A low ligand density cation exchange (CEX) chromatography resin, Eshmuno® CP-FT resin, was investigated for the removal of aggregates from monoclonal antibody (mAb) feeds using a continuous loading process. Removing mAb aggregates with a CEX resin using continuous loading is advantageous relative to a bind/elute loading process.
Trang 1j ou rn a l h om ep a ge :w w w e l s e v i e r c o m / l o c a t e / c h r o m a
EMD Millipore Corporation, United States
a r t i c l e i n f o
Article history:
Received 26 November 2018
Received in revised form 7 April 2019
Accepted 8 April 2019
Available online 9 April 2019
Keywords:
Cation exchange chromatography
Frontal chromatography
Continuous loading chromatography
Overloaded chromatography
Monoclonal antibody aggregates
a b s t r a c t
Alowliganddensitycationexchange(CEX)chromatographyresin,Eshmuno®CP-FTresin,was investi-gatedfortheremovalofaggregatesfrommonoclonalantibody(mAb)feedsusingacontinuousloading process.RemovingmAbaggregateswithaCEXresinusingcontinuousloadingisadvantageousrelative
toabind/eluteloadingprocess,becausetheresincanusenearlyitsfullcapacitytobindtheaggregates enablingmuchhigherloadings.TheremovalofmAbaggregateswithEshmuno®CP-FTresinusinga con-tinuousloadingprocesswasfoundtobeconsistentwithafrontalchromatographymechanismwherethe mAbmonomerinitiallybindstothecolumnandissubsequentlydisplacedbydimersandhigher molec-ularweightaggregates.TheremovalofmAbaggregateswithEshmuno®CP-FTresinusingacontinuous loadingprocesswascomparedwithsixothercommerciallyavailablestrongCEXchromatographyresins andfoundtocorrelatewiththeirionicdensities,butnottheirmAbstaticbindingcapacities.Theinfluence
ofpH,conductivity,residencetime,andmAbconcentrationontheremovalofaggregateswithEshmuno® CP-FTresinusingacontinuousloadingprocesswasalsoinvestigated.Finally,thepercentageof aggre-gatesinamAbfeedwasvariedtoexaminetheeffectontheremovalofaggregateswithEshmuno®CP-FT resinusingacontinuousloadingprocess
©2019TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-ND
license(http://creativecommons.org/licenses/by-nc-nd/4.0/)
Itiscriticaltoremoveaggregatesduringthedownstream
purifi-cationofmonoclonalantibodies(mAbs)astheycanincreasethe
riskofanimmunogenicresponseinpatients[1–4].Unlikeother
impurities,suchashostcellproteinsandDNA,mAbaggregates
con-tainanFcbindingdomainandarenottypicallyseparatedfromthe
monomericproductduringaproteinAchromatographycapture
step[5,6 ThechromatographicseparationofmAbaggregatesfrom
themonomerisparticularlydifficult,astheyhavenearlyidentical
isoelectricpointsandhydrophobicities.Aggregatesaremost
com-monlyremovedwithbind/elutechromatographyprocessesusing
ionexchange,mixed-mode,hydrophobicinteraction,or
hydroxya-patitemedia[5,7,8 However,thereisinterestinthedevelopment
ofCEXchromatographyprocessesthatusecontinuousloadingfor
theremoval of mAb aggregates rather than bind/elute loading
[9–12].CEXchromatographyusingcontinuousloadingallowsthe
resintobeloadedwiththemAbfeeduntilitiscompletely
occu-piedbytheaggregates,whichissignificantlyhigherthanbind/elute
∗ Corresponding author.
E-mail address: matthew.stone@emdmillipore.com (M.T Stone).
processeswheretheresinmustbindboththemonomerand aggre-gates.Forinstance,ifamAbfeedcontaining10%aggregatescan
beloadedupto50g/Lwithabind/eluteloadingprocessusinga CEXresin,thenithasthepotentialtobeloadedupto500g/Lby
acontinuousloadingprocessassumingtheCEXresinhasa sim-ilarcapacity forboththemAbmonomerandaggregates.Higher loadingsoftheCEXresinareadvantageousbecausetheyrequire significantlysmallervolumesofbothresinandbuffershrinkingthe footprintofthemAbdownstreampurificationprocess.Continuous loadingprocessesusingCEXmediatopurifymAbfeedshavebeen previouslyreportedasoverloadedchromatography[9,10,13].We suggestthemorepreciselydefinedtermfrontalchromatographyas hasbeendescribedbyRachinskii[14],Jonsson[15],Hilletal.[16], andAhuja[17]todescribethemechanismofseparationobserved
intheseprocesses
Frontal chromatography is characterized by the continuous loadingofthecolumnunderconditionswhereallthecomponents
ofamixturewillbindwiththeresin[17].Thismechanism sepa-ratesthecomponentsofamixtureintofrontsbasedontheirrelative strengthofinteractionwiththeresin[14,15].Theweakest interact-ingcomponentwillelutefromthecolumnfirstinapureform.The nextfrontelutedfromthecolumnwillbecomposedoftheweakest interactingcomponentplusthenextstrongestinteracting compo-https://doi.org/10.1016/j.chroma.2019.04.020
0021-9673/© 2019 The Authors Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.
Trang 2aggre-gates.Inpractice,somemonomerislikelytostillberetainedby
thecolumnaftertheaggregatesbegintoelute.However,agood
monomerrecoverycanstillbeachievedbythismethod,because
athigherloadingstheamountofmonomerretainedisonlyasmall
percentageofthetotalmonomerprocessed
Herein,wereportourinvestigationintoalowionicdensityCEX
resin,Eshmuno® CP-FTresin,fortheremovalofmAbaggregates
usingacontinuousloadingprocess.Tounderstandiftheremoval
ofaggregateswithEshmuno® CP-FTresinusingcontinuous
load-ingwasconsistentwithafrontalchromatographymechanismwe
measuredthecompositionofboththeelutedandretained
com-ponentsas theloadingoftheresin wasvaried.The removalof
aggregates froma mAb feedwithEshmuno® CP-FTresin using
acontinuousloadingprocesswasthencomparedwithsixother
commerciallyavailablestrongCEXchromatographyresinsandthe
percentageofaggregatesintheelutionpoolwerecomparedtothe
ionicdensityandmAbstaticbindingcapacityoftheresins.In
addi-tion,theinfluenceofseveraldifferentprocessconditionsincluding
pH,conductivity,residencetime,andmAbfeedconcentrationwere
examinedtounderstandhoweachofthesefactorsinfluencedthe
removalofaggregateswithEshmuno®CP-FTresinusinga
continu-ousloadingprocess.Finally,weexaminedhowtheremovalofmAb
aggregateswithEshmuno®CP-FTresinusingacontinuousloading
processwasinfluencedbythepercentageofaggregatesinthemAb
feed
2.1 Materials
2.1.1 EnrichmentofmAb05andmAb02feedswithaggregatesby
highpHhold
AhighpHholdprocesswasusedtoinduceaggregatesforboth
themAb05andmAb02feeds.Theprocesswasmodifiedfromthe
procedurereportedbyPottyandXenopoulos[18].First,internally
generated mAb-containing Chinese hamster ovary cell cultures
wereclarifiedandsubjectedtoproteinAcapturechromatography
TheresultingproteinAelutionpoolwasthenadjustedtoaneutral
pH.ThemAbconcentrationoftheelutionpoolrangedfrom10g/L
to20g/L.TheneutralizedmAbsolutionsweregentlystirredand
5MsodiumhydroxidewasaddeddropwiseuntilthesolutionpH
reached11.0.CarewastakentoavoidincreasingthesolutionpH
above11.0,whichcouldcausesignificantdegradationofthemAb
protein.ThemAbsolutionwasheldatpH11.0for30minandthen
1.0Mhydrochloricacidwasaddeddropwiseuntilthesolutionwas
reducedtopH5.0.Thepercentageofaggregatesintheresulting
solutionwasdeterminedbyanalyticalsize-exclusion
chromatog-raphy.ThepHcyclingprocedurewasrepeateduptofourtimesuntil
thedesiredpercentageofaggregateswasobtained.Theresulting
mAbsolutionwasthendialyzedintothedesiredbuffer.Thisprocess
wasfoundtogeneratevariablepercentagesofaggregates.The
per-lognumber:1335906,ThermoFisherScientificInc.,Waltham,MA,
02451,USA),Toyopearl® GigacapS-650M(100mL,partnumber:
0021833,TosohCorporation,Minato-Ku,Tokyo,105–8623,Japan), Capto®SImpAct(100mL,productnumber:17371702,GE Health-careBio-SciencesAB,Uppsala,Sweden),andSPSepharoseTMFast Flow (300mL,product number: 17072901, GE, Healthcare Bio-SciencesAB,Uppsala,Sweden)
2.2 Methods 2.2.1 Standardprocedureforremovalofaggregatesusingfrontal chromatography
Forallexperiments,aglasschromatographycolumn(Omnifit Benchmark Column6.6mm/100mm, 6.6mmdiameter, 100mm length, SKU: 006BCC-06-10-AF, Diba Industries, Danbury, CT
06810,US)waspackedtoaheightof3cmwith1.0mLoftheCEX chromatographyresin.Continuousloadingchromatography exper-imentswereperformedusingaGEHealthcareLifeSciencesÄKTA avant25.Beforeeachexperiment,thecolumnswereequilibrated withthesamebufferasthatofthemAbfeedsolutionfor10CV Beforeacolumnwasreusedforadditionalexperimentsitwasfirst washedwiththeloadingbufferfor10CV,strippedwiththeloading bufferalsocontaining1.0Msodiumchloridefor15CV,cleanedwith 0.5Msodiumhydroxidefor5CV,andequilibratedwithloading bufferfor15CV
2.2.2 Analyticalsizeexclusionchromatography Analyticalsize-exclusionchromatographyofproteinswas per-formedusingaWaters2695SeparationModule,aWatersDual AbsorbanceDetector,andaTosohBiosciencesTSKgelG3000SWxl column(partnumber:08541,columnsize:300×7.8mm,Tosoh BioscienceLLC,KingofPrussia,PA,USA).Theisocraticmobilephase wasasolutionof50mMsodiumphosphateand150mMsodium chlorideatpH7.0.Thecolumnwasrunataflowrateof1.00mL/min for20minandtheUVdetectorwassettoawavelengthof280nm Thepercentageofaggregateswascalculatedbasedontheareasof theHPLCpeaks
2.2.3 UVspectroscopicanalysisofproteinconcentration
UV spectroscopic analysis of protein solution concentration was performed with a Thermo Scientific GENESYS 10S UV–vis Spectrophotometer.TheconcentrationofthemAb05andmAb02 fractions was determined by measuring their absorbance at
280nmina disposableplasticcellhavinga 1.0cm pathlength Theabsorbancewasdividedbytheextinction coefficientof the mAbs (mAb05=1.419mL·g−1·cm−1,mAb02=1.467mL·g−1·cm−1) and the pathlength tocalculate the protein concentration.The monomerrecoverywascalculatedbasedontheconcentrationof themAbinafractionasdeterminedbyUVspectroscopic analy-sisandthepercentageofmonomerinafractionasdeterminedby analyticalsize-exclusionchromatography
Trang 3Fig 1. SEC chromatograms of individual fractions of a mAb05 feed processed through a 1.0 mL packed column of Eshmuno®CP-FT resin at a residence time of 3 min at various loadings (left) The mAb05 feed for this experiment examining the composition of proteins in the elution fractions had a concentration of 15 g/L with 10% total aggregates (5% dimers) and was dialyzed into a buffer composed of 100 mM sodium acetate at pH 5.0 and a conductivity of 5.0 mS/cm SEC chromatograms of high salt elutions from a 1.0 mL packed column of Eshmuno ®
CP-FT resin loaded with various amounts of the mAb05 feed at a residence time of 3 min (right) The mAb05 feed used for this experiment examining the composition of proteins that were retained by the column had a concentration of 16 g/L with 11% total aggregates (5% dimers) and was dialyzed into a buffer composed of 100 mM sodium acetate at pH 5.0 and a conductivity of 5.0 mS/cm.
2.2.4 DeterminationofCEXresinionicdensity
A1mLportionofgravitysettledCEXresinin20%ethanolwas
measuredinasmallplasticcolumn.Toremovetheethanolfromthe
CEXresinitwasthreetimessuspendedin5mLofwaterandthen
thewaterwasremovedbysuctionfromthecolumn.Thesulfonate
groupontheCEXresinwasconvertedtoasulfonicacidgroupby
threetimessuspendingtheresinin5mLof1.0Mhydrochloricacid
for5minandthenremovingthehydrochloricacidbysuction.To
removetheremaininghydrochloricacidtheresinsweresuspended
in5mLofwaterthatwasthenremovedbysuctionandthisprocess
wasrepeateduntilthepHofthewaterremovedwasneutral.The
CEXresinwasthentransferredintoa200mLglassbeaker.Tothe
glassbeakerwasalsoadded80mLof1.0Msodiumchlorideand
a1.0mLsolutionofphenolphthaleinataconcentrationof1%by
weightinethanol.Thesolutionwastitratedwitha0.01Msolution
ofsodiumhydroxideandtheend ofthetitration wasindicated
whenthesolutionchangedtoapinkcolor.Theionicdensityofthe
CEXresinwascalculatedbythefollowingformula:
ionicdenisity= VNaOH×CNaOH
Vresin
In this equation VNaOH is the volume of sodium hydroxide
titratedintothesuspensionofCEXresin,CNaOHistheconcentration
ofsodiumhydroxide,andVresinisthevolumeoftheresinthatwas
titrated.TheionicdensityoftheCEXresinsistheaverageoftwo
separatemeasurements
2.2.5 DeterminationofCEXresinstaticbindingcapacityfor
mAb05
A1mLportionofgravitysettledCEXresinin20%ethanolwas
measuredinasmallplasticcolumn.Toremovetheethanolfrom
theCEXresinitwasthreetimessuspendedinanacetatebuffer
composedof100mMsodiumacetateatpH5.0and5.0mS/cmand
thentheacetatebufferwasremovedbysuction.TheCEXresinwas
addedtoa50mLcentrifugetube.Tothecentrifugetubewasalso
added9mLoftheacetatebuffertogivea10%resinslurry.A1.0mL
portionofthe10%resinslurrywasaddedtoa15mLcentrifugetube
Tothe15mLtubewasalsoadded1.5mLoftheacetatebufferand
2.5mLofamAB05solutionataconcentrationof10g/Lwith0.4%
aggregatesthatwasdialyzedintotheacetatebuffer.Theresulting
slurrycontained0.1mLofCEXresinandmAb05ataconcentration
of5g/L.Controlsomittingtheresinwerepreparedbyadding2.5mL
oftheacetatebufferand2.5mLofthedialyzedmAB05solutionto
15mLtubes.The15mLtubescontainingCEXresinandthecontrol
tubeswererotatedfor4h.The15mLtubeswerethensubjected
tocentrifugeandaportionofthesupernatantmAb05solutionwas diluted20-fold.TheUVabsorbanceofthe20-folddilutedsolution
at280nmwasdeterminedasdescribedinSection2.2.3.Theprocess
ofpreparinga20-folddilutionandmeasuringtheUVabsorbance
ofthesolutionat280nmwasperformedintriplicateforeachtube andtheaverageofthethreemeasurementswasusedtocalculate themAb05concentration.ThestaticbindingcapacityoftheCEX resinformAb05wasthendeterminedbythefollowingformula: staticbindingcapacity
=(Vcontrol×Ccontrol)−(Vresintreated×Cresintreated)
Vresin
InthisequationVcontrolandVresintreatedarethetotalvolumeof themAb05controlsolutionsandthetotalvolumeofthemAb05 solutionstreatedwiththeresin,respectively.TheCcontrolandthe
Cresintreated aretheconcentrationofmAb05inthecontroltubes aftertheywererotatedfor4handtheconcentrationofmAb05in thetubes treatedwiththeresinaftertheywererotatedfor 4h, respectively.Vresin isthevolumeoftheresinaddedtotheresin treatedsolutions.ThestaticbindingcapacityoftheCEXresinsfor mAb05istheaverageoftwoseparatepreparationsoftheresin slurrythatwereeachmeasuredintriplicate
3.1 Mechanismfortheremovalofaggregates
WeinvestigatedtheremovalofaggregateswithEshmuno®
CP-FTresin usingacontinuousloadingprocesstodetermineifthe separation ofthemAbmonomer fromtheaggregates was con-sistentwithafrontalchromatographymechanism.AmAb05feed containing10% aggregates wasdialyzedinto a100mMsodium acetatebufferatpH5.0and5mS/cmthenloadedontoacolumnof theEshmuno®CP-FTresin.Thecompositionofthefractionseluted fromthecolumnwereanalyzedbysizeexclusionchromatography (Fig.1,left).Initially,onlythemonomerwasobservedtoelutefrom thecolumn.Ataloadingof600g/L,dimerswerealsodetectedin theelution.However,nohighermolecularweightaggregateswere observeduptoaloadingof1000g/Lwheretheexperiment was ended.Theseresultsareconsistentwithafrontalchromatography mechanisminwhichpuremonomerelutedinthefirstfrontand thenthedimerscoelutedwiththemonomerinasecondfront.The Eshmuno® CP-FTresinwasnotloadedwithasufficient amount
ofthemAb05feedtoobserveathirdfront thatwouldbe
Trang 4com-Fig 2.The cumulative percentage of aggregates in the elution pool as a function of mAb05 loading (left) and the cumulative percentage of aggregates in the elution pool as a function of the cumulative mAb05 monomer recovery in the elution pool (right) The mAb05 feed was processed through a 1.0 mL packed column of a CEX chromatography resin at a residence time of 3 min The mAb05 feed had a concentration of 15 g/L with 11% total aggregates (5% dimers) and was dialyzed into a buffer composed of 100 mM sodium acetate at pH 5.0 and a conductivity of 5.0 mS/cm CEX media legend: C1 - Eshmuno ®
CPX resin, C2 – Poros ®
XS, C3 – Poros ®
50 HS, C4 - Toyopearl ®
Gigacap S-650 M, C5 – Capto®S ImpAct, C6 – SP Sepharose TM Fast Flow The percentages included on the plots in black font indicate the cumulative monomer recovery at the last fraction before the cumulative percentage of aggregates exceeded 1%.
posedofthehighermolecularweightaggregatesalongthewith
themonomeranddimers
Next,weexaminedthecomponentsthatwereretainedbythe
Eshmuno® CP-FTresinafteritwasloadedwithvariousamounts
ofamAb05feedcontaining11%aggregatesthatwasdialyzedinto
a100mMsodiumacetatebufferatpH5.0and 5mS/cm.Aftera
specificloadingofthecolumnwascompletedthecomponentsof
themAb05feedretainedbythecolumnwereelutedina
subse-quentstepusingahighsaltbuffer.Wefoundthatastheloadingof
themAb05feedwasincreased,theamountofmonomerretained
bythecolumndecreasedandtheamountofaggregatesincreased
(Fig.1,right).Forexample,ataloadingof200g/L,the
composi-tionofmAb05feedthatwasretainedbythecolumnconsistedof
71%monomer whileata loadingof 1000g/L, thepercentage of
monomerretainedbythecolumndecreasedto9%.The
composi-tionoftheaggregatesthatwereretainedbythecolumnincluded
dimersandhighermolecularweightaggregates.Theseresultsare
consistentwithafrontalchromatographymechanism,inwhichthe
mAbmonomerisinitiallyretainedbythecolumnandsubsequently
displacedbyaggregates
3.2 RemovalofmAbaggregatesbycontinuousloadingofvarious
CEXchromatographyresins
TheabilityofEshmuno®CP-FTresintoremoveaggregatesusing
acontinuousloadingprocesswascomparedwithsix
commercially-available strong CEX chromatography resins having sulfonate
ligands.We selecteda solutionpHof5.0 anda conductivityof
5mS/cmbecauseasulfonateCEXresinwilltypicallyhaveahigh
capacityforthemAbmonomerandaggregatesunderthese
solu-tion conditions A mAb05 feed containing 11% aggregates was
dialyzedinto100mMsodiumacetateatpH5.0and5mS/cm,loaded
ontotheCEXcolumn,and thecompositionsoftheelution
frac-tionsweredetermined Under thesesolution conditionsfive of
theCEXresinsincludingEshmuno® CP-FTresin,Eshmuno® CPX
resin,Poros® XS,Poros® 50HS,andToyopearl® GigacapS-650M
showedagradualbreakthroughoftheaggregatesasisconsistent
withafrontalchromatographymechanism(Fig.2).Eshmuno®
CP-FTresinisanoutlierofthesefiveasitremovedsignificantlymore
aggregateswithabettermonomerrecovery.Theelutionpoolfor
Eshmuno®CP-FTresinhad0.8%aggregateswitha92%monomer
recoveryataloadingof741g/L.Bycontrast,theelutionpoolfor
theotherCEXchromatographyresinsallexceeded1%aggregates
beforetheirmonomerrecoveriesreached50%.Capto® SImpAct
andSPSepharoseTMFastFlowdidnotshowagradualincreasein
thecumulativepercentageofaggregatesintheelutionpoolasis
expectedforafrontalchromatographymechanism.This
observa-Table 1
Ionic density and static binding capacity for mAb05 determined for the CEX resins Note that the percentage of aggregates in the elution pool corresponds to a resin loading of approximately 1000 g/L However, there were variations in the final load-ing of the CEX resins which ranged from 967 g/L to 1000 g/L.
(eq/mL)
static binding capacity for mAb05 (g/L)
aggregates in elution at loading of
˜
1000 g/L Eshmuno ®
CP-FT resin
Capto ® S ImpAct
Eshmuno ®
CPX resin
Toyopearl® Gigacap S-650M
SP Sepharose TM Fast Flow
beads
ofaCEXresinwasdeterminedbyconvertingthesulfonategroups
tosulfonicacidsandthentitratingtheacidifiedresinwithsodium hydroxideinthepresenceofaphenolphthaleinindicator.Thestatic bindingcapacityoftheCEXresinswasmeasuredusingamAb05 feeddialyzedinto100mMsodiumacetateatpH5.0and5mS/cm
Weplottedthecumulativepercentageofaggregatesintheelution poolataloadingofapproximately1000g/Lasafunctionofboth variables(Fig.3)
Wefoundthattherewasaroughcorrelationbetweentheionic densityandthepercentageofaggregatesintheirelutionpool.For instance,Eshmuno®CP-FTresinhadthelowestionicdensityat37
eq/mLandhadthelowestpercentageofaggregatesinthe cumu-lativeelutionpoolat1.9%.Eshmuno® CPXresin,Poros® XS,and Poros® 50HShadintermediateionicdensitiesof70–81eq/mL andverysimilarpercentagesofaggregatesintheelutionpoolof 7.5–7.8% Toyopearl® GigacapS-650Mand SPSepharoseTMFast Flowhadthehighestionicdensitiesof188–210eq/mLandthe highestpercentagesofaggregatesintheelutionpoolof8.8–9.7%
Trang 5Fig 3. The cumulative percentage of aggregates in the elution pool at a loading of approximately 1000 g/L as a function of the ionic density of the CEX resin (left) and the cumulative percentage of aggregates in the elution pool at a loading of approximately 1000 g/L as a function of the static binding capacity of the CEX resin for mAb05 (right) The CEX static binding capacity was measured with a mAb05 feed at a concentration of 5 g/L with 0.4% aggregates that was dialyzed into a 100 mM sodium acetate buffer at
pH 5.0 and a conductivity of 5.0 mS/cm.
Fig 4.The cumulative percentage of aggregates in the elution pool as a function of mAb05 loading (top left) or mAb02 loading (bottom left) and the cumulative percentage
of aggregates in the elution pool as a function of the cumulative mAb05 monomer recovery in the elution pool (top right) or the cumulative mAb02 monomer recovery in the elution pool (bottom right) The feeds were processed through a 1.0 mL packed column of Eshmuno ® CP-FT resin at a residence time of 3 min The mAb05 feed had a concentration of 12 g/L with 10% total aggregates (6% dimers) and was dialyzed into a buffer composed of 50 mM sodium acetate at pH 4.5, pH 5.0, pH 5.5, or pH 6.0 The mAb02 feed had a concentration of 13 g/L with 6% total aggregates (4% dimers) and was dialyzed into 50 mM sodium acetate at pH 4.0, pH 4.5, or pH 5.0 The conductivities
of the acetate buffers for the mAb05 and mAb02 feeds were adjusted to 5.0 mS/cm and 2.5 mS/cm respectively by the addition of sodium chloride The percentages included
on the plots in black font indicate the cumulative monomer recovery at the last fraction before the cumulative percentage of aggregates exceeded 1%.
Capto®SImpActdoesnotfollowthistrendasithasan
intermedi-ateionicdensityof64eq/mL,butstillhasahighestpercentages
ofaggregatesintheelutionpoolof8.9–9.7%.However,itmightnot
beappropriatetocomparethedependenceofCapto®SImpActand
SPSepharoseTMFastFlowiftheyarenotoperatingaccordingto
afrontalchromatographymechanismasissuggestedbyshapeof
theircurvesinFig.2
WedidnotfindacorrelationbetweenthemAb05static
bind-ingcapacityoftheCEXresinsandthepercentageofaggregatesin
theirelutionpool.Forinstance,SPSepharoseTMFastFlow,Poros®
XS,Eshmuno® CP-FTresin,andEshmuno®CPXresinallhadvery
similarmAb05 staticbinding capacitiesof 66g/L, 68g/L,69g/L,
and 71g/L respectively, but varied significantly in the
percent-ageof aggregates in theirelutions Neither Poros® 50 HS that
had the lowest static binding capacity of 50g/L or Toyopearl®
GigacapS-650Mthat hadthehigheststaticbindingcapacity of
100g/Lshowedthelowestpercentageofaggregatesintheirelution pools
3.3 InfluenceofsolutionpH Theinfluence of solutionpHonremoval of aggregates with Eshmuno® CP-FTresin using a continuous loadingprocess was investigatedwithbothamAb05feedandamAb02feed.AmAb05 feedcontaining10%aggregatesandamAb02feedcontaining6% aggregatesweredialyzedintoacetatebuffersthatvariedinpH.We observedforboththemAb05andmAb02feedsthatasthe solu-tionpHwasloweredmoreaggregateswereremovedwithahigher monomerrecovery(Fig.4).Basedontheirisoelectricpoints,both mAb05(pI=8.1)andmAb02(pI=8.24)aremorestronglycharged
atalowersolutionpH.Thustheremovalofaggregateswasmost efficientatalowersolutionpHwheretheelectrostaticinteractions
Trang 6Fig 5. The cumulative percentage of aggregates in the elution pool as a function of mAb05 loading (top left) or mAb02 loading (bottom left) and the cumulative percentage
of aggregates in the elution pool as a function of the cumulative mAb05 monomer recovery in the elution pool (top right) or the cumulative mAb02 monomer recovery in the elution pool (bottom right) The feeds were processed through a 1.0 mL packed column of Eshmuno®CP-FT resin at a residence time of 3 min The mAb05 feed had a concentration of 12 g/L with 11% total aggregates (6% dimers) and was dialyzed into a buffer composed of 50 mM, 100 mM, 150 mM, or 200 mM sodium acetate at pH 5.0 having a conductivity of 2.8 mS/cm, 4.7 mS/cm, 6.2 mS/cm, or 8.2 mS/cm respectively The mAb02 feeds had a concentration of 13 g/L with 6% total aggregates (4% dimers) and was dialyzed into a 50 mM sodium acetate buffer at pH 4.0 having a conductivity of 2.5 mS/cm, 5.0 mS/cm, 7.0 mS/cm, or 9.0 mS/cm The conductivity of the buffers used for dialysis of the mAb02 feed were adjusted by the addition of sodium chloride The percentages included on the plots in black font indicate the cumulative monomer recovery
at the last fraction before the cumulative percentage of aggregates exceeded 1%.
betweenthepositivelychargedmAbmonomer/aggregatesandthe
negativelychargedresinarestrongest
3.4 Influenceofsolutionconductivity
Theinfluenceofsolutionconductivityontheremovalof
aggre-gates with Eshmuno® CP-FT resin using a continuous loading
processwasinvestigatedwithbothamAb05feedandamAb02
feed.AmAb05feedcontaining10%aggregatesandamAb02feed
containing6%aggregatesweredialyzedintoacetatebuffersthat
variedinconductivity.Wefoundthatasthesolution
conductiv-itywasdecreased,moreaggregateswereremovedfromboththe
mAb05feedandthemAb02feedwithahighermonomer
recov-ery(Fig.5).However,therewasasignificantdeparturefromthis
trendforthemAb05feedat2.8mS/cminwhichlessaggregates
wereremovedwithalowermonomerrecoverythanwasobserved
forthethreeothermAb05feedshavinghighersolution
conductiv-ities.Onepotentialexplanationforthisoutliercouldbethatthe
mAb05monomeris toostronglyboundtotheresinatthis low
solutionconductivityinhibitingdisplacementbytheaggregatesas
isrequiredforanefficientseparationwithafrontal
chromatogra-phymechanism.Nosuchexceptionwasobservedfortheremovalof
aggregatesfromthemAb02feedswherethemostefficientsolution
conditionwasatthelowestconductivityof2.5mS/cm
3.5 Influenceofresidencetime
Theinfluenceofresidencetimeontheremovalofaggregates
withEshmuno® CP-FTresinusing acontinuousloadingprocess
wasinvestigatedusingamAb05feedcontaining11%aggregates
Weobservedthatastheresidencetimewasincreasedmore
aggre-gateswereremovedfromthemAb05feedwithahighermonomer
recovery(Fig.6).Longerresidencetimesaretolikelyresultinthe moreefficientremovalofaggregateswithafrontal chromatogra-phymechanismbecausemasstransferoftheaggregatesintothe resinlimitsdisplacementoftheboundmonomers.However,longer residencetimesarenotdesirableastheywillrequirelonger load-ingtimes.Forinstance,increasingtheresidencetimefrom3min
to6minincreasedloadingtimeforthemAb05feedfrom3.3hto 6.7h
3.6 InfluenceofmAbfeedconcentration TheinfluenceofthemAbfeedconcentrationontheremovalof aggregateswithEshmuno®CP-FTresinusingacontinuousloading processwasinvestigatedwithamAb05feedcontaining10% aggre-gates.WeobservedthatastheconcentrationofthemAb05feedwas decreasedmoreaggregateswereremovedwithahighermonomer recovery(Fig.7).However,loweringtheconcentrationofthemAb feedisnotdesirable,becauselongerloadingtimesarerequiredto processthelargervolumesofthemAbfeed.Forinstance,decreasing theconcentrationofthemAb05feedfrom15g/Lto5g/Lincreased theloadingtimefrom3.3hto10h
3.7 InfluenceofthepercentageofaggregatesinthemAbfeed
Toexaminetheinfluenceofthepercentageofaggregatesinthe mAbfeedontheremovalofaggregateswithEshmuno®CP-FTresin usinga continuousloadingprocess,sixmAb05 feedswere pre-paredwithvaryingpercentagesofaggregatesrangingfrom1.9%
to14.6%.WeobservedforallsixmAb05feedsthataspecific load-ingofthefeedcouldbeselectedwherethelevelofaggregatesin theelutionpoolwasreducedbelow1%withamonomerrecovery greaterthan85%(Fig.8,topleftandright).Wealsoobservedthat
Trang 7Fig 6. The cumulative percentage of aggregates in the elution pool as a function of mAb05 loading (left) and the cumulative percentage of aggregates in the elution pool
as a function of the cumulative mAb05 monomer recovery in the elution pool (right) The feed was processed through a 1.0 mL packed column of Eshmuno®CP-FT resin at
a residence time of 1 min (180 cm/h), 2 min (90 cm/h), 3 min (60 cm/h), or 6 min (30 cm/h) The mAb05 feed had a concentration of 15 g/L with 11% total aggregates (5% dimers) and was dialyzed into a buffer composed of 100 mM sodium acetate at pH 5.0 and a conductivity of 5.0 mS/cm The percentages included on the plots in black font indicate the cumulative monomer recovery at the last fraction before the cumulative percentage of aggregates exceeded 1%.
Fig 7.The cumulative percentage of aggregates in the elution pool as a function of mAb05 loading (left) and the cumulative percentage of aggregates in the elution pool as a function of the cumulative mAb05 monomer recovery in the elution pool (right) The mAb05 feed was processed through a 1.0 mL packed column of Eshmuno ®
CP-FT resin
at a residence time of 3 min The mAb05 feed had an initial concentration of 15 g/L with 10% total aggregates (6% dimers) and was dialyzed into a buffer composed of 100 mM sodium acetate at pH 5.0 and a conductivity of 5.0 mS/cm Portions of the mAb05 feed were diluted to 5 g/L and 10 g/L with the dialysis buffer The percentages included on the plots in black font indicate the cumulative monomer recovery at the last fraction before the cumulative percentage of aggregates exceeded 1%.
Fig 8.The cumulative percentage of aggregates in the elution pool as a function of mAb05 loading (top left) and the cumulative percentage of aggregates in the elution pool as a function of the cumulative mAb05 monomer recovery in the elution pool (top right) The cumulative percentage of aggregates in the elution pool as a function of the loading of mAb05 aggregates (bottom) The mAb05 feeds were processed through a 1.0 mL packed column of Eshmuno ®
CP-FT resin at a residence time of 3 min The mAb05 feeds had a concentration of 15 g/L with 1.9%, 3.7%, 7.3%, 10.4% 12.2%, or 14.6% total aggregates and were dialyzed into a 100 mM sodium acetate buffer at pH 5.0 and a conductivity of 5.0 mS/cm The percentages included on the plots in black font indicate the cumulative monomer recovery at the last fraction before the cumulative percentage of aggregates exceeded 1%.
Trang 8plotshowsthatallsixfeedshadasimilarshapethatisconsistent
withafrontalchromatographymechanismwheretheaggregates
shouldnotbegintoeluteuntiltheyhaveexceededthecapacityof
theEshmuno®CP-FTresin
WealsonotedthatasthepercentageofaggregatesinthemAb05
feedwasincreased,theeffectiveloadingrangefortheremovalof
aggregates becamenarrower(Table2).For thisexperiment, we
definedtheeffectiveloadingrange asstartingwhen the
cumu-lativemonomerrecoveryin theelutionpool exceeded85%and
endingwhenthecumulativepercentageofaggregatesexceeded
1%.Theeffectiveloadingrangefortheremovalofaggregateswas
foundtoincreaseasthepercentageofaggregatesinthefeedwas
decreased.Theeffectiveloadingrangesforthe1.9%,3.7%,and7.3%
feedswerenotfullydetermined,asthepercentageofaggregates
intheelutionpoolremainedbelow1%ata loadingof1000g/L
Theeffectiveloadingrangeforthe1.9%and3.7%feedsarelikely
toextendsignificantlybeyond1000g/Lasthepercentageof
aggre-gatesintheelutionpoolwasonly0.2%and0.3%respectivelywhen
theexperimentended
First, wesought toconfirmthat Eshmuno® CP-FT resin was
removingaggregates froma mAb05 feedaccording toa frontal
chromatographymechanismwhenusingacontinuousloading
pro-cess.The composition ofthe mAb05 feedthat eluted fromthe
Eshmuno® CP-FTresinaswellasthecompositionofthemAb05
feedthatwasretainedbythecolumnwasdeterminedastheloading
wasvaried(Fig.1).WeobservedthatthemAb05monomereluted
fromthecolumnintheearliestfractionsandthedimersdidnot
begintoeluteuntil600g/L.Atlowerloadingsthecompositionof
mAb05feedthatwasretainedbythecolumnconsistedprimarilyof
monomer,butastheloadingwasincreasedtheretainedmonomer
wasdisplacedbydimersandhighermolecularweightaggregates
[14,16].Wealsonotedthatdimersweretheonlytypesof
aggre-gates observed in the elutionfractions while higher molecular
weightaggregateswerecompletelyretainedbythecolumn.This
suggeststhatthehighermolecularweightaggregatesareforming
athirdfrontthathasyettoelutefromthecolumnattheendof
theexperiment.Theresultsofbothexperimentsindicatethe
sep-arationofthemAbmonomerfromtheaggregateswithEshmuno®
CP-FTresinusingacontinuousloadingprocessisconsistentwitha
frontalchromatographymechanism
Next,wecomparedtheremovalofaggregateswithEshmuno®
CP-FT resin using a continuous loading process from a mAb05
feedwithsixcommerciallyavailablestrongCEXchromatography
resinshavingsulfonateligands(Fig.2).Wechosetocomparethe
strongCEXresinsat asolutionpHof 5.0 anda conductivityof
5mS/cm where they shouldhave a highcapacity for themAb
aggregates.Ifa CEXresinefficientlyremovesaggregates froma
tion conditions,then very highloadingsof theresin should be possible.Eshmuno®CP-FTresin,Eshmuno®CPXresin,Poros® XS, Poros®50HS,andToyopearl®GigacapS-650Mshowedagradual increaseinthepercentageoftheaggregatesintheelutionpoolas
isexpectedwithafrontalchromatographymechanism.Eshmuno® CP-FTresinremovedsignificantlymoreaggregateswithahigher monomer recovery than theother CEXchromatography resins Capto®SImpActandSPSepharoseTMFastFlowshowedan imme-diatebreakthroughofaggregatesintheelutionpoolindicatingthat theyarenotremovingaggregatesaccordingtoafrontal chromatog-raphymechanismunderthesesolutionconditions.ThesetwoCEX resinsarebothcomposedofanagarosebasebeadandwe spec-ulatethatthisfactormayberesponsiblefor inhibitinga frontal chromatographymechanismunderthesesolutionconditionsasall theotherCEXresinsarecomposedofpolymerbasebeads.The per-centageofaggregatesintheelutionpoolforallsevenCEXresins wasplottedasafunctionoftheirionicdensityandtheirmAb05 staticbindingcapacity(Table1,Fig.3).Alowpercentageof aggre-gatesintheelutionpoolwasfoundtocorrelatewithalowionic densitywhilenocorrelationwasobservedwiththestaticbinding capacitiesoftheCEXresinsformAb05.AlowerionicdensityCEX resinmayfacilitateefficientremovalofmAbaggregatesbyfrontal chromatographybecauseitlikelyhasfewerelectrostatic interac-tionswiththemonomerallowingittobemoreeasilydisplacedby theaggregates
Itisimportanttonotethatwecomparedtheremovalof aggre-gateswithCEXresinsusingacontinuousloadingprocessatasingle solutionconditionwhereCEXresinstypicallyhavehighcapacities forthemAbmonomerandaggregates.CEXresinswithhigherionic densitiesmayremoveaggregatesmoreefficientlyatahigher solu-tionpHand/orconductivitywherethestrengthoftheelectrostatic interactionbetweenpositivelychargedmAbmonomer/aggregates andthenegativelychargedresinwillbeweaker.However, operat-ingaCEXresinatahigherpH/conductivitywillreduceitscapacity formAbaggregatesandthuslimittheamountofthemAbmonomer thatcanbepurifiedbycontinuousloadingbeforeelutionof aggre-gateswilloccur
ThenweexaminedtheinfluenceofsolutionpHandconductivity
ontheremovalofaggregateswithEshmuno®CP-FTresinusinga continuousloadingprocessfrombothamAb05feedandamAb02 feed.Wefoundthatmoreaggregateswereremovedwithhigher monomerrecoveriesatlowersolutionpHs(Fig.4)and conductiv-ities(Fig.5 whichfavorstrongelectrostaticinteractionsbetween thepositivelychargedmAbmonomer/aggregatesandthe nega-tivelychargedresin.Oneexceptiontothistrendwasobservedwith mAb05 at2.8mS/cm,whereEshmuno® CP-FTresinwas signifi-cantlylessefficientfortheremovalofaggregatesthantheother higherconductivitiesmAb05feedsinvestigated.Oneexplanation
isthatatasolutionconductivityof2.8mS/cmthemAb05monomer
istoostronglyboundtotheEshmuno®CP-FTresinthuspreventing displacementbyaggregatesandinhibitingseparationbyafrontal chromatographymechanism[12].Liu,etal.alsoobservedthatthe removalofaggregatesfromamAbfeedusingPoros® 50HSCEX
Trang 9effi-cientasthesolutionconductivitywasdecreasedfrom18mS/cmto
5mS/cm,howeverat3mS/cmtheremovalofaggregateswas
sig-nificantlyworse[10].However,nosuchexceptionwasobservedfor
themAb02feedwheretheremovalofaggregateswasmostefficient
atthelowestconductivityof2.5mS/cm
Wealsoinvestigatedtheinfluenceoftheflow-rateandthemAb
feedconcentrationontheremovalofaggregatesfromamAb05feed
withEshmuno®CP-FTresinusingacontinuousloadingprocess.The
removalofaggregateswasfoundtobemostefficientatlonger
res-idencetimes(Fig.6)andlowermAbfeedconcentrations(Fig.7)
Liu,etal.alsoreportedthattheremovalofaggregateswithPoros®
50HSresinusingacontinuousloadingprocesswasmostefficient
atlongerresidencetimes[10] Longerresidentstimesarelikely
tobeadvantageousfortheremovalofaggregatesusingafrontal
chromatographymechanismbecausetheygivemoretimeformass
transferoftheaggregatesintotheresin.However,usinglonger
res-idencetimesandlowermAbconcentrationswillalsoincreasethe
resinloadingtimeandthuscoulddecreasetheproductivityofthe
resintoanunreasonablylowlevel
Finally,weinvestigatedhowthepercentageofaggregates in
themAb feed influenced theremoval of mAb aggregates with
Eshmuno® CP-FTresin using a continuousloading process.We
testedsix differentmAb05 feeds withlevelsof aggregates that
variedfrom1.9% to14.6% We foundthatas thepercentage of
aggregatesinthemAb05feedwasincreased,theaggregatesbegan
elutingfromtheresinatlowerloadings.Thelevelofaggregatesin
allsixmAb05feedscouldbereducedtolessthan1%withmonomer
recoveriesgreaterthan85%ataparticularloading(Fig.8).However,
purifyingmAb05feedscontaininghigherpercentagesofaggregates
usingacontinuousloadingprocessismorechallenging,becausethe
effectiveoperatingrangewasfoundtodecreaseasthepercentage
ofaggregatesinthefeedwasincreased(Table2).AmAb05feed
withahigherpercentageofaggregatesmustbeprocessedatlower
loadingsandovernarrowerrangestoremoveasufficientamount
ofaggregateswithagoodmonomerrecovery.WhileamAb05feed
awithlowerpercentageofaggregatescanbeprocessedathigher
loadingswithasignificantlywidereffectiveloadingrange
AlowionicdensityCEXchromatographyresin,Eshmuno®
CP-FT resin, was investigated for the removal of aggregates from
mAbfeeds using a continuousloading process.The removal of
mAbaggregates withEshmunoCP-FT® resinusingacontinuous
loadingprocesswasfoundtobeconsistentwitha frontal
chro-matographymechanism,wherebythemAbmonomersareinitially
retainedbythecolumnandaresubsequentlydisplacedby
aggre-gates.Eshmuno® CP-FTresinwasfoundtobesignificantlymore
effectivefortheremovalofmAbaggregatesusinga continuous
loadingprocesscomparedtosixcommerciallyavailablestrongCEX
chromatographyresinsundersolutionconditionswhereCEXresins
typicallyhavehighcapacitiesformAbmonomerandaggregates
Wefoundthattheefficientremovalofaggregatesusinga
continu-ousloadingprocesscorrelatedwithCEXresinshavinglowerionic
densitieswhilenocorrelationwasobservedwiththeirmAbstatic
bindingcapacities.Optimizationstudiesfoundthattheremovalof
aggregateswithEshmuno® CP-FTresinusingacontinuous
load-ingprocesswasmoreefficientatlowersolutionpHs andlower
solutionconductivities,whichfavor strongelectrostatic
interac-tionsbetweenthepositivelychargedmAbmonomer/aggregates
andthenegativelychargedEshmuno® CP-FTresin.Animportant
exceptiontothistrendinthesolutionconditionswasobservedat
thelowestconductivityforthemAb05feed.Optimizationstudies alsofoundthatEshmuno® CP-FTresinremovesmoreaggregates withhighermonomer recoveriesatlongerresidence timesand lowermAbfeedconcentrations.Eshmuno® CP-FTresinefficiently removedaggregatesfrommAbfeedscontainingbetween1.9%and 14.6%aggregatesusingacontinuousloadingprocess,howeverthe mAbfeedswithlowerpercentagesofaggregateshadmuchwider effectiveloadingranges
TheauthorsareemployeesofEMDMilliporeCorporationwhich sellsEshmuno®CP-FTresin
Acknowledgements
Theauthorsthank JamesHamzik, LarsPeeck, Dominic Zorn, RomasSkudas,PaulTuriano,LloydGottlieb,MichaelSchulte,David Beattie,andMatthiasJöhnckfortheirsupportandencouragement
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