SummaryAccuratecharacterizationofbiomacromoleculesises-sentialforsuccessfulprogramsofresearchanddevelop-mentinthelifesciencesandbiopharmaceuticals.Thebasicbiophysicalpropertiesofthesemoleculesincludemolecularweight,size,conformation,degreeofconjuga-tion,aggregationandcomplex-forminginteractions.

Size-exclusionchromatographyiscommonlyusedtosep-arateandanalyzeproteinsandotherbiomacromole-cules.However,inordertoreliablydeterminetheirbasicbiophysicalpropertiesinsolution,anabsolute,inde-pendentmeansofcharacterizationmustbeaddeddownstreamoftheseparationstep.

Multi-anglelightscatteringanddynamiclightscatteringinstruments,combinedwithUVandRIdetectors,fulfillthatneed,makingthemessentialineverylabthatpro-duces,usesorcharacterizesproteins,peptides,nucleicacids,polysaccharidesorbionanoparticlesconstructedofthesebuildingblocks.Thisarticleexploresthetech-nology,capabilitiesandapplicationsoflightscatteringpairedwithsize-exclusionchromatographyforbiophysi-calcharacterization.

Introduction

TheneedforbiophysicalcharacterizationReliableanalysisofthemolecularweight(MW)ofpro-teinsinsolutionisessentialforbiomolecularresearch1–4.MWanalysisinformsthescientistifthecorrectproteinhasbeenobtainedandifitissuitableforuseinfurtherexperimentation5,6.Asdescribedonthewebsitesofpro-teinnetworksP4EU7andARBRE-Mobieu8,proteinqualitycontrolmustcharacterizenotonlythepurityofthefinal

product,butalsoitsoligomericstate,homogeneity,iden-tity,conformation,structure,post-translationmodifica-tionsandotherproperties.

Biophysicalpropertiesdeterminedbylightscattering

Asolution-basedmeasurementofMWidentifiestheformoftheproteinthatispresentinanaqueousenvi-ronment.Whileformanyproteinsthegoalistoproducemonomers,forothersaspecificnativeoligomeriskeytobiologicalactivity9–12.Incorrectoligomericformorthepresenceofnon-nativeaggregateswilladverselyimpactstructuraldeterminationbycrystallography,NMRorsmall-anglex-rayscattering(SAXS);theymayalsocreateartifactsorinaccuraciesinfunctionalassaysthatquantifybindingandinteractions,e.g.isothermaltitrationcalo-rimetryorsurfaceplasmonresonance2,13.

Forbiotherapeuticssuchasmonoclonalantibodies(mAbs),solution-basedMWanalysisservesasimilarpur-poseofqualitycontrolandproductcharacterization.Ex-cessiveaggregatesandfragmentsareindicativeofanun-stableproductthatisnotsuitableforhumanuse.Regu-latoryagenciesrequirecarefulcharacterization,notonly

WP1615:SEC-MALSforabsolutebiophysicalcharacterizationDanielSome,Ph.D.,WyattTechnology

ofthetherapeuticmoleculebutalsopotentialdegra-dantsthatmaybepresentinthefinalproduct14–17.

SomeofthemostwidespreadmethodsforanalyzingproteinMWareSDS-PAGE,capillaryelectrophoresis(CE),nativePAGE,massspectrometry(MS),size-exclusionchromatography(SEC)andanalyticalultracentrifugation(AUC).Ofthese,SDS-PAGE,CEandMSarenotper-formedinthenativestateandtypicallyleadtodissocia-tionofoligomers,complexesandaggregates.Oftentheyareunabletocorrectlyanalyzeglycoproteinsandothermodifiedforms.

AlthoughnativePAGEdoes,theoretically,retainthena-tivestate,itisdifficulttooptimizeformanyproteins,andresultsarenotveryreliable.AUC,whetherbysedimenta-tionvelocityorsedimentationequilibrium,isquantita-tiveandcandetermineMWfromfirstprinciples,butitisquitecumbersome;AUCinvolvesmuchmanuallaborandrequiressignificantexpertiseindatainterpretation,longexperimenttimeandaveryexpensiveinstrument.

AnalyticalSEC:promising,withcaveatsSECisaquantitativeandrelativelyrobust,simpleandfastmethodforseparatingmacromolecules18–20.How-ever,separationofdifferentspeciesbySECdoesnotde-penddirectlyonMW;itdependsonsizeanddiffusionproperties21.

Size-exclusionchromatographyseparatesmoleculesbyhydrodynamicsize

InanalyticalSECacalibrationcurve,suchasthatinFig-ure1,isconstructedusingaseriesofreferencemole-cules,relatingtheMWofthemoleculetoitselutionvol-ume.Forproteins,thereferencemoleculesarewell-be-haved,globularproteinsthatdonotinteractwiththecolumnviachargeorhydrophobicsurfaceresidues.

Notably,theanalysisofMWinSECreliesontwokeyassumptionsregardingtheproteinstobecharacterized:

1. Theysharewiththereferencestandardsthesameconformationandspecificvolume(inotherwords,thesamerelationshipbetweendiffusionpropertiesandMW);

2. Likethereferencestandards,theydonotinteractwiththecolumnexceptbystericproperties—theydonotsticktothecolumnpackingviaelectrostaticorhydrophobicinteractions.

Figure1.SECcalibrationcurvesusereferencestandardstorelatemolecularweighttoelutionvolume,assumingglobularconfor-mationandidealstericinteractionswiththeSECcolumn.

Whentheseassumptionsarenotfulfilled,thecalibrationcurveisinvalidanditsusewillleadtoerroneousMWvalues.Manyclassesofprotein,includingtheADHte-tramerandkinasefragmentexamplesinFigure2,donotmeettheassumptions:

• Intrinsicallydisorderedproteinshavecomparativelylargehydrodynamicradiiduetotheirextensiveun-structuredregions22,23;

• Non-sphericalorlinearoligomericassemblies10are,bydefinition,non-globular;

• HeavilyglycosylatedproteinsarealsolargerthanpureproteinswiththesameoverallMW19,sincegly-cansaregenerallylinearratherthancompactlyfolded;

• Detergent-solubilizedmembraneproteinselutefromSECaccordingtothetotalsizeofthepolypeptide–

detergentor-lipidcomplexratherthantheoligo-mericstateandmolarmassoftheproteinalone24,25;

• Proteinswithchargedorhydrophobicsurfaceresi-duesmayinteractwiththestationaryphaseandelutenon-ideallydependingoncolumnchemistry,pHandsaltconditions26,27.

Figure2.Elutionvolumesofvariousproteinsandmolarmassdeter-minedbyMALS.ADHtetramereluteslaterthanBSAdimereventhoughithaslargermolarmass,whilealowermolarmasskinasefragmentelutesatthesamevolumeasBSAdimer.SeeAN1607.

Thesolution:lightscatteringSECbecomesmuchmoreversatileandreliableforMWdeterminationwhencombinedwithmulti-anglelightscattering(MALS),UV280anddifferentialrefractiveindex(dRI)detectors3,4,11,28–31.TheUVdetectormeasurespro-teinconcentrationviaabsorbanceatawavelengthof280nm.ThedRIdetectordeterminesconcentrationbasedonthechangeinsolutionrefractiveindexduetothepresenceoftheanalyte.TheMALSdetectormeasurestheproportionoflightscatteredbyananalyteintomultipleanglesrelativetotheincidentlaserbeam.CollectivelyknownasSEC-MALS,thisconfigurationde-terminesMWindependentlyofelutiontimesinceMWcanbecalculateddirectlyfromfirstprinciplesusingEquation1,

𝑀 = # $

%& '(')

* (1)

whereMisthemolecularweightoftheanalyte,R(0)thereducedRayleighratio(i.e.,theamountoflightscatteredbytheanalyterelativetothelaserintensity)determinedbytheMALSdetectorandextrapolatedtoanglezero,ctheweightconcentrationdeterminedbytheUVordRIdetector,dn/dctherefractiveindexincrementofthean-alyte(essentiallythedifferencebetweentherefractiveindexoftheanalyteandthebuffer),andKasystemcon-stant28.

Multi-anglelightscatteringmeasureslightscatteredbytheanalyteintoseveralanglesrelativetothelaserbeam.

InSEC-MALS,theSECcolumnisusedsolelytoseparatethevariousspeciesinsolutionsothattheyentertheMALSandconcentrationdetectorcellsindividually.Theactualretentiontimehasnosignificancefortheanalysisexceptasfarashowwelltheproteinsareresolved.Sincetheinstrumentsarecalibratedindependentlyofthecol-umnanddonotrelyonreferencestandards,SEC-MALSisconsideredan‘absolute’method.

MALScanalsodeterminethesize(physicaldimension)ofmacromoleculesandnanoparticleswithdiameterlargerthanabout25nmbyanalyzingtheangularvariationofthescatteredintensity28.Forsmallerspeciessuchasmonomericproteinsandoligomers,adynamiclightscat-tering(DLS)modulemaybeaddedtotheMALSinstru-mentinordertomeasureradiifrom0.5nmandup32.

WhileeitherUVordRIconcentrationanalysismaypro-videthevalueofcinEq.1,useofdRIispreferredfortworeasons:1)dRIisauniversalconcentrationdetector,suit-ableforanalyzingmoleculessuchassugarsorpolysac-charidesthatdonotcontainaUVchromophore;and2)theconcentrationresponsedn/dcofalmostallpurepro-teinsinaqueousbufferisthesametowithinoneortwopercent(0.185mL/g)33,sothereisnoneedtoguessorcalculatefromsequencetheUVextinctioncoefficient.

Instrumentation

SECSEC-MALSdetectorsgenerallyworkwithanygood-qual-itysizeexclusionchromatographincludingHPLC,UHPLCorFPLCsystems.InmostcasesthedetectorsmaybesimplyaddeddownstreamoftheLC’sUVdetectorwithappropriateinterfacingtotheUVanalogoutputsignalandanauto-injectcontactclosureswitch.Wyattdetec-torsaremostcommonlyusedwithHPLCorUHPLCsys-temsfromAgilent,Waters,Thermo,andShimadzu,andwithFPLCsystemsfromGEandBio-Rad.

TheDAWN18-angleMALSdetectorprovidesthehighestsensitivityandwidestmeasurementrangeforHPLC-,FPLCandFFF-MALS.

MALSdetectors

WyattTechnology’sDAWNâisthepremierMALSdetec-torforHPLCandFPLC,offeringthehighestsensitivity,widestmeasurementrangeandmostoptions:

• Rangeofmolarmass:200Da-1GDa(SECtypicallyworksforproteinsuptoafewmillionDaltons,buttheDAWNmaybeusedwithotherseparationtech-niquessuchasFFFtoaddresstheupperrange)

• RangeofrmsradiusRg:10–500nmusingtheangu-lardependenceofscattering

• Sensitivityrating:200nginjectedmassofmono-mericBSAinPBSonastandard7.8mmx300mmSECcolumn

• Numberofdetectionangles:18,whichdeterminethesizerangecoveredandaddbuilt-inredundancy

toovercomethemostcommonsourceofnoiseinSEC-MALS,particulatesshedbythecolumn

• Temperaturecontroloptions:ambient,-20°Cto+150°Candroomtemperatureto+210°C

Wyatt’sminiDAWNâisabasicMALSdetectorthatoffersaslightlylowermeasurementrangeandfeweroptionsthantheDAWN,butisstillappropriateformostSECwork:

• Rangeofmolarmass:200Da-10MDa

• RangeofrmsradiiRg:10-50nm

• Sensitivityrating:500ngofBSAmonomerinPBS,in-jectedonastandardSECcolumn.

• Numberofdetectionangles:3

TheminiDAWN3-angleMALSdetectorcoverstheentiresizerangeofstandardHPLC-orFPLC-SEC.

TheonlyMALSdetectordesignedspecificallyforUHPLC’slow-volumepeaksisWyatt’smicroDAWNâ.ItissimilartotheminiDAWNintermsofnumberofanglesandtherangesofmolarmassandsize,withasensitivityratingof70ngofBSAmonomerwheninjectedona4.6mmx150mmUHP-SECcolumnwithsub-2µmbeads.

ThemicroDAWN3-angleMALSdetectorworkswithUHP-SEC.

AdditionalMALSfeatures

AuniquefeaturecommontoallthreeMALSinstrumentistheForwardMonitor(FM)detectorwhichmeasureslighttransmittedthroughthecell.WhiletheFMhassev-eraluses,oneofthemostimportantisforanalysisofmoleculesthatabsorbattheinstruments’laserwave-lengthof660nm,e.g.heme-containingproteins.TheFMdetectsandcompensatesforthisabsorptionphenome-noninordertoreportthecorrectMW,whichotherwisewouldbeincorrect.

TheDAWN,miniDAWNandmicroDAWNallincludeabuilt-inultrasonicflowcellcleanertominimizemanualcellcleaning,andaremodularforrapidfieldservice.In-dicatorsonthefrontpanellettheuserknowwhentheSEC-MALSsystemisequilibrated,cleanandreadytomakehigh-qualitymeasurements.

TheCOMETmoduleappliesultrasonicagitationtodislodgeparticlesfromtheglass,reducingopticalnoise.

TheDAWNmayalsobefittedwithfluorescence-blockingfiltersincaseoffluorescently-taggedmoleculesorotheranalytesthatfluoresceunder660nmexcitation,inordertoprovideaccuratemolecularweights.

DLSdetectorsInordertominimizeflowpathsanddispersion,Wyatt’sonlineDLSdetectionoptionsutilizetheMALSflowcellandlaserbeam.DLSdetectionmaybeconfiguredintwoways:

1. AWyattQELSÔembeddedDLSmodule,connectedviaopticalfibertotheflowcell,residesinsidetheMALSdetector;or

2. Anexternal,stand-aloneDLSdetectorisreconfig-uredtoconnectviaopticalfibertotheMALSflowcell.BoththeDynaProâNanoStarâcuvette-basedDLSdetectorandtheMobiusâflow-throughDLS/PALSdetectorofferthisinteroperability.

dRIdetectorsThepreferreddRIdetectorforusewithSEC-MALSisWy-att’sOptilabâ.BenefitsoftheOptilab:

• Sensitivityrating:7.5×10-10RIU,equivalenttothebestHPLCdRIdetectorsonthemarket

• Wavelength-matchedtotheDAWNandminiDAWNformaximumaccuracyinmolarmassdetermination;

• ReaddigitallybyWyatt’schromatographysoftwareinordertotakefulladvantageofitssensitivity

• Range:±4.7×10-3RIU,10-20xmorethanstandardHPLCdRIdetectors,withnoneedtoswitchgainset-tingsorlossofsensitivity

• HardwaretimebasesynchronizationwithWyattMALSdetectorstoeliminatedriftbetweenthesignals

• Neverneedsrecalibration

Ahigh-concentrationversionoftheOptilabisavailableforspecializedmeasurementssuchassemi-preparativeMALSandcouplingofMALStoion-exchangechromatog-raphy34,35.ItissimilartotheOptilabsaveforahigherrange:-2.6×10-3to+3.4×10-2RIU,andslightlylowersensi-tivity,1.5×10-9RIU.

ForUHPLCSEC-MALS,WyattoffersthemicroOptilabâwhichissimilartotheOptilabsaveforreducedvolumeandslightlylowersensitivity,1.5×10-9RIU.ThemicroOp-tilabcoupleswiththemicroDAWNinUHPLCSEC-MALS.

InlieuofanOptilabormicroOptilab,standard(U)HPLCdRIdetectorsmaybeused.However,theyrequireuseofanalogoutputsignalswhichmayreduceeffectivesensi-tivity.Theywillusuallyuseabroadbandlightsourceoranarrow-bandsourceatawavelengthdifferentfromtheMALSdetectorandarenottimebase-synchedinhard-ware,reducingMWaccuracy.

Software

ASTRAâsoftwareforSEC-MALSisrequiredforusewithWyatt’sMALS,DLSanddRIdetectors.Itoffersrobustdataacquisition,straightforwarddataprocessingandacomprehensivesetofanalysesforbiophysicalcharacteri-zationincludingmolarmass,size,distributionsandaver-ages,percentaggregate,percentrecovery,conjugateanalysis,conformationalanalysisanddeterminationofextinctioncoefficient.Keyresultsformultiplesamplesmaybeconsolidatedintoonetable(EASITable)andthegraphicaldatasuchaschromatograms,absoluteMWorsizeversuselutionvolume,anddistributionsconsoli-datedintoonegraphforside-by-sidecomparison(EASIGraph).

ASTRAmaybesetuptocontrolselectHPLCmodulessuchaspumps,UVdetectorsandautosamplersormaybeusedside-by-sidewithnativeHPLCsoftware.

Reportsarecustomizable,allowingforasmuchoraslit-tleinformationasdesired.ForGMPuse,21CFR(11)dataintegrityandadministratorhierarchysupportisavailable.

AcompleteSEC-MALSexperimentalsetupforproteinanalysisincludesastandardHPLCorFPLCsystemwithUVdetector,anappropriatecolumn,aDAWNorminiDAWNMALSinstrumentandanOptilabdRIinstrument.ThemicroDAWNandmicroOptilabareusedwithUHPLC-basedSEC.

Controlofindustry-leadingHPLCmod-ulesisintegratedintoASTRAalong-sidecontrolofWyattinstruments.

ApplicationsofSEC-MALS

Monomers,oligomers,aggregatesandimpuritiesTheuseofSEC-MALSinproteinresearchisquiteexten-sive.Byfarthemostcommonapplicationsareestablish-ingwhetherapurifiedproteinismonomericoroligo-mericandthedegreeofoligomerization,andassessingaggregates3,10,11,17,30,36–38.

Qualitycontrol

Aproteinpurificationrunoftendoesnotcompletelyeliminateallundesirableformsorimpurities.AsshowninFigure3,SEC-MALSreadilyidentifiesandquantifiesthepurityandhomogeneityoftheprotein.Uniformmo-larmass,calculatedindependentlyateachelutionslice,isfoundacrossthemonomerpeakandthewell-resolvedsolubleoligomers.Wherethespeciesarenotfullyre-solvedbySEC,themolarmassesdeterminedbyMALSdecreasewithincreasingelutionvolume.

Ofparticularnoteistheshoulderonthetrailingedgeofthemonomerpeak.Suchshoulderscanarisefromafewcauses:

• Tailingresultingfromproteinstickingtothecolumn;

• Dynamicdissociationofcomplexesastheconcentra-tiondecreases;

• Low-molecular-weightspecies.

Figure3.BSAmonomer,solubleaggregatesandalow-molecular-weightshoulderidentifiedasafragmentusingFPLCandaGEIn-creaseSECcolumn.MolarmassesdeterminedbyMALSoverlaidwithUVchromatogram.

WhilesimpleSEC-UVcannotdeterminetowhichofthesetheshouldercorresponds,MALS-dRIimmediatelyprovidestheanswer–herea42kDafragment.ThoughtheproteinisunknownaprioriandhencetheUVextinc-tioncoefficientisunknown,dRIcanalwaysbeusedtoanalyzeunknownproteins.

Monoclonalantibodyaggregates

Theaggregatesproduceduponstressoragingofthera-peuticIgGmustbethoroughlycharacterizedforregula-toryfilingsandbiosimilarityassessments.ThisneedismetbyseparatingonUHP-SECandanalyzingonlinebyMALS.Inparticular,amicroDAWN-microOptilabsetuppairedwith30-cmBEHUHPLCcolumnprovidesexcellentcharacterizationcapabilities,asshowninFigure4,wheretheverylowdispersionoftheinstrumentspreservespeaksthatareverycloseinmolecularweight.

Theinstruments’sensitivitypermitsrobustcharacteriza-tionevenwhentheheightofeachaggregatepeakislessthan1%ofthemainmonomerpeak.Themolarmassesofthedistinctpeakscorrespondtothoseofacompletedimeraswellasdimersmissingoneheavychain(ortwolightchains),oneheavy+onelightchain,andtwoheavychains.

Figure4.High-resolutionUHP-SECseparationofaggregatesofastressedIgG,molarmassesdeterminedbyMALS(red)overlaidwithdRIchromatogram.Thevariouspeakscorrespondtofulldimer(307kDa)andcombinationsofdimerwithmissinglightandheavychains.

Monoclonalantibodyfragments

StressedIgGmaydegradeintofragmentsaswellasag-gregate.UHP-SECusing30-cmBEHcolumnsprovidesex-cellentresolutionofmonoclonalantibodies,aggregatesandfragments,demonstratedinFigure5.HerepeakselutinglaterthantheIgGmonomerat8minutesaresus-pectedtobefragmentsbasedontheirmolarmasses,whichcorrespondtodualheavychain,singleheavychainorduallightchain,andsinglelightchain.TheanalysisutilizesdRImeasurementsforconcentrationsincethespeciesarenotknownapriori.

Figure5.Fragmentsproduceduponstressingamonoclonalantibodyarewell-separatedbyUHP-SECanda30-cmBEHcolumnwith1.8µmbeads,molarmassesdeterminedbyMALS(red)overlaidwithdRIchromatogram.Theinsetshowsthelate-elutingportionmagnified50x.MolarmassesdeterminedbyMALSanddRIcorrespondtotheexpecteddegradationproducts.

Peak Mw[kDa] ExtinctionCoefficient[mL/(mg×cm)]

1 145 1.53

2 95 1.54

3 45 1.53

4 24 1.46

Table1.UV280extinctioncoefficientsdeterminedfromSEC-UV-dRIanalysisofthemonomerandpurportedfragmentpeaks.Thenearlyidenticalvaluesconfirmthattheseare,infact,fragmentsofthemonomer.

ConfirmationofthisassignmentisprovidedbyanalyzingtheUVextinctioncoefficient.TheanalysisconsistsofcomparingtheareasofthepeaksinUVanddRI.Table1liststhecalculatedextinctioncoefficients,showingthatthelate-elutingpeakshavethesameextinctioncoeffi-cientasthemonomerandthereforeare,infact,frag-ments.

Insulinoligomerizationunderdifferentbufferconditions

Figure6showstheresultsofanalyzinginsulinintwobuffers,oneofwhich(Sample1,red)maintainsmostlymonomerswhiletheother(Sample2,blue)promotesself-association39.MALSclearlyidentifiestheuniformmolarmassacrossthemainpeakofSample1,includingthetrailingedgewhichinthiscaseissimplytailing.Con-versely,forSample2,theprimarypeak—includingitstrailingedge—ishexameric,whichwouldnotbede-ducedfromtheUVtracealone.

Figure6.UVchromatogramsandmolarmassesfromMALSofinsulinundertwodifferentbufferconditions.Sample1(reddashedline)isprimarilymonomericwithsmallaggregatesthatreachhexamer.Sample2(bluesolidline)isprimarilyhexamericinform,withasmallamountofproteininmonomer-dimerequilibrium.SeeAN1605.

ThesecondarypeakofSample2isshownbyMALStotransitionfromdimertomonomer.Whileasingleexperi-mentcannotdetermineifthisshiftisaresultofpoorly-resolved,irreversibledimersordynamicequilibrium,afurtherexperimentpresentedintheapplicationnotein-jecteddifferentconcentrationsofSample2andshowed

unequivocallythattheequilibriumshiftswithconcentra-tion,ahallmarkofself-associationindynamicequilib-rium.

Largeaggregatesanddifferentconformation

Incontrasttothelow-molar-massproteinofFigure6thatdonotaggregatebeyondhexamer,Figure7pre-sentstheSEC-MALSanalysisoftwohigh-molar-massproteinsthataggregateextensively.BothapoferritinandIgMexhibitwell-resolvedmonomer,dimerandtrimerpeakswithuniformmolarmassesacrosseachasdeter-minedbyMALS,withunresolvedaggregatetailsextend-ingintothetensofmillionsofDalton.

Figure7.SEC-MALSanalysesoftwoproteinswithverydifferentcon-formationsthatexhibitextensiveaggregation,wellbeyonddimerandtrimer,intothetensofmillionsofDa.ThedimerofapoferritinelutesataverydifferentvolumethanthemonomerofIgMeventhoughtheyhaveapproximatelythesamemolecularweight,duetodifferentconformations.

Notably,theapoferritindimerhasaboutthesamemolarmassasIgMbutelutesataverydifferenttime.Thisisaconsequenceoftheirverydifferentconformations–apoferritinisglobularwhileIgMisextendedandpartiallyglycosylated.Despitethedifferentelutionbehavior,MALShasnoproblemascertainingthecorrectMWval-ues.

Aggregationduetolabeling

Labelingaproteincanoftenaffectitsbehaviorinsolu-tionandonSEC.AsdescribedindetailinAN1606:Pro-teinAggregateAssessmentofLigandBindingAssay(LBA)

ReagentsUsingSEC-MALS,ELISA-basedligand-bindingassaysusedtomeasurelevelsofbiologicdrugsoranti-drugantibodiesdependonreliablereagents.Therea-gentsareantibodieslabelledwithbiotinanddigoxigenin.SECmaybeusedforLBAreagentqualitycontrol,butSEC-MALSisrequiredforreliableinterpretationofthepurityandaggregateformspresent.Figure8showsthedifferenceinretentiontimeinducedbythelabel(despitemaintaininganidenticalandfullyhomogeneousmolarmass)aswellasdifferentaggregationlevelsandformspresent,relativetotheunlabeledantibody.

Figure8.SEC-MALSresultsforamonoclonalantibodydrug,unconju-gated(red),andthreedifferentlotsconjugatedtodigoxigeninforuseinELISA-basedligand-bindingassays(blue,purple,green).LSchromatogramsoverlaidwithMALSdata(symbols).Theconjugateincreasesretentiontimeofthemonomericspeciesandincreasesag-gregatelevels,affectingtheefficacyoftheassay.SeeAN1606.

ProteincomplexesSEC-MALSisusedproductivelyinstructuralbiologyandstructuralvirologytoinvestigatetheformationandabso-lutestoichiometryofbiomolecularcomplexes9–12,23,25,40–44.Akeybenefitistheabilitytodeterminethemolecularweightofalltypesofcomplexes,whethernon-globularorinherentlydisordered,evenifthecomponentsarenotentirelyproteinaceous;theformation(orlackthereof)andabsolutestoichiometry(asopposedtostoichio-metricratio)ofheterocomplexesincludingprotein-pro-tein,protein-nucleicacidandcomplexes23,41,42,44–47anddeterminingthemonomer-dimerequilibriumdissocia-tionconstant.41,43,48

Oligomerizationofwildtypeandmutants

Thenativeoligomericstateofmanyproteinsisdimeric,trimeric,tetramericorhexameric.Mutationsareoftenusedtoprobethespecificdomainresponsibleforoli-gomerization,exemplifiedinAN1610:StoichiometryofIntrinsically-DisorderedProteinComplexes.AsshowninFigure9,differentmutationscanmodifythenativeoligo-merfromtetramertodimerandevenmonomer.How-ever,thetetramerisnotextremelystableundertheseconditionsandtheSEC-MALS-derivedMWexhibitsdis-sociationatdecreasedconcentrations,ontheleadingandtrailingedgesofthepeak.

Figure9.Wild-typep53DNA-bindingproteinformstetramersinso-lutionwhiletheL344AandL344Pmutationsonlyformdimersandmonomers,respectively.Solidchromatogramsarelightscatteringin-tensitywhiledashedchromatogramsarerefractiveindexsignals.SymbolsindicatemolarmassfromMALS.Thepronouncedconcen-trationdependenceofthew.t.molarmassindicatesdynamicequi-librium,presumablybetweendimersandtetramers.SeeAN1610.

Protein-proteincomplexes

Whiletraditionaltitrationassayscanonlydeterminethemolarratioofproteinsinaheterocomplex,theaddi-tionalinformationprovidedbySEC-MALSenablestheconfirmationofabsolutestoichiometry,i.e.thenumberofcopiesofeachtypeofproteininthecomplex.ThisisaccomplishedbyincubatingdifferentratiosofthetwoproteinsandmeasuringtheresultingmolarmassesbySEC-MALS.AN1610:StoichiometryofIntrinsically-Disor-deredProteinComplexesfurtherdescribesaseriesofex-perimentsdesignedtostudythecomplexesformedbyp53wildtypeandmutants,withS100B,anativedimer.

Figure10presentstheSEC-MALSresultsfortheL344Pmutant.AtexcessL344P,substantialamountsofdimericS100BandmonomericL344Parefound,alongwithsmallamountsofcomplex.AstherelativeamountofS100Bin-creases,moreandmorecomplexesform,thoughinallcasesonlyonespeciesisidentified:onedimerofS100Bboundtoasinglemonomerofp53mutant.TheresultsofthecompletesetofexperimentsaresummarizedinTable2.

ForbothmutantsthecomplexconsistsofaS100Bdimerandap53monomer,eventhoughtheL344Amutantdi-merizesintheabsenceofS100B.ApparentlytheaffinityofL344AforaS100Bdimerismuchgreaterthanforan-otherL344Amutantprotein.

Figure10.FormationofS110B:L344PcomplexesuponincubationofvariousstoichiometricratiosofS100BandL344P.LSchromatograms(solidlines)overlaidwithMWdeterminedbyMALS(symbols).SeeAN1610.

Thoughwildtypep53bindstoS100Binthesamestoi-chiometricratioasthemutants,thecomplexthatformsismuchdifferent:fourS100Bdimersbindtoatetramerofp53,thefunctionaloligomer.TheoverallaffinityofS100Bforp53isnotveryhigh:relativelyweakdynamicequilibriumisindicatedbythedecreaseofmolarmassawayfromtheapexofeachpeak.

Nativestate:S100Bdimer

w.t.tetramer

L344Adimer

L344Pmonomer

Stoichiometry ComplexformswithS100B?

1:1 - - -

2:1 - Ö Ö

2:2 - - -

4:1 - - -

8:4 Ö - -

Table2.AbsolutestoichiometryofcomplexesthatformbetweenS100Bandp53,wildtypeandmutants.Forbothmutantsthecom-plexconsistsofaS100Bdimerandap53monomer,eventhoughtheL344AmutantdimerizesintheabsenceofS100B.

Protein-nucleicacidcomplexes

ASTRAoffersapowerfulmethodforanalyzingbinarycomplexes,ProteinConjugateAnalysis,describedinmoredetailbelow.ThismethodisapplicablewhenthetwocomponentsdiffersufficientlyineitherUVextinc-tioncoefficient,differentialrefractiveincrement,orboth.Whiletheanalysisisnotsuitableformostprotein-proteincomplexes,itoftenisforprotein-nucleicacidcomplexesbecauseofstrongabsorptionat280nmbynucleicacidsrelativetoproteins.

Figure11.AnalysisofprototypefoamyvirusintasomeboundtoU5DNAusingASTRA'sProteinConjugateAnalysismethod.UVchroma-togram(solidline)overlaidwithmolarmassvaluesofprotein,DNAandtotal,ateachelutionslice(symbols).SeeWP3001.

Theanalysisofacomplexbetweentheprototypefoamyvirusintegrase(PFVIN)proteinandaDNAsegment,U5,

isdescribedinWP3001:SEC-MALSandCG-MALScharac-terizeprotein-DNAinteractions.PFVINisanative~170kDatetramer.U5consistsof19basepairs,equivalentto11kDa.Figure11presentstheresultsoftheanalysis,in-dicatingthattheintasometetramerbindstwostrandsofU5toforma~200kDacomplex,thoughsomedissocia-tionispresentandthesmallerPFVINcomplexbindsjustoneU5strand.Similaranalysesmaybeperformedforsmallviruses49,orforlargervirusesusingFFFseparation.

Transientcomplexes

Asanaliquotofsolutioncontainingproteincomplexesindynamicequilibriumpassesthroughasize-exclusioncol-umn,thecomplexesaredilutedandpossiblysheared,re-sultinginpartialdissociation.Ontheonehand,thisphe-nomenoncomplicatesanalysisofthecomplexitself,butontheotherhandisbeneficialinprobingthepresenceofdynamicequilibriumandbindingaffinity.Insomein-stancesismaybeutilizedtoestimatethemonomer-di-merequilibriumdissociationconstant41,43,48asshowninFigure12anddemonstratedforadomainantibodyinAN1608:TransientProteinSelf-AssociationDeterminedbySEC-MALS.Formorerobustcharacterizationofself-associatingandhetero-associatingproteins,todeter-mineKdandabsolutestoichiometry,MALSisusedwithcompositiongradients,CG-MALS50.

Figure12.Analysisofatransiently-associatingdimerwithinjectionofthreeproteinquantities.Eachquantityresultsinadifferentcon-centrationprofileacrossthepeak.ThemolarmassesdeterminedbySEC-MALSreachamaximuminthevicinityoftheapexofthepeakanddecreasesoneitherside,indicatingdissociation.SeeAN1608

ConjugatedproteinsProteinsareoftenconjugatedtoothermaterials,whethernaturally(asinglycoproteins)orsynthetically(asinPEGylatedproteinsorantibody-drugconjugates,ADCs).ConjugationtypicallycausesgreatdeviationfromtheMW/Rhratioofunmodifiedglobularproteins,im-partinglargeuncertaintiestomethodssuchasanalyticalSEC,SDS-ornativePAGE.Conversely,theaddedmoietycouldinteractwiththeSECcolumnandchangetheelu-tionpropertiesforotherreasons.

Standardtwo-detectorSEC-MALScannotusuallyprovidethemostaccuratecharacterizationofsuchconjugatesbecausetheconcentrationresponseofthespecificde-tector(UVorRI)isdifferentforeachcomponent.Inthiscase,athree-detectortechnique,combiningMALS,UVandRIisapplied4,30.TheresultsprovideduponanalysisinASTRAarenotjustthemolecularweightoftheentirecomplex,butthemassesoftheproteinandmodifierin-dividuallyaswell.Theanalysisalsoprovidestheproteinfractionandtheoverallweight-averagespecificrefractiveindexincrementdn/dc.Thisanalysiscanbeappliedtoestablishingthedegreeofpost-translationalmodificationandpolydispersityofglycoproteins,lipoproteinsandsim-ilarconjugates4,30,46,51–53.Theabilitytoanalyzedetergent-solubilizedmembraneproteinsthatcannotbecharacter-izedbytraditionalmeansisespeciallyprized,andde-tailedprotocolsforthishavebeenpublished30,54–58.

Post-translationalmodificationsindifferentcelllines

Choiceofcelllineforproteinexpressioniscrucialforgly-coproteins,sincethedegreeofglycosylationwillvarywithcelltype.Figure13illustratesthedifferencesandsimilaritiesofaglycoproteinexpressedintwodifferentcelllines,oneinsectandtheothermammalian.ASTRA’sProteinConjugateAnalysismethodindicatesthattheproteincomponentsofbothsamplesare,asexpected,identicalinmolarmassanduniformityacrossthechro-matographicpeaks(solidlines).Ontheotherhand,thedegreeofglycosylationvariesabout50%betweenthecelllines,withmammaliancellsproducinghigherde-greesofglycosylation.Inadditiontothetotalamountofglycans,theheterogeneityisalsodeterminedthroughtheglycanmassateachelutionvolume.

Figure13.ConjugationanalysisbySEC-MALSofaglycoproteinex-pressedintwodifferenthostcells,insectandmammalian.UVchro-matograms(solidlines)overlaidwithmolarmassvalues(symbols).

Membraneproteins

Detergent-solubilizedmembraneproteinsarepartiallyenvelopedbyamphiphilicmoleculesthatenlargetheirhydrodynamicvolumegreatlyrelativetothemolarmassofthepureprotein.Thereforeitisimpossibletorelyoncolumncalibrationwithglobularproteins,ornativePAGE,todeterminethemolarmassandquaternarystateoftheprotein.ThesecomplexesmustbeanalyzedbymeansofSEC-MALS-UV-RI,whichcalculatesnotonlytheproteinmassbutalsothatofthedetergentorothermodifier.AnexampleisprovidedinFigure14,whichteststhemostappropriatedetergentforretainingthenative/functionaloligomericstateoftheprotein.

Intheanalysis,describedinmoredetailintheApplica-tionNote,LDAOisfoundtoleadtomonomericCorApro-tein.However,thefunctionalconfigurationisapen-tamer,whichwasmaintainedwithDDM(thoughsomedissociationisobserved).HenceDDMisasuitabledeter-gentforsolubilizingfunctionalCorA.ANadditionalexam-pleisprovidedinAN1602:Lipid-MembraneProteinComplexes.

Figure14.UVChromatogramsofCorAmembraneproteinsolubilizedinLDAO(left)andDDM(right)overlaidwithmolarmassvaluesoftheprotein,detergentandtotaldeterminedbyASTRA’sproteincon-jugateanalysis.DespitethesharpelutionprofileinLDAO,onlyDDMmaintainsthefunctional,pentamericform.SeeApplicationNote.

PEGylatedproteins

PEGylationisusedtoenhancePK/PDpropertiesofthera-peuticproteinsandpeptides,increasingthehalf-lifeinthebloodstream.SEC-MALS-UV-dRIanalysisisuniquelysuitedtoprovidequantitativeanalysisforprocessdevel-opmentandqualitycontrolofPEGylateddrugproduct,sinceitindicatesthenatureofthemoleculeineachelu-tionvolume(protein,PEGorPEGylatedprotein),thede-greeofPEGylation,andthemonomericoraggregationstate.

Figure15.SEC-MALSanalysisofaPEGylationprocess,showingtheLSchromatogram(solidline)overlaidwithmolarmassdeterminedateachelutionvolumebyMALS-UV-dRIanalysis(symbols).SeeAN1612.

TheresultsofsuchananalysisinthecourseofprocessdevelopmentispresentedinFigure15,withfurtherde-tailsprovidedinAN1612:ProteinPEGylationProcessesCharacterizedbySEC-MALS.Similaranalysesmaybeper-formedforprotein-polysaccharidecomplexes.45,47

ADCdrug-antibodyratio

Modifiersthatmakeupaslittle5%ofthetotalmassinaconjugatedproteinmaybequantifiedbySEC-MALS-UV-dRI.ApplicationnoteAN1609:ADCdrug-antibodyratiobySEC-MALSdescribestheresultsofanalyzingtwoanti-body-drugconjugates(ADC)samplesbasedonthesamemAbanddrug-linkersystembutdifferentconjugationprocesses.AsseeninFigure16,reproducedfromthatnote,themolarmassescalculatedforthemAbareiden-ticaltowellwithinexperimentalprecision.Thedrug-anti-bodyratio(DAR),calculatedfromtheknownlinker-drugmassof1260g/mol,is12.6forADC1and8.1forADC2.Separateexperiments,notshown,determinedthemodi-fier’sUVextinctioncoefficientanddn/dcvalueforuseintheconjugateanalysisalgorithm.

Figure16.Conjugateanalysisoftwoantibody-drugconjugates,withUVchromatograms(solidlines)overlaidwithprotein,drugandtotalmolarmasses(symbols).SeeAN1609.

ProteinconformationInformationprovidedbySEC-MALS-DLSisinvaluableinevaluatingoverallproteinconformationinsolution,evenifcirculardichroismdoesnotindicatechanges.59,60

Conformationalstabilizationbyligandbinding

Itisnotunusualforprotein-proteincomplexestoeluteearlierthantheconstituentproteinsduetothein-creasedsizeofthecomplex.Laterelutionisnotverycommon,butitdoesoccurandmayresulteitherfromnon-idealinteractionwiththecolumnmatrix,orfromareductioninoverallhydrodynamicsizewhentheligandstabilizesapartially-disorderedprotein.Thelatterbehav-iorisexhibitedbytheinterleuken-4trap:interleukin4(IL4)complex,depictedinFigure17.Thecauseoflaterelution—stabilizationofthepartially-disorderedtrapbythemuch-smallerIL4—maybededucedfromthesimul-taneously-acquiredDLSdatawhichshowasmallerhydrodynamicradiusforthecomplexthanforthetrap.

Figure17.Conformationalchangeininterleuken-4trapduetobind-ingofinterleuken4.Thecomplexeluteslaterdespiteitshighermo-larmass.Thiscounterintuitivebehaviorisexplainedbythedecreaseinhydrodynamicradius,measuredbyonlineDLS,ratherthancolumninteractions.

EvaluatingchromatographicconditionsOfthethreemAbpeaksshowninFigure18,acquiredasUVchromatogramsonUHP-SEC,onlyPeak1appearsintheelutionvolumecorrespondingtoitsexpectedmolec-ularweightwithanicelysymmetricshape.Peak2isde-layedandstretchedasaresultofhydrophobicadhesiontotheSECcolumnpacking,whilePeak3issymmetricbuteluteslateduetoelectrostaticrepulsionfromthecolumnmaterial.

Despitethenon-idealbehaviorsofPeaks2and3,SEC-MALScorrectlyidentifiestheirmolarmasses.SEC-MALSoftenaccompaniesmethoddevelopmentforoptimiza-tionoftheSECcolumnandbuffer,guaranteeingthatthe

elutingpeakscontinuetorepresentintact,unaggregatedandpureprotein(orothermacromolecule,asthecasemaybe).

Figure18.UVUHP-SECchromatograms(solidlines)ofthreemono-clonalantibodiesoverlaidwithmolarmassvaluesdeterminedbyMALS(symbols).Despitetheirdifferentelutionbehavior,allthreehavemolarmassesthatarecloseinvalue.

Figure19.SameasFigure18,withhydrodynamicradius(symbols)in-steadofmolarmass.AllthreemAbshavesameRh,indicatingthattheirdifferentelutionvolumesdonotderivefromdifferentconfor-mations.

Additionalinformationaboutthemolecularpropertiesandthepossiblecauseofnon-idealelutionisprovidedbyaddingonlinedynamiclightscattering,e.g.withaWyattQELSembeddedDLSmodule.AsseeninFigure19,thehydrodynamicradiiofallthreemAbsisthesame,confirmingthatthedifferentelutionvolumesarenot

relatedtodifferencesinconformation,buttoprotein-columninteractions.

AdditionalbiomoleculesBeyondproteins,SEC-MALSisinvaluableforcharacteri-zationofpeptides61,62,broadlyheterogeneousnaturalpolymerssuchasheparins63andchitosans.64,65

Smallpeptides

Multi-anglelightscatteringcoversaverybroadrangeofmolarmass,fromhundredsofDaltonstohundredsofmillions.Whilemostoftenusedtocharacterizeproteinsandpolymersabove10kDa,smallermoleculesareread-ilymeasuredaswell(aslongastheycanbeproperlysep-aratedonthecolumn).AN1613:PeptideCharacteriza-tionbySEC-MALSpresentstwoexamplesoftherapeuticpeptides,Bradykinin(a1060Dapeptideaccordingtose-quence)andLeucine-Enkephelin(556Daaccordingtosequence).

Figure20.SEC-MALSanalysisofamixtureoftwostandardproteinsandtwotherapeuticpeptides,BradykininandLeucine-Enkephelin.UVchromatogram(solidline)overlaidwithmolarmassvalues(sym-bols).SeeAN1613.

ThechromatogramsandmolarmassesareseeninFigure20.Themeasuredvaluesdifferedbyjustafewpercentfromthesequenceweights,possiblyaconsequenceofuptakeofcounterionsfromthesolution.Theresultswererepeatabletowithinjust2-3%.Sincesmallpeptidesdonotusuallyhavethesameuniversaldn/dcvaluesasproteins,theirrefractiveincrementsweremeasuredus-inganOptilab(theycouldalsohavebeencalculated

fromthesequence,justliketheUVextinctioncoeffi-cient).

Mono-anddisaccharides,low-andhigh-molecularweightpolysaccharides

Polysaccharidesare,bynature,quiteheterogeneousandspanabroadrangeinmolarmass.Analysisofthreein-jectedmassesofmaltodextrindemonstratejustapor-tionoftheDAWN’smeasurementrangeaswellasitsex-quisitesensitivity:eventhemonomermasscanbequan-tifiedwithamoderateinjectedmassof200µg(becauseofitslowmass,itscattersverylittlelightrelativetoitsconcentration).

Themolarmassesofallthreesampleloadingsoverlayquiteclosely.Thisisasignoftheidealityofthechroma-tography,theabsenceofintermolecularinteractionsandexcellentrepeatabilityofthedetectors.Theobservedloglinearity,togetherwiththeslopeoftheline,areindica-tiveofuniform,randomcoilconformationwithnobranching.Withanappropriateseriesofcolumnsorsep-arationbyAF4,theinstrumentscancoverarangeintothehundredsofmillionsofg/mol.

Figure21.Maltodextrinsolution,1mg/mL,injectedatthreevol-umestoassesssensitivity.Lightscatteringplotsaredashed,refrac-tiveindexplotsaresolid.Dotsindicatemolarmasses.Molarmassvaluesofthemonomerpeakwereonlyobtainedforthelargestin-jection,200µg.

SupportingQCofmultivalentpolysaccharidevaccines

Multivalentpolysaccharidevaccinescontainmanyimmu-nogeniccomponents,eachofwhichmustbecharacter-izedseparately.WhileSEC-MALSisnotsuitableforqual-itycontrolofthesemulti-componentmixtures,thefinalqualitycontroltechniquemustbetraceabletoreliableanalyticalmethodssuchasSEC-MALS.InAN1306:Poly-valentpneumococcalpolysaccharidevaccinebySEC-MALS,theuseofSEC-MALStocharacterizeindividualserotypesusedinMerck’sPNEUMOVAX23productisde-scribed.Theanalysisquantifiesthereductionofpolymerweight-averagemolarmassfrom270kDato110kDauponultrasonicationandtheirresultscorrelatedwithratenephelometry,anempiricalmethodappropriateforqualitycontrolpurposes.

SummaryMulti-angleanddynamiclightscattering,combinedwithsize-exclusionchromatography,areessentialbiophysicalcharacterizationtechnologiesapplicableacrossawiderangeofanalytes.SEC-MALSinstrumentationinformsre-searchanddevelopment,bothfundamentalandapplied,fromqualitycontroltounderstandinginteractions.

Theexamplesofbiomolecularcharacterizationmen-tionedinthisdocumentarejustafewofthousandsofpublishedinstances.Anextensivebibliographymaybefoundintheliterature66andonlineathttp://www.wy-att.com/bibliography,whileapplicationnotesareavaila-bleontheWyattwebsiteatwww.wyatt.com/AppNotes.

AcknowledgementsWegreatlyappreciatesharingofdataandexamplesofusingSEC-MALStoovercomecharacterizationchallengesintheapplicationnotescontributedbyourcustomers,including(butnotlimitedto)TesalDesai,JohnHennes-seyJr.,ChrisBroomell,DavidVeesler,ManishBurman,OliverSchoen,KusholGupta,JanvanDieck,JustinLow,MehrabanKhosraviani,Sylvia(Kyung-Joo)Lee,JihongYang,Per-OlofWahlund,JoeyPollastriniandShawnCao.

FurtherthankstoMarioLebendikerandHadarAmartelyforsuggestionsandreferences.

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