Platform manufacturing processes are widely adopted to simplify and standardize the development and manufacturing of monoclonal antibodies (mAbs). However, there are mAbs that do not conform to a platform design due to instability or other protein properties leading to a negative impact on product quality or process performance (non-platform mAb).
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
Adrian Mana, Haibin Luoa, Sophia V Levitskayab, Nathaniel Macapagala,
Kelcy J Newella,∗
a Purification Process Sciences, AstraZeneca, One MedImmune Way, Gaithersburg, MD, 20878, USA
b Analytical Sciences, AstraZeneca, One MedImmune Way, Gaithersburg, MD, 20878, USA
Article history:
Received 19 September 2018
Received in revised form 12 March 2019
Accepted 13 March 2019
Available online 20 March 2019
Keywords:
Monoclonal antibody purification
Automation
High throughput process development
Aggregation
Scale-down
Reversible self-association
Platformmanufacturingprocessesarewidelyadoptedtosimplifyandstandardizethedevelopmentand manufacturingofmonoclonalantibodies(mAbs).However,therearemAbsthatdonotconformtoa platformdesignduetoinstabilityorotherproteinpropertiesleadingtoanegativeimpactonproduct qualityorprocessperformance(non-platformmAb).Non-platformmAbstypicallyrequireprolonged developmenttimesandsignificantdeviationsfromtheplatformprocesstoaddresstheseissuesdueto theneedtosequentiallyoptimizeindividualprocesssteps
Inthisstudy,wedescribeanIgG2mAb(mAbA)thatissusceptibletoaggregationandreversible self-association(RSA)underplatformconditions.Inlieuofasequentialoptimizationapproach,we eval-uatedthesolutionstabilityofmAbAacrosstheplatformoperatingspace(solutionstabilityscreen).This screeningdesignwasusedtoidentifyinteractingparametersthataffectedthenon-platformmAb sta-bility.Asubsequentresponsesurfacedesignwasfoundtopredictanacceptableoperatingspacethat minimizedaggregateformationandRSAacrosstheentireprocess.Thisinformationguidedtheselection
ofoptimalparametersbestsuitedtoavoiddestabilizingconditionsforeachprocessstep.Substantial timesavingswasachievedbyfocusingdevelopmentaroundthesefactorsincludingprotein concentra-tion,bufferpH,saltconcentration,andexcipienttype.Inaddition,thisworkenabledtheoptimizationof
acationexchangechromatographystepthatremovedaggregatewithoutyieldlossesduetothepresence
ofreversibleaggregation.Thefinaloptimizedprocessderivedfromthisstudyresultedinanincreasein yieldof ˜30%overtheoriginalprocesswhilemaintainingthesamelevelofaggregateclearancetomatch productquality
Solutionstabilityscreeningisreadilyadaptedtohighthroughputtechnologiestominimizematerial requirementsandaccelerateanalyticaldataavailability.Implementationofhighthroughputapproaches willfurtherexpediteprocessdevelopmentandenableenhancedselectionofcandidatedrugsbyincluding processdevelopmentobjectives
©2019TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-ND
license(http://creativecommons.org/licenses/by-nc-nd/4.0/)
1 Introduction
Monoclonalantibodies(mAbs)haveemergedasarapidly
grow-ingclassoftherapeuticsincethemid-1990s.AccordingtotheFDA
databasetherearemorethan70modernantibody-based
thera-peuticagentsapprovedintheUSandmorethan500additional
∗ Corresponding author.
E-mail address: newellkj@medimmune.com (K.J Newell).
productsarecurrentlyinclinicaldevelopment[1 Many biophar-maceutical companies have adopted platform mAbpurification processes tosimplifyprocess developmentand manufacture of mAbs[2 Fig.1illustratesacommonplatformprocesswith com-monlyusedstepsincluding;(1)affinitypurificationcapture, (2) lowpHvirusinactivation,(3)anionexchangechromatographyfor processrelatedimpurityandvirusremoval,(4)cationexchange chromatographyforprocessandproductrelatedimpurityremoval (5)virusfiltration,and(6)formulationthatutilizes(a) ultrafiltra-tionand(b)diafiltrationtogeneratedrugsubstance
https://doi.org/10.1016/j.chroma.2019.03.021
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 2Table 1
Summary of non-ideal observations for a mAb in the platform process.
(mS/cm)
Protein conc.
(mg/mL) 1
Protein A
Chromatography
Low salt b
High protein concentration c
High aggregate in product 2
Low pH Inactivation
3
Anion Exchange
Chromatography
Low salt High protein concentration
Peak tailing
4
Cation Exchange
Chromatography
High salt Intermediate protein concentration
Peak splitting, aggregate formation,
low yield 5
Virus Filtration
Flux 6A
UF1
High salt High protein concentration
High feed pressure/Low Flux
6B
UF2
Low salt Very high protein concentration
High feed pressure/Low flux
a pH range was categorized using following criteria: Low (<4.6), Intermediate (4.6–6.5), Neutral (6.6–8.0), High (>8).
b Buffer ionic strength was categorized using following criteria: Low (<5 mS/cm), Intermediate (5–10 mS/cm), High (>10 mS/cm).
c Protein concentration was categorized using following criteria: Low (<1 mg/mL), Intermediate (1–10 mg/mL), High (11–100 mg/mL) and Very High (>100 mg/mL).
Fig 1.Standard platform manufacturing process for monoclonal antibodies.
A well-designed platform process is expected to robustly
removetheprocessandproductrelatedimpuritiesforthe
major-ityoftherapeuticmAbsusedforclinicaldevelopment.Whilemany
monoclonalantibodiesareeasilymanufacturedusingaplatform
processwithminoradjustments,difficultiesarisewithmAbsthat
do not fit intothe establishedplatform design due to
instabil-ityinsolution(non-platformmAbs).Thisproblemcanresultin
prolongeddevelopmenttimes,supplychainconcernsdueto
imple-mentationof noveltechnologiesand delayedclinical timelines
Proteininstabilityinsolutioncanbeduetomultiplecausesrelated
tomAb structuralheterogeneity and resultin theformation of productrelatedimpurities[3–7].Reversibleself-association(RSA)
isauniquesolutionpropertyinwhichnative,reversibleoligomeric species are formed as a result of non-covalent intermolecular interactions[8,9 Unlikeirreversibleaggregates(aggregates),RSA speciesexistatequilibriumwithmonomerandthemodification
ofsolutionconditionssuchaspH,ionconcentration,or tempera-turecandrivetheequilibriumtofavormonomeroverRSA.IfRSA hasaslowdissociationrate,itcanbedetectedbysizeexclusion chromatography.Ifdissociationconstantsareshort,theprimary methodsofRSAdetectionareanalyticalmethodsthatdetectthe hydrodynamicradiusofamixtureofspeciesorsedimentationrates
ofmultiplespeciessuchasdynamiclightscattering(former)and analyticalultracentrifugation(latter).TheprimarychallengeofRSA speciesisthattheyfrequentlybehavelikeaggregatesmakingit verychallengingtoremoveaggregateswhensolutionconditions favorRSAovermonomer.RSAhasbeendemonstratedtohavea largeimpact oncation exchange chromatography [10] and can alsoimpactsolutionviscosity[11].Specifically,Luoetal demon-stratedthatelutionsaltconcentrationsthatfavoredRSAresulted
inlowcolumnyieldsandtheformationofirreversibleaggregates thatappearedtobemediatedbythepresenceofthe chromatog-raphy resin The formation of RSA has thepotential to impact virtuallyeveryotherstepoftheplatformprocessparticularlyifthe RSAspeciesformsatconditionswheremAbmonomersare usu-allystable(Table1).Untilrecentlytheexperimentaltechniques employedtoaddressRSAeffectsupondownstreamprocesshave beenreactiveratherthanpredictivewithdevelopmenttimelines beingdelayedwhileprocesschangeswereevaluatedandforrobust solutions
Inthiswork,weusedanon-platformmAbthatexhibited atyp-ical solution behaviors when manufactured with the platform process(Table1).ADesignofExperiments(DoE)methodologywas usedtoscreenforsolutionconditionfactorsthatimpactthese atyp-icalbehaviorsandidentifymorestablesolutionconditions.The resultswereappliedtoeach processstepwiththegoalof iden-tifyingoperatingconditionsthatwouldenablegoodperformance forthenon-platform mAbwithonlyminordeviations fromthe platformprocess
Trang 32 Materials and MethodS
2.1 Chemicalsandreagents
2.1.1 Buffersaltsandexcipients
BuffersaltswereobtainedfromJ.T.Baker(CenterValley,PA)
andwereofreagentgradeorhigher.Concentratedstocksolutions
weremadebythedissolutionofthesolidcomponentsinreverse
osmosisdistilledwater(RODI).Aliquotsofthestocksolutionswere
mixedanddilutedwithRODIifnecessarytoobtainthetargetbuffer
concentrations
2.1.2 Reagents
ThemAb A was expressedin Chinese hamster ovary (CHO)
cellsmadebyAstraZeneca,Gaithersburg,MD.MAbAhasan
iso-electricpointrangeofpH8.5–9.0andanapproximatemolecular
weightof 147kDa Theexpressed mAbA was purified by
Pro-teinAchromatography,specificallyMabSelectSuReobtainedfrom
GEHealthcare(GEHealthcare,Piscataway,NJ).Whereapplicable,
themAbAaliquotswerebufferexchangedandconcentratedinto
theappropriate buffersolutions using tangentialflow filtration
(TFF)regeneratedcellulosemembranes(Millipore,Billerica,MA)
ConcentratedstockexcipientswerespikedintomAbAsampleas
required.High protein concentrationmAb A preparationswere
achievedusingapressurizedconcentratorcelldevice(Millipore)
andultrafiltrationdiscmembranes(Millipore)
2.2 Analyticalmethods
2.2.1 Proteinconcentration
ProteinconcentrationwasmeasuredusingaNanodrop
spec-trophotometerfromThermoFisherScientific(Waltham,MA).The
absorbanceofthesolutionwasmeasuredat280nmandthe
pro-teinconcentrationwascalculatedfromtheextinctioncoefficient
usingtheBeer-Lambertequation
2.2.2 Dynamiclightscattering(DLS)
ThehydrodynamicradiusfortheIgG2monoclonalantibodywas
analyzedatmultipleproteinconcentrationsusingahigh
through-put384-wellplateDynaProDLSinstrumentfromWyatttechnology
(SantaBarbara,CA)equippedwitha633nmlaser.Thescattered
lightwasmonitoredat173◦totheincidentlightbeamand
auto-correlationfunctionsweregeneratedusingadigitalautocorrelator
ThehydrodynamicradiuswascalculatedusingtheStokes-Einstein
equation
2.2.3 SizeexclusionHPLC
Analytical high performance size exclusion chromatography
(HPSEC)wasperformedusingaTSKgelG3000SWxlcolumnfrom
TosohBioscience(TosohBioscience,Montgomeryville,PA)withan
AgilentHPLC1200systemfromAgilentTechnologies(SantaClara,
CA,USA).Testsamplesweredilutedtoadesiredconcentrationon
iceafterstorage at2–8 ◦C tominimizeRSA dissociation inthe
proteinsamplepriortocolumnchromatography.TheHPLC
auto-samplerwasmaintainedat5◦C,andthecolumnwasrunatambient
temperature.Proteinsamplewasisocraticallyelutedusing0.1M
sodiumsulfate,0.1Msodiumphosphatebuffer,pH6.8,andelution
wasmonitoredbyUVabsorbanceat280nm.RSAwasmeasuredas
apercentoftheleadingshoulderofthemainpeakrelativetothe
totalpeakarea(Fig.2C)
2.3 Statisticaldesignofexperiments
Theexperimentaldesign,wasbuiltwithJMPstatisticalsoftware
version10.0.0(SASInstituteInc.,Cary,NC,USA).Aconcentrated
stockofmAbAwasadjustedtotargetpHandexcipient concen-trationsthroughadditionofstockbuffer/excipientsolutions.The timeofadditionwasdefinedasT=0s.Unlessspecifiedotherwise, theincubationtimewas≥1hpriortosampleanalysistoensure completionoftheaggregationorRSAreaction(Datanotshown) JMPsoftwarewasusedforstatisticalanalysisoftheexperimental dataandmodeldetermination
Screeningexperimentswereusedtoidentifyfactorsthathave
animpactonstabilityindicatingmeasurementsincluding aggre-gateandRSA.Factorstestedwereeitherordinal(salttype,buffer type)orcontinuous(pH,proteinconcentration,saltconcentration, excipientconcentration).Continuousfactorsweretestedatthree levels(−0+)withrangesandcenterpointvalueschosenbasedon anticipatedprocessconditionsorpriorstabilityinformationfrom literatureorin-housedata.Thescreeningdesignincorporatedruns
atcombinationsofhigh(+)andlow(−)valuesforthecontinuous factorsateachordinalfactorpluscenterpointrunsateachordinal factor
Responsesurfaceexperimentswereperformedtomeasurethe degreeofimpactthefactorshaveonthestabilityattributesand identifyinteractionsbetweencontinuousfactors.Anon-face cen-tralcompositedesignincorporatedrunsatcombinationsofhigh (+),centerpoint(0)andlow(-)valuesforeachfactor
2.4 Benchscalechromatographyexperiments 2.4.1 Chromatographyequipmentandmaterials Laboratoryscalechromatographyexperimentswereperformed using a GE Healthcare ÄKTA Explorer 100 using Unicorn soft-wareversion5.2(GEHealthcare,Piscataway,NJ,USA).Thecation exchangeresinusedinthisstudywasCaptoSPImpResobtained fromGEHealthcare(GEHealthcare,Piscataway,NJ,USA).Cation exchangechromatography(CEX)resinswerepackedinto1.15cm innerdiameter(ID)Vantagecolumns(Millipore,Billerica,MA,USA)
toabedheightofapproximately12cmandoperatedatalinear velocityof150cm/hour
2.4.2 Cationexchangechromatography TheCEXcolumnswere equilibratedwith3column volumes (CVs)of50mMsodiumacetatepH4.5.ThepHandconductivityof thecolumneffluentweremonitoredthroughbuilt-inÄKTAprobes
toconfirmcolumnequilibration.ThecolumnwasloadedwithmAb materialtoaresidencetimeof5min.Afterloading,thecolumnwas washedwith3columnvolumes(CV)of50mMsodiumacetate,pH 4.5.Theproteinboundonthecolumnwaselutedovera20CV lin-earsaltgradientfrom0to500mMin50mMsodiumacetatepH 4.5.Theelutionpeakwasfractionatedinhalf-CVfractionsbased
onA280collectioncriteriaof100mAU.Theabsorbanceofthe pro-teinwasmonitoredatA280bybuilt-inÄKTAprobes.In-linepHand conductivitywerealsomonitoredforalltestruns.Allrunswere carriedoutatroomtemperature(20–25◦C).Cleaningin-place(CIP) wasconductedusing3CV50mMsodiumacetate,1Msodium chlo-ride,pH5.0followedby1Nsodiumhydroxide.Thecolumnswere storedin20%ethanolaftereachrun.FinalCEXtestconditionswere demonstratedusinga20mMCaCl2stepelution
3 Results and discussion
3.1 Observationsofatypicalsolutionbehaviorsduring purificationofmAbAusingaplatformpurificationprocess MonoclonalantibodyA(mAbA)waspurifiedusingastandard platformprocess(Fig.1).Whileothermonoclonalantibodiescan
bepurifiedundertheseconditionswithacceptableprocess perfor-manceandproductquality,theprocessstandardsforperformance andqualitycouldnotbeachievedwithmAbA(Table1)
Trang 4Fig 2. Analytical methods employed to measure the purification process design space A) DLS data demonstrating the changes in hydrodynamic radius of a 10 mg/mL mAb
A sample at 25◦C (monomer) and at 5◦C (multimer) B) HPSEC data used to measure irreversible aggregate levels C) HPSEC data demonstrating RSA when using SEC method with refrigerated autosampler and higher protein concentration load.
AsshowninTable1,aggregateformationisasignificantsource
ofdecreasedproductpurityduringseveralplatformstepsincluding
ProteinAChromatography,LowpHinactivation,Cationexchange
chromatography andUltrafiltration and Diafiltration.For
exam-ple,observationsofmAbAduringlowpHactivationdemonstrated
aggregationratesupto0.9%aggregate/hour(SupplementalFig.1) Thisrateofaggregationis9timeshigherthanouractionlimitsfor aggregationrate(0.1%aggregate/hour)
Highproteinconcentrationconditionsarealsotypicalduring bindandelutechromatographystepsandultrafiltrationstepsofthe
Trang 5Fig 3.Design space for mAb A purification process [1] Protein A [2] Low pH [3] Anion exchange [4] Cation exchange [5] Virus Filtration [6a] UF1 [6b] UF2 indicating where aggregation and RSA occur A) Platform conditions, B) Platform condition with sucrose, C) Platform condition with calcium chloride.
platformprocess(steps1,4and6inTable1).Whileconventional
mAbs exhibited acceptable stability within these protein
con-centrations,Luoetal.demonstratedthatpeaksplittingobserved
duringmAbAcationexchangechromatographypurificationwas
duetoformationofadditionalaggregatesduringelutionfromthe
column The generation of aggregates results in lower product
purityandconsequentialyielddecreaseduringaggregateremoval
stepssuchasthecationexchangechromatographystep
Aggre-gation hasadditional impacts on process performance such as
reducedfluxduringviralfiltrationandultrafiltration(steps5and
6inTable1)
Early observations suggested that aggregate generation and
advanced aggregation rates might be the cause of the
perfor-mance issues (i.e peak tailing and splitting) observed during
cation exchangechromatography (step 4 ofTable 1).However,
theobservationsofpeaktailingandsplittingstilloccurredeven
when the feed aggregate levels were greatly reduced Further
investigationrevealedthatmAbAexhibitedon-columnreversible
self-association(RSA)aswellasirreversibleaggregateformation
[10] RSA and aggregatelevels canbemeasuredusingDLS and
HPSECrespectively(Fig.2).RefrigerationoftheHPLCauto-sampler
enabled theuseof HPSEC to visualize both RSA and aggregate
levels(Fig 2).RSA formation was demonstrated to be favored
at both refrigerated temperatures and high protein
concentra-tions
ItwashypothesizedthatmAbAnon-conformancetothe
plat-form process is due to susceptibility to RSA and aggregation
Development of a mAb A process therefore required
identifi-cation of solutionconditions where acceptablestability can be
achieved
3.2 Determinationofthedownstreamoperatingspace
A design of experiment (DoE) approach was used toscreen severalfactorsincludingpH,buffertype,salttypeandsalt con-centrationtodeterminetheimpact,ifany,onmAbAstabilityas measuredbyRSAandaggregateformation.Afteridentifying fac-torsthatimpactstability,afollow-upresponsesurfaceexperiment usingthesefactorsgeneratedapredictivemodeltodeterminethe operatingspacethatminimizesRSAandaggregateformation ThisoperatingspacewasdefinedatamAbAprotein concen-trationof10mg/mL.Whileproteinconcentrationisafactorthat wasshowntocauseaggregationandRSAwithincreasing concen-tration(Datanotshown),a10mg/mLconcentrationiswithinthe expectedrangeformostoftheplatformsteps(steps1–5inTable1) andthereforestatisticalmodelswerecreatedatthiscontrolprotein concentration
Fig.3aplotswhereRSAandaggregationispredictedtooccurat combinationsofsolutionpHandNaClconcentrationwhichwere determinedtobesignificantfromthescreeningstudy.To illus-trateapotentialpurificationdesignspace,limitsoflessthan40% RSAandaggregatewereassignedtoidentifyapHandNaCl con-centrationrangewherethenon-idealsolutionbehaviorswouldbe minimized.RSAandaggregateformationoccurunderdistinct con-ditionswithRSAoccurringatneutraltohighpHinasalt-dependent mannerandwithaggregatesformingatlowpHconditionsalsoin
asalt-dependentmanner.TheplotshowsanarrowpHrange,from approximatelypH5–6,andnarrowswithincreasingsalt concen-trationwhereRSAandaggregationareavoided.Consequently,only one(step6ainTable1)outofthesixplatformprocessstepsis suitableformAbApurification
Trang 6Fig 4.Impact of excipients on a) aggregation at pH 3.5 and b) RSA at pH 5.5 on a
50 mg/mL mAb A sample.
TheresultsinFig.3ashowthateitherthedesignspaceneeds
tobewidenedbytheadditionofstabilizingexcipientsorthatthe
purificationprocesswouldneedtobemodifiedtoshiftthemajority
ofprocessstepsintoanoperatingspacethatminimizesRSAand
aggregation
3.3 Excipientscanmodulatethepurificationdesignspace
Stabilityofproteinsmaybeenhancedinthepresenceof
excipi-entsthroughconformationalorcolloidalmechanisms[12].Sugars
suchastrehaloseandsucrosehavebeenshowntoprevent
attrac-tiveelectrostaticinteractionsbetweenmonomericmolecules[13]
ApatentwasfileddemonstratingthatmAbARSAwasmitigated
bytheadditionofsucrose[14],[15].Aggregationmediatedthrough
hydrophobicinteractionscanbemitigatedinthepresenceof
argi-nine and propylene glycol [16,17] Urea can stabilize proteins
throughhydrogenbonding[18].Manyoftheseexcipientsare
cur-rentlyusedinplatformformulationsbasedondemonstrateddrug
substancesafety,efficacyandstability
Ascreeningstudytodeterminetheeffectoffourexcipients,
sucrose,propyleneglycol(PG),ureaandarginine,uponRSAand
aggregationwasperformedandtheresultsshowninFig.4.Based
ondatafromtheresponsesurfacedesignmodelidentifyingpH,
NaClconcentrationandproteinconcentrationasfactorsimpacting
aggregationandRSA,twotestconditionsweredefined(50mg/mL
proteinconcentrationpH3.5orpH5.5inthepresenceof0.1M
NaCl).Excipientconcentrations testedweredemonstratedtobe
effectivewith othermonoclonalantibodies under development
(Datanotshown).MabAissusceptibletoaggregationandRSAat
pH3.5andpH5.5respectively.Theaggregateandreversibly
associ-atedspeciesweremeasuredandthepercentchangeineachspecies
relativetothenegativecontrol(mAbAintheabsenceofexcipient) calculated.ResultsfromthisstudyaresummarizedinFig.4 Sig-nificantmAbAaggregationoccursatpH3.5butthepresenceof sucrosemitigatestheincreaseby8%.Theotherexcipientstested appeartoexacerbateaggregation.RSAoccurringatpH5.5is miti-gatedbysucrose,propyleneglycolandargininebutismosteffective withsucrose.UreaisshowntobeineffectiveinstabilizingmAbAat eitheroftheriskconditionsforaggregationandRSA.Sucrosewas theonlyexcipienttestedthatmitigatedbothaggregationandRSA formation
ThedatainFig.4 suggeststhatsucrosehasthepotentialfor wideningthemAbApurificationdesignspace.Aresponsesurface designexperimentwasperformedwiththeinclusionofsucroseasa factorinadditiontopHandNaClconcentration.Applyingthesame criteriaforaggregationandRSAasthemodeldescribedinFig.3a,
bdemonstratesthat0.3Msucrosebroadensthedesignspace.The resultisthatfourofthesixplatformsteps(steps1,4,5and6in Table1)occurinthebroadersafeoperatingspace
Whileadditionofsucrosetoallprocessstepscanmitigatethe non-idealsolutionbehaviorsobservedwithmAbA,thereare pro-cess challenges and risks associated with theuse of sugars as
a manufacturingraw material.Thesechallengesinclude:higher viscosity liquids[19], a carbon sourcefor adventitious biologic contamination,andahigherriskforunacceptableendotoxin lev-elsin thedrugsubstance.Sucrose additionmaybeappropriate for unit operationslikelow pHinactivation (step2 inTable 1) whereexposuretimetosucrosewouldbelimitedandriskof micro-bialcontaminationlowatthispHcondition.Sucroseadditionto theProtein A chromatography (step1 in Table 1)low pH elu-tionbuffermayalsopreventaggregateformationunderplatform conditions However,filtrationsteps(steps 5 and 6in Table 1) withpressureconsiderationsorpolishingchromatographysteps (steps3and4inTable1)withpotentiallylongerproduct expo-suretime tosucrose are at greater risk tothe aforementioned challenges
3.4 Changingsaltsusedinthecationexchangechromatography stepcanmodulatethedesignspacelocation
Theresponsesurfacedesign showsthatRSAandaggregation increaseswithincreasingsodiumchlorideconcentration.Thesalt typewasnotasignificantfactorasdifferentsalts(Ex.calcium chlo-ride)hadasimilarimpactonRSAandaggregation(Supplemental Fig.2)atequivalentmolarconcentrations.While thisdata sug-geststhatselectionofsalttypecannotmodulatethedesignspace withrespecttoRSAandaggregationatequivalentmolarities,salt typecanimpactelutionconditionsduringcationexchange chro-matography [20] Cationexchange elution salt concentrationis dependentuponthestrengthofthecationiccharge.Becausethe bindingbetweenthemAbandtheresinligandiselectrostatic,the requiredconcentrationofsalttoelutethemAbvariesbasedonthe respectivechargeofthecation
Astudywasperformedtoevaluatetheefficacyofdifferentsalt typesinresolvingmAbAmonomericproductfromhigh molecu-larweight(HMW)impurityspeciesandtodeterminetherequired saltconcentrationtoachieveresolution.MabAwasloadedontoan equilibratedcationexchangecolumn,washedandelutedwithsalt
aspreviouslydescribed(Section2.4.2).Thischromatographic pro-cedurewasusedtoscreendifferentsalttypes,includingpotassium chloride,sodiumsulfate,sodiumacetate,sodiumcitrate,arginine hydrochloride,magnesiumchlorideandcalciumchloride,in addi-tiontotheplatformsodiumchloride.Thecationexchangeoutputs
ofstepyield,productvolumeandproductqualityweremeasured foreachrun
TheelutionchromatogramsforeachsalttypeareshowninFig.5 ThemAbAmonomer(peak1)iselutedfirstduringthesalt
Trang 7gra-Fig 5.Impact of Salt Species on Separation of Monomer and High Molecular Weight (HMW) Impurities during Cation Exchange Chromatography Monomer is the early eluting peak while the HMW impurities, annotated with the percent HMW, are late eluting.
Table 2
Summary of process modifications for mAb A purification and applicable HTPD tools for future non-platform mAb case studies.
Observations
Modified Conditions Potential HTPD methods 1
Protein A
Chromatography
High aggregate in product
Reduced aggregate in product
Low pH Low salt, High conc.
Sucrose
96-well stability study, DLS plate reader, HTSEC 2
Low pH Inactivation
Aggregate formation No aggregate formation 3
Anion Exchange
Chromatography
Low salt High conc.
Sucrose
TeChrom Robo-column purification
4
Cation Exchange
Chromatography
Peak splitting, aggregate formation, low yield
Peak splitting, No aggregate formation, Increased yield
Intermediate pH Low salt 5.
Virus Filtration
Low flux
Increased Throughput
Filterplate throughput experiments 6A.
Ultrafiltration 1
High feed pressure/Low flux
Reduced Feed pressure/
Increased flux
Intermediate pH Low salt High conc.
DLS flowthrough cuvette, High throughput tangential flow filtration 6B
Ultrafiltration 2
Intermediate pH Low Salt Arginine Very high conc.
dientfollowedbytheHMWspecies (peak2).Thisisdue tothe
HMWspecieshavingagreateraffinitytothecationexchange
lig-and.Theyieldpercentageofeachpeakcanbecalculatedfromthe
relativepeakareas.TheyieldoftheHMWspeciesisannotatedon
eachchromatograminFig.5.Thepoolofeachpeakwascollected
and analyzedfor product quality(Data not shown).Comparing
eachofthechromatographicrunswithrespecttomonomerpeak
purityandstepyield,calciumchlorideelutionachievedbothhigh
monomerpurity(98%)andhighyield(82%).Therunwithcalcium
chloridealsoshowsalowlevel(13.7%)oftheHMWspeciesandisan
indicationthatthiselutionsaltconditionminimizestheformation
ofHMWspecies.Thisobservationisconsistentwiththeresponse
surfacedesignmodelthatshowsRSAandaggregationoccurringat
highsaltconcentration.Peakelutionwithcalciumchlorideoccurs
atapproximately25mM whileother salts(Ex sodiumacetate)
thatrequireahighsaltconcentration(approximately150mM)for elutionshowa higheryield(36.4%) oftheHMWspecies While othersalttypescan achievesomecombinationof (1)highstep yield,(2)highproductpurityor(3)minimalHMWspecies forma-tion,calciumchlorideachievesallthreedesiredobjectives.Further confirmationofcalciumchlorideefficacyformAbApurification wasshownbyasubsequentcationexchangechromatographyrun usingacalciumchloride20mMisocraticstepelutionwherehigh yieldandhighproductqualitywereachieved(SupplementalFig.3) Additionally,theproductvolumewiththecalciumchlorideelution was25%lesscomparedtotheplatformsodiumchloridecondition allowingformanufacturingfacilityfitbenefits.Fig.3cillustrates howtheuseofcalciumchlorideshiftsthedesignspacelocationfor thecationexchangestep(step4inTable1)intoasafeoperating areatoavoidaggregation
Trang 8Fig 6.Optimized operating space for mAb A purification process [1] Protein A [2]
Low pH [3] Anion exchange [4] Cation exchange [5] Virus Filtration [6a] UF1 [6b]
UF2 indicating where aggregation and RSA is predicted to occur Proposed optimized
mAb A process using sucrose during Protein A chromatography and calcium chloride
during cation exchange chromatography.
4 Conclusion
Inthispaper,wedescribeacasestudywhereatypicalsolution
behaviors,determinedtobereversibleself-association(RSA)and
aggregation,wereobservedduringplatformprocessingofmAbA
resultinginunacceptableproductqualityandyieldforaplatform
commercialmanufacturingprocess.Anintensivestudywas
per-formedtobetterunderstandthesenon-ideal solutionbehaviors
andtodevelopadownstreamprocessforthisnon-platformmAb
Throughtheuseofscreeningandresponsesurfacedesignof
exper-iments,factorsimpactingthenon-idealsolutionbehaviorswere
identifiedandstatisticalmodelstopredictRSAandaggregation
lev-elswithinrangesofthesefactorsweregenerated.Itwasshownthat
pH,saltconcentration,proteinconcentrationandsucroseare
fac-torsthatdeterminewherenon-idealsolutionbehaviorsoccur.With
thisknowledge,anoptimizedprocessformAbAwasproposed
BecauseofthemAbAstabilizingpropertiesofsucrose,inclusion
ofsucrosetotheelutionbufferoftheProteinAchromatography
step(step1inTable2)andtotheacidificationtitrantofthelowpH
inactivationstep(step2inTable2)isexpectedtoreduceaggregate
formationcurrentlyobservedatplatformconditions
Manufactur-ingconcernsabouttheriskofadventitiousbiologiccontamination
associated withhighsugar concentrations aremitigated bythe
subsequentprocess stepswheresucrose would bediluted
dur-ingneutralizationafterlowpHtreatmentandflow-throughanion
exchangechromatography(step3inTable2)andremovedduring
cationexchangechromatography(step4inTable2).Knowledge
ofthesaltconcentrationeffectonRSAandaggregationdrove
fur-therexperimentstoscreensalttypesthatcanelutemAbAfrom
thecationexchangecolumnatalowmolarconcentration.Theuse
of20mMcalciumchlorideelutedmAbAproductof high
prod-uct quality and yield without aggregateinduced peak splitting
observed with the platform sodium chloride elution salt
Con-sequential lowersalt concentrationin themAbA producthave
additional process benefits where low salt concentrations may
improvefluxandreducefeedpressureduringviralfiltrationand
ultrafiltration(steps5and6inTable2)[21].Fig.6illustrateshow
leveragingthecombination ofexcipientandsalt typeresultsin
amanufacturingprocessthatisonlymodestlydifferentfromthe
platformbutnowallowsforfour(steps1,4,5and6inTable2) outofthesixplatformprocessstepstobewithinastableoperating spaceformAbApurification.WhilelowpHinactivation(step2in Table2)andanionexchangechromatography(step3inTable2) stilloccurwithintheaggregationandRSAriskspacerespectively, thehighmolecularweightspeciesformedatthesestepsarewithin theimpurityremovalcapabilitiesofthesubsequentdownstream steps
Thisstrategyforoptimizingaprocessforanon-platformmAb wasareactiveapproachtopurificationproblemsobserved dur-ingconventionalprocessdevelopment.Whilesuccessful,delayed timelinesandsignificantadditionalinvestmentinlabscale devel-opment time were an unintended consequence Most of the developmenttimewasattributedtoperformingthebenchscale experiments(section2.4)andanalyticalmethods(section2.3) For future mAband non-mAb drugproducts, theuseof the designofexperimentapproachdescribedherecouldbeusedina proactivemannerwherecandidatemoleculesareinitiallyscreened
toassesssolutionproperties.Thisapproachwillneedto(1) min-imize the amount of protein needed for thesestudies and (2) generatedataquicklytoenablecandidatedrugselection within theexistingdevelopmenttimelines.Bothobjectivescanbereadily accomplishedbyleveragingexistinghighthroughputscale-down modelsandanalytics,collectivelyreferredtoashigh-throughput process development(HTPD) Ourlab hasimplemented several HTPDmethodsthatgreatlyreduceexperimenttime[22].Someof thesetechniquesandtheirapplicabilitytoprocessstepsinthiscase studyareshowninTable2.Thisdatacanthendeterminesuitability forfurtherprocessdevelopmentandapplicabilitytoourplatform processthusreducingcostlydevelopmenttime.Themodelforour developmentlabofthefuturewilllinktheseHTPDtoolstogether
toenabletheproactivescreeningofallbiopharmaceuticalsthatfit withintheplatformparadigmtoassessthemolecule’sprocessfit andenablebetterplanningfordevelopmentresourcesand time-line
Acknowledgements
TheauthorswouldliketoacknowledgeMatthewDickson, Yul-ingLi,TimothyPabst,IrinaRamos,DavidRobbinsandMinZhu(all currentlyorformerlyatAstraZeneca)
Appendix A Supplementary data
Supplementarymaterialrelatedtothisarticlecanbefound,in theonlineversion, atdoi:https://doi.org/10.1016/j.chroma.2019 03.021
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