High throughput process development for the purification of rapeseed proteins napin and
cruciferin by ion exchange chromatography
Moreno-González, Mónica; Chuekitkumchorn, Pattra; Silva, Marcelo; Groenewoud, Roos; Ottens, Marcel
DOI
10.1016/j.fbp.2020.11.011
Publication date
2021
Document Version
Final published version
Published in
Food and Bioproducts Processing
Citation (APA)
Moreno-González, M., Chuekitkumchorn, P., Silva, M., Groenewoud, R., & Ottens, M. (2021). High
throughput process development for the purification of rapeseed proteins napin and cruciferin by ion
exchange chromatography. Food and Bioproducts Processing, 125, 228-241.
https://doi.org/10.1016/j.fbp.2020.11.011
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ContentslistsavailableatScienceDirect
Food
and
Bioproducts
Processing
jo u r n al ho m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / f b p
High
throughput
process
development
for
the
purification
of
rapeseed
proteins
napin
and
cruciferin
by
ion
exchange
chromatography
Mónica
Moreno-González,
Pattra
Chuekitkumchorn,
Marcelo
Silva,
Roos
Groenewoud,
Marcel
Ottens
∗DepartmentofBiotechnology,DelftUniversityofTechnology,vanderMaasweg9,2629HZDelft,theNetherlands
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received26May2020
Receivedinrevisedform9
November2020
Accepted22November2020
Availableonline30November2020
Keywords:
Canolamealproteins
Highthroughputexperimentation
Chromatography
IonExchange
Columnsimulation
a
b
s
t
r
a
c
t
Proteinsderivedfromplantresourcessuchasoilseedmeals,canolaandsunflower,are
con-sideredaviablealternativetoanimalproteinsforfoodconsumption.Thisworkpresentsa
rationalmethodology,usinghighthroughputexperimentation(HTE),fortheseparationof
cruciferinandnapin,thetwomajorproteinsofcanolameal,bychromatography.Eight
dif-ferentmixedmodeandionexchangeresinswereevaluatedatdifferentconditionswiththe
aimofcapturingnapinandidentifyingadsorption/desorptionbehavior,easeofdesorption
andselectivity.POROS50HSresultedasthemostpromisingresin.Theobtainedequilibrium
adsorptiondatafornapinandcruciferinwasusedinamechanisticchromatographymodel
andcomparedwithexperimentalresultsshowingaverygoodagreement.Themodelwas
usedtoidentifycolumnoperatingparametersthatleadto>98%yieldandpurityforboth
proteins.Subsequentlyaconceptualdownstreamprocessingwasproposed.
©2020TheAuthors.PublishedbyElsevierB.V.onbehalfofInstitutionofChemical
Engineers.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://
creativecommons.org/licenses/by-nc-nd/4.0/).
1.
Introduction
Globalproteindemandhasrisenasaresultofgrowing
pop-ulation and in order to satisfy this demand protein with
appropriatequalityneedstobegeneratedfromanimaland
plantsources(Wanasundaraetal.,2016).Valorizationof
agri-foodbyproducts,suchasoilseedmeal,couldhelptosatisfy
thedemandofthefuturefoodsupplyacrosstheworld.Canola
mealisacompetitiveplant-basedproteinsource,itcontains
∼40%proteincontent;∼12%ofcrudefiber,3%phenolic
com-poundsand3%ofphyticacidamongothers(Lomascoloetal.,
2012;Wanasundara,2011;Wanasundaraetal.,2017).Proteins
fromthismealhavepotentialapplicationsinbakeryproducts
asemulsifiers,indiaryand dressingproductsaseggwhite
proteinreplacement(Kodagodaetal.,1973),inbeveragesas
proteinsupplement,meatbinders,mayonnaiseandin
cheese-∗ Correspondingauthor.
E-mailaddress:M.Ottens@tudelft.nl(M.Ottens).
like products(Wanasundara et al., 2016). Moreover,canola
proteins are reported tohave antiamnestic, anti-oxidative,
antihypertensive, anorectic and antithrombotic properties
(Wanasundara,2011;Wanasundaraetal.,2016).
Napinandcruciferinarethetwomajorproteinsincanola
meal,theseare storageproteinswithdifferent
characteris-tics.Napin(2Salbumin)isabasicproteinwithanisoelectric
pointaround11andamolecularweightbetween12-14kDa.
HighlysolubleinwateratawidepHrange,stableathigh
tem-peratures (75◦C)(Jyothietal., 2007;Pereraet al.,2016)and
holdsfoamingproperties(AiderandBarbana,2011;Schmidt
etal.,2004;Wanasundaraetal.,2016a).Napinprotein
consti-tutesaround20%oftheproteincontentofcanolameal,while
cruciferinrepresent60%(Wanasundara,2011).Cruciferin(11S
globulin)hasanisoelectricpointofaround7.2anda
molec-ular weightbetween230−300kDa,muchhigherthan napin
protein. Thisproteinresemblesstructuralfeaturesofother
seedstorageproteins(Adachietal.,2003)andhaswell
orga-nizedstructurallevels(Wanasundaraetal.,2017).Incontrast
withnapin,cruciferinpossessesgelling,bindingand
emulsi-https://doi.org/10.1016/j.fbp.2020.11.011
0960-3085/©2020TheAuthors.PublishedbyElsevierB.V.onbehalfofInstitutionofChemicalEngineers.Thisisanopenaccessarticle
fyingpropertieswhichmakesitaninterestingproductforfood
applications(AiderandBarbana,2011;Wanasundara,2011).
Several methods have been described to successfully
extractproteinsfromdefattedoilseedmealsincludingsolvent
extraction,aqueousandalkalineextractionassistedwithsalt
andenzymes.Thereportedproteinyieldsvarybetween50%
and80% (Contreras etal., 2019; Fetzeretal., 2018; Pickardt et al., 2009; Vuorela et al., 2004). Despite the high protein
levelincanolamealextracts,thepresenceofglucosinolates,
phyticacidandphenolics,whicharealsoco-extracted,could
limitapplicationoftheproteinsinfoodproducts.Therefore,
effectiveseparationtechniquessuchasmembraneprocesses
(AkbariandWu,2015;Ghodsvalietal.,2005;XuandDiosady,
2002)arerequiredfortheseparationofsuchantinutritional
components(Wanasundaraetal.,2017).Eventhough
pheno-licsareconsideredantinutritionalcomponents,theirrecovery
mightpresentsomeeconomicpotential.Giventheir
antiox-idativeproperties,theyhavepotentialapplicationinthefields
of cosmetics, pharmaceutical and food products (
Moreno-Gonzálezetal.,2020).
Purificationoftheextractedproteinscanbeaccomplished
byseveralmethodssuchasisoelectricprecipitationfollowed
by membrane separation (Ghodsvali et al., 2005; Xu and
Diosady,2002)andproteinmicellarformation(Murrayetal.,
1980),beingisoelectricprecipitation(afteralkalineextraction)
themostapplied.However,extremealkalineconditionscan
havea negativeeffect on the functionalityof the proteins
due todenaturalization, loss ofessential amino acids and
lysinoalanineformation(Fetzeretal.,2018;Gerzhovaetal.,
2015;Houetal.,2017;Rommietal.,2015).Inaddition,high
pHmightcreateprotein-polyphenolscomplexes,whichmake
proteinproductsdarkandhavebitterflavor.Similarly,
pro-teinprecipitationmightreducethesolubilityofproductsand
promotesproteindenaturationcausedbyglobulinaggregates
(Bérotetal.,2005;RaabandSchwenke,1984).Moreover,
co-precipitationofbothproteinsmightoccurleadingtoprotein
mixturesratherthanindividualproteinisolates.Application
ofmore selective and milder conditions, such as aqueous
extractionandchromatographycouldbeappliedtokeep
pro-teinfunctionalityandimprovepurityofeachproteinproduct.
Studiesinpurificationofcanolaproteinsbychromatography
havebeenevaluated(Bérotetal.,2005;Pudeletal.,2015;Zhang
et al.,2007)using cationexchangeresins (CEX),
hydropho-bicinteraction (HIC) and size exclusion(SEC), showingthe
promisingpotentialofapplyingthistechnology.
Thisstudypresentsarationalstrategyfortheseparation
ofcruciferin and napin proteinsfrom canolameal extract
bypreparativeproteinchromatography.Proteinextractionis
assumedtobedoneatpH6andat0.3MNaCl.Thestrategy
involves theevaluation ofdifferentcation exchangeresins
(CaptoS,POROS50HS,CMSheparoseandMacroPrep50)and
mixedmoderesins(CaptoMMC,NuviacPrime,PPAHyperCel
andToyopearlMX-Trp-650M)atdifferentpHvaluesandsalt
concentrationstoidentifyadsorption/desorptionconditions
withthegoalofcapturingnapin.Resinscreeningwasdoneby
meansofhighthroughputexperimentation(HTE)
Addition-ally,resinselectionwasdonebyestablishingaresinselection
criteriabasedonnapincapacity,selectivity,easeofdesorption
and resinprice. Theobtainedequilibriuminformationwas
usedinacolumnadsorption/desorptionmodelwhichisthen
usedtosuggestaconceptualdownstreamprocessforthe
sep-arationofthetwomajorproteins(cruciferinandnapin)from
canolamealextract.
2.
Materials
and
methods
2.1. Chemicals
Forpreparationofthebuffersandsolutionsanalyticalgrade
chemicalswereused.Bis-tris(>98%),tris-HCl(>98%),
acetoni-trile(HPLCgrade),hydrochloricacidanalyticalreagent(37%),
trifluoroaceticacid(>99%)wereobtainedfromSigma-Aldrich,
theNetherlands.Sodiumchloride(>99%)waspurchasedfrom
J.T. Baker, Denmark, sodium hydroxide from Mallinckrodt
Baker, The Netherlands, and Ethanol:Emsure absolute for
analysiswasobtainedfromMerck,TheNetherlands.
Theusedproteinsare:napinisolate(>98%,ABIN1995012),
cruciferin isolate (>98%, ABIN1995013) and rapeseed
pro-tein mixture(57% napinand 43% cruciferin,ABIN1995014).
Theproductswere acquiredfromantibodies-online,GmbH,
Germany.As cruciferinisolatepresentedverylimited
solu-bilitypropertiesinwater,itwasdecidedtousetherapeseed
proteinmixturetoperformbinaryadsorptionexperiments.
Napinisolate,cruciferinisolateandrapeseedprotein
mix-ture were characterized by SDS-PAGE under non-reducing
conditions and reducing conditions (reducing agent TCEP
solution).SDS-PAGEwasperformedona4–12%Bis-TrisGel
(NuPAGETM Novex) at constant voltage (200V). The
non-reduced samplewas preparedwithNuPAGETM LDSsample
buffer. The reduced sample was incubated with LDS
sam-plebufferbeforeloadingongel.NuPAGEMark12TMwasused
asamolecularmarker. Theelectrophoresiswascarriedout
usingMESSDSasrunningbuffer.Afterrunning,thegelwas
stainedinGelCodeTM BlueSafeProteinStainanddestained
withMilli-Qwater.AllSDS-PAGEreagentswereobtainedfrom
ThermoScientific,TheNetherlands.
2.2. Resins
Threemixedmodecationresins:CaptoMMC(GEHealthcare,
Sweden), Nuvia cPrime(Bio Rad, USA) and Toyopearl
MX-Trp-650M(Tosoh,Japan);onemixedmodeanionresin,PPA
HyperCel(PallLifeSciences,France),twostrongcationresins,
Capto S (GE Healthcare, Sweden) and POROS 50HS
(Ther-moFisherScientific, TheNetherlands) andtwoweakcation
resins,CMSepharoseFastflow(GEHealthcare,Sweden)and
MacroPrep50CM(BioRad,USA),wereusedtoevaluatenapin
andcruciferinadsorption.Thecharacteristicsofallresinsare
showninTable1.
2.3. Buffersolutionandpreparations
For buffersatpH4,pH5,pH6andpH8,lacticacid,acetic
acid,bis-trisandtris-HClwereusedrespectively.Allbuffers
werepreparedbydissolvingtheamountofsaltcorresponding
to50mMinMilli-Qwater,adjustingthepHusing2MHClor
2MNaOH.Thesaltconcentrationwasadjustedto0.1M,0.3M,
0.7Mand1.0MbyaddingthecorrespondingamountofNaCl
beforecompletingbufferfinalvolume.Allbufferswerefiltered
previoustouseusingfilterswith0.45mporesize.
Napin stock solution was preparedby dissolving napin
protein(8g/L) inthe appropriatebuffer and filteredwitha
disposable0.22mPVDFfilter.Thisnapinstocksolutionwas
dilutedtodifferentnapinconcentrations(from1to8g/L)using
Milli-Qwater.
In binary component experiments, protein mixture
Table1–MixedmodeandIonexchangeresins.
Name/Type pKa Matrixcompositiona Liganddensitya
(mmol/L)a
CaptoMMC Mixedmodeweakcationexchanger ∼4.6b Highlycross-linkedagarose 80
NuviacPrime Mixedmodeweakcationexchanger ∼4.5b Macroporoushighlycross-linked
hydrophilicpolymer
65 ToyopearlMX-Trp-650MMixedmodeweakcationexchanger 2.9and9.4c Methacrylicpolymermatrix 110
PPAHyperCel Weakanionexchanger 8 Highporositycross-linked
cellulose
65
CaptoS Strongcationexchanger 1.2 Highlycross-linkedagarosewith
dextransurfaceextender
125
POROS50HS Strongcationexchanger 1.2 Cross-linked
polystyrene-divinylbenzene
104
CMSepharoseFastflow Weakcationexchanger 4.7 Cross-linkedagarose 80
MacroPrep50CM Weakcationexchanger 4.7 Methacrylatepolymerbased 210
a Providedbyresinsuppliers. b Basedon(ZhuandCarta,2016).
c BasedonthepKaofthetryptophanligand(NationalCenterforBiotechnologyInformation,2020).
Thesolutionwassonicatedatroomtemperaturefor30minin
ordertoincreasethesolubilityofcruciferin.Afterthat,
insol-ubilizedproteinwasremovedbyfilteringwithadisposable
0.22mPVDFfilter,thisstocksolutionwasdilutedtoevaluate
cruciferineffectonnapinadsorption.
2.4. Analyticalmethods
Insinglecomponentnapinexperiments,theconcentrationof
the proteinwasmeasured spectrophotometrically,
measur-ingthe absorbanceat280nmusingthe spectrophotometer
InfiniTePro200platereader(Tecan,Switzerland).The
mea-surementwas performed with100L of liquid solution in
a96-well half-area microplate (UV-STAR®, Greiner bio-one,
Germany)
To measure the concentration of both proteins, in the
proteinmixtureexperiments,reversephaseliquid
chromatog-raphy(RPC)wasapplied.TheanalysiswasdoneusinganUltra
High Performance Liquid Chromatography system (UHPLC
Ultimate3000)(ThermoScientific,USA)equippedwitha
Zor-bax 300 SB-C8 Rapid Resolution HD column (2.1×100mm,
1.8m)(Agilent,USA).Thecolumnwasequilibratedwith20%
acetonitrilesupplementedwith0.1%Trifluoroaceticacid(TFA)
at0.3mL/minkeepingcolumntemperatureat30◦C.The
sam-plewasinjectedandagradientofacetonitrilestartedfrom
20%to75%in7min,detectionwasdoneat280nm.Thenthe
columnwaswashedwith75%acetonitrilesupplementedwith
0.1%TFAfor5minbeforethenextinjection.Napin
calibra-tionlineswere evaluatedusingnapinproteinisolate.Since
cruciferinisolatedidnotshowtheexpectedcharacteristics,
asthe polypeptide profiledidnot show the corresponding
bands(Supplementarymaterial),astandardcurvecouldnot
beobtained.Therelativechangeofconcentrationwasusedto
evaluatecruciferin.Assumingthattheratiobetween
equilib-riumconcentrationandinitialconcentrationisproportional
topeakarearatio
CeCfeed =
Areapeakafteradsoprtion Areapeakoffeed
determinedby
UHPLC.
2.5. Batchadsorptionexperiments
Batch experiments were performed in order to determine
adsorptionequilibriumisothermsofnapinandcruciferinat
differentadsorption/desorptionconditionsondifferentmixed
modeandionexchangeresins,inordertoseparateboth
pro-teins.
2.5.1. Adsorptionequilibriumisotherms
Napinadsorptionequilibriumisothermswereevaluatedusing
aTecanEVOFreedom200roboticstation(Tecan,Switzerland)
equipped with an orbital mixer (Te-shake), an automated
vacuum system (Te-VacS),aplate reader(InfiniTe Pro200),
a roboticmanipulator (RoMa) arm(to movemicroplates to
thedifferentpositionsoftheroboticstation)andtwoliquid
handingarms(LiHaandMCA96).Theprocedureinvolvesthe
different stepsofthe chromatographyrununtiladsorption
(washing,equilibrationandadsorption).Aknownamountof
eachresin(15.6Lor23.4L)wasaddedtothewellsofa96
deep-wellfilterplate(catalognumber:MDRLN0410)from
Mil-lipore,USA.ResinswereaddedusingMediaScout®ResiQuot
resinloader(Atoll,Germany).Resinwerewashedtwotimes
withMilli-Q waterusing thevacuumsystem (Te-VacS)and
equilibratedwiththecorrespondingbufferfor20min.under
agitation(1200rpm).Equilibrationbufferwasremovedby
cen-trifugation with an Eppendorf centrifuge 5810 R (rotation
speed 4000rpmfor3min).Aftercentrifugation,resins were
contactedwith312Lofnapinsolutionsunderagitationuntil
equilibriumwasreached(2hat1200rpm).Onceequilibrium
was reached,the filterplate was centrifuged tocollect the
supernatant and equilibrium concentration was measured
spectrophotometrically (see2.4 Analytical methods).Napin
experimentswereperformedinduplicate.
EffectofpHandionicstrengthwereevaluatedfornapin
adsorptionbyperformingexperimentsat4differentpH(pH
4,5,6,8)keepingNaClconcentration(0.3M)constantandat
pH6varyingsaltconcentrationfrom0.1Mto1MNaCl.This
allowstoidentifydesorptionconditions.
Napinadsorptioncapacitywascalculatedaccordingtothe
massbalanceEq.(1)
qp,eq=
Cp,load∗Vload−Cp,eq(VH+Vload)
Vresin
(1)
Whereqp,eq istheadsorptioncapacity(mg/mLresin),Cp,loadis
theproteininitialconcentration(mg/mL),Vload isvolumeof
theliquidphase(mL)Cp,eqistheproteinequilibrium
concen-tration(mg/mL),VHistheholdupvolume(mL)andVresinisthe
Aftercentrifugation,someliquidcouldremainintheresin.
Thisliquidholdupwas determined usingthe method
sug-gested by Nfor et al. (2010). The resins were placed in a
deep-wellfilterplateandequilibratedwith350Lof1MNaCl.
After45minthefilterplatewascentrifugedwith5810R
cen-trifuge(Eppendorf,Germany) for3minat4000rpmandthe
flowthroughwascollectedtomeasureconductivity.Thiscycle
wasrepeateduntiltheconductivityofflowthroughwasequal
totheconductivityof1MNaClsolution. Consequently,the
equilibratedresinswerecontactedwith312LofMilli-Qwater
andincubatedovernightwithoutagitation.Aftercontacting,
thefilterplatewas centrifugedand theconductivityofthe
flow-through was measured.All the conditions were done
intriplicate.Theconductivity was measuredwitha
multi-parameteranalyzerC832(ConsortNV,Belgium).
Theholdupvolumewasmeasuredusingthesaltmass
bal-anceEq.(2)
CS,initial∗VH= CS,final∗ (VH+VW) (2)
Initialsaltconcentrationinallresinswas1MNaCl(CS,initial),
VHisholdupvolume(mL),CS,finalisthefinalsalt
concentra-tionaftercontactingwithMilli-QwaterandVW istheadded
volumeofMilli-Qwater.
Since cruciferin has low solubility in water, cruciferin
adsorptionisothermswereevaluatedusingtheprotein
mix-ture. A similar procedure to the one applied for napin
adsorptionisothermswasusedwiththeproteinmixturein
theliquidhandlingroboticstation.Theevaluatedconditions
were:pH6and 0.3MNaCl sincetheyare the same
condi-tionsintheproteinextract(adsorptioncondition).Cruciferin
experimentswereperformedinduplicate.
As the absolute cruciferin liquid concentration values
could notbemeasured,cruciferin adsorptioncapacitywas
determinedbydividingEq.(1)bythereferenceconcentration
(C0)(Eq.(3)),assumingthatCCp,eq
0 =
Areaatequilibirum Areaofreference
qp,eq=qCp,eq
0 =
Cp,load∗Vload−Cp,eq(VH+Vload)
VresinC0
(3)
Thereference cruciferin area peaks were the ones
cor-responding to the conditions of 0.3M NaCl and Napin
concentrationofaround6g/LforexperimentsatpH6(feed
condition).
2.5.2. Parameterestimation
Adsorptionisothermexperimentalresultsofnapinwere
fit-tedtoalinearisothermortoaLangmuirtypeisotherm(Eq.
(4)).Thistoidentifythe initialisotherm slope,which isan
indicationoftheaffinityofnapintotheresin.
qp,eq=
bp,iqmaxp,i Cp,i
1+bp,iCp,i
(4)
whereqmax
p,i isthemaximumadsorptioncapacity(mg/mLresin)
andbp,iistheLangmuirconstantalsoknownasequilibrium
constant (Carta and Jungbauer, 2010). Theinitial isotherm
slope,attheevaluatedconditions,wasusedtoevaluateresin
selection(see2.6Resinselection).
Themostsuitableresinequilibriumdata(POROS50HS)was
fittedtothemixedmodeisotherm,developedbyNforetal.
(2010).Thisisothermsisbasedonthethermodynamic frame-workofMollerupetal.(2008).Amoredetailexplanationofthis
isothermisfoundinsection2.8.1Mixedmodeisothermmodel
ofthispaper.Thefittedparametersofthisisothermmodel
were:1)thethermodynamicequilibriumconstant(Keq);2)the
stoichiometriccoefficientofsaltcounterion(v);3)the
param-eterthatdescribesthedifferencebetweenwater-proteinand
protein–protein interactions (Kp)and 4) the parameter that
describes the difference between water – protein and salt
–proteininteractions(Ks).Aspreviouslymentioned,binary
componentexperiments(napin+cruciferin)wereperformed
usingaproteinmixturepowder.Binarycomponent
adsorp-tionexperimentsshowedahighernapinadsorptioncapacity
thannapinsinglecomponentexperiments.Forisotherm
mod-ellingthis was adjusted byconsideringthe valueobtained
frombinarymixtureexperimentalresults,whichcorresponds
to44mg/mL.
DataregressionsweredoneusingMATLABR2017bandthe
functionlsqcurvefit.Inthemixedmodemodel,qpappearsin
both sidesofthe equation(Eq. (7)) Thenumericalsolution
ofthisequationwasfoundusingthefsolvefunctionof
MAT-LABanditwascombinedwiththeregressionusinglsqcurvefit
optimizer.Thus,theprocedureoftheparameterregressionis
applyinglsqcurvefitoptimizertominimizethesumofsquared
residualsbetweentheexperimentaldataandthenumerically
solvedadsorptiondatausingfsolvefunction.
Inaddition,asbinarymixtureexperimentpresentedavery
low change in cruciferin concentration. A linear isotherm
modelwasconsideredforthisproteinbyestimatingisotherm
slopebyfittingalinearcurvetotheexperimentaldata.
2.6. Resinselection
Thegeneratedequilibriumdatawasusedtoselectthemost
suitableresinfortheseparationofbothproteins.Thedesired
scenario is the adsorption of napin while cruciferin flows
through.Theselectioncriteriaweredefinedassuggestedby
Sevillanoetal.(2014),consideringnapinadsorptioncapacity
atfeedconditions(pH6and0.3MNaCl)andnapindesorption,
whichwasevaluatedusingtheinverseofthelowestisotherm
slopedeterminedattheevaluatedconditions.Thethird
crite-rion,selectivity,wasevaluatedusingtheratiobetweennapin
andcruciferinisothermslopesatadsorptionconditions.The
fourthcriterionwasthepriceoftheresinobtainedfromthe
differentsuppliers. Aweightbetween0and1wasgivento
eachcriterion,being0.5fornapincapacity,0.3fornapin
des-orption,0.1forselectivityand0.1forprice. Thereasonfor
givingalowweighttotheselectivitycriterionhadtodowith
theobservedpoorbindingofcruciferinontomostofthe
eval-uatedresins.
Eachcriterionwasnormalizedandresinscorewas
calcu-latedusingEq.(5).
Resinscore=
weight∗ criterionmaximumvalueofcriterion (5)
2.7. Columnadsorption/desorptionexperiments
The highestscoring resin,POROS 50 HS, was used to
per-formpulsecolumnexperimentsusingnapinandtheprotein
mixture(napin+cruciferin).Theexperimentswereperformed
inanAKTATMAvantsystem(GEHealthcareBio-SciencesAB,
Uppsala,Sweden)atroomtemperature(25◦C)operatedwith
Unicorn7.0software.Conductivity,pHandUVat280nm
sig-nalsweremonitoredduringtheexperiments.
AValiChrom11.3–100 POROS® 50 HS(Repligen,Sweden)
and the total bed heightwas 10cm, providing a total
col-umnvolumeof9.8mL.Extraparticleandtotalporositieswere
determinedbypulseinjectionsofbluedextranand1MNaCl
respectively.Theobtainedvalueswere0.83totalporosity(T)
and0.3forextraparticleporosity(b).
Fornapinand proteinmixtureexperiments,the column
was equilibrated with 50mM bis-tris pH6 with 0.3M NaCl
bufferfor5columnvolumes(CV)at2.5mL/min.Then1mL
ofnapinsolutionat6g/Lwasinjectedinthecolumn(10.1%
CV),columnwaswashedwiththeequilibrationbufferfor5CV
inordertoremovenon-bindingsubstratesoutofthecolumn.
Stepelutionwasdonebyapplying5CVofelutionbuffer,
Tris-HClpH8with0.7MNaCl.Aftereachexperiment,thecolumn
wasregeneratedwith1MNaClsolutionfor5CVas
recom-mendedbysupplier.
Forrapeseedproteinmixture,12g/Lofproteinmixturewas
dissolvedin50mMBis-TrispH6with0.3MNaClbuffer.This
correspondsto6.84g/Lofnapinand5.16g/Lofcruciferin.The
proteinmixture solution was then sonicatedfor 30min in
ordertoincreaseproteinsolubility.Thesolutionwasfiltered
with0.22mPVDFfilterbeforeinjectionincolumn.Afteruse,
thecolumnwasstoredin20%(v/v)ethanolassuggestedby
supplier.
2.8. Chromatographymodeling
2.8.1. Mixedmodeisothermmodel
ThemixedmodeadsorptionisothermmodelfromNforetal.
(2010) was appliedin this work. Themodel is a
combina-tionofHICandIEXmodelsfromMollerup(2007).Thismixed
modeisothermisbasedontheassumptionthataprotein(P)
bindsto namountofligand(L)byhydrophobicinteraction
andconcurrentlyexchangeswithvamountofsaltcounter-ion
(S)generatingaprotein-ligandcomplex(Ln+v)inafixed
stoi-chiometryasshowninEq.(6).Thestoichiometriccoefficient
ofsalt counterionisdefinedasthe ratiobetweenthe
pro-teinbindingchargedividedbythechargeofthesaltcounter
ion
v= zpzs
. Inthis work,sodium chloride wasused as a
saltcounterion.Thesinglecomponentadsorptionisotherm,
basedon reactioninEq.(6),resultsinthe isothermmodel
describedinEq.(7). P+vSL+nL ↔PLn+v+vS (6) qp cp = A∗
1−qqmaxp p v+n ∗˜p (7) whereA= ˜Keq()v+n 1 zscs v1 c n and˜p=exp (Kpcp+Kscs) (8)In Eq. (7), qp is the adsorbed protein concentration
(mg/mLresin), cp is the protein concentration in solution
(mg/mL),Keqisthermodynamicequilibriumconstantof
reac-tion(mL/mg),csistheconcentrationofsaltinliquidphase.c
ismolarityofthesolution(assumedtobewaterconcentration
(Mollerup,2007))andqmax
p ismaximumadsorptioncapacityof
protein(mg/mLresin),isliganddensityofthemixedmode
resinswhichareassumedtobeequalforHICandIEXligands
(mmol/L)andwasobtainedbasedonsupplierspecifications.
Theactivity coefficient ofthe protein ( ˜p) was determined
usingtheVan derWaalsequationofstateshowninEq.(8)
assuggestedbyMollerup(2006).Kpparameterdescribesthe
differencebetweenwater-proteinandprotein–protein
inter-actionswhileKsparameterdescribesthedifferencebetween
water– proteinand salt –proteininteractions (Nfor et al.,
2010).Inthe model,(1− qp
qmax
p )describesthe fractionoffree
ligands,whichis1ifnoproteinisboundanddecreases
asymp-toticallytozero.Thisisothermincorporatestheeffectofsalt
concentrationinthetermCs.
ThemixedmodeisothermdescribedinEq.(7)reducesto
theHICandIEXisothermmodelsderivedbyMollerup(2006)
whenv=0(electrostaticinteractionsnotpresent)andn=0(no
hydrophobicinteractions),respectively.
2.8.2. Columnchromatographymodel
Column chromatography was simulated based on the
transport-dispersive columnmodelwhich canbedescribed
percomponentasshowninEq.(9)
∂cp,i ∂t +
1−ε b εb ∂qp,i ∂t = −v ∂cp,i ∂x +DL,i ∂2cp,i ∂x2 (9)where,εbisthebedporosity,vistheinterstitialvelocityofthe
mobilephase(m/s),andDListheaxialdispersioncoefficient
(m2/s)
Masstransfercanbequantifiedbythe liquid-filmlinear
drivingforceapproximation.Thesolidstationaryphase
con-centrationterm ∂qp,i
∂t isdefinedasfollowsinEq.(10):
∂qp,i ∂t =kov,i
Cp,i−C∗p,eq,i (10)kov,iistheoverallmastranferscoefficient(1/s)andC∗p,eq.isthe
bulkequilibriumconcentrationwhichisobtainedusingthe
mixedmodeisotherm(Eq.(7)).Mixedmodeisotherm
param-eterswereusedtomodelnapinchromatographywhilealinear
isothermwasconsideredtomodelcruciferinchromatography.
The film masstransfer coefficient isdefined in Eq.(11)
(FelingerandGuiochon,2004). 1 kov,i= dp 6∗kf + d2 p 60∗εp∗Dp (11)
wheredpistheparticlediameter(m),kfisthefilmmass
trans-fer coefficient (m/s), εp isthe intraparticleporosity and Dp
is thepore diffusivity(m2/s). To determineadditional
rele-vantmodelparameters,correlationspresentedinTable2were
used.
ColumnboundaryconditionsaredescribedbyDanckwerts
fordispersivesystemsanditisassumedthatthecolumnis
notpreloadedwiththeproteinsCp(t=0)=0andqp(t=0)=0.
C (t,x=0)=Cinlet− DL uh · ∂C (t,x=0) ∂x (12) ∂C (t,x=L) ∂x =0 (13)
wherexistheaxialposition.Eq.(12)representstheboundary
attheinletofthecolumnandEq.(13)theboundarycondition
atthecolumnoutlet.Aspulseexperimentswereperformed,
theinjectionprofileismodelledasarectangularpulsewith
aconstantfeedconcentrationforagiventime,wheretpulse=
Vinj/˚v,where˚visthevolumetricflowrate(m3/s)andVinjis
theinjectionvolume.
Pulse Cinlet(t)=Cfeed,i at 0<t<tpulse (14)
Table2–Engineeringcorrelationsforcolumnmodelling.
Masstransferparameter Correlation Reference
Hydrodynamicradius StokesEinstein LeVanetal.(1999)
Freediffusivity Young Youngetal.(1980)
Filmmasstransfercoefficient Wilson&Geankoplis WilsonandGeankoplis(1966)
Poretortuosity Wakao&Smith WakaoandSmith(1962)
Porediffusivity Brenner&Gaydos BrennerandGaydos(1977)
Axialdispersioncoefficient Gunn Gunn(1987)
Pressuredrop Karman-Cozeny CartaandJungbauer(2010)
Eq.(9)isapartialdifferentialequation,dependentontime
andcolumnposition,thatcanbeapproximatedtoanordinary
differentialequationbyspatialdiscretization.Themethodof
lineswasusedtodiscretizeEq.(9)inspace.Thesetofordinary
differentialequationswassolvedinMATLABR2017busingthe
ODEsolverode15s.
3.
Results
and
discussion
3.1. Resinandproteinscharacteristics
Theusedproteinisolates,cruciferin,napinandprotein
mix-turewere analyzedbySDS-PAGE (Supplementarymaterial).
Napinandtheproteinmixtureshowedexpectedbandprofiles
thatwerecomparabletotheonesreportedelsewhere(Perera
etal.,2016).Cruciferindidnotshowntheexpectedprofileand
thesolubilityofthisproteinisolateinanyofthetested
condi-tionswasverylimited,thereforetheproteinmixturewasused
toevaluatecruciferinisotherms.
Toavoid an overestimation of the adsorbed phase
pro-teinconcentration, theresin liquid hold-upwasevaluated.
Theobtainedvalueswere9.3L,8.7L,7.0L,9.2L,10.0L,
9.8L,12.4Land8.5LforCaptoMMC,NuviacPrime,
Toy-opearlMX,PPAHyperCel,CaptoS,POROS50HS,CMSepharose
and MacroPrep50 CM respectively. These hold up volumes
correspond to ∼3−4% of the total volume applied in the
adsorptionexperiments(350L).
3.2. Napinadsorptionequilibriumisotherms
Napinadsorptionequilibriumisothermsweredeterminedfor
allresinsatfeedconditions,(0.3MNaClatpH6).Resultsare
shown inFig. 1, wherethe experimentalnapin adsorption
capacityisplotted againstthe equilibriumbulk
concentra-tion.Fromthefigure,onecanobservethatatfeedconditions,
thehighestadsorptioncapacityisobtainedusingresinCapto
MMC,followed byNuviacPrime,POROS HS, PPAHyperCel,
Toyopearl, Capto S, MacroPrep and CM Sepharose.Most of
theresinsshowedamaximumlimitintheamountadsorbed,
which can be described with a Langmuir isotherm model
(Eq.(4)).ResinsCaptoSandCMSepharosepresentedalinear
behavior.Theexperimentaldatawasfittedwithtwoisotherm
models-Langmuirorlineartype.
InadditiontotheresinisothermshapepresentedinFig.1,
theaffinityofnapintotheresinswasalsoobtainedby
deter-miningtheinitialisothermslopes.Theinitialisothermslopeis
anindicationoftheinteractionstrengthbetweentheprotein
andtheresin.Thelargertheisothermslope,thestrongerthe
interaction.Atfeedconditions,theisotherms’initialslopes,
foreachresin,canberankedfromlargesttosmallest:
Cap-toMMC,NuviacPrime,POROSHS, PPAHyperCel, Toyopearl,
Macro Prep, Capto S and CM Sepharose. Absolute values
(obtainedfromfitting)canbefoundinTable3.
Fig.1–Napinadsorptionisothermsonmixedmodeand ionexchangeresinsatpH6and0.3MNaCl.Symbols representexperimentalresults.Errorbarsresultedfrom duplicateexperiments.LinesrepresentLangmuirisotherm forCaptoMMC,NuviacPrime,POROSHS,Toyopearl,PPA HypercelandMacroPrepandlinearisothermforCaptoS andCMSepharose.
Thedifferencebetweenthenapinbindingstrengthonto
the different resins can be explainedbased on the pKaof
theresins(Table1),theisoelectricpointofnapinprotein-in
thiscasearound11-andtheproteinnetchargeatthe
eval-uated conditions.AtpH6,all resinsarenegativelycharged
and napin has a positive net charge. Therefore, attractive
electrostaticinteractionsarepossible.However,thereisalso
salt presentedinthe medium(NaCl) whichcould promote
hydrophobicinteractionsbetweennapinandthemixedmode
resins.Asaltconcentrationof0.3MNaCl–whichcanbe
con-sideredmedium-,provedtobealreadytoohighfortheweak
ion exchangers here evaluated (CM Sepharoseand
Macro-Perp)andevenforthestrongcationexchangerCaptoS.Asthe
preferablescenarioistheadsorptionofnapinatpH6and0.3M
NaCl,CMSepharose,Macro-PrepandCaptoSresinswere
dis-cardedassuitablecandidatesduetothepoorbindingofnapin
undersuchconditions.
TheeffectofionicstrengthandpHwasevaluatedforall
resinsinordertodeterminesuitabledesorptionconditions.
3.2.1. EffectofpHonnapinadsorption
Theionicstrength andpHmighthaveasignificant impact
onthe adsorptionofnapinonmixedmodecationicresins,
cationexchangeresinsandtheanionmixedmoderesinhere
evaluated.ExperimentswereperformedatdifferentpHvalues
keepingthesaltconcentrationconstantat0.3MNaCl,which
correspondtothesameionicstrengthasintheproteinextract.
NapinisothermresultsareshowninFig.2andFig.3.
From Fig. 2, one cannotice that for Capto MMC,Nuvia
Table3–NapinisothermslopeonmixedmoderesinandionexchangeresinatpH6andpH8anddifferentNaCl(Cs)
concentration.
Resin pH CS(M) Isothermslope
(mL/mLresin)
Resin pH CS(M) Isothermslope
(mL/mLresin) CaptoMMC 6 0.1 1185.4+245.2 Capto S 6 0.1 146.1+12 0.3 437.6+266.1 0.3 1.4+0.1 1.0 23.1+7.9 0.7 Notadsorbed 8 0.1 1114.4+248.8 8 0.1 25.2+3.8 0.3 99.3+25.6 0.3 0.2+0.0 0.7 17.8+6.8 0.7 Notadsorbed NuviacPrime 6 0.1 620.9+78.3 POROS50HS 6 0.1 453.8+76.2 0.3 67.5+17.23 0.3 52.5+14.9 1.0 1.3+0.01 0.7 0.7+0.9 8 0.1 276.6+57.5 8 0.1 108.9+22.5 0.3 20.2+3.5 0.3 6.0+1.8 0.7 2.1+0.2 0.7 Notadsorbed Toyopearl 6 0.1 97.4+13.6 CM Sepharose 6 0.1 8.5+3.0 0.3 5.9+5.02 0.3 0.3+0.0 1.0 0.4+0.2 0.7 Notadsorbed 8 0.1 52.4+9.1 8 0.1 13.5+1.9 0.3 1.8+0.3 0.3 0.2+0.0 0.7 0.5+0.1 0.7 Notadsorbed PPAHyperCel 6 0.1 1.9+0.1 MacroPrep 6 0.1 67.4+14.8 0.3 8.5+3.4 0.3 3.6+1.3 1.0 13.0+3.7 0.7 Notadsorbed 8 0.1 7.7+7.5 8 0.1 19.8+2.6 0.3 15.4+2.8 0.3 0.8+0.1 0.7 35.4+7.9 0.7 Notadsorbed
Fig.2–NapinadsorptionisothermsatdifferentpHkeepingsaltconcentrationat0.3MNaCla)CaptoMMCresin,b)Nuvia cPrimeresin,c)Toyopearlresinandd)PPAHyperCelresin.Symbolsrepresentexperimentalresults.Errorbarsresultedfrom duplicateexperiments.LinesrepresenttheLangmuirisotherm.
capacitywithincreasingpH.Thiscanbeexplainedbythe pro-tein’snetchargeandtheresinligand’spKa.Theweakcation exchangersarenegativelychargedatpHhigherthanthepKa, whiletheoppositeoccursforweakanionexchangers,which arepositivelychargedatpHlowerthanthepKa.Strongcation exchangersarepractically alwayschargedatanypH(Carta andJungbauer,2010).Theinteractionsbetweenmixedmode
resinsandproteinshavebeenexplainedbydifferentauthors
(Nforetal.,2010).TheinteractionisstrongestclosetothepKa
oftheligandanddecreasesifthepHisclosetothepIofthe
protein,whichisclearlyobservedhereforToyopearl,Capto
MMC,NuviacPrimeandPOROSHS.AtpH4,thenetcharge
ofnapinisaround12whileatpH8,thenetchargedecreases
significantlytoaround4.Thenetchargeofnapinat
Fig.3–EffectofionicstrengthonnapinadsorptiononCaptoMMC,POROSHSandPPAHyperCelatpH6andpH8.Symbols representexperimentalresults.Errorbarsresultedfromduplicateexperiments.Linesrepresentthemixedmodeisotherm thatthedatahasbeenfittedto.
sequence(obtainedfromtheRCSBProteinDataBank(PBD))
andEq.(16)(Supplementarymaterials).
znet=
i Nbasic,i 1+10pH−pKai − Nacidic,i 1+10pH−pKai (16)Whereznet isthe net chargeNbasic,i isthe numberofbasic
aminoacidsandNterminal,andNacidic,iisthenumberofbasic
aminoacidsandCterminal.
Theoppositebehavior isobservedwith theweak anion
mixed mode resin PPA HyperCel: an increase in
adsorp-tion capacitywithanincrease ofpH.Sincethe pKaofthe
PPA HyperCel resin ligand is around 8, the resin is
posi-tivelycharged.Becausenapinisalsopositivelychargedatall
theevaluatedpH values,thereisanelectrostaticrepulsion
betweenthe resin and the protein.The adsorptionis
con-sequentlyaresultofthehydrophobicinteractions between
napinandtheresinandtheelectrostaticrepulsion.ThepH
hasabiginfluenceontheadsorptionofnapininPPAresin,
astheisothermslopeandisothermshapechangewithpH:
thereisahighernapinaffinitytotheresinatahigherpH.
However,theadsorptioncapacityisstilllowerthanthe
max-imumobtainedatfeedconditionpH6and0.3MNaClwith
otherresins(e.g.40mg/mLbyCaptoMMC),whichdecreases
PPAresinscore.
Eventhough,thereisanotoriouschangeonnapin
adsorp-tionatdifferentpHvalues,theCaptoMMCresinistheleast
influenced,showingafavorableisothermevenatpH8.On
theotherhand,napinadsorptionstrengthonNuviacPrime,
POROS 50 HS and Toyopearl is significantly reduced. The
isothermslopesofallthe resinsatdifferentconditionsare
indicatedinTable3.
Theisothermsloperepresentstheaffinityoftheproteinto
theresin(thebindingstrength)whichisclearlymuchhigher
forCaptoMMCatalmostanypHvalue.However,thisisnot
necessary an advantage,as a higher pH might be needed
todesorbnapinwiththisresin,possiblyinterferingwithits
nativestructure,stablebetweenpH3–12(Wanasundara,2011),
Theseresultscanbeusedtoidentifydesorptionconditions
ofthebestperformingresin.
3.2.2. Effectofionicstrengthonnapinadsorption
Higher ionic strength is known to promote hydrophobic
interactionsduetotheso-called“salting-out”effect.Three
dif-ferentsaltconcentrations(ionicstrengths)weretestedattwo
differentpHconditions.Mostofthetestedresinsshowed
sim-ilartrends:adecreaseofnapinadsorptionstrengthwithan
increaseofionicstrength.Thiscanbeobservedbythelower
isothermslopevaluesandexperimentalcapacitiesobtained
athighersaltconcentrations(SeeFig.3andTable3).This
sug-geststhatnapinadsorptiononmixedmoderesinsismainly
controlledbyionicinteractions.
TheexceptionwasPPAresin,whichpresentedanincrease
innapin adsorptionwith increasing salt concentration. As
previouslymentioned,sinceforthisresinelectrostatic
inter-actionsarerepulsive,anincreaseinthebindingcapacityof
napinshouldbemainlyduetoanincreaseinthestrengthof
hydrophobicinteractions.Nosignificantimprovementinthe
adsorptionofnapinwas observedbetweenpH6and pH8,
atdifferentsaltconcentrations.Thismightsupportthefact
thatnapinadsorptiononPPAHyperCelischaracterizedonly
byhydrophobicinteractionsinthatrange.
Basedontheisotherm slope,onecannotice thatCapto
MMChasthelargestaffinityfornapin.However,thismight
complicatethedesorptionand recoveryoftheproteinafter
thecapturestep,asevenathighpHandsaltconcentrations
theisothermisfavorable.Asaconsequence,especiallyharsh
conditions(e.g.pHhigherthanthepIofthenapin)mightneed
tobeapplied.Thedeterminednapinisotherm slopesatall
testedconditionsareshowninTable3.
Fromtheobtainedresultsitisclearthattheeffectofionic
strengthismoresignificantthantheeffectofpH,especiallyfor
thePPAHyperCelresin.Whencomparingtheresultsforthat
resin(Fig.3eandf),theisothermsseemtooverlap.Thiswas
expected,astheadsorptionofnapinoccursmainlythrough
hydrophobicinteractions.Thiseffectcanbeevaluatedby
com-paringtheratiobetweentheisothermslopesattwodifferent
saltconcentration(ataspecificpH)andtheratiobetweenthe
isothermslopesattwodifferentpHconditions(ataspecific
saltconcentration).
Similar isotherm trendsto the onesdetermined inthis
workwereobservedintheresearchofNforetal.(2010).The
authorsevaluatedlysozyme(similarpIandmolecularweight
asnapin)adsorptiononCaptoMMCandPPAHyperCel.The
adsorptioncapacityoflysozymewasinthesameorderof
mag-nitudeastheoneobtainedfornapininthiswork.Inaddition,
similarisothermcurveswereobservedwhencomparingtwo
pHvaluesandonesaltconcentration.
Allthisinformationwasusedtoselectthemostsuitable
resinfornapincapture.
3.3. Proteinmixtureadsorptionexperiments
Batch adsorptionexperiments were performed using
solu-tionsofaproteinmixture(napin+cruciferin),toevaluateboth,
theeffect ofcruciferinonnapin’sadsorptionand to
deter-minecruciferin’sadsorptionisotherms.Atfeedconditions,it
isexpectedthatcruciferinpoorlybindstotheresins,asthepH
isclosetothepIofthisprotein(∼7).Acomparisonbetweenthe
expectednapinadsorptioncapacityandtheadsorption
capac-ityobtainedfromtheexperimentsusingtheproteinmixture
wasdoneandtheresultscanbefoundinFig.4.
Fig.4–Napinadsorptioncapacitycomparisonbetween expectedcapacityandexperimentallyobtainedfrom proteinmixtureexperimentsatpH6and0.3MNaCl. Expectedcapacitywascalculatedfromsinglecomponent isotherm.
Thepresenceofcruciferinseemstoenhancenapin
adsorp-tionatpH6and0.3MNaClforPOROSHSandCaptoSwhere
higher adsorption capacities than expected were obtained.
Thisbehaviormightbeexplainedbythechargeofthe
pro-teins.AtpH6,bothcruciferinand napinpossessapositive
netcharge,allowingcruciferintobindtotheresinsaswell.
However,cruciferinisconsideredalargeprotein,andin
solu-tion withnapin, the positivecharge oncruciferin’s surface
mighthaveresultedinastrongelectrostaticrepulsionofnapin
molecules.Bycreatingalessfavorablechemicalenvironment
intheliquidsolution,thepresenceofcruciferinmightthenbe
improvingtheadsorptionofnapinmoleculesontothetested
resins.
In all experiments (Supplementary material), cruciferin
showedaverysmallchangeinconcentrationafter
equilibra-tionwiththeresins,whichindicatedalowbindingstrength.
OnlyresinPPAHyperCelshowedarelativelyhighcruciferin
concentrationchange.Thecruciferin’sisothermslopevalues
were obtained by fitting a linear curve to the
experimen-tallyobtainedcapacities(asfunctionoftheequilibriumliquid
concentrations).Theobtainedvalueswere2.0+1.3,0.8+0.1,
2.3+1, 22.1+8.2, 1.6+0.5 and 0.9+0.7mL/mLresin for Capto
MMC,NuviacPrime,Toyopearl,PPAHyperCel,POROSHSand
Capto S respectively. Cruciferin’s isotherm slope is
signifi-cantly lower than the oneof napin for resin Capto MMC,
NuviacPrimeandPOROSHS,suggestingapreferential
bind-ingofnapintotheseresins.Napinisothermslopevalueswere
slightlyhigherforresinsCaptoSandToyopearlandcruciferin’s
isotherm slopewas higherthan napin’sisothermslopefor
resin PPA HyperCel. Asdiscussed before,for PPAHyperCel
resin,bindingismainlycharacterizedbyhydrophobic
inter-actions.Because,cruciferinisamorehydrophobicmolecule
thannapin,thismightexplainthehigheraffinitytowardsthis
resin.Alltheobtainedresultswereusedtoidentifythemost
suitableresinforthecaptureofnapin.
3.4. Resinselection
Themostsuitableresin fortheseparationofnapinan
cru-ciferin was selected using the previously definedselection
criteria (2.6 Resin selection). 1) Napin adsorption capacity
atfeedconditions(pH6with0.3MNaCl),2) bestdesorption
conditions(basedonthelowestisothermslopeobtainedfor
Table4–Valuesofeachcriterion(napincapacity,selectivity,resinpriceanddesorption)usedforresinselectionand resinscores.
Napincapacitya(mg/mL) Selectivityb(-) 1/slopedesorptionc(mL
resin/mL) Price/highestprice(-) Resinscore
CaptoMMC 40.0 219.9 0.1d 0.8 66%
NuviacPrime 34.3 81.3 0.8e 0.8 57%
Toyopearl 14.1 2.5 2.6f 1.0 37%
PPAHyperCel 26.6 0.4 0.5g 1.0 41%
POROSHS50 30.0 33.0 5.6h 0.5 75%
a Napincapacityat0.3MNaClandpH6.
b Ratiobetweennapinancrucferinisothermslopesat0.3MNaClandpH6. c Inverseofisothermslope:
d at0.7MNaClandpH8; e at1.0MNaClandpH6; f at1.0MNaClandpH6; g at0.1MNaClandpH6; h at0.7MNaClandpH8.
price.Adsorption(napincapacity)and desorptionwere the
two criteria with higher importance in the selection
pro-cess.Aspreviouslymentioned,resinsCaptoS,CMSheparose
andMacroPrepwherediscarded,astheyshowedsignificantly
lower napincapacity comparedtothe other resins atfeed
conditions.TheresultsareshowninTable4.
EventhoughresinCaptoMMCpossessesthehighestnapin
capacity(in allconditions tested)andselectivity,this resin
wasnotselectedasthemostpromising.Thereasonhasto
domainly withdesorption, possiblyrequiring a pHhigher
thannapin’spI,which,aspreviouslymentioned,canleadto
problemswiththestructuralstabilityofnapin.Therefore,
des-orbingatapHabovethepIisnotrecommended.Thebest
performing resin in terms of desorption performancewas
POROSHS, sinceitsresultsindicatethatdesorption canbe
performedathighsaltconcentrationwithoutincreasingthe
pHtoahigher valuethan napin’spI.Despite beingsecond
bestondesorptioncriteria.theToyopearlresinalsoshowed
amuchloweradsorptioncapacityfornapinwhencompared
toCaptoMMC andNuviacPrimeatfeedconditions.
There-fore,theToyopearlresinscoredthelowestintheadsorption
capacitycriterion.
PPAHyperCelandPOROSHSshowedcomparable
adsorp-tion capacities at feed condition. Nevertheless, from the
experiments performed with the protein mixture it was
clear that cruciferin also interacts with resin PPA
Hyper-Celhavinganevenlarger isothermslope than napin. This
wasobservedwiththeobtainedselectivityvaluewhichwas
lowerthanunity,indicatingthatatpH6and0.3MNaCl,
cru-ciferininteractsmorefavorablywithPPAHyperCelresinthan
napin.
Thelastevaluatedselectioncriterionwasresincost,with
resinPOROSHSbeingthe cheapestone,asthepricerange
isaroundhalfthepriceobtainedforthemixedmoderesins.
Eventhoughthiscriterionwasevaluatedwithalowerweight,
itwastakenintoaccount,asprocesseconomicsisasignificant
factorinprocessdevelopment.
3.5. Mixedmodeisothermparameterestimation
NapinadsorptiondataobtainedforPOROSHSresinwasused
inorder tofit theisotherm modeldescribed in 2.8.1Mixed
mode isotherm model. Since POROS HS is a strong cation
resin,therearenohydrophobicinteractionspresentedandthe
parameternfromEq.(7)wassettozero.Inordertoreducethe
complexityofsolvingthenon-linearsystem,themodelwas
linearizedbyapplyingthenaturallogarithminbothsidesof
theequationandtheregressionwasperformedasexplained
in2.5.2Parameter estimation.Theexperimentaldatafrom
thethreeevaluatedsaltconcentrationswerefittedto
deter-minethesetofparametersKeq,v,KpandKs,foreachpHvalue.
Themaximumadsorptioncapacity(qmax
p )wasobtainedfrom
adsorptionequilibriumexperimentsfromproteinmixtureand
theobtainedvaluewas44mg/mLresin.Theregressed
parame-tersareshowninTable5.
Thestoichiometriccoefficient(v) ofthe saltcounter ion
at pH6 was higher than at pH8, which was expected. For
thissystem,visequaltoproteinbindingcharge.AtpH
val-uesapproachingthepIoftheprotein(napinpI∼11),thenet
chargeoftheproteinapproacheszero(lower)andthe
electro-staticbindingstrengthbecomesweaker,resultinginalower
bindingcapacity.Kpistheparameterthatdescribesthe
differ-encebetweenwater-proteinandprotein–proteininteractions
(Mollerup,2006).TheregressedKpforbothpHvalues(6and
8)hadapositivevalue,suggestingthatinteractionsbetween
protein-water were stronger than protein-protein
interac-tions. Thisalso matches the observations obtainedin this
workandthedatafromliterature,indicatingahigh
solubil-ityofnapininwater.SimilartoKp,theparameterKsdescribes
thedifferencebetweenwater–proteinandsalt–protein
inter-actions (Mollerup, 2006). The obtained Ks values at both
evaluatedpHconditionswerearoundzero,implyingthatthe
strength ofprotein-saltinteractionsisnotsignificantly
dif-ferentthanprotein-waterinteractions.Byanalyzingthetwo
differentinteraction parameters-Kp andKs -,thestrength
ofwater-protein,protein-saltandprotein-proteininteractions
couldbeinferred.Inthissystem,giventhatKp>0andKs≈0,
protein-waterinteractionsweredominant.
ThepHvalueofmobilephasehasaninfluenceonthe
bind-ingchargeoftheproteinwhichconsequentlychangesthev
valueanditisexpectedtobelowerwhenthepHiscloserto
thepIoftheprotein.InordertoincludetheinfluenceofpHon
thestoichiometriccoefficient,approachesliketheonefrom
Pirrungetal.(2018)couldbeused.ThevforPOROSdecreases
withincreasingofpHwhichcorrespondstotheinfluenceof
pHonelectrostaticinteractions.
Alinearisothermmodelwasconsideredforcruciferin
Table5–IonexchangeisothermparametersofnapinproteininPOROSHSresinatpH6andpH8.
lnKeq v KP(mM-1) KS(mM-1) R2 SD
POROSHSpH6 6.6+0.3 1.9±0.4 2.3±2.7 −5×10-3+1×10-3 0.97 0.2
POROSHSpH8 5.8±0.2 1.2±0.2 0.8±1.0 −9×10-3+8x10-3 0.98 0.1
3.6. Columnchromatography
Twopulseexperimentswereperformedatsmallscale(∼10mL columnvolume)usingeitherasolutionofnapinora solu-tionofproteinmixture(napin+cruciferin).Theexperimental resultswere comparedwith the model output and can be foundinFig.5.Thedataobtainedfromthechromatography
stationwasnormalizedforeasiercomparisonwiththe
mod-elingresults.
AscanbeseeninFig.5,themodelisinexcellentagreement
withtheexperimentalresultsfornapinassinglecomponent
andalsowhennapinisinthepresenceofthesecondprotein,
cruciferin.Analyzing napinresults(Fig.5a),onecannotice
thatnodesorptionofnapinoccursafter5CVofwashingstep,
whichsuggestsastrongbindingattheseconditions(asshown
bythedeterminedisotherm).Elutionwithhighsalt
concen-tration(0.7MNaCl)and pH8showsaverysharp peak.This
wasexpectedasattheseconditions,napincapacityinPOROS
HSisclosetozero.HighsaltandpHconditionswerechosenin
ordertohavehighrecovery,whichcorrespondto100%based
onmassbalance(evaluatedbynumericalintegration).Note
thathighpHmightnotbeneededtofullydesorb this
pro-tein.Afterdesorption phase,the columnwaswashed with
aconcentratedsaltsolution(1MNaCl)untilnoabsorbance
at280nmwasdetected.Themaximumnapinconcentration
detectedwas6.8+0.1g/L.
Protein mixture chromatogram is presented in Fig. 5b.
Napinelutionprofileusingthisbinarymixturecorresponds
to the one obtained using single component napin. After
applyingtheelutionbuffer,againasharp peakisobserved
withapeakmaximumataround6.0CV(approximatelyone
columnvolumeaftertheswitchinconditions).Inthis
exper-iment, anextra peakisobserved duringthe washing step,
whichmightcorrespondtocruciferinprotein.Usingalinear
isothermmodelforcruciferinprotein(slope1.6mL/mL)and
performingthesimulation,onecanobservedapeakataround
1.8CV(washingstep).Comparingcruciferinsimulationresults
withexperimentalresults,itisclearthatcruciferinisotherm
slopewasoverestimated.Thisbecausetheexperimentalpeak
exitedthe columnearlier(∼1CV)andit issharperbut also
presentedtailing.Thistailingmightbecausedbythesizeof
cruciferin,whichisaround300kDa.Thedifferencebetween
theexperimentalandsimulatedresultsmightbeattributed
tocruciferinequilibriumparameter.Aspreviouslymentioned,
duringbinarymixtureexperimentscruciferindatapresented
verysmallchangeswhichweredifficulttoquantify.Besides
thesedifficulties, the model shows excellent agreement in
respecttonapinprotein.Themaximumnapinconcentration
obtainedinthechromatogramoftheproteinmixturepulse
experimentwas7.8+0.1g/L.Thisvalueishigherthantheone
obtainedintheexperimentwithpurenapinexperimentdue
totheslightlyhighernapinconcentrationapplied.
Using column experimental results from the protein
mixture, the slope of cruciferin was calculated to be
0.6mL/mLresin,whichconsequentlyincreasedtheselectivity
ofnapintoPOROSHSresinto87.0.
Thevalidatedmodelcanbethenusedforindustrialcolumn
designtoidentifycolumnsizingand operatingparameters.
Scaleupofchromatographyprocessesfromlaboratoryresults
isusuallydonebykeepingbedheightandvelocityconstant
andchangingcolumndiameter.However,thismakes
scalabil-ityinflexible,oftenresultingincolumnvolumesthatcannot
satisfythedesiredcapacityorthatdomarchthecapacities
foundatpilotand industrialscale,wherecompaniesoften
possessalreadyexistingequipment.Pre-determinedvolumes
(Stabyetal.,2017),withfixedcolumndiameterandadjustable
headers. The use of the previous model could reduce the
numberofexperimentsorinvestmentrequiredtosatisfy
pro-duction.
The following section describe the use of the previous
modeltodesignanionexchangecolumnfortheseparation
oftheseproteins.Themodelwasappliedtoahypothesized
casestudy consideringthe productionofrapeseedmealin
theNetherlands.Thiscasestudycanbeusedasbaseforlarge
scalepurificationofoilseedmeals.
3.7. Adsorptiveprocessdesignforthepurificationof napin,cruciferinandsinapicacidfromrapeseedmeal
Rapeseedproductionhassignificantlyincreasedinthe last
years, havinga globalproduction of75 million Mtin2018
where 25 million Mt were produced in Europe (Food and
AgricultureOrganizationoftheUnitedNations(FAO),2019).
Asmentionedbeforethemainproductobtainedfrom
rape-seedisoilforhumanconsumption, usuallyextracted from
theseedbymechanicalandsolventextraction(Fetzeretal.,
2018;Wanasundaraetal.,2017).Asbyproduct,rapeseedmeal
withahighproteincontent(∼40%drybasis),mainly
consist-ingofcruciferinandnapin(Contrerasetal.,2019;Lomascolo
et al., 2012; Wanasundara et al., 2017)isgenerated. In the
Netherlands,around3500MTofrapeseedmealwere
gener-atedin2018,correspondingtoaround1400MToftotalprotein.
Aconceptualdownstreamprocess(DSP)ofrapeseedmealfor
thepurificationofproteinsandpolyphenolsisshowninFig.6.
TheDSPincludes:anaqueousextractionassistedwithsalts
(Bérotetal.,2005)tosolubilizethedifferentcomponentsin
themeal,followedbyasmallmoleculeseparation(phyticacid,
polyphenolscompounds,carbohydrates)usingultrafiltration
with acut-off membraneof3kDa. Theprotein rich
reten-tateissenttoanionexchange(IEX)columnwherenapinis
capturedandcruciferinflowsthrough.Thepermeatecanbe
furtherprocessedtorecoversinapicacid(majorpolyphenolin
rapeseedmeal)usingadsorption(hydrophobicresins),as
rec-ommendedbyMoreno-Gonzálezetal.(2020)andSilvaetal.
(2018a) orliquid-liquid extraction(withorganic solventsor
ionicliquids)assuggestedbySilvaetal.(2018b).Thepurified
fractions of napin and cruciferin, obtained after the
chro-matographycolumn,arethensenttoanothermembraneunit
toremovethesaltsbeforebeingsenttothefinaldryingstage.
Following the adsorptionmethod forsinapic acid recovery
fromMoreno-Gonzálezetal.(2020),elutionandregeneration
mix-Fig.5–ModelpredictionvsExperimentaldataofNapin(a)andProteinmixture(b)onPOROS50HScolumn.Loading conditionpH6and0.3MNaCl,stepelutionatpH8and0.7MNaCl.Concentrationdatawasnormalized.
tureswhichcanberecoveredbydistillationandrecycledtothe
column.Toobtainsinapicacidcrystals,amultipleeffect
evap-orationcanbeusedtocrystalizethepolyphenol(Silvaetal.,
2018b).Theevaporatedsolventisdirectedtothedistillation
columnandsinapicacidcrystalsarethenfiltered,washedand
dried.
Consideringthattheannualproductionofrapeseedinthe
Netherlandsis5836tons(FoodandAgricultureOrganization
ofthe United Nations (FAO), 2019) and assuming that the
firststagesoftheprocess(aqueousextraction,centrifugation,
smallmoleculesseparation)accountanoverallproteinyield
of90%theinitialproteinextractflowratecanbeestimated.
Theionexchangecolumnforproteinpurificationcanbe
sizedusingthepreviousmodel.TheselectedresinPOROS50
HShasapolymericmatrix(rigidparticles)andaparticlesizeof
50mandbasedonsupplierspecification,thisresinpossesses
high mechanicalresistance (100bars). For astainless-steel
column,a maximumof 50barpressure drop was setfor a
columndiameterof1m(Schmidt-Traub etal., 2013).
Maxi-mumcolumnheightusingthisresincanbeestimatedusing
Karman-Cozenyequation,anditcorrespondsto1m.
Thecolumnmodelisthenusedtodeterminetheoperation
oftheionexchangecolumn,inspecifictoidentifytheloading
volume,whichisthiscaseistwocolumnvolumes.The
load-ingvolumeislowbecausetheconcentrationofeachproteinin
theproteinrichextractisrelativelyhigh(higherthan8g/L)and
theresingetstoequilibriumsaturationwithrelativelylow
vol-ume.Forthissimulation,anisothermslopeof0.6mL/mLresin
wasconsidered forcruciferin equilibrium.Themodel
indi-catesaresinutilizationof82%andanapinyieldof98%with
>99%purity.Intheflowthrough,cruciferinisrecovered(>99%
yieldand>98%purity).Theproductivitycalculatedwiththis
columnsize(1mdiameterand1mheight)is26.3gNapin/LR/h,
whichconsequentlyproduces52.1MT/a.Thisaccountsto12%
ofthetotalamountofnapinthancanberecoveredfromthe
rapeseedmealgeneratedintheNetherlandsusing oneion
exchangecolumn.
Itispossibletopurifytheannualproductionofrapeseed
mealbycalculatingthenumberofcolumnsneededto
oper-ateinparallel andthe number ofcyclesthat each column
can berun. It isimportant tokeep in mind that this is a
preliminaryevaluationandthatbreakthroughcurve
experi-mentsfornapinwillbestillneededtocorroboratetheselected
loading.Inaddition,forthisestimationasimilarcolumn
oper-ationintermsofdesorptionwasselected(5CV)whichcould
bereduced,asbasedonthecolumnexperiments,fullnapin
recoverymightbeachievedwithless volume.Operation of
theionexchangecolumncanalsobeadaptedtocontinuous
(Simulatedmovingbed)orsemi-continuous(CaptureSMBand
PeriodicCountercurrentChromatography,PCC)withmultiple
columnsconnectedinseriesoroperatinginparallel.In
addi-tion,economic feasibilityshould beassessedwith adetail
economicevaluationtoidentifyoverall capitalexpenditure
(CAPEX)andtheoveralloperationalcost(OPEX)and
perform-ingforinstanceacashflowanalysis.
4.
Conclusions
Highthroughputprocessdevelopment(HTPD)generates
reli-ableandsignificantinformationinashorttimeperiod.Inthe
caseofadsorptiveprocesses,HTPD,differentresinsand
condi-tionscouldbeassessedinparallelwhichallowsproperresin
choice.Thegeneratedinformationcanbeusedin
combina-tionwithmechanisticmodelstoevaluatetechnicalfeasibility
ofaprocessdesignwhichinvolvesprocessunderstandingand
contributestofastprocessdevelopment.
Declaration
of
interests
The authors declare that they have no known competing
financialinterestsorpersonalrelationshipsthatcouldhave
appearedtoinfluencetheworkreportedinthispaper.
Acknowledgments
ThisworkwassupportedbytheISPT(InstituteofSustainable
ProcessTechnology)undertheprojectCM-20-07inAdsorption
ofnon-volatilesfromfoodproducts.Thankstotheindustrial
partnersDSM,Unilever,FrieslandCampinaandRoyalCosun
fortheirvaluableinputthroughtheproject.Theauthorsare
alsothankfultoYiSongfromDelftUniversityofTechnology
foralltheprovidedlabsupport.
Appendix
A.
Supplementary
data
Supplementary material related to this article can be
found, in the online version, at doi:https://doi.org/10.
1016/j.fbp.2020.11.011.
References
Sevillano,D.M.,vanderWielen,L.A.M.,Hooshyar,N.,Ottens,M.,
2014.Resinselectionfortheseparationofcaffeinefromgreen teacatechins.FoodBioprod.Process.92,192–198.
Adachi,M.,Kanamori,J.,Masuda,T.,Yagasaki,K.,Kitamura,K.,
Mikami,B.,Utsumi,S.,2003.Crystalstructureofsoybean11S
globulin:glycininA3B4homohexamer.Proc.Natl.Acad.Sci. U.S.A.100,7395–7400.
Aider,M.,Barbana,C.,2011.Canolaproteins:composition,
extraction,functionalproperties,bioactivity,applicationsasa foodingredientandallergenicity–apracticalandcritical review.TrendsFoodSci.Technol.22,21–39.
Akbari,A.,Wu,J.,2015.Anintegratedmethodofisolatingnapin
andcruciferinfromdefattedcanolameal.LWT-FoodSci. Technol.64,308–315.
Bérot,S.,Compoint,J.P.,Larré,C.,Malabat,C.,Guéguen,J.,2005.
Largescalepurificationofrapeseedproteins(Brassicanapus L.).J.Chromatogr.B.818,35–42.
Brenner,H.,Gaydos,L.J.,1977.Theconstrainedbrownian
movementofsphericalparticlesincylindricalporesof comparableradius:modelsofthediffusiveandconvective transportofsolutemoleculesinmembranesandporous media.J.ColloidInterfaceSci.58,312–356.
Carta,G.,Jungbauer,A.,2010.ProteinChromatography:Process
DevelopmentandScale-up.JohnWiley&Sons.
Contreras,Md.M.,Lama-Mu ˜noz,A.,ManuelGutiérrez-Pérez,J.,
Espínola,F.,Moya,M.,Castro,E.,2019.Proteinextractionfrom
agri-foodresiduesforintegrationinbiorefinery:potential techniquesandcurrentstatus.Bioresour.Technol.280, 459–477.
Felinger,A.,Guiochon,G.,2004.Comparisonofthekinetic
modelsoflinearchromatography.Chromatographia60 (Suppl.1),S175–S180.
Fetzer,A.,Herfellner,T.,Stäbler,A.,Menner,M.,Eisner,P.,2018.
Influenceofprocessconditionsduringaqueousprotein extractionuponyieldfrompre-pressedandcold-pressed rapeseedpresscake.Ind.CropProd.112,236–246.
FoodandAgricultureOrganizationoftheUnitedNations(FAO),
2019.FAOSTATProductionQuantity(accessed24March2019)
http://www.fao.org/faostat/en/#data/QC.
Gerzhova,A.,Mondor,M.,Benali,M.,Aider,M.,2015.A
andconventionalextractionmethodontheextractability, compositionandphysicochemicalpropertiesofcanola proteinconcentratesandisolates.FoodBiosci.11,56–71.
Ghodsvali,A.,Khodaparast,M.H.H.,Vosoughi,M.,Diosady,L.L.,
2005.Preparationofcanolaproteinmaterialsusing
membranetechnologyandevaluationofmealsfunctional properties.FoodRes.Int.38,223–231.
Gunn,D.J.,1987.Axialandradialdispersioninfixedbeds.Chem.
Eng.Sci.42,363–373.
Hou,F.,Ding,W.,Qu,W.,Oladejo,A.O.,Xiong,F.,Zhang,W.,He,R.,
Ma,H.,2017.Alkalisolutionextractionofriceresidueprotein
isolates:influenceofalkaliconcentrationonprotein
functional,structuralpropertiesandlysinoalanineformation. FoodChem.218,207–215.
Jyothi,T.,Sinha,S.,Singh,S.A.,Surolia,A.,Rao,A.A.,2007.Napin
fromBrassicajuncea:thermodynamicandstructuralanalysis ofstability.BiochimicaetBiophysicaActa(BBA)-Proteinsand Proteomics1774,907–919.
Kodagoda,L.P.,Nakai,S.,Powrie,W.D.,1973.Somefunctional
propertiesofrapeseedproteinisolatesandconcentrates. CanadianInstituteofFoodSci.Technol.J6,266–269.
LeVan,D.M.,Carta,G.,Yon,C.M.,1999.Adsorptionandion
Exchange.In:Perry,R.H.,Green,D.W.(Eds.),Perry’SChemical Engineers’Handbook.,sevened.McGraw-Hill,NewYork,pp. 16–19.
Lomascolo,A.,Uzan-Boukhris,E.,Sigoillot,J.-C.,Fine,F.,2012.
Rapeseedandsunflowermeal:areviewonbiotechnology statusandchallenges.Appl.Microbiol.Biotechnol.95, 1105–1114.
Mollerup,J.M.,2006.Appliedthermodynamics:anewfrontierfor
biotechnology.FluidPhaseEquilibr.241,205–215.
Mollerup,J.M.,2007.Thethermodynamicprinciplesofligand
bindinginchromatographyandbiology.J.Biotechnol.132, 187–195.
Mollerup,J.M.,Hansen,T.B.,Kidal,S.,Staby,A.,2008.Qualityby
design—thermodynamicmodellingofchromatographic separationofproteins.J.Chromatogr.A1177,
200–206.
Moreno-González,M.,Girish,V.,Keulen,D.,Wijngaard,H.,
Lauteslager,X.,Ferreira,G.,Ottens,M.,2020.Recoveryof
sinapicacidfromcanola/rapeseedmealextractsby adsorption.FoodBioprod.Process.120,69–79.
Murray,D.E.,Terrence,M.J.,Barker,L.D.,Myers,C.D.,US4208323A,
USApatent1980.ProcessforIsolationofProteinsUsingFood
GradeSaltSolutionsatSpecifiedpHandIonicStrength.
NationalCenterforBiotechnologyInformation,2020.PubChem
Database.Tryptophan,CID=6305(accessedonJan.16,2020)
https://pubchem.ncbi.nlm.nih.gov/compound/Tryptophan.
Nfor,B.K.,Noverraz,M.,Chilamkurthi,S.,Verhaert,P.D.,vander
Wielen,L.A.,Ottens,M.,2010.High-throughputisotherm
determinationandthermodynamicmodelingofprotein adsorptiononmixedmodeadsorbents.J.Chromatogr.A1217, 6829–6850.
Perera,S.P.,McIntosh,T.C.,Wanasundara,J.P.,2016.Structural
Propertiesofcruciferinandnapinofbrassicanapus(canola) showdistinctresponsestochangesinphandtemperature. Plants.5,36.
Pickardt,C.,Neidhart,S.,Griesbach,C.,Dube,M.,Knauf,U.,
Kammerer,D.R.,Carle,R.,2009.Optimisationofmild-acidic
proteinextractionfromdefattedsunflower(Helianthusannuus L.)meal.FoodHydrocoll.23,1966–1973.
Pirrung,S.M.,ParrucadaCruz,D.,Hanke,A.T.,Berends,C.,Van
Beckhoven,R.F.W.C.,Eppink,M.H.M.,Ottens,M.,2018.
Chromatographicparameterdeterminationforcomplex biologicalfeedstocks.Biotechnol.Prog.34,1006–1018.
Pudel,F.,Tressel,R.P.,Düring,K.,2015.Productionandproperties
ofrapeseedalbumin.LipidTechnol.27,112–114.
Raab,B.,Schwenke,K.D.,1984.Simplifiedisolationprocedurefor
the12Sglobulinandthealbuminfractionfromrapeseed
(BrassicanapusL.).Nahrung28,863–866,
http://dx.doi.org/10.1002/food.19840280820.
Rommi,K.,Ercili-Cura,D.,Hakala,T.K.,Nordlund,E.,Poutanen,
K.,Lantto,R.,2015.Impactoftotalsolidcontentand
extractionphonenzyme-aidedrecoveryofproteinfrom defattedrapeseed(BrassicarapaL.)presscakeand
physicochemicalpropertiesoftheproteinfractions.J.Agric. FoodChem.63,2997–3003.
Schmidt,I.,Renard,D.,Rondeau,D.,Richomme,P.,Popineau,Y.,
Axelos,M.A.-V.,2004.Detailedphysicochemical
characterizationofthe2sstorageproteinfromrapeseed (BrassicanapusL.).J.Agric.FoodChem.52,5995–6001.
Schmidt-Traub,H.,Schulte,M.,Seidel-Morgenstern,A.,2013.
PreparativeChromatography,seconded.JohnWiley&Sons, Incorporated,Weinheim,Germany.
Silva,M.,Castellanos,L.,Ottens,M.,2018a.Captureand
purificationofpolyphenolsusingfunctionalizedhydrophobic resins.Ind.Eng.Chem.Res.57,5359–5369.
Silva,M.,García,J.C.,Ottens,M.,2018b.Polyphenolliquid-liquid
extractionprocessdevelopmentusingNRTL-SAC.Ind.Eng. Chem.Res.57,9210–9221.
Staby,A.,Rathore,A.S.,Ahuja,S.,2017.Preparative
ChromatographyforSeparationofProteins.JohnWiley& Sons,Incorporated,NewYork.
Vuorela,S.,Meyer,A.S.,Heinonen,M.,2004.Impactofisolation
methodontheantioxidantactivityofrapeseedmeal phenolics.J.Agric.FoodChem.52,8202–8207.
Wakao,N.,Smith,J.M.,1962.Diffusionincatalystpellets.Chem.
Eng.Sci.17,825–834.
Wanasundara,J.P.,2011.ProteinsofBrassicaceaeoilseedsand
theirpotentialasaplantproteinsource.Crit.Rev.FoodSci. Nutr.51,635–677.
Wanasundara,J.P.D.,McIntosh,T.C.,Perera,S.P.,
Withana-Gamage,T.S.,Mitra,P.,2016.Canola/rapeseed
protein-functionalityandnutrition.OCL23(4),D407.
Wanasundara,J.P.D.,Tan,S.,Alashi,A.M.,Pudel,F.,Blanchard,C.,
2017.Chapter18-proteinsfromcanola/rapeseed:current
status.In:Nadathur,S.R.,Wanasundara,J.P.D.,Scanlin,L. (Eds.),SustainableProteinSources.AcademicPress,San Diego,pp.285–304.
Wilson,E.J.,Geankoplis,C.J.,1966.Liquidmasstransferatvery
lowReynoldsnumbersinpackedbeds.Ind.Eng.Chem. Fundam5(1),9–14.
Xu,L.,Diosady,L.L.,2002.Removalofphenoliccompoundsinthe
productionofhigh-qualitycanolaproteinisolates.FoodRes. Int.35,23–30.
Young,M.E.,Carroad,P.A.,Bell,R.L.,1980.Estimationofdiffusion
coefficientsofproteins.Biotechnol.Bioeng.22,947–955.
Zhang,S.B.,Wang,Z.,Xu,S.Y.,2007.Downstreamprocessesfor
aqueousenzymaticextractionofrapeseedoilandprotein hydrolysates.J.Am.OilChem.Soc.84,693.
Zhu,M.,Carta,G.,2016.Proteinadsorptionequilibriumand
kineticsinmultimodalcationexchangeresins.Adsorption22, 165–179.
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