Applications of modern SEC to the characterization of 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].IntactmAbswereseparatedfromsolubleandinsoluble aggre-gatesusingsizeexclusionchromatography,undernon-denaturing conditions,andtheindividualcomponentsofthemixturewere identifiedwithaUHPLC-TOF-MSplatform andquantifiedvs.an unstressedcontrol.Aninternalstandard wasaddedtothe mix-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.

AvolatileammoniumacetatesaltwasusedtogenerateanMS com-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].The capil-larySECmethodenabledpicogramsensitivitywithanRSDofless than8%fortherelativepeakareaofHMWoftheFabfragments.

Fig.8showsrepresentativechromatogramsobtainedwith capil-larySECbyinjectingantibodyFabfragments.Anotherstudyalso

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

(ReprintedfromRef[93]).

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

(ReprintedfromRef[96]).

demonstratedtheenhancedsensitivityofcapillarySECforthe anal-ysisofmAbspurifiedfromharvestedcellculturefluid[99].

ThedevelopmentofafastandeasySECmethodunder mod-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 sodiumacetateandsodiumsulfatethatseparatesIgGinto aggre-gates,monomer,andfragments.Themoderatesaltconcentration resultedinasecondaryseparationmodebasedon hydrophobic-ity,resolvingamonomerpre-peakfromthemonomermainpeak.

MALSdeterminedthatthepre-peakhadasimilarmassastheIgG monomer.Characterizationofthepurifiedpre-peakfractionusing MS,andbioactivityrevealedthis degradanttobea tryptophan-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,thesmallercharge vari-ants,namelyreducedfragments(i.e.,lightchainsandheavychains withdifferentdrugloads)weresuccessfullycharacterizedbyRPLC usinggenericconditions[104].AsfornakedmAbs,mass spectrom-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-HuEPOaggregatesinformulations con-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-HuEPOmonomerandthe phar-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,and Reo-Pro.Thereportedmethodcouldsimultaneouslydetermineboth thequantitiesandthemolecularweightsofmacromoleculesfrom asingleinjection[107].

Amethodologybasedonon-linecouplingofSECwith mixed-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,anddetectedbyanevaporativelight scat-teringdetector(ELSD).AvolatilebufferwasusedforbothSECand mixed-modeseparation.Thismethodfacilitatedthedetectionof differentexcipientsbyELSDandprovidedpotentialforonline char-acterizationoftheproteinbymassspectrometry(MS).Themethod wasfinallyappliedtoquantifyproteinandexcipientsindifferent biopharmaceuticalproducts, includingantibodydrugconjugates (ADC)andvaccines[108].

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

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