized/controlsamplesofFab1obtainedbycapillarySEC.Themainpeakheightisnormalizedacrosssamples.The percentageofoxidizedsampleinthesamplemixturecorrespondingtoeachchromatogramisindicated.

(ReprintedfromRef[96]).

demonstratedtheenhancedsensitivityofcapillarySECfortheanal- ysisofmAbspurifiedfromharvestedcellculturefluid[99].

ThedevelopmentofafastandeasySECmethodundermod- erately hydrophobic conditions (mixed mode) to monitor the heterogeneity in drug product samples was recently reported [100]. The best separation was obtained on a column packed withsub-2␮mparticlesalongwithamobilephaseconsistingof sodiumacetateandsodiumsulfatethatseparatesIgGintoaggre- gates,monomer,andfragments.Themoderatesaltconcentration resultedinasecondaryseparationmodebasedonhydrophobic- ity,resolvingamonomerpre-peakfromthemonomermainpeak.

MALSdeterminedthatthepre-peakhadasimilarmassastheIgG monomer.Characterizationofthepurifiedpre-peakfractionusing MS,andbioactivityrevealedthis degradanttobeatryptophan- oxidizedIgGmonomer,withsignificantlyreducedbioactivity[100].

SimilartowhatiscommonlyperformedforintactmAbs,the determinationofAntibodyDrugConjugates(ADC)iscarriedout using SEC. However, it is worth mentioning that regular SEC usinga phosphate-bufferedmobilephase providesa poorpeak shape of ADC and unacceptable resolution between aggregates andmonomericADCproducts[101].Thisresultcouldprobablybe explainedbynon-specificinteractionsbetweenthehydrophobic cytotoxicdrugsandthesurfaceofthestationaryphase.Tosolve thisproblemandimprovepeakshape,variousorganicmodifiers wereaddedtotheSECmobilephase,suchas25%propyleneglycol [102]or10%DMSO[103].Itisalsoconceivablethatalcohol-type organicmodifierscouldbesuccessfullyemployedtoimproveSEC performanceofADCproducts.Inaddition,thesmallerchargevari- ants,namelyreducedfragments(i.e.,lightchainsandheavychains withdifferentdrugloads)weresuccessfullycharacterizedbyRPLC usinggenericconditions[104].AsfornakedmAbs,massspectrom- etryisakeytechniquetogaininsightsintotheoligomericnature oftheSEC-isolated“multimeric”fractionofADCs[105].

7.2. ApplicationofSECforthecharacterizationofotherproteins The gain in analysis time and resolution power achieved using narrow-bore SEC columns packed with 1.7␮m particles

was demonstrated for insulin aggregate analysis [106]. Com- paredtoaregular300mm×7.8mm,10␮mstandardSECcolumn, theanalysistime wasshortenedby afactor of3 when usinga 300mm×4.6mm,1.7␮mcolumn.

The development and validation of a sensitiveSEC method forthequantitationofr-HuEPOaggregatesinformulationscon- taining 0.03% polysorbate 80 were described by Gunturi et al.

[73]. A conventional column (300mm×7.8mm, 250 ˚A) packed with 5␮m particles and fluorescence detection was used. The mobilephaseconsistedofisopropylalcohol–potassiumphosphate (0.1M)/potassium chloride buffer (pH 6.8, 0.2M) (25:75, v/v).

Themethodwasshown tobespecificfor r-HuEPOtotal aggre- gates(dimer and oligomers)and allowedfortheirquantitation at80ng/mlinthepresenceofr-HuEPOmonomerandthephar- maceuticalexcipients,namelyglycine(5mg/ml),sodiumchloride (4.3mg/ml)and0.03%polysorbate80[73].

The interest in SEC–MALLS to investigate aggregation and degradationofglycosylatedandnon-glycosylatedproteins,under variousconditions,suchasadditionofdetergent,pHmodification, variationoftheproteinconcentrationandheatstresstemperature wassystematicallyexaminedbyYe[107].Thecharacterizationof proteinsandtheiraggregateswasperformedbyonlineUV,refrac- tive index, and MALLS detectors. Aggregation and degradation wereexaminedundervariousconditionsandquantitativeresults werepresented for bovineserumalbumin,choriogonadotropin, glyceraldehyde-3-phosphatedehydrogenase,Herceptin,andReo- Pro.Thereportedmethodcouldsimultaneouslydetermineboth thequantitiesandthemolecularweightsofmacromoleculesfrom asingleinjection[107].

Amethodologybasedonon-linecouplingofSECwithmixed- modeliquidchromatographyhasbeenreportedbyHeetal.[108].

Themethodallowedforthesimultaneousmeasurementofawide rangeofcomponentsinbiopharmaceuticaldrugproducts,includ- ing the active pharmaceutical ingredient (protein) and various typesofexcipients,suchascations,anions,nonionichydrophobic surfactantandhydrophilicsugars.Dual,shortSECcolumnswere usedtoseparatesmallmoleculeexcipientsfromlargeproteins.

TheseparatedproteinwasquantifiedbyUVdetectionat280nm.

Fig.9. SECanalysisofthermallyaggregatedinterferonalpha-2bsample.Separation ofinterferonalpha-2b(0.1mg/ml)containingmonomer(peak1,53%),dimer(peak 2,6%)andHMWaggregate(peak3,38%).Thesamplewasincubatedinawaterbath at70^◦Cfor30min.Fluorescencedetectionwasperformed.

(ReprintedfromRef[7]).

Theisolatedexcipientswereswitchedonline,toamixed-mode columnforseparation,anddetectedbyanevaporativelightscat- teringdetector(ELSD).AvolatilebufferwasusedforbothSECand mixed-modeseparation.Thismethodfacilitatedthedetectionof differentexcipientsbyELSDandprovidedpotentialforonlinechar- acterizationoftheproteinbymassspectrometry(MS).Themethod wasfinallyappliedtoquantifyproteinandexcipientsindifferent biopharmaceuticalproducts, includingantibodydrugconjugates (ADC)andvaccines[108].

SEC methods coupled with intrinsic fluorescence detection weredevelopedforevaluatingthestabilityanddegradationpro- filesofinterferonalpha-2drugsubstancesanddrugproducts[7].

The method allowed baseline resolution of the active ingredi- entfromtheexcipientscontainedinthefinalproduct,including largeamountsofalbumin.HMWaggregateswithapparentmolec- ularweightof ∼650kDa, aswellas dimersand denatured and reducedvariants,weresuccessfullyidentifiedandseparatedfrom thenativeproteins.Thischromatographicmethod,whichquan- titativelymeasuredphysicalandchemicalchangestakingplacein solutionformulations,wasfoundtobecapableofmonitoringinter- feronalpha-2stability[7].Fig.9showstheSECprofilesofthermally aggregatedinterferonsamples.

8. SomefuturetrendsinSEC

8.1. InterlacedandparallelinterlacedSEC

Usingtwo SEC columnsinparallel cansignificantly increase theanalysisthroughput.Afurtherreductionofanalysistimecan beobtainedbycombininginterlacedsampleinjectionswithpar- allelizationof twonarrowborecolumnspackedwithsub-2␮m particles.Withthisstrategy,bothlagtimesbeforeandafterthe peaksofinterestcansuccessfully beeliminated,resulting inan assaytimebelow2min[27].

Farnanetal.[109]havedescribedthemethodologyofinterlaced SECindetail.Insummary,themethodologyisbasedoninjectinga newsamplebeforetheongoinganalysisofaprevioussamplehas ended.Thesubsequentinformationphasebeginsimmediatelyafter thesaltfractionoftheprecedingsamplehaseluted.Fig.10Ashows theschematicviewofaninterlacedSECinjection.Thetotaltime requiredfortheanalysisofnsamplescanbeexpressedas:

t_total=t_lag+n·(t_inf+t_hold+t_salt) (6)

Fig.10.Schematicviewofinterlaced(A)andparallelinterlaced(B)SECseparation.

(ReprintedfromRef[27]).

wheret_totalisthetimerequiredforthewholeanalysis,t_lagisthefirst partoftheelutionwindowaftertheinjection(withoutanycom- poundofinterest),t_infistheinformationphasethatcorrespondsto thetimewindowinwhichaggregatesandmonomerelute,t_holdis thethirdpartofthechromatogrambetweenthemonomerandsalt peaksandt_saltistheelutionregionofsaltspecies(lowmolecular weightcompoundselutingatt0).

Afurtherincreaseinthroughputcanbeachievedwhenapply- ing interlaced injections on two columns operating in parallel (Fig.10.B).Comparedtointerlacedchromatography,theassaytime inparallelinterlacedSECisfurtherreducedbyt_hold.Twoswitch- ing valvesare used to direct the flow alternately betweenthe autosampler, thetwo columns and the detector, thus enabling theeliminationoft_lag,t_holdandt_salt.Theuseoftwocolumnsand switchingvalvesrequiretwodistinctprogramsonwhichpumps, autosamplerandcolumn compartment,includingtheswitching valves,arecontrolled.

8.2. Sub-3mparticles,ultrahigh-pressureSEC

Sincetheintroductionofultra-high-performance/pressureliq- uidchromatography(UHPLC)systemsandcolumns,whichenabled higherresolution,sensitivityandpeakcapacity,therehasbeena varietyofnewanalyticaladvancementsinLCandLC–MSbased ontheuseoflowdispersionsystems[110].Becausecolumnpeak dispersion–andthereforetheapparentefficiency–dependson the column volume, the achievable plate numbers and solute retentionfactor(k),theimportanceofsystemdispersionisworth mentioning. In theSECmode, there is noretention;thus,rela- tivelylowcolumnpeakvarianceisexpected.However,achievable platenumberswithlargemoleculesarerelativelylowandmore- overthevolumeof150mm×4.6mmcolumnsisreasonablylarge (∼1700␮l). Therefore, these two contributions compensate for thelowretention.Finally,theexpectedcolumnpeakvarianceis approximately200–400␮l².Usingstate-of-the-artUHPLCinstru- ments (possessing extra-column peak variance between 2 and 30␮l²),thesystemdispersionisnegligibleanddoesnotimpact thecolumnefficiency.

TheUHPLCtechnologywasoriginallydevelopedforRPLCappli- cations,butitisnowalsoavailableforSECoperationduetothe availabilityof1.7and2.5␮mBEHsilicaparticleswithvaryingpore

Fig.11.AveryfastSECseparationofpanitumumabaggregatesbyusingnarrow- borecolumnpackedwith1.7␮mparticles.Peaks1and2areaggregatedformsof thenativeantibody.

(ReprintedfromRef[21]).

sizes.Today,columnsforaqueousandnon-aqueousSECapplica- tionswithporesizesof125–900 ˚Aarecommerciallyavailable[110].

Veryfastseparationsofpeptide,myoglobulinandinsulinaggre- gatesweredemonstratedwith1.7␮mSECcolumns[111].These veryefficientcolumnswerealsoappliedforthecharacterization ofrecombinantmAbs[26].Fig.11presentsa4minseparationof panitumumabaggregates.

Itappearsthatapplying1.7and2.5␮mparticlesinSECopened anewlevelofSECseparations,butithastobekeptinmindthaton veryfineparticles,theseparationqualityisimprovedatthecostof pressure(andfrictionaltemperaturegradients).Therefore,there isariskofcreatingon-columnaggregateswhenanalyzingsensi- tiveproteinsunderhighpressure(i.e.,>200bar)conditions[26]

asdiscussedinSection6.Theotherdisadvantageofsub-2␮mSEC separationsisthatcurrentlythereareonlyaverylimitednumber ofcommerciallyavailablestationaryphases.

8.3. CapillarySEC

Toimprovethesensitivityofaggregatedeterminationorhan- dleverysmallamountsofavailablesamples,theuseofcapillary columnsin SEC appearstobe a promisingapproach. However, severalkeymodificationstoacommerciallyavailableliquidchro- matographysystemarerequiredtoreducethesystemvolumeand associatedextra-columnbandbroadening,whichcouldbecritical forcapillarySECoperation.Untilnow,thenumberofapplications inthisfieldisratherlimited,but300mm×300␮mI.D.SECcapil- larycolumnsweresuccessfullyappliedfortheseparationofmAbs fragments[98,99].

9. Conclusion

Toconclude,SECremainsanimportantstrategyforthedetailed characterizationofproteinaggregation.Indeed,duetotheirpoten- tialimmunogenicity,theamountofaggregateshastobethoroughly controlledduringtheproductionandstorageoftherapeuticpro- teins.

Duringrecentyears,therehavebeenanumberofadvancesin SECthatimprovethequantityofinformationthatcanbegained from a single injection. Among them, we can cite the use of shorterand narrowercolumnspackedwithsmaller-sizedparti- cletoimprovethethroughputandresolution,butcareshouldbe

takentoavoid(oratleastlimit)theriskofsheardegradation.To furtherdecreaseanalysistime,parallelinterlacedSECcanalsobe implemented.

VariousdetectorsmaybemultiplexedinSEC,includingrefrac- tiveindex(RI),ultraviolet(UV),multi-anglelaserlightscattering (MALLS)andviscometer(IV),forextensivecharacterizationofpro- teinsamples.Itisalsoimportanttonotethatmassspectrometry (MS)playsapivotalroleinthestructuralelucidation;forthisrea- son,someeffortswererecentlymadetosuccessfullycoupleSECto MSbychangingthenatureofmobilephaseconstituents.

The sale of biologics grew at an incredible rate during the lastthreeyears.Thenewdrugscurrentlyinpreclinicalandclin- ical phases are very difficult to characterize; for example, the caseoftherapeuticmAbsorADCsaswellasbispecificantibodies [112–114].Thus,therewillbeanincreasingneedforhighlyefficient SECmethods.Inthiscontext,theuseofa2D-LCplatformincluding SECinthefirstdimensionandRPLCintheseconddimensionmay beofinterestto(i)furtherimprovetheresolvingpowerofthechro- matographicstepand(ii)improvethecompatibilityofSECwithMS duetotheintermediateRPLCstep.

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