Monoclonal antibodies (mAbs) dominate themarketfor biopharmaceutical proteins because they provide active and passive immunotherapies for many different diseases. However, for most mAbs,two expensive manufacturing platforms are required.
Trang 1jou rn al h om ep a g e : 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
C Rühla, M Knödlera, P Opdensteinena, J.F Buyela,b,∗
a Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstraße 6, 52074 Aachen, Germany
b Institute for Molecular Biotechnology, Worringerweg 1, RWTH Aachen University, 52074 Aachen, Germany
Article history:
Received 3 June 2018
Received in revised form 19 July 2018
Accepted 5 August 2018
Available online 7 August 2018
Keywords:
Affinity chromatography
Design of experiments
Fluorescent protein carrier
HIV-neutralizing monoclonal antibody
Plant molecular farming
Transient protein production
Monoclonalantibodies(mAbs)dominatethemarketforbiopharmaceuticalproteinsbecausetheyprovide activeandpassiveimmunotherapiesformanydifferentdiseases.However,formostmAbs,twoexpensive manufacturingplatformsarerequired.Thesearemammaliancellculturesforupstreamproductionand ProteinAchromatographyforproductcaptureduringdownstreamprocessing.Herewedescribeanovel affinityligandbasedonthefluorescentproteinDsRedasacarrierforthelinearepitopeELDKWA,which cancapturetheHIV-neutralizingantibody2F5.WeproducedtheDsRed-2F5-Epitope(DFE)intransgenic tobacco(Nicotianatabacum)plantsandpurifieditusingacombinationofheattreatmentandimmobilized metal-ionaffinitychromatography,resultinginayieldof24mgkg−1at90%purity.Usinga design-of-experimentsapproach,wecoupledupto15mgDFEpermLSepharose.Theresultingaffinityresinwas abletocapture2F5fromtheclarifiedextractofN.benthamianaplants,achievingapurityof97%,a recoveryof>95%andaninitialdynamicbindingcapacityat10%productbreakthroughof4mgmL−1after
acontacttimeof2min.Theresincapacitydeclinedto15%ofthestartingvaluewithin25cycleswhen 1.25Mmagnesiumchloridewasusedforelution.Weconfirmedthebindingactivityofthe2F5product
bysurfaceplasmonresonancespectroscopy.DFEisnotyetoptimized,andacostanalysisrevealedthat boostingDFEexpressionandincreasingitscapacitybyfourfoldwillmaketheresincost-competitivewith someProteinAcounterparts.Theaffinityresincanalsobeexploitedtopurifyidiotype-specificmAbs
©2018TheAuthor(s).PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCC
BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/)
1 Introduction
Antibodiesdominatethebiopharmaceuticalmarket,withmore
than50approvedproductsandmorethan300candidatesinthe
developmentpipeline[1 Thetotalsalesvolumewasmorethan
D40billionin2013,whichisabout33%ofallbiopharmaceutical
proteinsales.Mostproductsaremonoclonalantibodies(mAbs)that
aretypicallyproducedinmammaliancells,suchasChinesehamster
ovary(CHO)cells,withtitersregularlyexceeding∼5gL−1 inthe
culturesupernatant[2 Despitethesehighproducttiters,upstream
Abbreviations: CV, column volume; DoE, design of experiments; IMAC,
immobi-lized metal-ion affinity chromatography; SPR, surface plasmon resonance; TSP, total
soluble protein.
∗ Corresponding author at: Fraunhofer Institute for Molecular Biology and Applied
Ecology IME, Forckenbeckstraße 6, 52074 Aachen, Germany.
E-mail addresses: clemens.ruehl@gmail.com (C Rühl),
johannes.buyel@rwth-aachen.de , matthias.knoedler@ime.fraunhofer.de
(M Knödler), patrick.opdensteinen@ime.fraunhofer.de (P Opdensteinen),
johannes.buyel@ime.fraunhofer.de (J.F Buyel).
productioninmammaliancellsisexpensiveduetothecostofmedia andtheneedforsterileconditions.Alternativeexpressionsystems arethereforebeinginvestigated,includingyeastsuchasPichia pas-toris[3]andplants,thelatterofferingascalableandsafeproduction platform[4 Plant-derivedmAbshavealreadybeentestedin clin-icaltrials,includingtheHIV-neutralizingmAb2G12[5
Regardlessoftheexpressionhost,anothermajorcostdriverfor mAbmanufacturingistherelianceofmostprocessesonaProteinA capturestep,whichhasbecomethegoldstandardforinitial purifi-cation[6 Althoughtheproductionofthisprotein-basedaffinity ligandinbacterialsystemsiscost-effective,theresinis neverthe-less expensivegiven theneed forqualificationbeforeitsusein processesthatcomplywithgoodmanufacturingpractices(GMP) and alsothesubstantialmarginwhich reflectsthelacksuitable alternatives.Dependingontheproductionscale,thecostsforthe resinalonecanamountto10millioneuros(assuming6×15,000-L bioreactors,anda10-tonoutputofmAbproductperyear)[7 This correspondstomorethan25%ofthetotalprocesscosts[8 The impactoftheProteinAresinonthecostofgoodsisonereasonfor thehighmarketprices,oftenexceeding2000eurospergpurified
https://doi.org/10.1016/j.chroma.2018.08.014
0021-9673/© 2018 The Author(s) 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/
Trang 2mAb[9 Suchpricesareamajorburdenforhealthcaresystemsand
canbeprohibitiveindevelopingcountries,especiallyiflargedoses
ofproductarerequired.Forexample,upto12gofmAbperpatient
isrequiredforalymphomatherapy[10],andupto3gannuallyper
personforaprophylacticanti-HIVtreatment[5 Therefore,several
inexpensivenon-proteinligandshavebeendevelopedthatcouldin
principlereplaceProteinA[11].Manyofthempreferentially
tar-gettheconstantregionsofmAbs,e.g.theMEPligandbindstothe
CH2domain[12],facilitatingrapidprocessdevelopmentduetothe
uniformelutionconditions[13].However,theperformanceofsuch
alternativeresinsintermsofrecoveryandpurityhasbeen
incon-sistentcomparedtoProteinA,e.g.bothhighandlowpuritieshave
beenreportedfollowingmAbelutionfromMEP[14–16],whereas
>95%purityistypicallyachievedwhenusingProteinA[17,18]
Herewehavedevelopedanalternativeapproachforthe
affin-itypurificationofmAbsbasedontheuseoflinearepitopes,inthis
caseELDKWA(one-letteraminoacidcode)fortheHIV-neutralizing
antibody2F5 [19,20] We fused this epitope tothe fluorescent
protein DsRed [21] as a carrier, generating the fusion protein
DsRed-2F5-Epitope(DFE) We thenproduced DFE intransgenic
tobacco (Nicotiana tabacum) plants and purified it by
single-stepimmobilizedmetal-ionaffinitychromatography(IMAC).We
optimizedthecouplingofDFEtoaSepharoseresinusinga
design-of-experiments(DoE)approach,resultinginanovelaffinityresin
whichweusedtopurifymAb2F5(transientlyexpressedinN
ben-thamiana)fromclarifiedleafextracts.Wediscusstheoptimization
ofelutionconditionsandprovideaninitialcostevaluation,
com-paredwithaProteinA-basedprocesscounterpart
2 Materials and methods
2.1 Designofexperiments
DesignExpertv10(Stat-Ease,Minneapolis,MN,USA)wasused
tosetupandevaluateallexperimentaldesigns.Thefactorsand
levelsarepresentedinthesupplementarydata(TableS1),andthe
detailedDoEmethodisdiscussedelsewhere[22]
2.2 Expressionvectorsandbacterialcultures
Thenucleotidesequence ofDsRed(a redfluorescentprotein
fromDiscosomasp.[23]) wasextended byPCR using
appropri-ateprimerstoadd thesequenceencoding theELDKWA epitope
(towhichmAb2F5binds)atthe3 end.Theresultingconstruct
wastransferredtovectorpTRAforexpression[24],yieldingthe
DFEfusionproteinconsistingofDsRed,the2F5epitope,aHis6tag
andaKDELsequenceforretentionintheendoplasmicreticulum
(Fig.S1).Thecodingsequencesfortheheavyandlightchainsof
mAb2F5[19]wereclonedasindividualexpressioncassettesand
werealsointroducedintopTRA[25].Accordingly,theexpression
ofallpolypeptideswasdrivenbythedoubleenhancedCauliflower
mosaic virus35S promoter The vectorsfor DFE and mAb 2F5
wereintroducedseparatelyintoAgrobacteriumtumefaciensstrain
GV3101:pMP90RKbyelectroporation.TheDFEconstructwasused
togeneratetransgenictobacco(N.tabacum)plantsandthe2F5
con-structwasusedfortransientexpressioninN.benthamianaleaves
asdescribedbelow.Ahomologymodelofthe3DstructureofDFE
basedon1ZGO[26]wasbuiltusing3D-JIGSAW(https://bmm.crick
ac.uk/∼populus/)[27]
2.3 Plantmaterial,infiltrationandexpression
TransgenictobaccoplantsexpressingDFEweregenerated as
previouslydescribed[28].Fortransientexpression,N
benthami-anaplantswereinfiltrated withA.tumefacienscarryingthe2F5
constructusingeitherthevacuuminfiltrationmethod[29]or man-ualinjectionintoleaves[30].Wholeplantsorleafsectionswere infiltratedwithA.tumefaciens(OD600nm=1.0)ininfiltrationbuffer (0.5gL−1FertilizerMEGA2(PlantaDüngemittelGmbH,Regenstauf, Germany),200M acetosyringone,pH5.6)andcultivated fora further5daysbeforeharvesting[30]
2.4 Proteinextractionandclarification Proteinswere extractedfromplants byblade-based homog-enization in 3mL extractionbuffer (50mM sodium phosphate,
500mMsodiumchloride,10mMsodiumbisulfite,pH8.0)pergram wetbiomass,followed byclarification usinga sequenceof bag, depthandsterilefilters[31].TobaccoextractscontainingDFEwere heattreatedbeforeclarification[28]
2.5 Immobilizedmetal-ionaffinitychromatography DFEwaspurifiedbyimmobilizedmetal-ionaffinity chromatog-raphy (IMAC) on an ÄKTApure system (GE Healthcare, Little Chalfont,UK)usinganXK-26columncontaining53mLof chelat-ingSepharosefastflowIMACresinloadedwithnickelions.After loadingtheclarifiedextractontoaconditionedcolumn(extraction bufferwithoutsodiumbisulfite),theresinwaswashedwith10 col-umnvolumes(CVs)ofbufferwithoutimidazolefollowedbyelution
inbuffercontaining300mMimidazoleataflowrateof50cmh−1 Theconcentrationsofproteinandnucleicacidweremonitoredat
280and260nm,respectively
2.6 CouplingDFEtoSepharoseresin The purified DFE affinityligand wasimmobilized onHiTrap NHS-activated[32]SepharoseHPcolumns(GEHealthcare)witha bedvolumeof1mL.Beforecoupling,thecolumnswerewashed with 6mL ice-cold 1mM hydrochloric acid at a flow rate of
<1mLmin−1.Immediatelyafterwashing,1.5CVsofaffinity lig-andsolution(0.15–15mgmL−1)wereinjectedusinga2-mLsyringe (Braun, Melsungen, Germany), and the flow-through fractions weremonitoredusingaTE6101precisionscale(Sartorius, Göttin-gen,Germany).Thecolumnswerethensealedandincubatedfor 15–45minat22◦C,followedbythoroughwashingtoremove resid-ualNHSesters.Thisinvolvedthree cyclesofwashing,firstwith
6mLofdeactivationsolution(0.5Methanolamine,0.5Msodium chloride,pH8.3)injectedataflowrateof<1mLmin−1followed
by6mLofalow-pHsolution(0.1Msodiumacetate,0.5Msodium chloride,pH4.0).Thecolumnswereleftfor15minafterthethird washingcycleandwerethenstoredin0.05Mdisodiumphosphate containing0.1%(m/v)sodiumazide(pH7.0)at4◦C.Forthe simul-taneouswashingofmultiplecolumns,anIsmatecIPC24-channel peristalticpump(Cole-ParmerGmbH,Wertheim,Germany) was usedataconstantflowrateof0.6mLmin−1.Thecoupling proce-durerequired∼2hintotal
2.7 AffinityresincharacterizationandpurificationofmAb2F5 DFE-coupledcolumnsweremountedonanÄKTApuresystem andequilibratedwith5CVsofequilibrationbufferataflowrate
of1mLmin−1.Upto80mLofextractcontaining2F5wasloaded ontothecolumnatarateof0.5mLmin-1ensuringacontacttimeof
2min.Thecolumnswerewashedwith6CVsofequilibrationbuffer beforeeluting2F5in5CVsofelutionbufferwithlowpH(0.05M citrate,0.05Msodiumchloride,pH4.0–3.25)orslightlyalkalinepH (1.0–4.0Mmagnesiumchloride,0.1MHEPES,pH8.0).The
Trang 3ontheimmobilizedamountofDFEusingEq.(1)
SBCtheor.=Mw,mAb
Mw,DFE ×mDFE
whereSBCtheor isthetheoreticalstaticbindingcapacity[gL−1],
Mw,mAb isthemolarmassofmAb2F5(154.6kDa),Mw,DFE ishe
molarmassoftheDFEmonomer (28.4kDa),mDFEis the
immo-bilizedmassofDFE (3–10mg),andVresin isthecolumnvolume
(1mL)
Approximately80mLofclarifiedplantextractcontaining2F5
wasloadedunderthesameconditionsasabovetoobtainsigmoidal
breakthroughcurves.Thevolumeatwhich10%oftheplateau
prod-uctconcentrationwasdetectedintheflow-throughfractionwas
multipliedbytheproductconcentrationintheloadtodetermine
thedynamicbindingcapacityat10%productbreakthrough
2.8 Proteinquantitationandactivitytesting
Theconcentrationoftotalsolubleprotein(TSP)wasdetermined
usinga microtiterversion oftheBradford methodas described
before [33] and thesample protein composition was analyzed
bystaininglithiumdodecylsulfate(LDS)polyacrylamidegelswith
Coomassie Brilliant Blue [29] DFE and 2F5 werequantified by
fluorescencespectroscopyandsurfaceplasmon resonance(SPR)
spectroscopy,respectively[34].Theamountofproteinpergram
wet biomass was calculated as described elsewhere [35] The
bindingofDFE-purified 2F5(elutedbypHshiftortheaddition
of magnesium chloride)to the 13.5-kDa trimeric HIV-1 fusion
inhibitorFuzeon(enfuvirtid)containingthe2F5epitope(Roche,
Basel,Switzerland)wasusedtoassessthebindingactivityof2F5
Approximately270responseunits(RU)of2F5werecapturedona
ProteinAchipusingaBIAcoreT200instrument(GEHealthcare)at
25◦CinHEPES-bufferedsalinecontaining0.05%(v/v)Tween-20as
arunningbuffer.EightdilutionsofFuzeoninthe0.16–20.00nM
rangewereinjected individuallyand capturedby2F5boundto
ProteinA.Thekineticbindingconstantska,kdandkDwere
calcu-latedbasedona1:1stoichiometricmodelusingtheBIAevaluation
software(GEHealthcare)
3 Results and discussion
3.1 TheDFEfusionproteinisexpressedathighlevelsinplants
andcanbepurifiedeasily
The28.4-kDafusionproteinDFE(Fig.S1)wasexpressedwith
a yieldof ∼120mgkg−1 leafbiomass, equivalentto∼42mgL−1
extract (Fig 1A), which is in the middle range compared to
otherrecombinantproteinsexpressedintransgenictobacco,e.g
0.9mgkg−1 formAbCO17-1A[36],∼500mgkg−1 formAbM12,
and∼400mgkg−1ofunmodifiedDsRed[31].ThepurityofDFEin
thecrudeextractwas<5%ofTSP,butourDoEapproachrevealed
thatblanchingthetobaccoleavesat70◦Cfor1.5minbefore
extrac-tionincreasedthepuritytoalmost40%becausemostofthehost
cellproteins(HCPs)wereprecipitated(Fig.1B,Fig.S2,TableS2)
Approximately 50% of the product was lost, regardless of the
blanchingtemperature,resultingintherecoveryof∼65mgkg−1
(∼22mgL−1).Theseresultswereingoodagreementwith
previ-ous studiesusing heat precipitation, indicating that more than
90%oftheTSPcanberemovedbyblanchingpriorto
chromatog-raphy [28,37] Removing HCPs early in a process can prevent
productdegradation,asshownforotherfusionproteinstransiently
expressedinN.benthamiana[29].Wethereforeusedblanchingfor
allsubsequentDFEpurificationsdespitetheproductlossandthe
availabilityofanaffinity-basedpurificationstep(IMAC),giventhe
lattercanalsocapturenonspecificplantHCPs[35,38].After homog-enizationandtheremovalofcoarseparticlesusingapolypropylene needle-feltbagfilter,aPDH4two-layerdepthfilter(nominalpore sizesof∼10mand∼1m)wasusedtoclarifytheplantextract, achievinganaveragecapacityof135±36Lm-2(±SD,n=3)anda productrecoveryof∼70%uptothisstep,whichwasequivalentto
45mgkg−1biomass(15mgL−1).Thesevalueswereingood agree-mentwithpreviousstudies,whichreportedcapacitiesof∼70Lm-2
andrecoveriesof∼75%[31].Theuseoffilterlayerslacking diatoma-ceousearthmayimproveDFErecovery,aspreviouslyshownfor
amulti-domainfusionprotein[39].SubsequentDFEpurification
byIMAConaresincontainingNi2+increasedthepurityofDFEto almost90%(Fig.1A),atypicalpurityachievedforplant-derived recombinantproteinswhenusingthistechnique[40–42].The tar-getproteinconcentrationintheelutionfractionwas20-foldhigher thanintheload,buttherecovery(basedonfluorescenceanalysis) wasonly55%,correspondingtoanoverallyieldof23.5mgkg−1and substantialfluorescencewasobservedin theflow-through frac-tions.However,westernblotsofthesefractions(Fig.1B)didnot revealdetectableamountsofDFEwhenusingaprimaryantibody directedagainsttheC-terminalHis6tagofthefusionprotein.We speculatethatatleasttheC-terminalHis6andKDELpartsofthe fusionproteinwerecleavedoffeitherinplantaorafterextraction, whichexplainsthepresenceofDFEvariantsintheflow-through fractionsbecausetheywillnothavebeenabletobindtheIMAC resin.SimilardegradationeffectshavebeenreportedformAbsand vaccinecandidatesexpressedinplants[29,39,43]andweare cur-rentlyinvestigatingthisphenomenoninmoredetail
3.2 DFEcanbecoupledtoSepharoseresinataloadingofupto
7mgmL-11
Inaninitialscreen,wedeterminedthequantityofDFEthatcan
becoupledtoNHS-activatedHiTrapcolumnsandfoundthatthe couplingefficiencydeclinedfrom80to90%to<70%whenweused morethan15mgDFEpermilliliterresin(Fig.1C).Interestingly,
wefoundthatHEPESbuffer,insteadofthebicarbonatebuffer rec-ommendedbythemanufacturer,increasedtheaveragecoupling efficiencyfrom78±9%(±SD,n=3)to89±6(±SD,n=3)atpH8.3
Wealsoobservedamoreintenseredcoloratthetopofthe col-umnwhenHEPESwasusedinsteadofbicarbonate,indicatingthat thecouplingcapacitybecamesaturatedwithlessDFEligandinthe presenceofbicarbonate(Fig.S3A).Thepkavaluesofcarbonicacid are3.6and10.3[44],implyingthatatpH8.3mostofthebicarbonate buffermoleculesshouldbepresentinthehydrogencarbonateform (HCO3–)andonlyasmallamountinthecarbonateform(CO32–)
Wespeculatethatthefreeelectronpairsinthesespeciesmayallow themtoactasnucleophiles,whichcompetewiththeaminogroups
oftheproteinforinteractionwiththeactivatedNHSestersas pre-viouslyreportedforotherfunctionalgroups[45].HEPESbufferwas thereforeusedinallsubsequentexperiments
WethenusedaDoEapproachtooptimizetheconditionsforDFE coupling(TableS1)andfoundthattheamountoffusionprotein boundtothecolumnincreasedasmoreDFEwasbroughtinto con-tactwiththeresin,reachingaplateauat∼15mgDFEpermilliliter resinandresultingin∼10mgofboundDFE,or∼0.35molmL−1 (Fig.2A).However,ifmorethan10mgDFEwasbroughtinto con-tactwiththeresin, thecouplingefficiencydropped from∼90%
tolessthan50%,dependingonthepH(Fig.2B).Also,increasing theamountofcoupledDFEincreasedthecostpercolumnbecause morepurifiedfusionproteinwasconsumed(Fig.2C).Wetherefore usedthenumericaloptimizationtoolbuiltintotheDoEsoftware
toidentifytheidealconditionsforDFEcoupling,i.e.theconditions combininghighcouplingefficiency,thegreatestquantityof cou-pledDFEandthelowestcosts,givingeachoptimizationcriterion
anequalweighting.Theseconditionswerebestmetbycoupling
Trang 4Fig 1. DFE expression, purification and coupling (A) DFE concentration and purity as a fraction of the total soluble protein in untreated plant extracts (control) and after blanching of the leaf material (Hom, homogenate) as well as in the subsequent clarification and purification steps (Adj – pH adjusted, Bag – bag filtrate, DF – depth filtrate, Load – filter-sterilized extract loaded onto the IMAC column, FT start – initial flow-through fraction, FT pool – pooled flow-through fractions) (B) LDS-PAGE analysis (top) and western blot (bottom) of samples from panel A The dominant plant host cell proteins (RuBisCO large and small subunits) are highlighted by green arrows whereas the DFE product is indicated by red arrows Note the apparent oligomerization of DFE despite the denaturing and reducing conditions (C) Coupling efficiency of DFE to NHS-activated Sepharose HP as a function of the injected amount of purified DFE DFE concentrations were determined based on fluorescence analysis (circles) and Bradford assay (diamonds) results for verification purposes.
7.0mgofDFEatpH9.0for45min.Weidentifiedabroadandlargely
pH-independentdesirabilityplateauin therange 6–12mgmL−1
resinDFEloading(Fig.2D),whichmadethecouplingarobust
pro-cess.Interestingly,thefusionproteinretaineditsredcoloreven
aftercouplingandtheinactivationofunusedinteractionsites,and
thecolorcorrelatedwiththeabsoluteamountofDFEboundtothe
resin(Fig.2E).ThisindicatedthatDFEwaspresentinthenative
tetrameric stateof DsRed despitethe low-pH inactivation step
(pH4.0)whichwaspreviouslyfoundtocausethedenaturationof
DsRedandanearpermanentlossoffluorescence[46].Thecolorof
theresincouldthereforebeusedforqualitycontrolduringlater
manufacturingstages
BasedonthetetramericstructureofDFE[21],itsmolecularmass
of28,411gmol−1andthecoupledmassofupto∼10gL−1resin,we
calculatedtheliganddensityoftheresultingaffinityresinusing
Eq.(1).Thepredictedvalueof0.35molmL−1wasabout0.4%of
the50–250molmL−1reportedforion-exchangeresins[47],but
wassimilarinmagnitudetootheraffinityresinssuchasProteinA
(2–11gL−1)[48].Theeffectivenumberof2F5-epitopedomainson
thefusionproteinthatareavailablefor2F5bindingmaybelower
dueto(i)sterichindranceresultingfromthebindingorientation
ofthecoupledDFEmolecules,(ii)thedirectinvolvementofthe
epitope’slysineresidueinthecouplingreaction,and(iii)shielding
oftheepitopesbymAbsboundtoadjacentligands
Oneoption to reduce column costsin the future, especially
whenhighconcentrationsofDFEareneededforcouplingto
NHS-activatedresin,istherecyclingofuncoupledDFErecoveredduring
resininactivationaftercoupling.Forexamplewerecovered∼2mg
(∼13%) of DFE when loading 15mg of the fusion protein per milliliterresin
3.3 Magnesiumchlorideisasuitablereplacementforthelow-pH elutionof2F5
WeusedDFEcolumnswith∼7mgcoupledfusion proteinto capturemAb2F5fromaclarifiedplantextract(Fig.S3B).Wethen testedalow-pHelutionapproachasusedwithProteinAandfound thatgreaterquantitiesof2F5werereleasedasthepHfellbelow 4.5(Fig.3A).Thehighestantibodyrecoveryof∼35%(91%purity) wasachievedatpH3.25accordingtowesternblotanalysisand densitometry,but whenweanalyzed thesamesamplesbySPR spectroscopywefoundthatthemAbelutedatthispHwasunable
tobindtotheProteinAsurfaceofthesensorchip.Weconcluded that2F5wasprobablyirreversiblydenaturedduringelutionatpH 3.25butthathigher-pHelutionconditionswereuneconomicaldue
totheevenlowermAbrecovery.Furthermore,weobservedthat thedistinctredcolorofthecolumnresultingfromDFEcoupling fadedastheelutionpHfellbelow5.0(Fig.3B).Weattributedthis effecttothedenaturationofthefusionprotein,whichhasbeen reportedforDsRedatpH<4.0[21,49].Althoughthe2F5epitope
islinear[19]andshouldthereforebedetectedby2F5evenafter denaturation,wespeculatethattheconformationalchangemight reducethebindingcapacityoftheresinbecausepolypeptidechains
oftheDFEtetramerthathadnotbeencovalentlylinkedtotheresin matrixmaydissociateintotheliquidphase,reducingthenumber
ofepitopeligandsinthecolumn.Indeedwefoundthattheresin capacityfelltozeroafterthreecyclesofelutionatpH3.0
Trang 5Fig 2. DFE coupling efficiency to NHS-activated Sepharose resin (A) Absolute amount of coupled DFE, showing the dependence on pH and the DFE mass brought in contact with the activated resin (B) Coupling yield of DFE calculated as the fraction of fluorescence remaining on the column, showing the dependence on pH and the DFE mass brought in contact with the activated resin (C) Column costs based on the amount of immobilized DFE and the manufacturing costs for the affinity ligand as well as the activated resin (D) Desirability of coupling conditions, showing the dependence on pH and the DFE mass The optimization target was a combination of a large quantity
of coupled DFE, a high coupling efficiency, and low costs, with each optimization criterion given equal weighting The optimal condition is highlighted by a red dot (E) Photographs of columns containing the DFE affinity resin after coupling The numbers beneath the photographs correspond to the conditions highlighted in panels A C.
Table 1
Kinetic parameters and absolute binding capacity of mAb 2F5 transiently expressed in N benthamiana and purified by DFE or Protein A affinity chromatography.
M r,mAb [Da] 154,600 154,600 154,600 159,383 150,814
M r,Fuzeon [Da] 13,476
a values according to [ 25 ].
Wethereforetestedmagnesiumchlorideasanalternative
elu-tionagentbecauseithasbeenusedtoeluteantibodiesfromother
affinityresins[50–52].Inaninitialtest,wefoundthat1.0M
mag-nesiumchloridepredominantlyelutednonspecificallyboundHCPs,
whereas2.0Mmagnesiumchloridewassufficientforthecomplete
elutionof2F5(Fig.3C).Interestingly,4.0Mmagnesiumchloride
forelutioncausedsimilarcolorfadingasobservedfor the
low-pHelution(Fig.3D).Inasubsequentrefinementweobservedthat
even1.25Mmagnesiumchloridewassufficienttoelute2F5from
theDFEcolumnsandtheantibodywasconsistentlydetectedby
westernblottingandSPRspectroscopy(Fig.4A).Underthese
con-ditions,weachieved105±11%recovery(±SD,n=3)and97±3%
purity(±SD,n=3)(Fig.S3C).Althoughweachievedasimilarpurity
(∼96%)usingProteinA,therecoveryof2F5droppedtoonly∼50%
whenitwaselutedincitratebufferatpH3.0.However,recoveries
of∼90%[53]andpuritiesof>95%[17]havebeenreportedforother
antibodies.Weassumedthat2F5issensitivetoacidicdenaturation, andthereforedeterminedthebindingconstantsforthe interac-tionbetween2F5andthesynthetictrimericpeptideFuzeon,which containsthe2F5epitope[25],followingthepurificationof2F5by conventionalProteinAchromatography,DFEaffinity chromatogra-phywithelutionatpH4.0,andthesametechniquewithelutionin 1.25Mmagnesiumchloride.Theabsoluteactivityinallthree prepa-rationswashigh(Table1)andsimilartothosereportedpreviously [25].Valuesaboveunitymayreflectproteinglycosylation,which
wedidnotinvestigateinthisstudy,hencetheirexclusionfromour calculations.Incontrast,thekonweobservedwasonlyhalfofthat reportedformAb2F5expressedineitherCHOcellsortobacco, pos-siblyreflectingthedifferenthostspeciesandexpressionplatform However,allthreepreparationsshowedsimilarkinetic parame-ters,soweconcludedthatthepurificationmethodsdidnothavea negativeimpactonthefunctionalityof2F5
Trang 6Fig 3. Elution of 2F5 from the DFE affinity resin using low-pH buffer or magnesium chloride (A) Total soluble protein and 2F5 concentrations in pH elution fractions after DFE affinity purification as determined using the Bradford assay and SPR spectroscopy, respectively (B) Photographs of DFE columns following exposure to different pH buffers and for repeated bind-and-elute cycles (C) Total soluble protein and 2F5 concentrations in magnesium chloride elution fractions after DFE affinity purification as determined using the Bradford assay and SPR spectroscopy, respectively (D) Photographs of DFE columns after exposure to different magnesium chloride concentrations and for repeated bind-and-elute cycles.
DFEaffinitycapturealsoachievedalogreductionvalueof∼3for
HCPs,similartothevaluereportedforProteinA[53].Thismay
indi-catethatDFEandProteinAresinshavesimilarlevelsofselectivity
However,theHCPconcentrationinourloadwas59mgmg−1mAb
andthus200-foldhigherthanfortypicalCHO-basedprocessesfor
themanufacturingofmAbs[17]
3.4 Thedynamicbindingcapacityremainsat15%after25
bind-and-elutecycles
TheinitialspecificDBC10%oftheaffinityresinformAb2F5was
0.70mg2F5perimmobilizedmgofDFE,or∼4mgmL−1resin.This
wasdeterminedusingoptimizedelutionconditionscombinedwith
loadingatpH7.5 in0.05Mphosphatebuffer, a conductivityof
∼48mScm−1,aresidencetimeof2min,andalinearflowrateof
75cmh−1.TheDBC10%valuecorrespondedto∼12.5%ofthe
the-oreticalstaticbindingcapacitycalculatedbasedontheamountof
coupledDFE(Fig.4B)andwas∼13%ofthe25–60mgmL−1recently
reportedfornovelProteinAresinsundersimilarconditions[17,48]
butsimilartothe0.76–4.80mgmL−1 observedforothercustom
resins[54] Overthecourseof25cycles,theDBC10%oftheDFE
resindeclinedlinearly(adj.R2=0.99)to0.10mgmg−1(∼15%ofthe
initialvalue)(Fig.4C)
WespeculatethattheobservedlossinDBC10%wasduetothe
lossofDFEmoleculesthatwerenotcovalentlyboundtothebase
matrixbut were onlyretained onthecolumn through
associa-tionwithotherDFEmoleculesformingthecharacteristicDsRed
tetramer[21].Thislimitationcouldthereforebeaddressedbyusing
amonomericderivativeofDsRedasanepitopecarrier
3.5 Thepotentialbenefitsoflinearepitopeligandscanoutweigh thecurrentdrawbackscomparedtoProteinA
We used our DFE expression levels and the simple one-steppurificationprocedureasinputparametersforapreviously reportedcostmodel[34]toestimatetheproductioneffortforDFE, andcombinedtheseresultswiththecostsofaffinityresin manufac-turingtoenableacostcomparisonwithProteinA.Thecurrentcost perrunfortheDFEaffinityresinwasfoundtobe∼170-foldhigher thanaconventionalProteinAresin,particularlyreflectingthelower DBC10%andfewerre-usecycles(Table2).However,theProteinA resinselectedfor comparisonrepresentsmorethan45 yearsof intensivedevelopment[55,56].Wethereforeperformedaneffect analysisfortheDFEresincostsincludingpotentialimprovements
totheresinthatseemedwithinreachgiventhecurrentbodyofdata Basedonthelatestreportsofhighlevelproteinexpressioninplants [57],wepredictthatDFEexpressioncanbeincreasedto2.0gkg−1 biomass,whichwillreducetheproductioncostsfortheligandby morethan85%perunitmass.Thecostscanalsobereducedthrough
anincreaseinDFErecoveryduringpurification,whichcouldbe achievedbyoptimizingtheblanching proceduretoreduce pro-teolyticdegradationorthermaldenaturation[39],bothofwhich
we observed for DFE (Fig 1) We predict that thesemeasures wouldincreasetheDFErecoveryfactorfrom0.5to0.7 Further-more,increasingtheliganddensitycaninsomecasesimprovethe DBC10%asshownforion-exchangeresins[58,59].However,when
weinvestigatedthesizeoftheDFE–2F5complexcomparedtothe typicalporediameterof∼80nmreportedforSepharoseHPresin [60],wefoundthatthecomplexis∼29nmindiameterinitsmost
Trang 7Fig 4.DFE resin characteristics (A) Typical chromatogram of a bind-and-elute cycle for a DFE affinity column used to capture mAb 2F5 from a clarified plant extract The axis dimensions of the inset are the same as in the main panel (B) Breakthrough curves of mAb 2F5 using DFE affinity resin after multiple bind-and-elute cycles (C) Dynamic binding capacity for 10% product breakthrough compared to the load referring to the amount of immobilized DFE (D) Schematic representation of the DFE (red) 2F5 (green) complex at full extension in an idealized pore with circular perimeter and a pore radius (r pore ) of 40 nm The 2F5 epitope (orange) is indicated by an orange arrow, and the theoretical minimal effective remaining pore radius (r min,eff ) is shown by a size bar The resulting minimal pore size is shown as a gray circle.
Table 2
Calculation of DFE affinity resin costs compared to Protein A, including two hypothetical scenarios for feasible improvements of the DFE setup assuming the immobilization
of 7 mg DFE per milliliter of resin.
Setup
a Values according to [ 69 ].
b DBC 10% – dynamic binding capacity at 10% product breakthrough.
extendedstate,leaving onlyaneffectiveminimalporeradiusof
∼11nmforproteindiffusionintoandoutoftheresinpores,which
wouldbetoosmallforadditionalantibodiestopass(Fig.4D).Even
thoughtheorientationofthecomplexisflexibleandnotall
com-plexeswillbepresentinthemostextendedform,thismaylimitthe
effectivebindingcapacity.Othershavereporteda poreblocking
effectforion-exchangeliganddensitiesexceeding400molg−1
[59] and we assume that such an effect would occurat lower
densitiesforDFEduetothelargersizeoftheaffinityligand
Fur-thermore,increasingtheliganddensityabove50molmL−1does
notimprovetheDBC [47].Therefore,densitiesinthe2–11gL−1
rangeasforProteinAaremorelikelytobeeffective[48]and matri-ceswithlargerporesizesforDFEaffinityresinpreparationmay helptoimproveligandaccessandthusthebindingcapacity.The useofrecently-developedmonomericvariantsoftheDsRed car-rierprotein[61,62] mightreducethelossofDFEligandsdue to thewash-outofnon-covalentlyboundmoleculesfromtetrameric DFE,whichwespeculateisonereasonforthedecliningcapacity
weobservedoverseveralbind-and-elutecycles.Thesemonomeric variantsoftheDsRedcarrierhavealsobeendesignedforminimal cytotoxicity,enablingthemtobeusedwidelyfortheanalysisof proteinlocalizationandinteractioninlivingcells,sotheyshould
Trang 8impurities[63,64].Proteinengineeringmayalsofacilitaterational
increasesinthestabilityofDFE,asachievedforProteinA[65,66],
andmayalterthepreferredcouplingorientationoftheDFEligand
[67].Thelattercanincreasethelikelihoodthatthe2F5epitopeis
exposedtothecenteroftheresinporesand maythusfacilitate
antibodybinding,resultinginahigherbindingcapacity.A
simi-lareffectcouldbeachievedbyincreasingthenumberofrepeats
ofthe2F5epitopeontheDFEC-terminus,asdemonstrated for
ProteinA[53].Furthermore,increasingthecurrentcontacttime
from2to4mincoulddoubletheDBC10%asreportedforseveral
ProteinAresins[68].Wespeculatethatthesemodificationscould
cumulativelyincreasetheDBC10%fromcurrently4gL−1to15gL−1
(whichisabouthalfoftheDBC10%ofProteinA[69])andfacilitate
50insteadof6cyclesoftheaffinityresin.Bygradually
incorporat-ingthesemodificationsinourcostcalculations,wefindthatthe
DFEresincanbecomecompetitivewithaProteinA-based
counter-part(Table2).EvenwithmoderateDFEproductioncostsavingsand
smallincreasesincolumnperformance,thepriceforthebaseresin
wasthemajorcostdriver(Fig.S4).Wepredictthatbulk
produc-tionoftheaffinityresinwouldreducethebasematrixpricebyup
to75%,whichwouldreducethecostofgoodsfortheDFEresinto
D35pergramofantibodyforthemoderateimprovementscenario
andto<D5pergramofantibodyforthesubstantialimprovement
scenario,thelatterrepresentinga12%savingcomparedtoProtein
A.Inadditiontothedirectresincosts,DFEmayalsobe
economi-callyadvantageousbecausetheamountof2F5recoveredwasabout
twicethatachievedduringconventionalProteinA
chromatogra-phy
Costbenefitsaside,theDFE resinhasthegeneraladvantage
that only mAbs specific for the epitope will be purified This
feature could be exploited to facilitate the purification of
cer-tainidiotype-specificantibodiesfromapolyclonalmixtureorto
improvein-processquality byensuringthatonlymAbisoforms
witha functionalantigen-bindingmoietyareenriched
Further-more,antibodyderivativesthatlacktheFccomponent(e.g.the
scFv, Faband diabodyformats) can bepurified using this new
approach,andbycombiningtwoepitope-basedaffinityligandsina
two-stagebind-and-eluteprocess,bispecificantibodiescouldalso
bepurifiedfromabulkextractorcellculturesupernatant
contain-ingamixtureofmonospecificandbispecificmAbs.Additionally,
mAbscontainingFcdomainswhichexhibitonlyaweak
interac-tionwithProteinAordonotbindtotheresin(e.g.humanIgAand
IgG3ormouseIgG1)caneasilybepurifiedusingDFEorsimilar
ligandscarryingtheaccordingepitope
Sofar,wehaveshownthatDFEhasthepotentialcompetewith
ProteinAorprovidenovelpurificationmodes.Itwillbeinteresting
toinvestigatehowwelltheepitope-fusionapproachcanbe
trans-ferredtoothermAbswithlinearepitopes,giventhattheexpression
levelsofnewaffinityligandproteinsmayvarydependingonthe
natureoftheepitopesequence.However,wehaveworkedwith
severalDsRedfusionproteinsinthepast20years,andhave
reg-ularlyachievedexpressionlevelsexceeding100mgkg−1biomass
[70], makingit likely that novelepitope fusion proteinscanbe
expressedatsimilarlyhighlevels.Furthermore,giventhat
tran-sientproteinexpressioninplantshasagene-to-producttimescale
ofonly2–4weeks[71],itshouldbepossibletoprepare
individ-ualresinsformAbsbindingtodifferentepitopes.Thesequenceof
thelinearepitopemustbeknowninordertogeneratesuchnovel
affinity–ligandfusionproteins,butthisshouldnotrequirefurther
workbecausesequence characterizationistypically requiredas
partofregularproduct andprocess development,notonly due
toregulatoryrequirementsbutalsotoensurefreedomtooperate
andtopreventlegalissues[72,73].Evenifepitopecharacterization
isnotpartoftheprocessdevelopment,a DsRed–epitopefusion
proteinlibrarycanbegeneratedrapidlyusingtechniquessuchas
random-primerPCRcombinedwithappropriatescaffoldsto iden-tifysuitableaffinityligands
4 Conclusions
We have shown that the fluorescent protein DsRed can be usedasacarrierforantibodyepitopes,resultinginfusionprotein expressionlevels exceeding0.1gkg−1 biomass Thesubsequent purificationofDFEwassimplifiedbytheincorporationof blanch-ingandIMACsteps,facilitatingthecost-effectiveproductionofa novelaffinityligand.Theoptimizedcouplingprocedureensured
aDBC10% thatwasonlyoneorderofmagnitudelowerthanthe well-establishedindustrystandardProteinA.Moderate improve-mentsinexpression, purificationandcouplingcouldmake DFE economicallycompetitivewithProteinA,anditsengagementwith epitope-specificcontacts(paratopes)ontheantibodymeansthat DFE and similarligands would be particularlybeneficial when dealingwithmixturesofdifferentantibodies,suchasthose encoun-teredduringthemanufacturingofbispecificmAbs.Ourfuturework willfocusonthefurtherimprovementof DFEstability, epitope densityandbindingaffinity
Acknowledgements
TheauthorsacknowledgeIbrahimAlAmediforcultivatingthe plantsusedinthisinvestigationandDr.ThomasRademacherfor providingthepTRAvector.WearegratefultoMarkusSackfor fruit-fuldiscussionsontheDFEligandstructure.WewishtothankDr RichardMTwymanforeditorialassistance.Thisworkwasfunded
bytheFraunhofer-GesellschaftInternalPrograms,Germanyunder Grant No.Attract125-600164.Theauthorshave noconflictsof interesttodeclare
Appendix A Supplementary data
Supplementarymaterialrelatedtothisarticlecanbefound,in theonlineversion, atdoi:https://doi.org/10.1016/j.chroma.2018 08.014
References
[1] D.M Ecker, S.D Jones, H.L Levine, The therapeutic monoclonal antibody market, mAbs 7 (2015) 9–14.
[2] R.A Rader, E.S Langer, 30 years of upstream productivity improvements, Bioprocess Int 13 (2015) 10–14.
[3] O Purcell, P Opdensteinen, W Chen, K Lowenhaupt, A Brown, M Hermann,
J Cao, N Tenhaef, E Kallweit, R Kastilan, A.J Sinskey, P Perez-Pinera, J.F Buyel, T.K Lu, Production of functional anti-ebola antibodies in pichia pastoris, ACS Synth Biol 6 (2017) 2183–2190.
[4] J.F Buyel, R.M Twyman, R Fischer, Very-large-scale production of antibodies
in plants: the biologization of manufacturing, Biotechnol Adv 35 (2017) 458–465.
[5] J.K Ma, J Drossard, D Lewis, F Altmann, J Boyle, P Christou, T Cole, P Dale, C.J van Dolleweerd, V Isitt, D Katinger, M Lobedan, H Mertens, M.J Paul, T Rademacher, M Sack, P.A Hundleby, G Stiegler, E Stoger, R.M Twyman, B Vcelar, R Fischer, Regulatory approval and a first-in-human phase I clinical trial of a monoclonal antibody produced in transgenic tobacco plants, Plant Biotechnol J 13 (2015) 1106–1120.
[6] C.M.C The, Biotech Working, Group, A-Mab: a case study in bioprocess development, A-Mab: a Case Study in Bioprocess Development, CASSS - An International Separation Science Society (2009) 1–278.
[7] B Kelley, Very large scale monoclonal antibody purification: the case for conventional unit operations, Biotechnol Prog 23 (2007) 995–1008.
[8] M Pathak, G Ma, D.G Bracewell, A.S Rathore, Re-use of protein a resin fouling and economics, BioPharm International, BioPharm Int 28 (2015) 28–33.
[9] B Kelley, Industrialization of mAb production technology: the bioprocessing industry at a crossroads, mAbs 1 (2009) 443–452.
[10] P Chames, M Van Regenmortel, E Weiss, D Baty, Therapeutic antibodies: successes, limitations and hopes for the future, Br J Pharmacol 157 (2009) 220–233.
[11] S Kabir, Immunoglobulin purification by affinity chromatography using protein a mimetic ligands prepared by combinatorial chemical synthesis, Immunol Invest 31 (2002) 263–278.
Trang 9[12] D.Q Lin, H.F Tong, H.Y Wang, S.J Yao, Molecular insight into the ligand-IgG
interactions for 4-mercaptoethyl-pyridine based hydrophobic
charge-induction chromatography, J Phys Chem B 116 (2012) 1393–1400.
[13] S Ghose, M Allen, B Hubbard, C Brooks, S.M Cramer, Antibody variable
region interactions with Protein A: implications for the development of
generic purification processes, Biotechnol Bioeng 92 (2005) 665–673.
[14] V.B Brochier, V Ravault, High throughput development of a non protein A
monoclonal antibody purification process using mini-columns and bio-layer
interferometry, Eng Life Sci 16 (2015) 152–159.
[15] T Arakawa, M Futatsumori-Sugai, K Tsumoto, Y Kita, H Sato, D Ejima, MEP
HyperCel chromatography II: binding, washing and elution, Protein Express.
Purif 71 (2010) 168–173.
[16] S Ghose, B Hubbard, S.M Cramer, Evaluation and comparison of alternatives
to Protein A chromatography Mimetic and hydrophobic charge induction
chromatographic stationary phases, J Chromatogr A 1122 (2006) 144–152.
[17] T.M Pabst, R Palmgren, A Forss, J Vasic, M Fonseca, C Thompson, W.K.
Wang, X Wang, A.K Hunter, Engineering of novel Staphylococcal Protein A
ligands to enable milder elution pH and high dynamic binding capacity, J.
Chromatogr A 1362 (2014) 180–185.
[18] R.L Fahrner, D.H Whitney, M Vanderlaan, G.S Blank, Performance
comparison of protein A affinity-chromatography sorbents for purifying
recombinant monoclonal antibodies, Biotechnol Appl Biochem 30 (Pt 2)
(1999) 121–128.
[19] T Muster, F Steindl, M Purtscher, A Trkola, A Klima, G Himmler, F Rüker, H.
Katinger, A conserved neutralizing epitope on gp41 of human
immunodeficiency virus type 1, J Virol 67 (1993) 6642–6647.
[20] C.E Parker, L.J Deterding, C Hager-Braun, J.M Binley, N Schulke, H Katinger,
J.P Moore, K.B Tomer, Fine definition of the epitope on the gp41 glycoprotein
of human immunodeficiency virus type 1 for the neutralizing monoclonal
antibody 2F5, J Virol 75 (2001) 10906–10911.
[21] G.S Baird, D.A Zacharias, R.Y Tsien, Biochemistry, mutagenesis, and
oligomerization of DsRed, a red fluorescent protein from coral, Proc Natl.
Acad Sci U S A 97 (2000) 11984–11989.
[22] J.F Buyel, R Fischer, Characterization of complex systems using the design of
experiments approach: transient protein expression in tobacco as a case
study, J Vis Exp 1 (2014) e51216.
[23] M.V Matz, A.F Fradkov, Y.A Labas, A.P Savitsky, A.G Zaraisky, M.L Markelov,
S.A Lukyanov, Fluorescent proteins from nonbioluminescent Anthozoa
species, Nat Biotechnol 17 (1999) 969–973.
[24] M Sack, T Rademacher, H Spiegel, A Boes, S Hellwig, J Drossard, E Stoger, R.
Fischer, From gene to harvest: insights into upstream process development
for the GMP production of a monoclonal antibody in transgenic tobacco
plants, Plant Biotechnol J 13 (2015) 1094–1105.
[25] D.M Floss, M Sack, J Stadlmann, T Rademacher, J Scheller, E Stoger, R.
Fischer, U Conrad, Biochemical and functional characterization of anti-HIV
antibody-ELP fusion proteins from transgenic plants, Plant Biotechnol J 6
(2008) 379–391.
[26] J.L Tubbs, J.A Tainer, E.D Getzoff, Crystallographic structures of Discosoma
red fluorescent protein with immature and mature chromophores: linking
peptide bond trans-cis isomerization and acylimine formation in
chromophore maturation, Biochemistry-Us 44 (2005) 9833–9840.
[27] P.A Bates, L.A Kelley, R.M MacCallum, M.J Sternberg, Enhancement of
protein modeling by human intervention in applying the automatic programs
3D-JIGSAW and 3D-PSSM, Proteins (Suppl 5) (2001) 39–46.
[28] J.F Buyel, H.M Gruchow, A Boes, R Fischer, Rational design of a host cell
protein heat precipitation step simplifies the subsequent purification of
recombinant proteins from tobacco, Biochem Eng J 88 (2014) 162–170.
[29] S Menzel, T Holland, A Boes, H Spiegel, J Bolzenius, R Fischer, J.F Buyel,
Optimized blanching reduces the host cell protein content and substantially
enhances the recovery and stability of two plant-derived malaria vaccine
candidates, Front Plant Sci 7 (2016) 1–15.
[30] J.F Buyel, T Kaever, J.J Buyel, R Fischer, Predictive models for the
accumulation of a fluorescent marker protein in tobacco leaves according to
the promoter/5’UTR combination, Biotechnol Bioeng 110 (2013) 471–482.
[31] J.F Buyel, R Fischer, Scale-down models to optimize a filter train for the
downstream purification of recombinant pharmaceutical proteins produced
in tobacco leaves, Biotechnol J 9 (2014) 415–425.
[32] P Cuatrecasas, I Parikh, Adsorbents for affinity chromatography Use of
N-hydroxysuccinimide esters of agarose, Biochemistry-Us 11 (1972)
2291–2299.
[33] J.F Buyel, R Fischer, Flocculation increases the efficacy of depth filtration
during the downstream processing of recombinant pharmaceutical proteins
produced in tobacco, Plant Biotechnol J 12 (2014) 240–252.
[34] J.F Buyel, R Fischer, Predictive models for transient protein expression in
tobacco (Nicotiana tabacum L.) can optimize process time, yield, and
downstream costs, Biotechnol Bioeng 109 (2012) 2575–2588.
[35] B.B Gengenbach, C.R Müschen, J.F Buyel, Expression and purification of
human phosphatase and actin regulator 1 (PHACTR1) in plant-based systems,
Protein Express Purif 151 (2018) 46–55.
[36] K Ko, Z Steplewski, M Glogowska, H Koprowski, Inhibition of tumor growth
by plant-derived mAb, Proc Natl Acad Sci U S A 102 (2005) 7026–7030.
[37] J.F Buyel, J Hubbuch, R Fischer, Comparison of tobacco host cell protein
removal methods by blanching intact plants or by heat treatment of extracts,
J Vis Exp e54343 (2016) 9.
[38] J.A Bornhorst, J.J Falke, Purification of proteins using polyhistidine affinity
[39] S Menzel, T Holland, A Boes, H Spiegel, R Fischer, J.F Buyel, Downstream processing of a plant-derived malaria transmission-blocking vaccine candidate, Protein Expr Purif 27 (July (152)) (2018) 122–130, http://dx.doi org/10.1016/j.pep.2018.07.012 [Epub ahead of print] PMID: 30059744 [40] J.F Buyel, J.A Bautista, R Fischer, V.M Yusibov, Extraction, purification and characterization of the plant-produced HPV16 subunit vaccine candidate E7 GGG, J Chromatogr B 880 (2012) 19–26.
[41] F Sainsbury, P.V Jutras, J Vorster, M.C Goulet, D Michaud, A chimeric affinity tag for efficient expression and chromatographic purification of heterologous proteins from plants, Front Plant Sci 7 (2016) 141.
[42] Y Kim, G Babnigg, R Jedrzejczak, W.H Eschenfeldt, H Li, N Maltseva, C Hatzos-Skintges, M.Y Gu, M Makowska-Grzyska, R.Y Wu, H An, G Chhor, A Joachimiak, High-throughput protein purification and quality assessment for crystallization, Methods 55 (2011) 12–28.
[43] M Benchabane, C Goulet, D Rivard, L Faye, V Gomord, D Michaud, Preventing unintended proteolysis in plant protein biofactories, Plant Biotechnol J 6 (2008) 633–648.
[44] N.N Greenwood, A Earnshaw, Chemistry of the Elements, 2 ed., Elsevier, New York, NY, 1997.
[45] S Madler, C Bich, D Touboul, R Zenobi, Chemical cross-linking with NHS esters: a systematic study on amino acid reactivities, J Mass Spectrom 44 (2009) 694–706.
[46] A Sacchetti, V Subramaniam, T.M Jovin, S Alberti, Oligomerization of DsRed
is required for the generation of a functional red fluorescent chromophore, FEBS Lett 525 (2002) 13–19.
[47] A.M Hardin, C Harinarayan, G Malmquist, A Axen, R van Reis, Ion exchange chromatography of monoclonal antibodies: effect of resin ligand density on dynamic binding capacity, J Chromatogr A 1216 (2009) 4366–4371.
[48] A.D Tustian, L Laurin, H Ihre, T Tran, R Stairs, H Bak, Development of a novel affinity chromatography resin for platform purification of bispecific antibodies with modified protein a binding avidity, Biotechnol Prog (Feburary) (2018), http://dx.doi.org/10.1002/btpr.2622 [Epub ahead of print] PMID: 29464924.
[49] P.V Vrzheshch, N.A Akovbian, S.D Varfolomeyev, V.V Verkhusha, Denaturation and partial renaturation of a tightly tetramerized DsRed protein under mildly acidic conditions, FEBS Lett 487 (2000) 203–208.
[50] V.C Tsang, P.P Wilkins, Optimum dissociating condition for immunoaffinity and preferential isolation of antibodies with high specific activity, J Immunol Methods 138 (1991) 291–299.
[51] A Ben-David, M.A Firer, Immunoaffinity purification of monoclonal antibodies In search of an elution buffer of general applicability, Biotechnol Tech 10 (1996).
[52] M.A Firer, Efficient elution of functional proteins in affinity chromatography,
J Biochem Biophys Methods 49 (2001) 433–442.
[53] E Muller, J Vajda, Routes to improve binding capacities of affinity resins demonstrated for Protein A chromatography, J Chromatogr B 1021 (2016) 159–168.
[54] T Besselink, M Liu, M Ottens, R van Beckhoven, A.E Janssen, R.M Boom, Comparison of activated chromatography resins for protein immobilization, J Sep Sci 36 (2013) 1185–1191.
[55] H Hjelm, K Hjelm, J Sjöquist, Protein A from Staphylococcus aureus Its isolation by affinity chromatography and its use as an immunosorbent for isolation of immunoglobulins, FEBS Lett 28 (1972) 73–76.
[56] G.R Bolton, K.K Mehta, The role of more than 40 years of improvement in protein A chromatography in the growth of the therapeutic antibody industry, Biotechnol Prog 32 (2016) 1193–1202.
[57] J Zischewski, M Sack, R Fischer, Overcoming low yields of plant-made antibodies by a protein engineering approach, Biotechnol J 11 (2015) 107–116.
[58] A Franke, N Forrer, A Butte, B Cvijetic, M Morbidelli, M Johnck, M Schulte, Role of the ligand density in cation exchange materials for the purification of proteins, J Chromatogr A 1217 (2010) 2216–2225.
[59] H.L Lu, D.Q Lin, M.M Zhu, S.J Yao, Effects of ligand density and pore size on the adsorption of bovine IgG with DEAE ion-exchange resins, J Sep Sci 35 (2012) 2131–2137.
[60] B.C To, A.M Lenhoff, Hydrophobic interaction chromatography of proteins I The effects of protein and adsorbent properties on retention and recovery, J Chromatogr A 1141 (2007) 191–205.
[61] R.E Campbell, O Tour, A.E Palmer, P.A Steinbach, G.S Baird, D.A Zacharias, R.Y Tsien, A monomeric red fluorescent protein, Proc Natl Acad Sci U S A.
99 (2002) 7877–7882.
[62] T.M Wannier, M.M Moore, Y Mou, S.L Mayo, Computational Design of the beta-Sheet Surface of a Red Fluorescent Protein Allows Control of Protein Oligomerization, PLoS One 10 (2015), e0130582.
[63] R.L Strack, D.E Strongin, D Bhattacharyya, W Tao, A Berman, H.E.
Broxmeyer, R.J Keenan, B.S Glick, A noncytotoxic DsRed variant for whole-cell labeling, Nat Methods 5 (2008) 955–957.
[64] I.I Shemiakina, G.V Ermakova, P.J Cranfill, M.A Baird, R.A Evans, E.A Souslova, D.B Staroverov, A.Y Gorokhovatsky, E.V Putintseva, T.V.
Gorodnicheva, T.V Chepurnykh, L Strukova, S Lukyanov, A.G Zaraisky, M.W Davidson, D.M Chudakov, D Shcherbo, A monomeric red fluorescent protein with low cytotoxicity, Nat Commun 3 (2012) 1204.
[65] K Minakuchi, D Murata, Y Okubo, Y Nakano, S Yoshida, Remarkable alkaline stability of an engineered protein A as immunoglobulin affinity ligand: C domain having only one amino acid substitution, Protein Sci 22 (2013)
Trang 10[66] S Hober, K Nord, M Linhult, Protein A chromatography for antibody
purification, J Chromatogr B 848 (2007) 40–47.
[67] O Koniev, A Wagner, Developments and recent advancements in the field of
endogenous amino acid selective bond forming reactions for bioconjugation,
Chem Soc Rev 44 (2015) 5495–5551.
[68] R Hahn, P Bauerhansl, K Shimahara, C Wizniewski, A Tscheliessnig, A.
Jungbauer, Comparison of protein A affinity sorbents II Mass transfer
properties, J Chromatogr A 1093 (2005) 98–110.
[69] B Lain, Protein a, Bioprocess Int 11 (2013) 29–38.
[70] A Boes, H Spiegel, N Voepel, G Edgue, V Beiss, S Kapelski, R Fendel, M.
Scheuermayer, G Pradel, J.M Bolscher, M.C Behet, K.J Dechering, C.C.
Hermsen, R.W Sauerwein, S Schillberg, A Reimann, R Fischer, Analysis of a
multi-component multi-stage malaria vaccine candidate–tackling the cocktail
challenge, Plos One 10 (2015), e0131456.
[71] Y Shoji, J.A Chichester, M Jones, S.D Manceva, E Damon, V Mett, K Musiychuk, H Bi, C Farrance, M Shamloul, N Kushnir, S Sharma, V Yusibov, Plant-based rapid production of recombinant subunit hemagglutinin vaccines targeting H1N1 and H5N1 influenza, Hum Vaccin Immunother 7 (2011) 41–50.
[72] X Deng, U Storz, B.J Doranz, Enhancing antibody patent protection using epitope mapping information, mAbs 10 (2018) 204–209.
[73] C.G Sandercock, U Storz, Antibody specification beyond the target: claiming
a later-generation therapeutic antibody by its target epitope, Nat Biotechnol.
30 (2012) 615–618.