Fractionation of whey proteins with high-capacity superparamagnetic ion-exchangers

Journal of Biotechnology 113 (2004) 247–262

Fractionation of whey proteins with high-capacitysuperparamagnetic ion-exchangers

Anders Heebøll-Nielsen1, Sune F.L. Justesen1, Owen R.T. Thomas∗

Center for Process Biotechnology, BioCentrum-DTU, Technical University of Denmark, DK-2800, Kgs. Lyngby, Denmark

Received 12 August 2003; received in revised form 26 May 2004; accepted 1 June 2004

Abstract

In this study we describe the design, preparation and testing of superparamagnetic anion-exchangers, and their use togetherwith cation-exchangers in the fractionation of bovine whey proteins as a model study for high-gradient magnetic fishing.Adsorbents prepared by attachment of trimethyl amine to particles activated in sequential reactions with allyl bromide andN-bromosuccinimide yielded a maximum bovine serum albumin binding capacity of 156 mg g−1 combined with a dissociationconstant of 0.60�M, whereas ion-exchangers created by linking polyethylene imine through superficial aldehydes bound upto 337 mg g−1 with a dissociation constant of 0.042�M. The latter anion-exchanger was selected for studies of whey proteinfractionation. In these, crude bovine whey was treated with a superparamagnetic cation-exchanger to adsorb basic protein species,and the supernatant arising from this treatment was then contacted with the anion-exchanger. For both adsorbent classes of ion-e the elutionb ved withs teins byd cantlyp reased to0 .©

K s adsor-b

ydrin;e;

rmingham,

0

xchanger, desorption selectivity was subsequently studied by sequentially increasing the concentration of NaCl inuffer. In the initial cation-exchange step quantitative removal of lactoferrin (LF) and lactoperoxidase (LPO) was achieome simultaneous binding of immunoglobulins (Ig). The immunoglobulins were separated from the other two proesorbing with a low concentration of NaCl (≤0.4 M), whereas lactoferrin and lactoperoxidase were co-eluted in signifiurer form, e.g. lactoperoxidase was purified 28-fold over the starting material, when the NaCl concentration was inc.4–1 M. The anion-exchanger adsorbed�-lactoglobulin (�-LG) selectively allowing separation from the remaining protein2004 Elsevier B.V. All rights reserved.

eywords: Activation/functionalization chemistry; High-gradient magnetic fishing; Lactoperoxidase; Polyethylene imine; Non-porouents; Tentacular ligands

Abbreviations:AB, allyl bromide; AGE, allyl glycidyl ether; BSA, bovine serum albumin; DEAE, diethyl amino ethyl; ECH, epichlorohHGMF, high-gradient magnetic fishing; Ig, immunoglobulin;�-LA, �-lactalbumin; LF, lactoferrin;�-LG,�-lactoglobulin; LPO, lactoperoxidasNBS,N-bromosuccinimide; PEI, polyethyleneimine; PG, polyglutaraldehyde; TMA, trimethyl amine

∗ Corresponding author. Present address: Department of Chemical Engineering, School of Engineering, The University of BiEdgbaston, Birmingham B15 2 TT, UK. Tel.: +44-121-414-5278; fax: +44-121-414-5377.E-mail address:[email protected] (O.R.T. Thomas).

1 Both authors contributed equally to this work.

168-1656/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.jbiotec.2004.06.008

248 A. Heebøll-Nielsen et al. / Journal of Biotechnology 113 (2004) 247–262

1. Introduction

The fractionation of bovine whey, a by-productfrom cheese manufacture produced in vast quanti-ties, is of great interest (McIntosh et al., 1998), asthis cheap feedstock contains a range of valuable pro-teins with health benefiting qualities. Among theseare broad-range antimicrobial functions of lactoperoxi-dase (LPO) and lactoferrin (LF) and specific antimicro-bial effects of immunoglobulins (Ig), (Ekstrand, 1989;Vasavada and Cousin, 1993; Shimazaki, 2000), as wellas potentially anti-hypertensive effects of hydrolysatesof �-lactalbumin (�-LA) and �-lactoglobulin (�-LG,Pihlanto-Leppala, 2001). Whey processing ideally re-quires robust scaleable techniques capable of high vol-umetric throughput with minimal pre-treatment of thefeedstock. The technology known as ‘high-gradientmagnetic fishing’ (HGMF;Hubbuch, 2001; Hubbuchet al., 2001; Heebøll-Nielsen, 2002; Heebøll-Nielsenet al., 2003), possesses many desirable qualities forwhey fractionation, and we have recently designed andprepared superparamagnetic cation-exchangers and ap-plied these in HGMF for the separation of basic pro-teins from sweet bovine whey (Heebøll-Nielsen et al.,2004a).

The fundamental properties of the most importantprotein species in whey (seeTable 1) imply thatanion-exchange separation may be suitable for wheyfractionation. However, despite the importance ofanion-exchange separation in downstream processing( r-p otein

TA heirr

P

T�

�

IBLLL

capture exist (e.g.Xue and Sun, 2002). Furthermore,column-based ion-exchange chromatography com-monly employs gradient elution to achieve separationand resolution of adsorbed protein species, but as themajority of protein purification work done with super-paramagnetic adsorbents until now has involved affin-ity adsorption principles (O’Brien et al., 1996, 1997;Zulqarnain, 1999; Hubbuch, 2001; Hubbuch et al.,2001; Hubbuch and Thomas, 2002; Heebøll-Nielsen,2002; Heebøll-Nielsen et al., 2003, 2004b), resolutionduring desorption has not been studied thoroughly.

In this work we extend on our previous studies em-ploying magnetic adsorbents, HGMF and whey. Ouraims have been two-fold: first, to fabricate and evalu-ate a range of anion-exchangers and second, havingidentified the best candidate to employ this adsor-bent with magnetic cation-exchangers developed ear-lier (Heebøll-Nielsen et al., 2004a) to fractionate wheyproteins in a tandem separation process, involving se-quential step changes in eluting conditions to desorbbound proteins.

2. Materials and methods

2.1. Materials

The iron chloride salts, FeCl2·4H2O andFeCl3·6H2O, used in the preparation of super-paramagnetic core particles, were obtained fromM ds)a Thef urea iumt ys de),a ,e ),d des e( es reb nem 1.7,f yS ny).B SO( AG

Bonnerjea et al., 1986), only few instances of supearamagnetic anion-exchangers designed for pr

able 1pproximate concentration of major proteins in bovine whey, t

elative molecular weights (Mr) and isoelectric points (pI)

rotein Concentration (g L−1) Mr (103) pI

otal 7b – –-LG 3b 18.6a 5.3a

-LA 0.7b 14.2a 4.8a

g 1b 150–900a 5.5–6.8a

SA 0.3b 66.3a 5.1a

F 0.02–0.35c 78.5d 9.5d

PO 0.01–0.03c 77d 9.8d

ysozyme <0.001c 14d 10.7d

a Kinsella and Whitehead (1989).b Creamer and MacGibbon (1996).c Vasavada and Cousin (1993).d Ekstrand (1989).

allinckrodt Baker B.V. (Deventer, The Netherlannd Merck (Darmstadt, Germany), respectively.

ollowing materials (employed in the manufactnd subsequent testing of adsorbents)—sod

etraborohydride (NaBH4), 3-aminopropyl triethoxilane, glutaraldehyde (50%, photographic grallyl bromide (AB), allyl glycidyl ether (AGE)pichlorohydrin (ECH),N-bromosuccinimide (NBSiethyl amine ethyl (DEAE) chloride (hydrochlorialt), trimethyl amine (TMA), polyethylene iminPEI; polymer-size (Mr) 600,000–1,000,000), bovinerum albumin (BSA; fraction V powder, 96% puy SDS-PAGE, A 9647), lactoferrin (LF; from boviilk, L 9507), and lactoperoxidase (LPO; EC 1.11.

rom bovine milk, L 2005)—were all supplied bigma–Aldrich Chemie GmbH (Steinheim, Germaromoethane sulphonic acid (sodium salt) and DM

99.5%) were purchased from Fluka Chemie

A. Heebøll-Nielsen et al. / Journal of Biotechnology 113 (2004) 247–262 249

(Buchs, Switzerland) and Mallinckrodt Baker B.V.,respectively, whereas polyethylene imines (PEIs)with Mr ’s of 600; 10,000 and 70,000 were acquiredfrom Polysciences Inc. (Warrington, PA, USA).The bicinchoninic acid (BCA) protein assay kit andTMB ONE ready-to-use substrate were, respectively,obtained from Pierce Chemicals (Rockford, IL, USA)and Kem-En-Tec A/S (Copenhagen, Denmark), whilepre-cast SDS-PAGE gels and Novex Colloidal Blueprotein staining solutions were supplied by Invitrogen(Groningen, the Netherlands). The whey used forfractionation studies, a calf stomach rennet digestof pasteurised bovine milk, was a gift from E.O.W.Nielsen (The Royal Veterinary and AgriculturalUniversity, Copenhagen, Denmark). Prior to fraction-ation the liquid portion was crudely separated fromremaining casein precipitates and lipids by aspiration.All other chemicals were of analytical grade.

2.2. Base matrix preparation

A powerful neodymium-iron-boron magnet block(B ≤ 0.7 T) from Danfysik A/S (Jyllinge, Denmark)was used for separation of magnetic particles duringall stages of the particle preparation and derivatisa-tion. Superparamagnetic iron oxide crystals were pre-cipitated from mixed iron chloride (FeCl2 and FeCl3)solutions under strongly alkaline conditions and sub-sequently coated with an amino-silane layer by reac-tion with 3-amino propyl triethoxy silane according top 2)T witha d byO rtsw wa-t dw thenii re-d xyls(

2

eact-i onica ibedp

2.4. Preparation of anion-exchangers

A range of chemistries was examined in the prepara-tion of anion-exchange supports. The reactions occur-ring in the manufacture of these materials are shownin Figs. 1–4and summarised inTable 2. During allreactions described below the supports were kept insuspension by shaking (on a vibrating table), by in-version of the reaction vessels on a test tube rotator,or by stirring. Unless indicated otherwise the supportswere washed extensively with distilled water and thenwith storage buffer (20 mM sodium phosphate, pH 6.8,1 M NaCl) after each manufacturing step. The supportswere stored at 4◦C until required.

2.4.1. Activation chemistriesHydroxyl-bearing supports were activated with AB,

AGE or ECH prior to ligand functionalisation. Activa-tion with AB and AGE (Fig. 1) were performed es-sentially as described byBurton and Harding (1997a).All operations in the AB and AGE-activation proce-dures were performed in glass vessels. For reactionswith AB, 250 mg aliquots of particles were suspendedin 5 mL 2.5 M NaOH with 1 mL DMSO. Followingaddition of 1 mL of AB to each vial, reactions were al-lowed to proceed for 18–20 h at ambient temperature.Activation with AGE was performed on 250 mg por-tions of supports in 10 mL 2.0 M NaOH in 70% ethanolby adding 2.0 mL AGE and then permitting the reac-tion to take place at room temperature for 18–20 h. Thed henc sup-pw d, as-p BS( ) ata gt ture.

en-t1 mL0i asa

2ated

p (to

rocedures described byHubbuch and Thomas (200.he amine-terminated supports were then coatedthin layer of polyglutaraldehyde (PG) as describe’Brien et al. (1996), and the final PG-coated suppoere subsequently washed 10 times with distilled

er, twice with 1 M NaCl, and finally twice with distilleater. Some PG-coated particle preparations were

ncubated (at 50 g L−1 particles) with 18 mM NaBH4n 0.50 M NaOH for 1 h at room temperature touce the surface carbonyl groups to primary hydrohydroxyl-bearing supports).

.3. Preparation of cation-exchangers

The cation-exchangers used were prepared by rng PG-coated supports with bromoethane sulphcid under reducing, alkaline conditions, as descrreviously (Heebøll-Nielsen et al., 2004a).

ouble bonds of the introduced allyl groups were tonverted to bromohydrin moieties by reacting theorts with NBS under aqueous conditions (Fig. 1). Thisas done by resuspending the magnetically settleirated particle slurries in unbuffered solutions of Ncontaining up to 8% acetone to dissolve the NBSn NBS charge of 2.25 mmol g−1 support, and allowin

he reaction to proceed for 1 h at ambient temperaEpoxy-activation with ECH was performed ess

ially as described byHermanson et al. (1992). For thisg quantities of particles were suspended in 20.50 M NaOH containing 18 mM NaBH4, and follow-

ng the addition of 5 mL of ECH the reaction wllowed to proceed for 6 h at room temperature.

.4.2. Derivatisation with DEAEReduced base supports and AB- or AGE-activ

articles were functionalised with DEAE groups


Fig. 1. Reactions in the activation of reduced PG-coated particles with ECH, AB or AGE.

make adsorbents types I–III, respectively) by adding2 g quantities of DEAE·Cl (hydrochloride salt) to40 mL suspensions of particles (25 g L−1) in 0.5 MNa2CO3, 1.0 M NaCl, pH 11.5, and allowing the re-action to proceed for 15 h at 60◦C (Fig. 2). Followingmagnetic separation the DEAE-derivatised adsorbentswere washed three times with distilled water.

2.4.3. Derivatisation with TMAStrongly charged quarternary amine groups were in-

troduced by coupling TMA to AB- or AGE-activatedsupports resulting in supports IV and V, respectively.In this final preparation step, the bromine atoms of thereactive halohydrins were substituted with TMA to in-troduce positively charged groups (Fig. 3) by adding50�L TMA to 50 mg aliquots of supports suspended in1.0 mL of 2.5 M NaOH with 200�L DMSO, and thenallowing the reactions to proceed for 18 h at room tem-perature. Finally, the functionalised adsorbents werefirst washed with distilled water, then with 1 M aque-ous NaCl, and finally 70% ethanol.

2.4.4. PEI-coupling to ECH-activated supportsCoupling of long-chained PEI (Mr 600,000–

1,000,000) to ECH-activated particles (Fig. 4) was ini-tially done simply by adding 5.0 mL 50% (v/v) PEI(in distilled water) to the activation suspension (stillcontaining ECH), and letting this react for 0.5 h at60◦C. The resulting gelatinous substance (support type

VI) was then disrupted by homogenisation for 1 h at8000 rpm in a Polytron PT 6000 device (KinematicaAG, Switzerland) to create type VII adsorbent particles.

In a different approach, ECH-activated supportswere reacted with PEI directly in the homogeniser unit.For this the activated particles were washed sequen-tially with distilled water (four times) and then 1 MNaCl (two times) before resuspending 1 g portions in1% (v/v) PEI in 0.2 M NaOH containing 6 mM NaBH4and incubating for 3 h at 60◦C in the high shear envi-ronment created by the homogeniser unit. Homogeni-sation at 6000 or 10,000 rpm resulted in type VIII andIX supports, respectively.

2.4.5. Attachment of PEI by reductive aminationShort-chained polymers of PEI (withMr ’s of 600,

10,000 or 70,000) were coupled directly to aldehydegroups of PG-coated particles by reductive amination(Fig. 4) as suggested byHermanson et al. (1992). Thereactions were performed in 60 g L−1 solutions of PEIcontaining 50 mM NaCNBH3 at 10 g L−1 supports for60–70 h at room temperature. The pH-values of thePEI-solutions used for the reactions were∼11.

2.5. Adsorbent characterisation

Side-pull permanent magnet racks (PerSeptive Di-agnostics, Cambridge, MA, USA) were used in sep-aration of magnetic supports during millilitre-scale

A.H

eebøll-N

ielse

netal./Jo

urnalofB

iotechnolog

y113(2004)247–262

Fig. 2. Reaction schemes for the preparation of anion-exchange support types I, II and III starting from reduced base particles or activated supports(seeFig. 1). As both DEAE·Cland bromohydrins are susceptible to hydrolysis (creating additional hydroxyl groups) other structures for types II and III may also result. For types II and III, all Br− and Cl−moieties were assumed to be hydrolysed, i.e. to hydroxyls.

251


Fig. 3. Reaction schemes for the preparation of types IV and V anion-exchange adsorbents from activated supports.

binding and desorption studies. Routinely, the anion-exchangers were equilibrated to binding conditions bywashing three times with a 50 mM Tris/HCl buffer, pH8. The particle concentration of the resultant suspen-sion was then determined by measuring the dry-weightcontent (seeSection 2.7), and adsorbent aliquots of

chang cles.

1–3 mg size were pipetted into eppendorf tubes. Thealiquoted suspensions were settled magnetically andthe supernatants replaced with 1 mL of a solution ofBSA in the equilibration buffer. A range of BSA con-centrations from 0 to 2 g L−1 was used. After 600 s ofbinding, or 120–1200 s for type X supports, the total

Fig. 4. Reaction schemes for the preparation of anion-ex
e support types VI–XII from ECH-activated or PG-coated parti


Table 2Summary of the preparative routes applied in the design of anion-exchangers and BSA adsorption characteristics estimated with the Langmuirequation

Support Activation Ligand Size (�m) Qmax (mg g−1) Kd (�M) Qmax/Kd (L g−1)

I Reduction DEAE <2 111 0.49 3.43II AB DEAE <2 92 0.91 1.52III AGE DEAE <2 107 1.1 1.48IV AB TMA <2 156 0.60 3.89V AGE TMA <2 122 0.88 2.08VI ECH PEIa 10–50 943 0.17 85.6VII ECH PEIa 5–10 553 0.69 12.2VIII ECH PEIa 3–4 385e ndf ndIX ECH PEIa <2 228e ndf ndX – PEIb <2 197 0.027 109XI – PEIc <2 243 0.036 101XII – PEId <2 337 0.042 120

a Mr 600,000–1,000,000.b Mr 600.c Mr 10,000.d Mr 70,000.e The isotherms were essentially rectangular, andQmax-values were not estimated from the Langmuir equation, but simply calculated as the

averages ofQ∗-values corresponding to non-zeroC∗-values.f Kd-values could not be determined (nd).

protein concentration of the supernatants and that ofthe added solutions were determined, and the adsorbedprotein was calculated from the difference. Adsorptionof BSA to the various adsorbents was fitted to the Lang-muir equationEq. (1)

Q∗ = QmaxC∗

C∗ + Kd(1)

whereQ∗ is bound protein (mg g−1), C∗ the proteinin solution (g L−1 or �M), both at equilibrium con-ditions,Qmax the maximum binding capacity andKdthe dissociation constant. The values of the parame-ters,Qmax andKd, were estimated using the non-linearleast squares fitting function of Origin 4.1 (MicrocalSoftware Inc., Northampton, MA, USA). Desorption ofbound BSA from support type X was examined by firstbinding BSA as above, and then replacing the super-natant with 0.50 mL of an elution buffer (either 1.0 MNaCl in the equilibration buffer or 100 mM sodium cit-rate, pH 3.5).

2.6. Whey fractionation

The pH of the crudely clarified whey was adjustedto 6.0 with 2 M HCl, and 20 mL of the resulting liq-uid was mixed with 121 mg of cation-exchange adsor-

bents (previously equilibrated by washing three timeswith binding buffer, 20 mM sodium phosphate, pH6.0). Following 600 s of incubation at ambient tem-perature the adsorbents were settled magnetically, be-fore removing the supernatants. Two 5 mL aliquots ofthe supernatant were adjusted to pH 7.0 or 8.0 us-ing 2.0 M NaOH. Subsequently 5 mL portions of thesepH adjusted (i.e. to pH 6, 7 and 8) supernatants weremixed with 118–166 mg of appropriately equilibratedtype XII anion-exchange particles (Table 4) and incu-bated for 600 s at room temperature. Conductivities ofthe feedstock were∼5 mS cm−1 in all cases. Prior tocommencing desorption, loosely bound protein and in-terstitially trapped material was removed by washingthe adsorbents in 10 mL of the respective equilibrationbuffers. Proteins adsorbed to the ion-exchangers were

Table 3Elution of BSA from type X supports

Desorption cycle BSA desorbed (%)

50 mM Tris/HCl,1 M NaCl, pH 8.0

50 mM Sodiumcitrate, pH 3.5

First 77.9 51.5Second 10.4 2.8

Total 88.3 54.3


then eluted stepwise with increasing NaCl in the equi-libration buffers, by settling the supports, aspiratingthe supernatant and replacing it with the next elutionbuffer. The concentrations of NaCl employed were: 0.1,0.2, 0.4, 0.6, 0.8 and 1.0 M for the cation-exchangestage; and 0.1, 0.2, 0.3, 0.4, 0.5 and 1.0 M for theanion-exchange step. The elution volumes employedwith the cation-exchangers and the anion-exchangerswere 5 mL and 3 mL, respectively. All fractions wereanalysed for protein composition and total protein con-centration, and for the cation-exchange fractionationexperiment LPO activity was measured as well (as de-scribed inSection 2.7).

2.7. Analytical methods

A Cobas Mira robot spectrophotometer (Roche Di-agnostic Systems, Rotkreutz, Switzerland) was used inanalysis of total protein concentrations with the BCAprotein assay (used as recommended by the manufac-turer). All protein concentrations were expressed asmilligram BSA equivalents.

The activity of LPO was monitored usingTMB-ONE ready-to-use substrate, using the robotspectrophotometer, exactly as described byHeebøll-Nielsen et al. (2004a).

The conductivities of buffers, whey and eluates weremeasured with a CDM210 conductivity meter (Ra-diometer, Copenhagen, Denmark).

Reducing SDS-PAGE (Laemmli, 1970) was per-f -l esedgl sito-m 0,B theh io-R s int units(

erec ro-s

ingsp I,U ;H

3. Results

3.1. Characterisation of the anion-exchangeadsorbents

A variety of superparamagnetic anion-exchangesupports, prepared via numerous different preparationroutes, were compared for their ability to adsorb BSA(Table 2). Two general strategies were chosen for thepreparation of these anion-exchangers, the first involv-ing attachment of simple single charge ligands (Table 2,supports I–V), and the second featuring derivatisationwith highly charged PEI polymers of various sizes(Table 2, supports VI–XII).

3.1.1. DEAE and TMA-linked anion-exchangersAdsorbents prepared following the strategy of at-

taching ligands each carrying a single charge (typesI–V, Fig. 5a and b) generally yielded adsorbentswith maximum BSA binding capacities from∼90 to160 mg g−1 and sub-micromolar dissociation constants(Table 2). For this group, supports activated with ABfollowed by coupling of TMA (type IV,Fig. 5b) showedbetter BSA binding characteristics than all of the DEAEbased supports (types I–III,Fig. 5a). This can probablybe explained by a higher density of charges (at the pHof binding) resulting from a combination of stronglycharged quarternary amines (e.g. type IV versus typeI) and/or more efficient derivatisation with TMA, giventhe low solubility of DEAE·Cl at the pH of coupling.

3on-

e for-m withe I,F dt os-i typeV ar ypesV e-d , al-t IIIw atedp e IXs ateds

ormed using Novex® 4–20% Tris–glycine polyacryamide gels. The protein bands in the electrophorels were visualised by staining with Novex Col-

oidal Blue, and then analysed by scanning denetry using Quantity One® software (version 4.1.io-Rad Laboratories, Hercules, CA, USA) withelp of a GelDoc2000 documentation system (Bad Laboratories). Contents of individual specie

he sample lanes were then expressed in arbitraryAU).

Particle sizes of the magnetic ion-exchangers wompared microscopically using an Optiphot 2 miccope (Nikon GmbH, Dusseldorf, Germany).

Dry-weight contents were examined by filteramples through pre-dried and pre-weighed 0.2�more size filters (PALL Corporation, Ann Arbor, MSA) as described previously (Heebøll-Nielsen, 2002eebøll-Nielsen et al., 2004b).

.1.2. Anion-exchangers prepared with PEIThe preliminary approach to prepare ani

xchangers using long-chained PEI resulted in theation of magnetically responsive adsorbents

xtremely high BSA binding capacity (type Vig. 5c). However, particle sizes up to 50�m suggeste

hat this material was of a highly porous nature. Expng these supports to high shear rates (resulting inII, Fig. 5c) or performing the coupling reaction with

educed concentration of PEI in the homogeniser (tIII and IX, Fig. 5c) produced materials with both ruced particle sizes and BSA binding capacities

hough the latter were still high. Types VII and Vere both of increased sizes compared to PG-coarticles, whereas microscopic examination of typupports indicated a very similar size to the PG-cotarting material (Table 2).


Fig. 5. Equilibrium adsorption isotherms for BSA on anion-exchange supports types: (a) I (�), II (�), and III ( ); (b) IV (�) and V (©);(c) VI (�), VII (), VII (�), and IX (�), and (d) X (�), XI (♦) and XII ( ). The lines through the data represent the fitted Langmuir curveswith the parameter values cited inTable 2.

As an alternative to performing coupling reactions inthe homogeniser attachment of lower molecular weightPEI was tested. When PEI ofMr ’s of 600–70,000 wascoupled directly to the PG-coated supports throughreductive amination, high-capacity anion-exchangerswere created (types X–XII,Fig. 5d), and in this in-stance microscopic analysis did not reveal any cross-linking of the PEI-derivatised supports. BSA bindingcapacities for the supports were proportional to the sizeof the attached PEI, and in all three cases significantlyimproved over the adsorbents (types I–V) created byattachment of DEAE or TMA (Table 2). Furthermore,binding to the adsorbents was very tight, and the valuesfor the initial slopes (Qmax/Kd) of the binding isothermswere increased >30-fold over support types I–IV.

3.1.3. Binding kineticsThe time to reach equilibrium between BSA and

support type X was examined and the results (data not

shown) indicated that binding was very fast with equi-librium being attained well inside 120 s. It was deemedunnecessary to conduct any further investigations ofthe exact adsorption kinetics since this time frame wasconsidered highly satisfactory for practical operation.

3.1.4. Desorption from the adsorbentsComparison of two different eluents (1 M NaCl in

equilibration buffer and buffer at pH 3.5) showed thatthe high ionic strength system proved more efficientfor desorption than decreasing the pH (Table 3), andelution with increasing concentrations of NaCl was se-lected as the method of choice for fractionation of whey.

3.2. Fractionation of whey

3.2.1. Protein adsorptionIt was chosen to initially bind whey proteins to

cation-exchange adsorbents followed by a second


Table 4Mass balance for the ion-exchange fractionation of whey

Separation principle pH Supports (mg) Q∗ (mg g−1) Protein (mg) Balance (%)

Feed Unbound Wash Eluate

Cation-exchange 6 121 93 128 116 7.38 18.8 111Anion-exchange 6 166 68 29.1 17.8 5.85 11.3 120Anion-exchange 7 132 79 28.6 18.2 5.28 8.91 113Anion-exchange 8 118 91 27.5 16.7 3.85 9.43 109

adsorption step of the unbound acidic proteins using thetype XII anion-exchanger. In order to bind LPO, LF andIgs the cation-exchange adsorption step was done at pH6. Three different binding pH-values were tested forthe subsequent anion-exchange procedure. From ourprevious studies employing superparamagnetic cation-exchangers and whey it was evident that LPO couldbe removed quantitatively using a support concentra-tion of 6 g L−1 (Heebøll-Nielsen et al., 2004a). Thisvalue was also used in the present investigation result-ing in an initial adsorption of 99% of the LPO alongwith total protein of 93 mg g−1 adsorbent (Table 4),which corresponded to an 11-fold purification of LPOin the binding stage (calculated from mass-balances ofLPO and protein, respectively, in the whey feedstockand in the supernatant after binding). In the followinganion-exchange adsorption step the support concentra-tions were increased approximately five-fold given themuch higher concentration of acidic proteins presentin whey compared to the basic ones. As expected,increasing the binding pH for the anion-exchangestep resulted in higher amounts of adsorbed protein(Table 4).

3.2.2. Elution of bound proteinsDesorption of proteins was performed in a stepwise

manner by sequentially raising the NaCl concentrationof the eluent. The volumes of the added elution buffersin the anion-exchange operation were reduced to ap-proximately one half of the original feedstock volumesi ro-c o-t allf -p vol-u teinc ivelyl the

adsorbents after removal of the feedstock supernatantfrom the settled support slurry. Thus, it is conceivablethat the protein measured in the wash fractions was ac-counted for twice due to the batchwise nature of opera-tion. If the protein in the wash fractions is disregardedthe mass balances close within 5%.

The compositions of the whey, unbound proteinand eluate fractions are illustrated inFig. 6. Thecation-exchanger adsorbed basic proteins with goodselectivity and efficiency as expected. From the lanesrepresenting whey and unbound protein (Fig. 6a, lanes2 and 3), complete removal of LPO and LF was ob-served. A species with anMr of 15,000 (most probablylysozyme) was eluted at NaCl of≥0.4 M, but becauseits apparent molecular weight was close to that of�-LG this band could not be detected in the lanes rep-resenting whey and unbound protein. Some�-LG andIg were captured as well, as seen from the eluate frac-tions ofFig. 6a. The highest�-LG concentrations werefound in early eluates, which may suggest that most ofthis protein can be removed by repeated washing withequilibration buffer.

At pH 8 the anion-exchanger preferentially ad-sorbed�-LG (60% of the total added�-LG was ad-sorbed) and BSA, whereas below pH 8 only�-LG wasbound selectively as seen fromFig. 6b. Although ex-amination of eluate protein compositions clearly in-dicated that both types of ion-exchangers adsorbedother proteins, none of these species were bound insufficient amounts to show differences between feed-s ma-j H 8( ya

n ofo den-sa ieved

n order to more closely resemble a typical HGMF pess (seeHubbuch, 2001). Mass balances for the tal protein were consistently 10–20% too high inour fractionation schemes (Table 4). When the suports were settled magnetically the majority of theme consisted of interstitial liquid, and the high prooncentrations of the feedstocks may lead to relatarge amounts of protein being trapped between

tock composition and unbound material. Severalor whey proteins were negatively charged at pTable 1), yet despite this�-LG was preferentialldsorbed.

LPO was assayed enzymatically, and resolutiother bound proteins was analysed using scanningitometry. The results are presented inFig. 7. Betterpparent separation of adsorbed proteins was ach


Fig. 6. Reducing SDS-PAGE of fractions from (a) the cation-exchange of whey, and the anion-exchange of the unbound fraction from the cation-exchange experiment with binding pH at (b) 7 and (c) 8. For all three panels lanes 1, 2, 3 and 4, respectively, show the protein compositions of themolecular weight marker, feedstock, unbound protein and wash fractions. The remaining lanes contain eluates with the following concentrationsof NaCl in the added buffer: panel (a) lane 5, 0.1 M; lane 6, 0.2 M; lane 7, 0.4 M; lane 8, 0.6 M; lane 9, 0.8 M and lane 10, 1.0 M; panels (b) and(c) lane 5, 0.1 M; lane 6, 0.2 M; lane 7, 0.3 M; lane 8, 0.4 M; lane 9, 0.5 M and lane 10, 1.0 M.


Fig. 7. Contents of selected proteins in the eluates from the ion-exchange fractionation of whey versus the NaCl concentration in thedesorption buffers. (a) For the cation-exchange step Ig (� , heavy andlight chains), LPO/LF (�), LPO activity (♦), and LPO purificationfactor (PF, +) are depicted. LPO activity and purification factorsare plotted on the right axis. (b) From the anion-exchange step thecontents of�-LG (�), �-LA (©), and BSA (�) were plotted. Exceptfor LPO activity and purification factor, the values were obtainedfrom scanning densitometry of the SDS-PAGE gels ofFig. 6a and c;the contents are given in arbitrary units.

from the cation-exchanger, where a broader range ofsalt concentrations was necessary to desorb all boundspecies, and the majority of the Igs were desorbed priorto the more basic proteins, LPO, LF and lysozyme.LPO and LF could not be resolved by simple manipu-lation of ionic strength as indicated by the similarity ofthe concentration profiles of the LPO/LF band and theLPO activity (Fig. 7a). For the anion-exchange exper-iment most of the BSA was eluted before the majorityof the �-LG, though a very broad peak was observedfor the latter, and no fraction was free of either. In theconducted separation experiments only one desorptionstep was done at each concentration of NaCl. However,generally two to three identical washes are required toequilibrate these types of adsorbents to new conditions,and resolution may be improved by performing one ortwo extra cycles of elution at each ionic strength, and

also help remove loosely bound proteins in the washingphase.

4. Discussion

The uncoupling of protein binding and subsequentadsorbent capture in magnetised filters in HGMF al-lows for separate optimisation of the two steps, andfurthermore model studies conducted at smaller scalewith permanent magnets for (open gradient) particlecollection can easily be transferred to actual HGMFprocessing (Hubbuch, 2001; Hubbuch et al., 2001). Thefocus of this study has been to: (i) prepare high-capacitymagnetic adsorbents; and (ii) employ these in fraction-ation studies as a model for HGMF.

4.1. Adsorbent design

4.1.1. Binding kineticsThe fast equilibrium adsorption (<120 s) is in keep-

ing with the essentially non-porous nature of the fi-nal adsorbents (Hubbuch, 2001; Hubbuch et al., 2001;Hoffman, 2002; Heebøll-Nielsen et al., 2003). Simi-larly, Anspach et al. (1989)have demonstrated thatequilibria for the adsorption of proteins onto non-porous glass beads were reached in less than 60 s, com-pared to binding times of >1 h for the slowest poroussorbents of their study. The advantages of the PG-coated base material employed for the support designa cro-pao

4hy

c ingg ufferr wast d ont ratea alueb edt theP ver,t at am ffectd r.

re further emphasised by comparison with the maorous superparamagnetic anion-exchangers ofXuend Sun (2002)for which binding equilibrium onlyccurred after >2 h.

.1.2. Desorption from the magnetic supportsElution from ion-exchange chromatograp

olumns is commonly performed with increasradients in ionic strength, but due to the ease of beplacement with the magnetic adsorbents ithought that gradual changes in elution pH basehe pI of the bound species could efficiently sepadsorbed proteins. By changing the pH to a velow the pI of the adsorbed protein it was intend

o create electrostatic repulsion between BSA andEI-ligand, and thereby mediate desorption. Howe

he poor performance observed may indicate thuch higher buffering capacity was needed to eesorption and this route was not examined furthe


4.1.3. Protein binding of DEAE andTMA-derivatised adsorbents

By activating cellulose-based chromatography ma-trices with AB and AGEBurton and Harding (1997a)reported much higher activation levels than could bereached with ECH or 1,4-butanediol diglycidyl etherchemistries, due to a near-quantitative coupling effi-ciency of the allyl chemistries (Burton and Harding,1997b), and it was expected that these two chemistrieswould yield good results in the preparation of anion-exchangers. Comparison of support types I and IVconfirmed the advantages of the AB-preparation routeover direct derivatisation with DEAE. Moreover, thecapacities of support type IV were comparable tothose found for affinity adsorbents created from verysimilar magnetic starting materials byHubbuch andThomas (2002)and Zulqarnain (1999)for trypsinbinders (125 mg g−1) and yeast dehydrogenase adsor-bents (110–120 mg g−1), respectively.

4.1.4. PEI polymers as ligands for proteinadsorbents

In the design of cation-exchangers there was anindication that tentacular ligands on the surface ofnon-porous adsorbents could increase the surface areaavailable for protein adsorption (Heebøll-Nielsen et al.,2004a), and a similar effect was expected from posi-tively charged PEI polymers. PEI is not widely used asa chromatographic ligand, though the employment hasbeen reported. For example,Navarro et al. (1996)im-m s tore on-e ons.P ered -p o sepap iss om-m ec-o i-Kd a-b ionicc 300t ncew ause

the polymers tended to fill smaller pores (Vanecek andRegnier, 1982). Our results demonstrated that the BSAbinding capacity was proportional to the size of PEI,and given the non-porous nature of the adsorbents pore-filling effects of PEI can be neglected.

4.1.5. Long-chained PEITwo general approaches were explored in the prepa-

ration of anion-exchangers with large size PEI (Mr600,000–1,000,000). The first involved formation ofa PEI-gel ‘seeded’ with magnetic particles followedby homogenisation to reduce the size of the supports,whereas in the second, PEI was attached directly toactivated supports under conditions to prevent cross-linking. ECH served to link PEI to the supports as wellas to cross-link the PEI-strands in the former case. Inthe latter approach, ECH-activation was chosen over re-ductive amination for coupling the polymers since thenumber of active groups was determined to be∼5-foldlower on ECH-activated supports compared to the PG-coated starting material (Heebøll-Nielsen et al., 2004a),which was thought to limit inter-particle cross-linkingvia PEI, while the size of the polymers would still besufficient to achieve high-charge densities.

The extreme protein binding capacities seen fortypes VI and VII adsorbents were highly interesting,though the porosity and broad size distribution wereboth unwanted characteristics for HGMF-supports.However, this method may be of use for manufacture ofanion-exchangers for other applications (such as wastew

di-t h thec d byd uchs typeX

4d at

6 o-l ups( tes ions.I ofc loset o tot s at

obilised PEI on macroporous cellulose supportemove heavy metals from waste water, andMcNefft al. (1994)prepared zirconia-based HPLC anixchangers for separation of small, organic aniEI-derivatised silica supports for use in HPLC wescribed byAlpert and Regnier (1979), and such suports have since been used as anion-exchangers trate oligonucleotides (Pearson and Regnier, 1983) androteins (Kennedy et al., 1986). The PEIs used in thtudy were branched, water-soluble molecules conly quoted to consist of 25% primary, 50% sndary, and 25% tertiary amines (Andersson and Hattaul, 1999). Vanecek and Regnier (1982)comparedifferent sizes of PEI for the preparation of silicased ion-exchangers and found no difference inapacity when the polymer-size was varied fromo 60,000. However, the protein binding performaas found to depend on the matrix pore size bec

-

ater purification).By coupling PEI directly under high shear con

ions, support agglomeration was prevented, thougapacities were no better than what was achieveerivatisation with smaller PEI-molecules in a mimpler procedure (e.g. support type IX versusII).

.1.6. Short-chained PEIReaction with short-chained PEI was conducte

g g−1 support, which constituted a >100-fold mar excess of PEI nitrogen atoms to aldehyde groHeebøll-Nielsen et al., 2004a) and ensured compleurface coverage during reductive amination reactn order to obtain the highest possible densityharges the coupling was done at pH 11, which is co the point of zero charge of the polymer and alshe point at which physical extension of molecule i


a minimum due to minimal intramolecular electrostaticrepulsion (Horn, 1980).

Human serum albumin is a soft protein and maxi-mal adsorption will be dependent on pH (Lyklema andNorde, 1996). Typical values for monolayer coveragewith human serum albumin reach 1–2 mg m2 at pH 8,as has been shown for a range of materials (Lyklemaand Norde, 1996). From N2-adsorption studies a spe-cific surface area of 110 m2 g−1 for PG-coated supportsidentical to those used in this work has been determined(Zulqarnain, 1999; Hoffman, 2002). TheQmax-valuesfound for support types X–XII, thus surpassed the re-ported values for plateau (monolayer) adsorption, andit can be hypothesised that the increase in PEI size led toan increasing tendency for positively charged strandsof PEI to protrude from the surface of the adsorbent(Fig. 8), effectively yielding a larger specific surfacearea for interaction with protein.

In comparison to cation-exchange adsorbents,which also possessed tentacular ligand structure inthe form of ECH-oligomers (Heebøll-Nielsen et al.,2004a), the anion-exchangers (type XII) displayed afour-fold higher initial slope of the isotherm curve(Qmax/Kd ∼ 100 L g−1 versus 26 L g−1). This may beexplained with a higher number of charges on the ad-sorbents due to more densely charged polymers for PEIcompared to the derivatised ECH-oligomers. The lig-and tentacles further yielded better adsorption than hasbeen observed for various affinity adsorbents wherevalues forQmax/Kd of 10–20 L g−1 are common forw he øll-N

4

n-e pture

F ge bea Adsorbentt ateria

the valuable basic proteins with minimum losses andthis was then followed by anion-exchange separation ofthe remaining species. During the cation-exchange stepthe supports adsorbed protein corresponding to 40%of the maximum lysozyme binding capacity (Heebøll-Nielsen et al., 2004a), whereas the type XII anion-exchangers could only bind protein up to 27% of theBSA capacity at pH 8. The ethane sulphonic acid ligandof the cation-exchanger is a ‘strong’ cation-exchangeligand, unlike PEI, which is best described as a ‘weak’ion-exchanger (at low pH up to 75% of the aminesof PEI will be charged,Horn, 1980), which may ex-plain the more efficient binding of the former in thewhey feedstock. Moreover, the whey had a higher con-ductivity (∼5 mS cm−1) than the buffered BSA solu-tions, which may also influence the binding efficiency.The very high concentration of acidic proteins in wheywas problematic for the anion-exchange processing as acorrespondingly high-adsorbent concentration was re-quired, and the eluates from these steps were, therefore,more dilute than the feed.

The fast adsorption to the superparamagnetic ma-trices was advantageous as seen by comparison to thework of Dionysius et al. (1991)in which LPO and LFwere recovered by batch adsorption to porous cation-exchange matrices. In this case a binding time of 1 hwas used. Moreover, five elution cycles (all at 1 MNaCl) were needed for complete desorption of boundprotein.

A complete procedure for fractionation of whey pro-t dc ara-t LFw d step� tica pre-v ion.S rs

ell-functioning systems (Zulqarnain, 1999; Hubbuct al., 2001; Hubbuch and Thomas, 2002; Heebielsen, 2002; Heebøll-Nielsen et al., 2003).

.2. Whey fractionation

In the whey fractionation experiments catioxchange separation was done first in order to ca

ig. 8. Schematic comparison of the relative thickness of charypes VI–IX are not included as these are considered porous m

ring layers of the non-porous adsorbent types I–V and X–XII.ls (see text).

eins was suggested byYe et al. (2000), who also useation-exchange followed by anion-exchange sepion in column chromatography. Initially LPO andere adsorbed and separated, and in the secon-LA and �-LG were separated. As for the magnedsorption system the high protein concentrationented concentration of the proteins during elutimilar effects were seen byGeberding and Bye


(1998) for an optimised packed bed anion-exchangeexperiment with concentrated, bovine sweet whey,although good resolution of�-LG and �-LA wereachieved by desorbing with a complex series of stepchanges in elution conditions, involving both pH andconcentration of the buffer component.

In our previous study of cation-exchange HGMFof whey proteins LPO was purified 36-fold (Heebøll-Nielsen et al., 2004a), whereas in the current work amaximum purification factor of 28 was reached. Fac-tors such as a lower binding pH used in the currentexample, and bound Igs most probably contributed tothe lowering of the purification factor. From the currentresults an improved HGMF fractionation procedure canbe suggested, in which adsorption is done at a decreasedpH of 6 to capture part of the whey Igs. Desorption intwo separate cycles at 0.3 M and 1.0 M NaCl from thecation-exchangers, respectively, will provide fractionswith purified Ig and LPO/LF with LPO-purificationfactors expected to reach 30 or more for an optimisedsystem. Not surprisingly, it has not been possible tocompete with packed bed chromatography in terms ofseparation of LPO and LF, however resolution of Igsfrom a LPO/LF fraction is competitive, and for LPOand LF a good yield is further expected.

The selective binding of�-LG during the subse-quent anion-exchange can be used in a HGMF process,though further fine-tuning of adsorption pH as well asadsorbent concentration will be required to fully op-timise the process. This process will suffer from thes teinc aphy.H entsh raphyt rtic-u arifyf fromp ss-i sorp-t tes( re(

5

signa nion-

exchange supports. When molecules with a single, pos-itive charge were attached to the surface of the basematrix employed (types I–V) maximum BSA bindingcapacities up to 156 mg g−1 were obtained with disso-ciation constants of∼1�M. In contrast, derivatisationwith PEI-molecules ofMr 70,000 (type XII) yielded ad-sorbents with tentacular ligands, each carrying multiplecharges, capable of binding BSA up to 337 mg g−1 withextremely tight interactions (Kd ∼ 40 nM,Qmax/Kd ∼100 L g−1).

The fractionation of sweet bovine whey usingcation-exchangers as well as the prepared adsor-bents (type XII) was studied. After sequential adsorp-tion to first cation- and then anion-exchangers boundproteins were desorbed with stepwise increases inNaCl-concentration in the elution buffers. From thecation-exchangers Igs could be resolved from LPO andLF, which were eluted simultaneously, by using NaClof 0.3 and 1.0 M. In contrast, adsorption to the anion-exchangers was surprisingly selective for�-LG, but lit-tle resolution could be obtained during elution, though�-LG of high-purity was obtained. The observation ledto a suggestion for an improved procedure for large-scale fractionation of Igs, LF and LPO from bovinewhey in a HGMF process.

Acknowledgement

We thank the Danish Technical Research Coun-c rS

R

A opar-ns. J.

A t of

A .T.,cids,esti-nd

B ifica-58.

B n ofand

ame limitations imposed by the high target prooncentration as seen for packed bed chromatogrowever, fractionation-based on magnetic adsorbas the advantage over packed bed chromatog

hat it can handle bioprocess liquors containing palate contaminants, thus far less pre-treatment to cl

eedstocks is necessary. A further benefit gainedurification by HGMF is the potential for fast proce

ng, which arises as a direct consequence of fast adion/desorption combined with the very high flow rae.g.∼100 m h−1) permissible during particle captuHubbuch et al., 2001).

. Conclusions

Two general strategies have been followed to dend construct superparamagnetic non-porous a

il (Statens Teknisk-Videnskabelige Forskningsad,TVF) for funding this work.

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Fractionation of whey proteins with high-capacity superparamagnetic ion-exchangers