Towards a simple on-line coupling of ion exchange chromatography and native mass spectrometry for the detailed characterization of monoclonal antibodies

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Article

Reference

Towards a simple on-line coupling of ion exchange chromatography and native mass spectrometry for the detailed characterization of

monoclonal antibodies

MURISIER, Amarande, et al .

MURISIER, Amarande, et al . Towards a simple on-line coupling of ion exchange

chromatography and native mass spectrometry for the detailed characterization of monoclonal antibodies. Journal of chromatography , 2021, vol. 1655, p. 462499

DOI : 10.1016/j.chroma.2021.462499 PMID : 34487883

Available at:

http://archive-ouverte.unige.ch/unige:155153

Disclaimer: layout of this document may differ from the published version.

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ContentslistsavailableatScienceDirect

Journal of Chromatography A

journalhomepage:www.elsevier.com/locate/chroma

Towards a simple on-line coupling of ion exchange chromatography and native mass spectrometry for the detailed characterization of monoclonal antibodies

Amarande Murisier

^a^,^b

, Bastiaan L. Duivelshof

^a^,^b

, Szabolcs Fekete

^a^,^b

, Julien Bourquin

^c

, Andrew Schmudlach

^c

, Matthew A. Lauber

^c

, Jennifer M. Nguyen

^c

, Alain Beck

^d

, Davy Guillarme

^a^,^b

, Valentina D’Atri

^a^,^b^,^∗

aInstitute of Pharmaceutical Sciences of Western Switzerland (ISPSO), University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland

bSchool of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland

cWaters Corporation, 34 Maple Street, Milford, Massachusetts 01757-3696, United States

dIRPF - Centre d’Immunologie Pierre-Fabre (CIPF), 5 Avenue Napoléon III, BP 60497 Saint-Julien-en-Genevois, France

a rt i c l e i nf o

Article history:

Received 20 May 2021 Revised 20 August 2021 Accepted 22 August 2021 Available online 26 August 2021 Keywords:

Ion exchange chromatography CEX-MS

Monoclonal antibody Mab biosimilars

a b s t r a c t

Thisworkdescribesthe directhyphenationofcationexchangechromatography(CEX)withacompact, easy-to-usebenchtop TimeofFlightmass spectrometer(ToF/MS) forthe analyticalcharacterizationof monoclonalantibodies(mAbs).Forthispurpose,awiderangeofcommercialmAbproducts(includingex- piredsamplesandmAbbiosimilars)wereselectedtodrawreliableconclusions.Fromachromatographic pointofview,variousbuffersandcolumndimensionsweretested.WhenconsideringpHresponse,buffer stabilityovertimeand MScompatibility,thebestcompromiseisrepresented bythefollowingrecipe:

50mMammoniumacetate,titratedtopH5.0(mobilephaseA)and160mMammoniumacetate,titrated topH8.5(mobilephaseB).Despitethebroaderpeaksobservedwiththe2.1mmi.d.CEXcolumn,this waspreferentiallyselectedforCEX-MSoperation,sincetheeﬃciencyloss(causedbyextra-columndispersion)wasstillacceptablewhileMScompatibilitywasstronglyenhanced(thankstolowﬂowrate).In termsofMS,itwasimportanttoavoidtheuseofglass-bottledmobilephases,laboratoryglasswareand glassvialstominimizelossofMSresolution,sensitivity,andmassaccuracyduetometalcontaminants.

WiththisnewCEX-MSsetup,straightforwardandrapidanalysis(inlessthan10min)ofchargevariants waspossible,allowingtheseparationandidentiﬁcationofseveralchargevariants.

ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/)

1. Introduction

Based on the therapeutic and commercial success of monoclonal antibodies(mAbs),manynewantibody-baseddrugformats havebeendevelopedinthelastdecade.[1]Asresultoftheirpro- duction usingrecombinant DNAtechnologyandexpressioninliv- ing systems,antibody-baseddrugsareproneto severalenzymatic and chemical post-translational modifications (PTMs) throughout any stage of their life cycle up to the formulated product. PTMs resultinproteinvariationsthatcanbeconsideredascriticalqual- ityattributes(CQAs)andpotentiallyinfluencetheclinicaloutcome ofthefinaldrugproduct.[2]Therefore,theassessmentandmoni-

∗Corresponding author.

E-mail address: valentina.datri@unige.ch (V. D’Atri).

toringofsuchCQAsisrequiredtoensurethedesiredqualitytarget productproﬁle(QTPP).

Acidic and basic variants are created by the molecular surface charge distribution of proteins, that can result from PTMs.

[3] Deamidation of Asn, glycation and the sialic acid content contribute to the creation of acidic species. [4,5] Incomplete cy- clization of N-terminal Gln or Glu, C-terminal Lys truncation, C- terminal amidation, Met oxidation or succinimide formation, on theotherhand,leadtobasicspecies.[5]

Ionexchangechromatography(IEX),andinparticularcationex- changechromatography(CEX),constitutesa keytechniqueforthe analysisofchargevariants.[6]InIEX,theretentionofmAbcharge variantsisdependant ontheir interactionwiththeionexchanger functionalgroups.[7]Fortheelution,eithersaltorpHgradientcan beusedtodisrupttheionicinteractions.[8,9]Recently,theuseof saltmediatedpHgradientshasalsoemergedasapowerfultoolto

https://doi.org/10.1016/j.chroma.2021.462499

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achievebetterseparationeﬃciency,bycombiningthetwocomple- mentaryelutionmechanisms(i.e.,saltandpHgradient).[10–12]

CEX is widely used forthe monitoring of changes in product quality. [4,13,14] However, to analyse which variation has led to thecreationoftheacidicorbasicspecies,massspectrometry(MS) detectionisultimatelyrequired.Duetoitshighspecificity,MS al- lowstheidentificationofunknownspeciesbasedonthemeasure- mentoftheirmassoverchargeratio(m/z).[11,15]Atfirst,MSwas predominantly coupled to denaturingchromatographytechniques (e.g., RPLC and HILIC),but more recentlyinterest hasshifted towards thecouplingofnon-denaturingchromatographytechniques suchasIEX,SECorHICtoMSdetection.[11,12,16]Thiscouplingal- lowstheanalysisoftherapeuticproteinsinnativeconditions,pre- serving their natively folded state and decreasing, therefore, the riskoflosingcriticalPTMs.[11,17,18]

Historically,IEX isconsideredto beincompatiblewithMS due theuseofnon-volatilesaltsinthemobilephase,e.g.,sodiumphos- phateandsodiumchloride,athighconcentrations.Therefore,mul- tidimensional chromatographic systems (mD-LC) coupled to MS havebeendevelopedtoenablethedirecthyphenationofIEXtoMS in a heart-cutting 2D-LC system. [11,19,20] However, this comes withtherisk oflosinglow abundantcharge variantsthat arenot selected asfractionsofinterestin theﬁrstdimension and,therefore,arenot analysedinthe seconddimension thatiscoupledto theMS.Moreover,mD-LCmethodscanbehardtodevelop,time- consuming and require dedicated instruments and well-trained users. Consequently, 2D-LC is not well adapted for routine ap- plications. [16] Hence, the strategy of using MS-compatible mobile phasesfor IEX separation has emerged. This allows a direct coupling of IEX to MS that can potentially allow unbiased and straightforward characterization ofcharge variants within a rela- tivelyshortanalysistime.[16,21–24]

However, IEX-MS methods present several issues. The chro- matographicseparationmightbestronglycompromisedwhenonly volatile salts are used, asthey possess a lower buffering capacity than non-volatilesalts. [24] The ionization process isanother challengetoovercome,aslowsignalintensitiesarecommonlyob- served in IEX experiments. [12] A trade-off has to be found be- tweenahighamountofvolatilesaltsinthemobilephaseincreas- ing theseparationquality,andtheriskofhamperingtheMS ionization. [22] Therefore, several solutions have been proposed by differentresearchgroups.First,toenhanceionization,theaddition oflowpercentagesoforganicsolventinthemobilephaseandﬂow splittingwastried.[25]Then,aninnovativestrategywasproposed by Füssl etal. consistingof a pHgradient combined witha neg- ative saltgradientwhich resultedin better MS compatibilityand optimizationofmassresolutionanddesolvation.[16]Recently,Yan et al. proposed a promising salt mediated pH gradient that pro- videdbetterMSsensitivitycomparedtopreviousstudies,allowing theidentiﬁcationofminorvariants.[26]

However,inmostofthepublishedstudies,highresolutionMS, especially Orbitrap technology, was used. [4,16,17,21,23,27] Orbi- trapinstrumentsofferaresolvingpowerupto100,000,abletore- solve near-isobaric isoformsandprovideexcellent massaccuracy, butinstrumentationcostandtheneedforqualiﬁedusersarelim- iting factorsforwidespreaduseofthosemethods.[4,17]Here,we demonstrate a straightforward method to couple CEX to a com- pactbenchtop MSsystem consistingofa high-resolution Timeof Flight (ToF)massanalyser,offeringaresolvingpowerof10,000.A wide rangeofantibodies, withacidicandbasicisoelectric points, was used.Unlikemostpublishedarticles, wherelarge amount of protein(i.e.,50–100μg)areinjectedtoobtainasuﬃcientMSsen- sitivity, the protein loadwaslower inthe presentstudy (10 μg).

[4,17,22,26–28] Three MS-compatible mobile phase compositions usedinrecentIEX-MSpublicationswerecompared.[29,30]ThepH responsewastakeninaccounttoincreasechromatographicresolu-

tion,asitwasdemonstratedtobeakeyparameterinCEXanalysis ofmAbs.Bufferstabilityovertimewasalsocontrolled.Onceopti- mumchromatographicconditionswereachievedforeachantibody, thehyphenationtoMS wasevaluated.The goalwastoobtainthe bestachievable resolutionwitha ToFinstrumentandthehighest signal intensitiesof thetestedmAbs innativeconditions.Thanks to this new CEX-MSsetup, straightforward andrapid analysis of chargevariantswasmadepossible(inlessthan10min),allowing theseparationandidentiﬁcationofseveralchargevariantswitha newlevelofthroughput.

2. Experimental

2.1. Chemicalsandreagents

Type 1 water was provided by a Milli-Q puriﬁca- tion system from Millipore (Bedford, MA, USA). 2-(N- morpholino)ethanesulfonic acid monohydrate (MES) (≥99.0%), sodium chloride (NaCl) (≥99.5%), ammonium hydroxide solution (28%NH3inH₂O, ≥99.99%),ammoniumcarbonate(LC-MSgrade), ammoniumbicarbonate(BioUltra,≥99.5%)andammoniumacetate solution(BioUltra, formolecular biology, ∼5 M)were purchased from Sigma-Aldrich (Buchs, Switzerland). Sodium hydroxide (NaOH) was purchased from VWR Scientiﬁc (Radnor, PA, USA) andaceticacid(LC-MSgrade) fromBiosolve(Dieuze,France). The commercialionexchangebuffersIonHanceCX-MSpHconcentrate AandBwereobtainedfromWaters(Milford,MA,USA).

2.2. Samplepreparation

TherapeuticIgGmonoclonalantibodies, includingadalimumab, belimumab,dalotuzumab,eculizumab,elotuzumab, inﬂiximab,ip- ilimumab,palivizumab,rituximabandtrastuzumabwereobtained asEuropeanUnionpharmaceutical-gradedrugproductsfromtheir respective manufacturers. NIST mAb reference material waspur- chased from the National Institute of Standards and Technology (Gaithersburg, MD,USA). Samples were diluted to 1 mg/ml with Milli-QwaterforIEXexperimentsandto10mg/mLforIEX-MSex- periments.

2.3. IEXexperiments:instrumentationandchromatographic conditions

IEX experiments were performed on a Waters Acquity UPLC H–Class Bio system equipped with a quaternary solvent deliv- erypump, an autosamplerwithflow-through needle(FTN) injection system, a fluorescence detector (ʎex 280 nm, ʎem 350 nm) anda pH/C-900online pHmetre (Amersham Pharmacia Biotech) equipped with a pH electrode (General Electric, Baden, Switzer- land). BioResolve SCX mAb (3 μm, 2.1 × 50 mm) strong cation exchanger column fromWaters (Milford, MA, USA) wasused, as well as MabPac SCX-10 RS (5 μm, 2.1 × 50 mm) column from Thermo Fisher Scientific (Waltham, MA, USA). Column tempera- turewasset at30°Cfor allmeasurements and1 μLsamplehas beeninjected,unlessstatedotherwise.ForeachmAb,alineargra- dient wasrun from0 to 100%of B in 10 min on both columns ata flow rateof0.2mL/min. Gradientprograms were then optimized forinfliximab, eculizumab, elotuzumab andNIST mAbus- inga^%Bôf^20% ⁱⁿ¹⁰^min^followed^by⁵^minequilibrationwith thedifferentbuffer systems(55%to75% Bforthe NISTmAband dalotuzumab, 45% to 65% B for infliximab and 35% to 55% B for elotuzumab).The influence of columndiameter wasalso studied witha BioResolve SCX mAb column(3 μm,4.6 × 50mm). With thiscolumn,theflowratewassetto0.96mL/minaftergeometri- cal methodtransfer calculationsand2μl wasinjected. ThreeMS

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compatiblemobilephasebuffersystemswerecomparedtoaclas- sical salt-gradientbuffer composed of 10mM MES in MilliQ water atpH 6.0(mobilephase A) and 10mM MES+0.25M NaClat pH6.0(mobilephaseB). First,thebuffer systemproposedbyYan etal.composedof20mMammoniumacetate,pHadjustedto5.6 with acetic acid (mobile phase A) and 140 mM ammonium ac- etateand10mMammoniumbicarbonatepH7.4(mobilephaseB) wasprepared[26].Second, anin-housebufferwasusedwithaﬁ- nal composition of10mM ammonium acetateand10 mM acetic acid(mobilephaseA)and50mMammoniumacetateand25mM ammoniumcarbonate(mobilephaseB). Last,thecommercialIon- Hance CX-MSpHconcentrateAandBwerediluted10timeswith Milli-Q water to reach a composition of 50 mM ammonium acetate with2% of acetonitrile,titrated topH 5.0(mobilephase A) and 160 mM ammonium acetate with 2% of acetonitrile,titrated to pH 8.5 (mobilephase B). This dilutionprocedure is suggested by the vendor[30]. Instrument control and data acquisition were performedby EmpowerPro 3software(Waters).Excel(Microsoft OﬃceProfessionalPlus2016)andSigmaPlot14.0(SystatSoftware, Inc)wereusedfordatatreatment.

2.4. pHmeasurements

For the evaluation of buffer systems stability of non MS- compatible (MES) and MS-compatible buffer systems (Yan, In- House and Commercial), each mobile phase (A andB) was pre- paredtwotimesandstoredeitheratroomtemperatureorat4°C.

ThepHofeachbuffersystemwascontrolledafter3,8and10days toevaluatebufferstability.On-linepHresponsewasmeasuredon the H-class Bioinstrument by using the narrow bore BioResolve and MabPac columns, while off-line pH response was measured without column. pH responses were then corrected for gradient delayandcolumndeadtime,tobecomparable.

2.5. IEX-MSanalyses:instrumentationandexperimentalconditions

IEX-MSexperimentswereperformedusinganACQUITYUPLCI- ClasssystemcoupledtoaTUVdetectorforUVdetection(280nm) and to a high-resolution BioAccord ToF mass spectrometer from Waters.Flowratewassetat0.1mL/minand10

μ

^g^of^mAb^sample

wasinjected onto narrow borecolumnof2.1 mmi.d. BioResolve and MabPaccolumns (1 μL injection volume). Selected mAbs for IEX-MS analyses were NIST mAb,adalimumab, dalotuzumab, elotuzumab, infliximab (the originator Remicade® and the biosimi- larsRemsima® andInflectra®),andtrastuzumab(originatoranda 7-years expired sample). Optimized gradient consisted of a ^%B of 20% in 10 min, for a total analysis time of 20 min including columnwashandequilibration.WiththeBioResolve column,gradients were 35% to 55% B forelotuzumab, 48%to 68% B for infliximab, 50% to 70%for trastuzumab55% to 75% B forthe NIST mAb, and60% to 80% B foradalimumab and dalotuzumab. With theMabPaccolumn,gradientswere 35%to55% Bforelotuzumab, 45% to55% B fortrastuzumaband infliximab, and55% to 75% B forNISTmAb,dalotuzumabandadalimumab.Themassspectrom- eter wasused in ESI positive mode withan acquisition range of 400to7000m/z.Thesystemwascalibratedbyusinga200pg/μL sodium iodide solutiondiluted in amixture ofwater/isopropanol 50/50 (v/v) with 0.1% FA. FormAb analysis in their native state, thedesolvationtemperaturewassetat350°C,sourcetemperature at 120°C,the cone voltageto 150Vand thecapillaryvoltageto 1.5kV.UNIFIwasusedasdataacquisitionsoftware.Nativeprotein mass spectradata treatment wasperformedwithMassLynx software(Waters).

3. Resultsanddiscussion

3.1. Evaluationofbufferperformanceandstability

TofindtheoptimalmobilephasecompositionforIEX-MSanal- ysis, three promisingMS-compatible buffer systems (namely, Yan buffer, Commercial buffer, and In-house buffer) were compared with a classical salt-gradient buffer that is widely used for IEX mAb analysis(MES buffer). The differentmobile phaseswere all usedincombinationwiththeBioResolveSCX (2.1×50mm)col- umnand a generic gradient of 0% to 100%B in 10 min(gradient delaytimeofapprox.2min).Tocomparetheperformance ofthe differentbuffersystems,tendifferentmAbshavingpIvaluesrang- ingbetween6.1and9.4wereanalysed.Fig.1showstheobtained resultsfor theten differentmAbs analysed withthe four mobile phasecompositions.Itwasobservedthat forboththeMES buffer andtheYan buffer,the mAbwiththe lowestpI(i.e.,eculizumab, pI6.1)waspoorly retainedonthecolumn. Thiscanbe explained bytherelatively highstartingpHoftheusedbuffers, 5.6and6.0 for the Yan buffer and MES buffer, respectively. This effect was lesspronouncedwiththe Commercialbuffer andIn-Housebuffer that both have a starting pH of 5 or less. Further evaluation of thedifferentbuffer systemsshowedthatchargevariantsofinflix- imabandelotuzumabwerebetterseparatedwhenusingtheMES, YanorIn-Housebuffercomparedto theCommercialbuffer.How- ever,applicationoftheIn-HousebuffertothemAbsanalysedafter elotuzumab (i.e., pIvalues of 8.6and higher) wasof limiteduse duetothe lossofselectivity betweentheanalytes. Itwasshown that,all mAbselutedatthe samepointoftheanalytical gradient andthepotentialseparation ofchargevariants waslostwiththis buffer. Thiseffectcan be explainedby a lackofionic strengthin theBeluentthatispreventingthemAbsandtheirchargevariants to elutewithin the givenanalytical gradient. This effectwas not presentfor the other three buffers. The mAbs withpI values up to9.4were indeedsufficientlyretainedon thecolumn. Forthose mAbs, it was observed that better separation of charge variants wasobtainedwiththeYanbufferandMESbuffer.Itisworthmen- tioning thatintheseexperiments,a genericgradient wasapplied to compare the differentbuffer systems. The obtained selectivity can therefore be further improved by individual optimization of thegradientprograms.

For further assessing the performance of the different eluent systems,thepH-stabilityofeachbuffer compositionover 10days wasevaluated.As showninFig.2,thebeststabilitywasobtained withthe reference MES buffer that kept a constant pH through- outthe10days.Onthecontrary,theYanbufferdemonstratedthe largestinstability with0.25unitsofdifference inpHafter3days and up to 0.5 units after10 days. A similar trend was observed fortheIn-Housebufferthatpresenteddifferencesofover0.25pH units after 8 days. In addition, it was observed that the storage temperaturehadaminorinﬂuenceonthebufferstabilitysinceno cleardifferenceswerefound forthebufferskeptatroom temper- atureandat4 °C. Thisis important,sincethe buffersaremostly keptatroomtemperatureduringanalysis.Moreover,thevariations inpHvalueswerepredominantlyfoundintheBcomponentofthe elutionsystemthat representsthehigh-pHgradientendpointand strongeluentsolution.amongsttheMS-compatiblebuffersystems, theCommercialsolutionsprovidedthebeststabilitywithlessthan 0.25unitsofpHvariationafter10daysatambienttemperature.It shouldbe notedthat theCommercial buffercompositionconsists of50 mM ammonium acetate with 2% ofacetonitrile, titrated to pH5.0(mobilephaseA)and160mMammoniumacetatewith2%

ofacetonitrile,titratedtopH8.5(mobilephaseB).Indeed,thead- ditionof2%ACNisusedforbacteriostaticpropertiesandshelflife andhasnoeffectontheseparationperformances(FigureS1).

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Fig. 1. Comparison of non MS-compatible salt gradient buffer (MES buffer + NaCl) and MS-compatible buffers (Yan, Commercial and In-House buffers) for the analysis of 10 mAbs. Chromatograms obtained with the BioResolve SCX mAb 3 μm, 2.1 ×50 mm column with a generic gradient from 0% B to 100%B on the H –Class Bio system.

Fig. 2. Evaluation of buffer systems stability of non-MS-compatible (MES) and MS-compatible buffer systems (Yan, In-House and Commercial). pH was measured with a pH- metre after 3, 8 and 10 days (noted as D3, D8, and D10, respectively). Bars filled in grey and black correspond to the mobile phases A (MPA) stored at room temperature or at 4 °C, respectively. Bars filled in light blue and dark blue correspond to the mobile phases B (MPB) stored at room temperature or at 4 °C, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Based on the presented results, the Yan buffer and Commer- cial buffershowedthe mostappropriateperformance interms of chromatographicbehaviourandstability.However,eachmAbhasa speciﬁc retention behaviourthat requires individual optimization.

Therefore,theselectionoftheeluentsystemisstronglyconnected with the used columnandthe resulting on-columnpH response thatcanbeusedtooptimizetheseparationofchargevariants.

3.2. PracticalconsiderationsonpHresponse

InIEX,thepHgradientmodebecomesmoreandmorepopular todayespeciallywhenperformingIEX-MS.Thisway,relativelylow ionicstrength(saltconcentration)issuﬃcienttoelutemostmAbs.

The mostcommonly used volatile mobile phase components are

ammonium-acetateandammonium-carbonateorthecombination ofthetwo.[31]Itwasfoundthatdespitethefactthatsomebuffer mixtures provide suﬃciently highbuffer capacity andnearly linear pH responses inthe range ofthe pIvalues ofmostcommon mAbs, their chromatographic applicability is not always guaran- teed, asproteins mayelute in tailed and broadpeaks or on the contrary,in extremely sharppeaks whichsuffer fromthe lackof selectivity.[10]Ideally,a linearpH responseisdesiredduringthe gradienttocontrolselectivityandofferthepossibilityforanalytes toeluteacrossthewidestpossibleretentiontimerange.IfthepH response of the eﬄuent is too steep (large pH change within a shorttimeduringthegradient),thepeakselutinginthatpHrange willbepoorlyseparatedduetothelargechangeofeluentstrength (related topH). On theother hand,thecolumn itself canhave a

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significantimpactontheeffluentpH.[16]Thisphenomenonisat- tributed to the ratios ofthe exchangeable ions betweenthe mobile phase andthe functionalgroupsofthestationaryphase.The stationary phase hasa buffer capacity, providing highamount of exchangeableionstostabilizetheratiosofionconcentrations.[32] The ionexchange capacityofthecolumndependsonthe surface chemistryandcoverageofthestationaryphase.Theadsorption of the counter-ionson the surfacereleases H⁺ and OH⁻ ions, thus changingthemobilephasepH.[16]Therefore,itmayhappenthat aselectedbuffersystem(mobilephase)canworkwellononecol- umn, butcan failon anothercolumn. It isworth mentioning too that column volume and mobile phase flow rate may also have an impacton theeffluentpHresponse.[32]Theratioofgradient steepnessandcolumnvolumeindeedhasimportantconsequences intermsofcolumnequilibrationand^pHôver^theâmountôf^col- umnvolumeinwhichthegradientisdelivered.

Based on some preliminary experiments, we have selected three promising buffer systems and two columns which might provide appropriate selectivity,peak shape andMS sensitivityfor our samples. The buffer system proposed by Yan et al., the In- HouserecipeandtheCommercialbuffer systemswererun onthe strong cationexchanger BioResolveSCX mAbandMabPacSCX-10 columns. [26] Fig. 3 shows the experimentally measured pH responses for all combinations, and also in absence of column. In absenceofcolumn(Fig.3c),theYanrecipeprovidesaconcavepH responseintherangeof5.6– 7.9andthepHremainsnearlycon- stantbeyond 50%B mobilephase composition suggestingthat selectivity can be tuned only between0 and 50%B eluent. The In- House systemresulted in pHresponse range of 4.6– 8.1, being nearly linear between 4.6 and 7.6. The Commercial system pro- videdapHrangeof4.9– 8.2,showinganinﬂectionpointatpH∼7.

Intherangeof6.3to7.8, itspHresponseisthesteepestamongst all the three buffers. When runningthe buffer gradients through thecolumns,theeffluentpHresponsesweresignificantlymodified (Fig.3a-b).ThemostpromisingcombinationsweretheCommercial buffer withtheBioResolvecolumninthepHrangeof4.9and6.8 (correspondingto0– 75%Bgradient)andalsowiththeYanbuffer forpH=5.6– 6.9(correspondingto0– 50%Bgradient).Inaddi- tion,theMabPaccolumnalsoprovidednearlylinearpHresponses withtheCommercialandYanbuffers,inthepHrangeof4.9– 6.6 and5.7– 7.8,respectively.ThepHresponseoftheIn-Housebuffer systemwasthemostaffectedbythecolumns.

It isimportanttokeep inmindthat inaddition tothepH response, thepeak shape,selectivity andMS sensitivityall needto be considered to selectthemostappropriate combinationofcol- umnandmobilephasebuffer.

3.3. Selectionofcolumn

It wasdemonstratedearlierthat thedifferentcombinations of stationary andmobilephasesmayresultin diverseretention,se- lectivityandefficiency.Therefore,thewholephasesystem(combi- nationofstationaryandmobilephase)shouldbeconsideredwhen developinga method.[7]Inaddition,retention behaviourismAb dependantandshouldbeindividuallyoptimized.Afterhavingdis- cussedtheselectionofmobilephasesystem,herewewilldiscuss the choiceofcolumn. Preliminaryexperimentsto analysevarious mAbs were run with BioResolve SCX andMabPac columns. Both standard(4.6mmi.d.)andnarrowbore(2.1mmi.d.)columnswere tested and compared in terms of selectivity, efficiency and peak shapes. Fig. 4shows acomparison of2.1and 4.6mm i.d. BioRe- solve columnsfor fourdifferentmAbs applyingthe samegeneric gradient (geometrical method transfer rules were systematically applied). As expected, thenarrow borecolumnshowedlower efficiency(resolution),which isprobablyduetoextra-columnpeak dispersion.Indeed,stateoftheartion-exchangersarepackedwith

non-porousparticlessuchthattheypossessquitelowporosity.For theBioResolveandMabPaccolumns,undertheappliedconditions, a total porosity of

ε

= 0.35 – 0.40 was measured. Such poros- itycorresponds toV₀ =60– 70μLinthecaseofthe2.1mmi.d.

column and to V₀ = 290 – 330 μL forthe 4.6 mm i.d. column.

When considering that the extra-column volume of the Acquity H–Class Biosystem is about V_ext = 15 μL, it becomes clear that for the 2.1 mm i.d. column, the apparent peak width should be strongly affectedby extra-columnpeak dispersion, since the sys- temvolume isabout21 – 25%of thecolumnvolume. While,in caseofthe4.6mm i.d.column, it isonlyabout4– 5%.Despite thesignificantlybroaderpeaksobservedwiththe2.1mmi.d.column, we selected the narrow bore column, since this efficiency loss was still acceptable and more importantly MS compatibility wasstronglyenhanced(lowflowrateleadstohigherMSsensitiv- ity).Moreover,additionalresolutionwasregainedwiththeswitch toanevenlowerdispersionsystem(AcquityI-Classsystemhaving anextra-columnvolumeofaboutVext=7μL).Ontheotherhand, theselectivityremainedcomparablebetweenthe2.1and4.6mm i.d.columns.

3.4. Hyphenationtomassspectrometry:bufferperformance evaluation

MS performance of the three IEX mobile phases (Yan, Com- mercialandIn-Housebuffer)wasevaluated byanalysingtheNIST mAb referencematerial onthe BioResolve column, using the LC- MS conﬁguration and settings describedin Section 2.5. Totalion chromatogramsandthe integratedspectraofthemainNIST mAb peakobtainedwitheachbuffercompositionareshowninFig.5.It should be notedthat both the Yan and In-House buffer systems contain ammonium carbonate or ammonium bicarbonate in the solution. Onthe contrary, the Commercial solutiononly contains ammoniumacetateasamobilephase additive.Theuseofammo- niumcarbonateandbicarbonateisoftenpreferredovertheuseof ammoniumacetate froma chromatographicpointofview,dueto the improved bufferingcapacity at pH 7.However, severalprob- lems mightoccur whenusing carbonate-based buffers.As shown inFig. 5b (redline),NIST mAb wasdetectedin nativeconditions only withthe Commercial buffer, whichis evident by signal and acharge enveloperanging onlybetween5000and7000m/z values. In native conditions,the protein keeps its folded conforma- tionandhasfewerchargestates,thereforeprovidingmassspectra withahighspatialresolutionthat allows easierdeconvolutionof thechargevariants.ThisbehaviourwasnotmaintainedwhenNIST mAbwasanalysedwitheithertheYanorIn-House buffer(Fig.5b, greenandblueslines). Instead,thesebuffersystemsintroduced a substantial shiftofionsignal towards lower m/z values(between 2500and4500m/z)andundesirablymorecomplicatedmassspec- tra. The widening ofthe charge envelope indicates that the pro- teinhasundergonedenaturation,resultingintheformationofhigh chargestatesthatmightleadtoanoverlapofisoformsofadjacent chargedstates,makingdiﬃcultthedeconvolutionprocessandde- tectionoflowabundanceproteoforms.

The observeddenaturingeffects can be directlyrelatedto the presenceofthe(bi)carbonateinthemobilephaseduringtheelec- trosprayionization(ESI)process.When theprotein-containing solutions of (bi)carbonate are heated prior to ionization, CO₂ gas bubbles arereleased,and theycan induce adenaturing effecton the protein. [33] The subsequent unfolding of the protein in the ESI droplets leads to the formation of highly charged ions like those observed in the described MS spectra (Fig. 5b, green and blues lines). Additionally, thepresence of CO₂ during the ioniza- tionprocessresultsintheformationofCO2adducts(+44Da)that canleadtobroaderdeconvolutedpeaksandlowermassaccuracies.

[16,34]Theseeffectsareabsentwhenusingammoniumacetatein

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Fig. 3. Evaluation of pH response with the BioResolve 3 μm 2.1 ×50 mm column (a), the MabPAC 5 μm 2.1 ×50 mm column (b), and without column (c). Black traces corresponds to the Yan buffer, blue traces to the Commercial buffer and grey traces to the In-House buffer. pH was measured on-line on the H-class Bio system equipped with an online pH metre when running a generic gradient from 0% B to 100% B in 20 min. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

the buffer solutions,because the additive isreleasedas NH3 and CH₃–COOHgasandthereforethenegativeeffectsofCO₂ gasbub- blesareavoided.

Therefore, based on the presented results,the buffer contain- ingonlyammoniumacetateprovedtobethebestmobilephaseto keep the mAbin its nativeformand obtainaccurate mass infor- mationwhencouplingIEXtoMSdetection.

3.5. IEX-MSchargevariantidentiﬁcationonselectedsamplesand casestudies

IEX-MS experiments were acquired by using speciﬁcally selected conﬁgurations, consisting of narrow bore (2.1 mm i.d.) BioResolveandMabPaccolumnsandtheCommercialbuffer,made of 50 mM ammonium acetate with2% of acetonitrile,titrated to pH5.0(mobilephaseA)and160mMammoniumacetatewith2%

ofacetonitrile,titratedtopH8.5(mobilephaseB).First,MSsource

conditionswerecarefullyoptimizedtoallowthedetectionandMS deconvolution of minor charge species. The NIST mAb reference materialwasusedfortheseexperiments.Glassmobilephasebot- tles and vials were replaced with trace metal certified thermo- plastics to minimize loss of MS resolution, sensitivity, and mass accuracydueto metalcontaminants. [35] Similarly,specialatten- tionwaspaidduringthemobilephasepreparationbyalsoreplac- ing the laboratory glassware with plasticware. As highlighted in Fig.6a, the charge profile ofthe NIST mAbwas characterized by amainspecies(peaklabelledasM)andtwominorspecieseluting inthebasicvariantsregion (peakslabelledasB1andB2),inline withtheNIST mAbCEXprofilealreadyreportedinthe literature.

[17,26]DespitethemassspectraofB1andB2showingintensities 10-and96-foldlower thanM(Fig.6b),respectively,thedeconvo- lutionofthesepeakswasfeasibleandallowedtheidentiﬁcationof the C-terminal Lys modiﬁcations (∼128 Da shifts,responsible for the separationof thetwo basic charge variants), andthe protein

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Fig. 4. Comparison of mAbs chromatograms with the BioResolve columns 3 μm 2.1 ×50 mm and 4.6 ×50 mm on the H-class Bio system with the Commercial buffer.

Selected mAbs were NIST mAb (light blue), inﬂiximab (dark blue), elotuzumab (grey) and dalotuzumab (black). On the narrow bore column, the ﬂow rate was set at 0.2 ml/min and geometrically adapted to 0.96 ml/min on the 4.6 mm ID column. Injection volume was 1 μl and 2 μl for 2.1 mm ID and 4.6 mm ID column, respectively.

Gradient were optimized for a %B of 20% in 10 min for each mAb (55% to 75% B for NIST mAb and dalotuzumab, 45% to 65% B for inﬂiximab and 35% to 55% B for elotuzumab). (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

Fig. 5. MS performances by using different IEX buffers. (a) Total ion chromatograms (TICs), and (b) integrated spectra of the main peak of NIST mAb analysed by using Yan (green), In-House (blue), or Commercial (red) buffer. Cps stands for counts per second. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

glycoforms associated ateach charge variant (Fig. 6cand MS assignment providedinTable S1). Ofnote, the qualityofthe water usedforthepreparationofthemobilephaseplayedakeyroledur- ing theLC-MSoptimization process.Infact, theswitch fromMS- gradeglassbottledwatertoMilliQwatercollectedinthermoplas- ticcontainersresultedtobefundamentalforobtaining theproper resolutionoftheglycoforms(FigureS2).

Then,ﬁveadditionalmAbswereanalysed,namelydalotuzumab, adalimumab, elotuzumab, trastuzumab, and inﬂiximab (Fig. 7).

AnalyseswereperformedwiththeBioResolvecolumnandthepro- ﬁling of the charge variants wasobtained after speciﬁc gradient optimizationforeach mAb. However, fordalotuzumab andadali- mumab,acompressedpeakshowingpoorselectivitywasobserved (Figure S3), most probably dueto the fact that thesetwo mAbs

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Fig. 6. Identiﬁcation and peak assignment of charge variants, illustration with NIST mAb. (a) Total ion chromatogram (TIC) showing the main peak (M) and two basic variants (B1-B2). (b) Mass spectra of M, B1 and B2 peaks with their related (c) deconvolution and glycoform annotation. 0 K – 2 K refers to the lack/presence of both C-terminal K.

were elutedduringthesteepestpartofthepHgradient.Thispar- ticularbehaviour wasalreadyobservedby Farsangetal., whenit wasreportedthatinacertainpHrange,somecolumn/buffercom- binations might providelow selectivity duetothe very steeppH response curve. [32] To avoid this issue,one could further opti- mize the elution conditionsor change the column/buffer combi- nation.TheMabPaccolumnwasthereforeusedfortheanalysisof dalotuzumab andadalimumabreportedinFig.7,since thepH re- sponsewasnotthesamewiththiscolumn(differentbuffercapac- itycomparedtotheBioResolvecolumn),asalsoreportedinFig.3. Forsakeofcompleteness,thechargevariantsproﬁlingofallmAbs obtainedwiththeMabPaccolumnhavebeenreportedintheSup- plementaryInformation(FiguresS4-S7).

Forprotein isoformidentiﬁcation,theoretical referencemasses were calculated basedon theaminoacidsequences oftwo identical heavy chains and two identical light chains, andby assuming the presence of 16 disulphide bonds (12 intra-chains and 4 inter-chainsbonds, withlossof∼2Daforeach disulphidebond).

AsshowninFigureS8a,theformationofpyroglutamate(pE)from theN-terminalGlu(E)orGln(Q) mayrespectivelyresultinaba- sicoranacidicvariantdependingontheprecursorofthecycliza- tion process. Inaddition, isomerization of Asp (D) and the presence of succinimide, which is the common intermediate for Asn (N)deamidationandAsp (D) isomerization(FigureS8b), canalso contributetothegenerationofbasicspecies.[13]

On the other hand, deamidation andthe presence of charged glycans (in the form of sialylated glycans) have been reported asthe main modiﬁcationsresponsible forthe formationofacidic chargevariants.[13,36,37]

Inthiscontext,itshouldbe notedthatinacommonmAbgly- can proﬁle,sialylatedglycans aregenerallypresentat avery low abundance. Therefore, the identiﬁcation of the proteoforms containing sialylatedglycans mightbe extremely challengingby MS, despitethefact thatthesespeciesmightbe wellseparatedatthe chromatographic level. Similarly, the unambiguous assignment of

adeamidation(only∼1Dashift,asreportedinFigure S8b)isof- tennotachievedwhentheMSanalysisisperformedatintactlevel (massshiftfallingintoassignment error),andpeptidemappingis generally applied on collected peaks to support the assignment.

[16,38] However, it should be noted that the change of the ap- parentpIofa deamidatedmAb variant,responsible ofthereten- tion shift through the acidicregion ofthe CEX profile, might be sufficientto indirectlyestablishthe presenceof adeamidation, if supportedbytheMSdatathatexcludethepresenceofothermod- ifications.

Charge variant retention times and mass assignments performed by CEX-MS analysis of NIST mAb, dalotuzumab, adalimumab,elotuzumab,trastuzumab,andinfliximab,arerespectively reported in Tables S1-S6. Specifically, dalotuzumab CEX profile (Fig.7a,pinkline)consistedofamainpeak(M)showinghomoge- nousN-terminalmodifications (Q/pEformation), 2acidic variants (A1– A2)emergingfromthepossibleincreasednumberofdeami- dations, and2 basicvariants (B1 – B2) dueto the presence of one andtwo C-terminal K,respectively. The major proteinglyco- forms(Fig.7b,pinkline) identifiedforthe mainvariant (namely, G0F/G0F,G0F/G1F,andG1F/G1ForG0F/G2F)wereconsistentlyde- tected forall thecharge variants (Table S2). Similarly, thecharge profileof adalimumab(Fig.7a, orangeline) wascharacterized by a mainpeak (M) withno N-terminalmodifications (homogenous presenceofE),anacidicvariant(A1)duetothepossiblepresence ofadeamidation, andtwo basicvariants(B1– B2)linked tothe presenceofanincreasednumberofC-terminalK.Alsointhiscase, theglycosylationprofile(majorglycancombinationbeingG0F/G0F, G0F/G1F,asshowninFig.7b,orangeline)wasconsistentamongst allthechargevariantsandinlinewithresultsreportedinthelit- erature(TableS3).[4,16,38]

A more interesting CEX proﬁle was provided by elotuzumab (Fig. 7a, blue line). In this case, the N-terminal region of the mAbwasnothomogeneous,showingthreedifferentcombinations of E/pE formation, namely E-E, E-pE (∼18 Da shift), and pE-pE

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Fig. 7. Charge variant analysis of five mAbs by CEX-MS analysis. (a) Total ion chromatograms (TICs), and (b) deconvolution of the mass spectra of the main variant. TICs peaks identified as main (M), basic (B) or acidic (A) variant. For sake of simplicity, mAb cartoons were added to the top of each peak and dots of different colours were used to highlight the presence of modifications responsible for the charge variant, such as deamidation (D, blue dot), isoAsp (iD, cyan dot), N-terminal Glu (E, yellow dot) or pyroGlu (pE, pink dot), and C-terminal Lys (K, green dot). Glycan proteoforms in deconvoluted spectra are labelled. ^∗stands for unknown. Experiments acquired by using optimized LC conditions with the Commercial buffer and the BioResolve column, unless stated otherwise. (For interpretation of the references to colour in this figure legend,

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Fig. 8. Charge variants profile of mAb case studies. a) Comparison between trastuzumab (light green line) and a 7-years expired trastuzumab sample (purple line) and b) between infliximab Remicade® (red line) ant its two biosimilars infliximab Remsima® (grey line) and inflecta® (cyan line). Total ion chromatograms (TICs) acquired by using optimized LC conditions with the Commercial buffer and the BioResolve column. Peaks were identified as main (M), basic (B) or acidic (A) variant by MS. For sake of simplicity, mAb cartoons were added to the top of each peak and dots of different colours were used to highlight the presence of modifications responsible for the charge variant, such as deamidation (D, blue dot), isoAsp (iD, light blue dot), and C-terminal Lys (K, green dot). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

(∼36Dashift). AsshowninFigure S8,whentheformation ofpE isoccurringfromE,thechargevariantcontainingthepEresultsto be morebasicthanthat onecontainingtheE,whiletheopposite behaviourisobservedwhenthepEderivesfromaQ.Theimpactof thisN-terminalmodificationiswelldemonstratedfortheCEXpro- file ofelotuzumab that ischaracterized by amain peak(M) con- tainingthe E-pEcombination, an acidicvariant(A1)withtheE-E combinationasN-terminalmodifications,andthreebasicvariants (B1– B3).Interestingly,thefirsttwobasicvariants(B1– B2)are both containingthepE-pEcombination, butdiffer fromthepres- enceofaC-terminalK.Incomparison,themostbasicchargevari- ant(B3)isinsteadcharacterizedbythepresenceoftheE-pEcom- bination(astheMspecies)butwiththeadditionoftwoC-terminal K. Being positively charged,the two K residues are thereforere- sponsiblefordrivingtheshiftinretentionofthischargevariantin the deeper basicregion ofthe chromatogram. Regardingthe gly- cosylationprofile,theelotuzumabsamplewasquitehomogeneous, showing amajor glycoform (G0F/G0F, Fig. 7c,blueline) that was detectedforallchargevariants(TableS4).

Unlike all samples discussed so far, the CEX profile of trastuzumab (shownin greenin Figs.7a and8a)wasquite chal- lenging to interpret. As already reported in the literature, it has beenextensivelystudiedby bottom-upLC-MSanalysis,becauseit is mainly characterized by variants containingAsn deamidations andAsp isomerizations.[4,16,21,39,40] AsreportedinFigure S8b, thismeansthat amassshiftof∼1Daisexpectedforthedeami- dation processandnoshiftatall fortheisomerization.Therefore, the intact level CEX-MS analysis would not be sufficient for the unambiguous interpretation ofthis profilebecause all the charge variants wouldhavealmostthesame masses.However, assuming that deamidation and isomerization are responsible for the two distinct variationsoftheapparentpIofthemAbvariants(deami- dationtowardsmoreacidicvaluesandisomerizationtowardsmore basic values), the assignment of thetrastuzumab charge variants might be achieved based on the retention shifts of the variants andaccordingtobottom-updatapublishedintheliterature.[39– 41]Therefore,inaccordancewithdatapublishedbySchmidetal.

(CEX proﬁle obtained by using sodium phosphate buffer as mobile phase), and by Bailey et al. (CEX proﬁle obtained by using

ammonium acetate buffer as mobile phase), the CEX proﬁle of trastuzumab(Figs. 7a and8a, green lines) ischaracterized by an unmodiﬁedmain species(M), an acidicvariant(A1) containinga deamidation, and a basic variant (B1) bearing an isomerization.

[21,41] In addition, asshown in Fig. 7b (green line), four major protein glycoformswere identiﬁed for the main variant(namely, G0F/G0F,G0F/G1F,G1F/G1ForG0F/G2F, andG1F/G2F)andconsis- tentlydetectedforallthechargevariants(TableS5).A7-yearsex- piredtrastuzumabsamplewasalsoanalysed(Fig.8a,purpleline).

Interestingly, this CEX proﬁle was characterized by 4 additional acidicspecies(A2– A5),andoneadditionalbasicvariant(B2).

InagreementwiththeCEXproﬁles publishedby Schmidetal.

(trastuzumab samplessubjected to several stress conditions), the acidicvariantsA2andA4wereidentiﬁedasbearinganisomeriza- tion,andoneandtwodeamidations,respectively,whiletheacidic variantsA3andA5were assignedashavingoneandtwo deamidations,respectively.[41]

AlsoaccordingtothedatapublishedbyBailey etal.,thebasic variantB2wasinsteadassignedashavingtwoisomerizations.[21] In all cases,the major protein glycoforms, consistingof G0F/G0F, G0F/G1F,G1F/G1ForG0F/G2F,andG1F/G2F,wereconsistentlyde- tectedforallthechargevariants(TableS5).Finally,theCEXprofile ofinfliximab(showninredinFigs.7aand8b)presentedthemost complex charge variant profile, consisting of four acidic variants (A1– A4),amainspecies(M),andthreebasicvariants(B1– B3).

Overall,theobservedCEXprofilewasinagreementwithwhathas alreadybeenreportedintheliteratureforvariousinfliximabsam- ples.[36,37,42,43] In addition,two infliximabbiosimilars,namely Remsima® and Inflectra® (Fig. 8b, grey and cyan lines, respectively),were alsoanalysedincomparisontotheoriginator(Remi- cade®)andshowedsimilarCEXprofilesintermsofdetectedvari- ants.Goingintothedetailsofthepeakassignment(TableS6),the threemainspecies(M,B1,andB3)correspondedtoadifferentex- tent of C-terminal Kmodifications (namely, 0,1 and2 K), while the acidicvariant A3correspondedto a deamidated variant.[36] Interestingly,thebasicvariantB2waselutedinbetweenthevari- antscontaining1and2K,althoughitwasbearing2Kitself.This behaviour was dueto the fact that the variant B2 wasaddition- allyseparatedbased on its“acidic” nature impartedby thepres-

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ence of sialylated glycans, as also reported elsewhere. [36,37,42] SimilarshiftsinretentionwerefoundforthevariantsA1(unmod- ified), A2 (1 C-terminal K), and A4 (deamidated), all due to the presence ofnegatively chargedglycans. Ofnote, the majordiffer- ence between the originator andthe two biosimilars wasrepre- sentedbytheextentoftheC-terminalKmodification.Infact,both biosimilarsshoweda higheramountof1Kspecies(increment of A2 andB1peaks) anda lower amountof 2Kspecies (slightde- crease ofB2 andB3 peaks) with respectto the originator. These findingswerealsoingoodagreementwithdatareportedbyBeyer atal.(comparisonoftheCEXprofiles ofRemicade® versusInflec- tra® and Remsima®). [37] Interestingly,based on theresults obtained byreleasedN-glycans analysis, itwaspossibleto establish thattheextentoftheglycansialylationwasdifferent,withthetwo biosimilarscontainingalargeramountofsuchspecies(FigureS9).

Indeed,itwouldseemthenthatthehigherpercentageofsialylated glycansinthebiosimilarsamplesweredetectedattheintactlevel.

4. Conclusions

Inconclusion,asimple,easy-to-useCEX-MSmethodwasdevel- opedfortheanalyticalcharacterizationofvariousbiopharmaceuti- calproducts.Ideally,amodern,shortCEXcolumnof50×2.1mm i.d. should be preferentially selected and used with equipment havingminimalextra-columndispersion.DespitethegoodMSsen- sitivityofferedbysuchcolumndimensions,itisimportanttokeep inmind thatthe columnvolume isverylimited, duetothenon- porous nature of the particles in CEX, so it is essential to work with a modern UHPLC system offering low extra-columnvolume to limitpeakbroadening. Intermsofmobilephase, ahighpurity buffercomposedof50mMammoniumacetatewith2%ofacetonitrile,titratedtopH5.0(mobilephaseA)and160mMammonium acetatewith2%ofacetonitrile,titratedtopH8.5(mobilephaseB) offersthebestcompromiseintermsofpHresponse,bufferstabil- ityovertimeandMScompatibility.Finally,theimportanceofusing tracemetalcertiﬁedthermoplasticstominimizelossofMSresolu- tion,sensitivity,andmassaccuracyduetometalcontaminantswas alsohighlighted.

Undertheseconditions,astraightforwardandrapidanalysis(in lessthan10min)ofchargevariantsbecomespossiblewithasim- ple, compact benchtop ToF/MS device. Even some relatively mi- nor peaks representing only one or two% relative abundance of the main species can be deconvoluted and accurately identified withthissetup.Varioustypesofmodifications wereidentifiedon commercialmAbproducts,suchastheformationofpyroglutamate fromN-terminalGluorGlnresidues,thepresenceofsialylatedgly- cans,andthetruncationofC-terminalLys.Ontheotherhand,the intactlevelCEX-MSanalysiswasnotsufficient forthe unambiguous characterizationofAsndeamidation(1 Dashift) andAsp isomerization (no massshift), andpeptide mapping analysisshould ideally be performedforconfirmation. However, an effectiveCEX separation infrontofthemassspectrometer couldhelp totenta- tively identifythesemodifications, sincedeamidationandisomer- izationareresponsiblefortwopredictableanddistinctchangesto theapparentpIofamAb.

DeclarationofCompetingInterest

Theauthorsdeclarethattheyhavenoknowncompetingﬁnan- cialinterestsorpersonalrelationshipsthatcouldhaveappearedto inﬂuencetheworkreportedinthispaper.

CRediTauthorshipcontributionstatement

Amarande Murisier: Investigation, Visualization, Writing – original draft. BastiaanL. Duivelshof: Investigation, Writing –

original draft. Szabolcs Fekete: Investigation, Writing – original draft, Writing – review & editing. Julien Bourquin: Re- sources, Writing – review & editing. Andrew Schmudlach: Re- sources.MatthewA.Lauber: Resources,Writing – review&editing.JenniferM. Nguyen: Resources.AlainBeck: Resources,Writ- ing – review & editing. Davy Guillarme: Conceptualization, Su- pervision, Writing – original draft, Writing – review & editing.

Valentina D’Atri: Project administration, Investigation, Visualiza- tion,Writing– originaldraft,Writing– review&editing.

Acknowledgements

The authors wish to thank Henri Kornmann and Kudzai Mu- tenda(FerringPharmaceuticals,Saint-Prex,Switzerland),fortheﬁ- nancialsupportofthiswork,andJean-Luc VeutheyfromtheUni- versityofGenevaforhisfruitfulcommentsanddiscussions.Rowan Moore andGuillaume Bechade (Waters Corporation) are also ac- knowledged for the loan of the BioAccord system used in this work.

Supplementarymaterials

Supplementary material associated with this article can be found,intheonlineversion,atdoi:10.1016/j.chroma.2021.462499. References

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