B

Ea

b

a

ARRA

KCBA

1

ilvassDamamnbCfiodbTa

1d

Process Biochemistry 45 (2010) 1713–1719

Contents lists available at ScienceDirect

Process Biochemistry

journa l homepage: www.e lsev ier .com/ locate /procbio

acterial cellulose nanofibers for albumin depletion from human serum

mel Tamahkara, Ceyhun Babaca, Tülin Kutsala, Erhan Piskina, Adil Denizli b,∗

Bioengineering Division and Chemical Engineering Department, Hacettepe University, Ankara, TurkeyDepartment of Chemistry, Biochemistry Division, Hacettepe University, Ankara, Turkey

r t i c l e i n f o

rticle history:eceived 8 April 2010eceived in revised form 5 July 2010ccepted 7 July 2010

eywords:ibacron Blue F3GA

a b s t r a c t

Cibacron Blue F3GA (CB) was covalently attached onto the bacterial cellulose (BC) nanofibers for humanserum albumin (HSA) depletion from human serum. The BC nanofibers were produced by Acetobacterxylinum in the Hestrin–Schramm medium in a static condition for 14 days. The CB content of the BCnanofibers was 178 �mol/g. The specific surface area of the BC nanofibers was determined to be 914 m2/g.HSA adsorption experiments were performed by stirred-batch adsorption. The non-specific adsorptionof HSA on the BC nanofibers was very low (1.4 mg/g polymer). CB attachment onto the BC nanofiberssignificantly increased the HSA adsorption (1800 mg/g). The maximum HSA adsorption was observed

acterial cellulose, Cellulose nanofibers,lbumin depletion, Proteomics at pH 5.0. The HSA adsorption capacity decreased drastically with an increase of the aqueous phase

concentration of sodium chloride. The elution studies were performed by adding 1 M NaCl to the HSAsolutions in which adsorption equilibria had been reached. The elution results demonstrated that thebinding of HSA to the adsorbent was reversible. The depletion efficiencies for HSA were above 96.5%for all studied concentrations. Proteins in the serum and eluted portion were analyzed by SDS-PAGE for

SA d

testing the efficiency of H

. Introduction

Serum proteins may often serve as indicators of disease ands a rich source for biomarker discovery. One of the biggest chal-enges to the proteomic studies is that a few proteins comprise theast majority of the protein mass of serum [1]. High abundance oflbumin and immunoglobulins which comprise about 80% of totalerum protein, is a major problem in proteome studies which useerum, plasma, cerebrospinal fluid or synovial fluid samples [2].epletion of abundant serum proteins will help in the discoverynd detection of less abundant proteins that may prove to be infor-ative disease markers [3]. A variety of depletion methods for high

bundant proteins from body fluids have been developed. Severalajor strategies are available concerning the depletion mecha-

isms of albumin and immunoglobulins. Albumin depletion cane achieved by either dye-ligands such as the widely recognizedibacron Blue F3GA [4], or specific antibodies [5]. The high speci-city of antibodies provides excellent selectivity. However, in spitef its high selectivity, antibody carrying adsorbents also has some

rawbacks. The cost of antibodies tends to be very high. The anti-odies are difficult to immobilize in the proper orientation [6].hey are also susceptible to degradation during the sterilizationnd cleaning procedures. A dye-affinity resin for albumin deple-

∗ Corresponding author.E-mail address: denizli@hacettepe.edu.tr (A. Denizli).

359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.oi:10.1016/j.procbio.2010.07.007

epletion from human serum. Eluted proteins include mainly HSA.© 2010 Elsevier Ltd. All rights reserved.

tion has the advantage of high loading capacity as compared toan antibody based system but has been shown to lack of speci-ficity [6–8]. Reversible adsorption could provide the possibility ofusing such dyes in an immobilized form and, in this way, having theadvantages of the use of dye-affinity adsorbents, saving time andcost. Other methods reported include a proprietary polypeptideaffinity matrix that removes albumin together with immunoglob-ulins (IgGs), but is now apparently unavailable [9], and a methodbased on the size separation in a centrifugal filtration device thatwas, perhaps predictably, unsuccessful [10]. The next challengein serum protein analysis is the depletion of the high concentra-tion of IgGs. The IgG depletion is commonly achieved by proteinA affinity resins. Protein A binds to the Fc region of the IgG [11].In addition, the depletion of IgG in human plasma is employedfor the treatment of immune disorders including systemic lupuserythematosus, rheumatoid arthritis, myasthenia gravis, alloim-munization and cancer [12–17].

The nanomaterials could present very large surface area to vol-ume ratio which biomolecules could be adsorbed in high amount[18–21]. Due to these reasons, nanofibers have been gaining moreattention for the affinity purification of biomolecules [22]. A majoradvantage of the non-porous nanofibers is that significant intra-

particle diffusion resistances are absent; this is particularly usefulfor the rapid analysis of proteins with high efficiency and reso-lution [23]. The rapid separation makes it very useful for qualitycontrol, on-line monitoring, and purity check of biomolecules suchas peptide mapping of recombinant products.

1 iochem

iisBrMiclpissmm

ffstBaao

2

2

6Cs1Ueanwkdiuab

2

mysidatap1

2

3tsttwbcIiw

714 E. Tamahkar et al. / Process B

Cellulose is a good affinity matrix because of its biocompatibil-ty with biomolecules [24]. Besides cellulose from plants, celluloses also secreted extracellularly as synthesized cellulose fibers byome bacterial species, which is called bacterial cellulose (BC) [25].C which is used produced by Gram-negative, acetic acid bacte-ia Acetobacter, offers a unique alternative to plant cellulose [26].any unique features of BC have been exploited. One of the most

mportant features of BC is its high purity, which distinguishes thisellulose from plant, usually associated with hemicelluloses andignin, removal of which is inherently difficult [27]. The uniqueurity of BC enables its successful applications in fields of biomed-

cal applications. BC is relatively inexpensive and it has a highlywollen nanofiber network structure which depends on the exten-ive interior surface area of the interstitial spaces of the driedatrix. Also, it has some properties such as high chemical andechanical stability.The aim of this study is to prepare the dye-affinity BC nanofibers

or human serum albumin (HSA) depletion from human serum. Soar, no nanofiber based adsorbents were reported for depletiontudies. BC nanofibers were produced by Acetobacter xylinum inhe Hestrin–Schramm medium in static condition for 14 days. TheC-CB nanofibers were characterized by FTIR, SEM and elementalnalysis. Then, human serum albumin (HSA) depletion studies fromqueous solutions and human serum were performed. Reusabilityf the BC-CB nanofibers was also tested.

. Experimental

.1. Materials

Human serum albumin (HSA, 98% pure by gel electrophoresis, fatty acid free,7 kDa) was purchased from Octapharma (Lachen, Sweden) and used as received.ibacron Blue F3GA (CB) (Molecular mass: 840.11 g/mol) was obtained from Poly-cience (Warrington, USA) and used without further purification. A. xylinum (ATCC0245) was supplied from Agricultural Research Service Culture Collection (ARS,SA) in lyophilized form. Growth medium components, d-glucose, peptone, yeastxtract, K2HPO4 and KH2PO4 were obtained from Merck (Darmstadt, Germany) innalytical grade and used without further purification. Commasie Blue and silveritrate (AgNO3) were purchased from Sigma (St. Louis, USA). All other chemicalsere of reagent grade and were purchased from Merck. Laboratory glassware was

ept overnight in a 5% nitric acid solution. Before use the glassware was rinsed witheionized water and dried in a dust-free environment. All water used in the exper-

ments was purified using a Barnstead (Dubuque, IA) ROpure LP® reverse osmosisnit with a high flow cellulose acetate membrane (Barnstead D2731) followed byBarnstead D3804 NANOpure® organic/colloid removal and ion exchange packeded system.

.2. Production of bacterial cellulose nanofibers

The production of BC was performed by growing A. xylinum in Hestrin–Schrammedium, pH 5.1. The medium containing 20 g/l glucose, 10 g/l bactopeptone, 10 g/l

east extract, 4 mM KH2PO4 and 6 mM K2HPO4 is used for the production of BC intatic culture. The pH of the medium was adjusted to pH 5.1–5.2 using 1 M HCl. Thenoculum was prepared by growing A. xylinum at 30 ◦C using a rotary shaker for 3ays. The BC nanofiber formation was allowed to occur over a period of 14 daysfter inoculating subculture in the proportion 1:10 in petri dishes statically. Then,he formed BC nanofibers were redispersed within 10 ml of ethanol and centrifugedgain under similar conditions. Ethanol washing was repeated three times for com-lete removal of bacterial impurities. Finally, the BC nanofibers were redispersed in0 ml of water (0.10%, by weight) and stored at room temperature.

.3. Cibacron Blue F3GA attachment to bacterial cellulose nanofibers

CB was covalently attached to the BC nanofibers. First, CB was dissolved in0 ml of water (CB concentration: 1.6 mg/ml). The CB solution was transferredo BC nanofibers in 30 ml distilled water. The medium was heated to 60 ◦C in aealed reactor and stirred magnetically for 30 min. This was followed by the addi-ion NaCl (750 mg) in order to stimulate the deposition of CB on the surface ofhe BC nanofibers. After 1 h, temperature was increased to 70 ◦C and the solution

as treated for 30 min. Then, Na2CO3 (75 mg) was added to accelerate the reaction

etween CB and the BC nanofibers. Under the experimental conditions, a chemi-al reaction took place between the chlorine of CB and hydroxyl groups of the BC.n order to remove the non-specifically attached CB molecules, an extensive clean-ng procedure was applied, which was as follows: The BC-CB nanofibers were first

ashed with deionized water. Then, the nanofibers were dispersed in methanol,

istry 45 (2010) 1713–1719

and the dispersion was sonicated for 2 h in an ultrasonic bath. At the last stage, thenanofibers were washed again deionized water. The BC-CB nanofibers were storedat 4 ◦C with 0.02% sodium azide to prohibit microbial contamination.

The release of CB from the BC-CB nanofibers was investigated at different pHvalues in the range of 4.0–6.0. It should be noted that these mediums were thesame which were used in the HSA adsorption experiments. CB release was alsodetermined in the elution medium. The medium and the BC-CB nanofibers wereincubated for 24 h at room temperature. Then, the BC-CB nanofibers were sepa-rated from the medium, and CB concentration in the supernatant was measured byspectrophotometry at 630 nm.

2.4. Characterization of bacterial cellulose nanofibers

Water-uptake ratio of the BC nanofibers was determined in distilled water. Thedry BC nanofibers were carefully weighed before being placed in a 50 ml vial con-taining distilled water. The vial was put into an isothermal water bath with a fixedtemperature (25 ◦C) for 2 h. The BC nanofibers were taken out from the water andweighed. The weight ratios of dry and wet samples were recorded. The water con-tent of the BC nanofibers was calculated using the weight ratios. The amount of CBattached on the BC nanofibers was determined by elemental analysis using the sulfurpercentage. The specific surface area and porosity of samples were investigated fromnitrogen (N) adsorption isotherm at 77 K (ASAP 2020, Micromeritics, USA). Using asessile drop method, static water contact angle was measured at room temperatureon a contact angle goniometer (KRUSS-DSA10-MK) equipped with video capture.Microscopic observations of the BC nanofibers were performed using a scanningelectron microscopy (SEM, JEOL, JEM 1200 EX, Tokyo, Japan). Nanofibers were driedat room temperature and coated with a thin layer of gold (about 100 Å) in vacuumand photographed in the electron microscope. Fourier transform infrared (FTIR)measurements were performed on a Shimadzu FTIR 8000 Series spectrometer innormal transmission mode using a KBr detector over the range of 400–4000 cm−1

at a 2 cm−1 resolution averaged over 64 scans. All spectra were base-line correctedand normalized to a thickness of 1 �m. The nanofibers were degassed overnight ina vacuum oven maintained at 60 ◦C before FTIR measurements.

2.5. HSA depletion from aqueous solutions

The HSA adsorption amounts of HSA of the BC-CB nanofibers were measuredbatch-wise. In this study, the effects of HSA concentration, pH and ionic strengthon the adsorption capacity of the BC-CB nanofibers were studied. HSA concentra-tion was varied between 0.5 and 10 mg/ml. The pH of the adsorption medium wasvaried between 4.0 and 6.0 using 0.1 M CH3COONa–CH3COOH buffer system. Thesestudies were performed with a salt-free buffers. Ionic strength was changed in therange of 0.01–0.1 by adding NaCl. In a typical adsorption experiment, HSA was dis-solved in 10 ml of buffer solution, and 20 mg of BC nanofibers were added. Then, theadsorption experiments were performed for 2 h at 25 ◦C at a stirring rate of 20 rpm.At the end of this equilibrium period, HSA adsorption was determined by measur-ing the initial and final concentration of HSA within the adsorption medium usingCoomassie Brilliant Blue. The protein adsorption amount was calculated by massbalance.

2.6. Elution studies

The elution of HSA was carried out using 1 M NaCl at 25 ◦C. The HSA adsorbednanofibers (20 mg) were placed in the elution medium and stirred for 1 h at a stirringrate of 20 rpm. The final HSA concentration in the elution medium was determinedby using Coomassie Brilliant Blue. The elution ratio was calculated from the amountof HSA adsorbed on the BC nanofibers and the amount of HSA eluted into themedium. In order to test the reusability of the BC nanofibers, HSA adsorption–elutionprocedure was repeated ten times by using the same polymeric adsorbent. It shouldbe also noted that, after elution of HSA, possible CB release was also monitored.

2.7. HSA depletion from human serum

HSA adsorption from human serum on the BC-CB nanofibers was studied inbatch-wise. The blood is collected from thoroughly controlled voluntary blooddonors. Each unit separately controlled and found negative for hepatitis B spe-cific antigen and HIV I, II and hepatitis C antibodies. No preservatives are addedto the samples. Blood samples were centrifuged at 500 g for 3 min at room tem-perature to separate the serum. The serum samples were filtered using 0.45 �mcellulose acetate microspin filters (Alltech, Deerfield, IL, USA). The original serumof the healthy donor contained 47 mg HSA/ml as determined by bromocresol green(BCG) dye method at 628 nm. Total protein content of crude and depleted serumsamples were determined using the DC Protein Assay (Bio-Rad) according to themanufacturers instructions with bovine serum albumin (BSA) as standards (Pierce,

Rockford, IL, USA). Total protein concentration in crude serum was 62 mg/ml. Inorder to deplete IgG, the freshly separated human serum (100 ml) was pumped intothe Protein A-Sepharose column (10 cm × 0.9 cm inside diameter) equipped with awater jacket for temperature control. Equilibration of the Protein A-Sepharose 4Bcolumn (Sigma) was performed by passing four column volumes of sodium acetatebuffer (pH: 5.2) before injection of the serum. When serum passes through the col-

E. Tamahkar et al. / Process Biochemistry 45 (2010) 1713–1719 1715

Table 1The physical and morphological properties of the nanofibers.

Items Property

Surface area 914 m2/gPorosity 84%Nanofibril diameter 50–100 nmThickness 100 �mWater uptake 2000%CB content 178 �mol/g

utsscri2eHfi0e

3

3

ntdoTcFrsaHwds

Blfsoaae

pswiuasimtmw

Fig. 1. SEM photograph of BC nanofibers.

Contact Angle 28◦

The results were means of duplicate determination on tripleindependent measurements.

mn, the IgG molecules adsorbed on the Protein A-Sepharose 4B adsorbent. Thereated serum which passed from the column consists mainly of HSA and othererum proteins. After that the serum is ready for affinity depletion of HSA. Analy-is of IgG was performed by a nephelometer assay (Beckman Array 360, USA). Theoncentration of IgG in serum was determined to 8.5 mg/ml. The concentration ofemaining IgG in serum sample was very low. This stated a depletion ratio of 99% IgGn serum sample. Then, 10 ml of the IgG depleted human serum was incubated with0 mg of BC nanofibers pre-equilibrated with acetate buffer (pH 5.0) for 2 h. Thesexperiments were conducted at 20 ◦C and a stirring rate of 100 rpm. The amount ofSA adsorbed by the BC-CB nanofibers was determined by measuring the initial andnal concentration of HSA in serum. Phosphate buffered saline (PBS, pH: 7.4, NaCl:.9%) was used for dilution of human serum. In order to test the performance, gellectrophoresis was carried out as described in details previously [28].

. Results and discussion

.1. Characteristics of cellulose nanofibers

The main physical and morphological properties of theanofibers were presented in Table 1. Fig. 1 shows SEM pictures ofhe BC nanofibers. As seen here, the porous structure of the freeze-ried BC nanofibers with three-dimensional non-woven structuref nanofibrils (50–100 nm) which are highly uniaxially oriented.his unique nano-morphology results in a very high water uptakeapacity (Fig. 2). This is due to both chemical and physical structure.or chemical structure, BC nanofibers are hydrophilic nanomate-ial that is expected to uptake more water molecules. For physicaltructure, BC is three dimensional non-woven network with largemount of pores which was maintained by freeze-drying method.igh water uptake ability is an indication of high surface areahich is important for an affinity support. Intact bacteria andebris were not found in the polymer structure after washingtep.

FTIR spectroscopy was used to show attachment of CB on theC nanofibers. These spectra were thickness normalized and base-

ine corrected and no other processing was performed. There areour main vibrational modes: aromatic C C vibration (1080 cm−1),ymmetric stretching of S O (1058 cm−1), asymmetric stretchingf S O (1112 cm−1) and aromatic C–N vibration (3410 cm−1) aslso pointed out on the chemical structure of the CB (Fig. 3). Thesebsorption bands may be considered as an indication of the pres-nce of CB within the BC nanofibers.

CB loading was calculated as 178 �mol/g polymer (149.54 mg/golymer). The visual observations (colour of the BC nanofibers)howed the attachment of CB molecules. The BC-CB nanofibersere extensively washed with methanol until to ensure that there

s no CB leakage from any of the BC-CB nanofibers and in any mediased at adsorption–elution steps. The release of CB molecules waslso measured in three different kinds of media. There was no mea-urable CB release into the acidic medium (pH 3.0). CB was released

n the neutral medium while some was released in the alkaline

edium too. The release in the strongly alkaline medium indicateshe existence of strong ionic interactions. The release in neutral

edium might just be the physically occluded CB along with anyeakly/physically bonded CB. It can be said that there was not a Fig. 2. Water uptake behaviour of BC nanofibers.

1716 E. Tamahkar et al. / Process Biochemistry 45 (2010) 1713–1719

sw

3

3

ttttmianmcsaahao

cgb

q

Fp

Fig. 3. Chemical structure of CB.

ignificant increase in the amount of CB released (more than 20eeks).

.2. HSA depletion from aqueous solutions

.2.1. Effect of HSA concentrationFig. 4 shows the effects of HSA concentration on HSA adsorp-

ion amount of the BC nanofibers and the BC-CB nanofibers. Notehat one of the main requirements in affinity chromatography ishe specificity of the used adsorbent [4]. The non-specific interac-ions between the BC nanofibers and the HSA molecules should be

inimum in order to consider the interactions as specific. As seenn this figure, non-specific HSA adsorption was 1.4 mg/g. While CB-ttachment significantly increased the HSA binding capacity of theanofibers (up to 1800 mg/g). The amount of HSA adsorbed per unitass of the BC-CB nanofibers increased first with the initial con-

entration of HSA then reached a plateau value which representsaturation of the active adsorption sites (which are available andccessible for HSA) on the BC-CB nanofibers. This effect can resultcooperative effect of different interaction mechanisms such as

ydrophobic, electrostatic and hydrogen bonding caused by thecidic groups and aromatic structures on the CB and by the groupsn the side chains of the amino acids on the HSA molecules [29].

The Langmuir adsorption isotherm is expressed by Eq. (1). Theorresponding transformations of the equilibrium data for HSAave rise to a linear plot, showing that the Langmuir model could

e applied in this system and described by the equation:

= qmaxbCeq

(1 + bCeq)(1)

ig. 4. Effect of HSA concentration on adsorption capacity; CB loading: 178 �mol/g;H: 5.0; T: 25 ◦C.

Fig. 5. Effect of pH on adsorption capacity; CB loading: 178 �mol/g; HSA concen-tration: 2.0 mg/ml; T: 25 ◦C.

where q is the adsorbed amount of HSA (mg/g), Ceq is the equi-librium HSA concentration (mg/ml), b is the Langmuir constant(ml/mg) and, qmax is the maximum HSA adsorption capacity (mg/g).This equation can be linearized so that;

(2) Ceqq = 1

(qmaxb) + Ceqqmax

.

The plot of Ceq versus Ceq/q was employed to generate the inter-cept of 1/qmax.b and the slope of 1/qmax.

The maximum adsorption capacity (qmax) was obtained from theexperimental data. The correlation coefficient (R2) was 0.986. TheLangmuir model can be applied in this dye-affinity system. It shouldbe also noted that the maximum adsorption capacity (qmax) and theLangmuir constant were found to be 2333 mg/g and 0.04 mg/ml,respectively.

3.2.2. Effect of pHThe pH was changed from 4.0 (below the isoelectric point of

HSA) to 6.0 (above the isoelectric point of HSA) with a salt-freebuffer. Fig. 5 shows the effects of pH on the adsorption of HSA ontothe BC-CB nanofibers. No significant effect of pH was observed onthe physical adsorption of HSA onto the BC nanofibers. In all thecases investigated, the maximum adsorption of HSA was observedat pH 5.0. Significantly lower adsorption capacities were obtainedin more acidic and in neutral pH regions. The decrease in the HSAadsorption capacity in more acidic and more alkaline pH regionscan be attributed to electrostatic repulsion effects between theidentical charged protein molecules on the surface of the BC-CBnanofibers. As it has been shown that proteins have no net chargeat their isoelectric points and so the protein solubility in aque-ous media decreases. Thus the maximum adsorption from aqueoussolutions is usually observed at their isoelectric points. The isoelec-tric pH of HSA is 4.9. However, acidic and basic medium caused theprotein to be positively or negatively charged, increasing the solu-bility of protein in media. These results are in agreement with theliterature [30].

3.2.3. Effect of ionic strengthIt is clearly shown in Fig. 6 that, increasing the NaCl concentra-

tion, the adsorption capacity of the BC-CN nanofibers drasticallydecreased. A 80% reduction in the adsorption capacity was foundby changing the experimental conditions from 0 to 0.1 M NaCl,showing that the addition of salt, even at relatively low concen-tration, can disrupt electrostatic interactions between HSA and

E. Tamahkar et al. / Process Biochemistry 45 (2010) 1713–1719 1717

Fc

tfcw[ahabtfwC

3

aHaoewwatar

aua

TH

Tm

in the analysis of serum proteins, and therefore we attempted todeplete HSA from human serum. In the first step of this study,the depletion of IgG class proteins was achieved by using ProteinA-Sepharose 4B column. Protein A binds to the Fc region of the

ig. 6. Effect of ionic strength on adsorption capacity; CB loading: 178 mmol/g; HSAoncentration: 2.0 mg/ml; pH: 5.0, and T: 25 ◦C.

he CB molecules. The adsorption capacity decreased significantlyrom 548 to 108 mg/g polymer with the increase of the NaCloncentration from 0 to 0.1 M. The ionic interactions decreasedith increasing ionic strength due to the Debye screening effect

31]. In addition, increasing the ionic strength could promote thedsorption of the CB molecules to the BC-CB nanofibers surface byydrophobic interactions [32]. Moreover, the hydrophobic inter-ctions between the attached CB molecules themselves would alsoecome strong, because it has been observed that the salt additiono a CB solution caused the stacking of the free CB molecules [33]. Inact this could cause a decrease in available CB molecules to interactith the HSA molecules. Thus, the binding of the HSA to attachedB became difficult.

.2.4. ElutionThe elution of HSA from the BC-CB nanofibers was studied in

batch system. The BC-CB nanofibers loaded different amounts ofSA were placed in an elution medium containing 1 M NaCl and themount of HSA released in 60 min was determined. More than 95%f the adsorbed HSA was eluted in all cases when NaCl was used forlution. Cl−, being a chaotropic ion, disorganized the structure ofater, thus stimulating the elution of protein [34]. Note that thereas no CB release in this case which shows that CB molecules are

ttached to the nanofibers surface by strong chemical bond. Withhe elution data given above we concluded that 1 M NaCl is a suit-ble elution agent especially for the BC-CB nanofibers, and allowsepeated use of the affinity nanofibers developed in this study.

Reuse of the BC-CB nanofibers was tested with a series of 10dsorption–elution cycles. The BC-CB nanofibers were regeneratedsing a 1 M NaOH solution after every 3 cycles. After the ten runslight decrease of ca. 10% in HSA adsorption capacity has been

able 2SA depletion from the serum of a healthy donor; CB loading: 178 �mol/g; T: 25 ◦C.

HSA concentration (mg/ml) Achieved depletion (%)

0.5 96.5 ± 1.80.7 95.3 ± 2.11.1 95.1 ± 1.62.3 93.3 ± 1.74.7 95.2 ± 2.0

he results were means of duplicate determination on triple independent measure-ents.

Fig. 7. Repeated use of BC nanofibers. CB Loading: 178 �mol/g; HSA concentration:2.0 mg/ml; pH: 5.0 and T: 25 ◦C.

observed with reuse of the nanofibers (Fig. 7). The possibility toregenerate the BC-CB nanofibers was also considered as a majoradvantage. Moreover, no obvious changes of the BC nanofibers werefound in the recycling process. These results demonstrated that theBC nanofibers had high stability during the depletion process.

3.3. HSA depletion from serum

Depletion of additional abundant proteins can be beneficial

Fig. 8. SDS-PAGE of serum fractions. The fractions were assayed by SDS-PAGE using10% separating gel (9 cm × 7.5 cm), and 5% stacking minigels were stained with 0.25%(w/v) Coomassie Brillant R 250 in acetic acid–methanol–water (1:5:5, v/v/v) anddestained in ethanol–acetic acid–water (1:4:6, v/v/v). Lane 1, 1/10 diluted serum;Lane 2, 1/10 diluted serum after adsorption; Lane 3, eluted sample; Lane 4 biomarker(Sigma). Equal amounts of samples were applied to each line.

1 iochem

IifbslraitDDDPsutHabn

4

ti[dwtaethaeopittatahdtothTibfacdtfaUesa

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

718 E. Tamahkar et al. / Process B

gG [35]. The depletion efficiency for IgG class proteins was 99%n serum sample. Then, in the second step, the depletion of HSArom human serum was performed with the BC-CB nanofibers in aatch-wise. The depletion efficiency for HSA was above 87% for alltudied concentrations. Results, shown in Table 2, indicate that aarge portion of the HSA was bound by the BC-CB nanofibers. Theseesults are consistent with published studies [36–38]. Björhal etl. used five different commercially available depletion columnsncluding Aurum Serum Protein Minikit (Bio-Rad, USA), ProteoEx-ract HSA/IgG Depletion kit (Merck, Germany), Multiple Affinityepletion Column (Agilent Technologies, USA), POROS Affinityepletion Cartridges (Applied Biosystems, USA) and HSA-IgGepletion Kit (Amersham Biosciences, Sweden) [1]. Aurum Serumrotein Minikit (Bio-Rad, USA) contained CB as a ligand and theyhowed minimum achieved HSA depletion was 96.3%. We reachedp to 96.5% HSA depletion amount and it may be concludedhat the BC-CB nanofibers are sufficient in terms of efficiency ofSA depletion. The purity of HSA eluted from BC nanofibers wasssayed by SDS-PAGE. As clearly seen in Fig. 8, the presence of onlyand at Lane 3 indicates the purity of HSA after elution of the BC-CBanofibers.

. Conclusions

The serum proteome has been shown to contain informationhat directly reflects patho-physiological states and represents annvaluable source of diagnostic information for a variety of diseases39]. Unfortunately, the dynamic range of protein abundance ren-ers complete characterization of this proteome nearly impossibleith current analytical methods [40]. To study low abundance pro-

eins, which have potential value for clinical diagnosis, the highbundant species, such as HSA and immunoglobulins, are generallyliminated as the first step in many analytical protocols. But, deple-ion of HSA from serum is problematic because of its extremelyigh concentration [41]. Monoclonal antibodies to HSA are avail-ble from many commercial suppliers. These antibody ligands arextremely specific. But, they very are expensive, due to high costf production and/or extensive purification steps [42–44]. In therocess of the preparation of specific adsorbents, it is difficult to

mmobilize antibodies on the supporting matrix with retention ofheir original biological activity and proper orientation. Precau-ions are also required in their use (at adsorption and elution steps)nd storage. However, HSA is present in human serum at concen-rations in the range of 35–45 mg/ml and very large quantities ofntibody are required for its quantitative depletion. In addition,igh capacity adsorbent is required. Synthetic dye-ligands shouldisplay not only reduced process costs, but also increased resis-ance to chemical and biological actions, reduction in the amountf contaminants of biological nature and high capacity. Our goal iso find a cost effective and reusable dye-affinity adsorbent havingigh adsorption capacity for depletion of HSA from human serum.he amount of HSA adsorbed per unit mass of the BC-CB nanofibersncreased then reached a plateau value. This increase in the HSAinding capacity may have resulted from cooperative effect of dif-erent interaction mechanisms such as hydrophobic, electrostaticnd hydrogen bonding. With an increase of the aqueous phase con-entration of sodium chloride, the adsorption capacity decreasedrastically due to the Debye screening effect. More than 95% ofhe adsorbed HSA was eluted in all cases when NaCl was usedor elution. After the ten run a slight decrease of ca. 11% in HSA

dsorption capacity has been observed with reuse of the nanofibers.p to 96.5% HSA depletion amount was achieved. Based on ourvaluations of depletion efficiency, reproducibility and bindingpecificity of the BC-CB nanofibers offered the promising depletionpproach.

[

[

istry 45 (2010) 1713–1719

References

[1] Björhall K, Miliotis T, Davidsson P. Comparison of different depletion strate-gies for improved resolution in proteomic analysis of human serum samples.Proteomics 2005;5:307–17.

[2] Kocourek A, Eyckerman P, Thome-Krome B. The combined removal of albuminand immuno-globulins from human serum. Bio Tech Int 2005;17:24–5.

[3] Steel LF, Trotter MG, Nakajima PB, Mattu TS, Gonye G, Block T. Efficient andspecific removal of albumin from human serum samples. Mol Cell Proteomics2003;2:262–70.

[4] Denizli A, Piskin E. Dye-ligand affinity systems. J Biochem Biophys Methods2001;49:391–416.

[5] Wang YY, Cheng P, Chan DW. A simple affinity spin tube filter methodfor removing highabundant common proteins or enriching low-abundantbiomarkers for serum proteomic analysis. Proteomics 2003;3:243–8.

[6] Yavuz H, Denizli A. Immunoadsorption of cholesterol on protein a orientedbeads. Macromol Biosci 2005;5:39–48.

[7] Altıntas EB, Denizli A. Efficient removal of albumin from human serum bymonosize dye-affinity beads. J Chromatogr B 2006;832:216–23.

[8] Boto REF, Anyanwu U, Sousa F, Almeida P, Queiroz JA. Thiacarbocyanine as lig-and in dye affinity chromatography for protein purification. II. Dynamic bindingcapacity using lysozyme as a model. Biomed Chromatogr 2009;23:987–93.

[9] Yavuz H, Denizli A. Dye Affinity hollow fibers for albumin purification. Macro-mol Biosci 2004;4:84–91.

10] Lollo BA, Harvey S, Liao J, Stevens AC, Wagenknecht R, Sayen R, et al. Improvedtwo-dimensional gel electrophoresis representation of serum proteins by usingProtoClearTM. Electrophoresis 1999;20:854–9.

11] Georgiou HM, Rice GE, Baker MS. Proteomic analysis of human plasma: fail-ure of centrifugal ultrafiltration to remove albumin and other high molecularweight proteins. Proteomics 2001;1:1503–6.

12] Odabası M, Denizli A. Polyhydroxyethylmethacrylate-based magnetic DNA-affinity beads for anti-DNA antibody removal from systemic lupus erythemato-sus patient plasma. J Chromatogr B 2001;760:137–48.

13] Yılmaz E, Uzun L, Rad AY, Kalyoncu U, Unal S, Denizli A. Specific adsorption ofthe autoantibodies from rheumatoid arthritis patient plasma using histidine-containing affinity beads. J Biomat Sci Polym Ed 2008;19:875–92.

14] Odabası M, Özkayar N, Özkara S, Ünal S, Denizli A. Pathogenic antibody removalusing magnetically stabilized fluidized bed. J Chromatogr B 2005;826:50–7.

15] Haas M, Mayr N, Zeitihofer J, Goldammer A, Derfler K. Long-term treatment ofmyasthenia gravis with immunoadsorption. J Clin Apher 2002;17:84–7.

16] Alkan H, Bereli N, Baysal Z, Denizli A. Antibody purification with pro-tein A attached supermacroporous poly(hydroxyethyl methacrylate) cryogel.Biochem Eng J 2009;45:201–8.

17] Denizli A, Rad AY, Piskin E, Protein. A immobilized polyhydroxyethyl methacry-late beads for affinity sorption of human immunoglobulin-G. J Chromatogr B1995;668:13–9.

18] Karakoc V, Yılmaz E, Türkmen D, Öztürk N, Akgöl S, Denizli A. Selective sep-aration of human serum albumin with copper(II) chelated poly(hydroxyethylmethacrylate) based nanoparticles. Int J Biol Macromol 2009;45:188–93.

19] Türkmen D, Ozturk N, Akgol S, Elkak A, Denizli A. Phenylalanine con-taining hydrophobic nanospheres for antibody purification. Biotechnol Prog2008;24:1297–303.

20] Kim TG, Park TG. Surface functionalized electrospun biodegradable nanofibersfor immobilization of bioactive molecules. Biotechnol Prog 2006;22:1108–13.

21] Akgöl S, Öztürk N, Denizli A. New generation polymeric nanospheres forlysozyme adsorption. J Appl Polym Sci 2010;115:1608–15.

22] Zhang H, Nie H, Yu D, Wu C, Zhang Y, White CJB, Zhu L. Surface modifica-tion of electrospun polyacrylonitrile nanofiber towards developing an affinitymembrane for bromelain adsorption. Desalination 2010;256:141–7.

23] Che AF, Liu ZM, Huang ZJ, Wang ZG, Xu ZK. Chitosan modified poly(acrylonitrile-co-acrylic acid) nanofibrous membranes for the immobilization of con-canavalin. Biomacromolecules 2008;9:3397–403.

24] Putra A, Kakugo A, Furukawa H, Gong JP, Osada Y. Tubular bacterial cel-lulose gel with oriented fibrils on the curved surface. Polymer 2008;49:1885–91.

25] Wan YZ, Luo H, He F, Liang H, Huang Y, Li ZL. Mechanical, moisture absorption,and biodegradation behaviours of bacterial cellulose fibre-reinforced starchbiocomposites. Compos Sci Technol 2007;69:1212–7.

26] Sakairi N, Asano H, Ogawa M, Nishi N, Yokura S. A method for direct harvestof bacterial cellulose filaments during continuous cultivation of Acetobacterxylinum. Carbohydr Polym 1998;35:233–7.

27] Verschuren PG, Cardona TD, Nout MJR, De Gooijer KD, Van Den Reuvel JC. Loca-tion and limitation of cellulose production by Acetobacter xylinum establishedfrom oxygen profiles. J Biosci Bioeng 2000;89:414–9.

28] Steel LF, Shumpert D, Trotter MG, Seeholzer SH, Evans AA, London WT, et al. Astrategy for the comparative analysis of serum proteomes for the discovery ofbiomarkers for hepatocellular carcinoma. Proteomics 2003;3:601–9.

29] Uzun L, Yavuz H, Say R, Ersöz A, Denizli A. Poly(ethylenedimethacrylate–glycidyl methacrylate) monolith as a stationary phasein dye-affinity chromatography. Ind Eng Chem Res 2004;43:6507–13.

30] Denizli A, Tuncel A, Kozluca A, Ecevit K, Piskin E. Cibacron Blue F3GA attachedpoly(vinyl alcohol) particles for specific albumin adsorption. Sep Sci Technol1997;32:1003–15.

31] Basar N, Uzun L, Güner A, Denizli A. Lysozyme purification with dye-affinitybeads under magnetic field. Int J Biol Macromol 2007;41:234–42.

iochem

[

[

[

[

[

[

[

[

[

[

[

E. Tamahkar et al. / Process B

32] Yu Y, Sun Y. Macroporous poly(glycidyl methacrylate–triallylisocyanurate–divinylbenzene) matrix as an anion-exchange resin forprotein adsorption. J Chromatogr A 1999;855:129–36.

33] Karakoc V, Yavuz H, Denizli A. Affinity adsorption of recombinant humaninterferon-alpha on a porous dye-affinity adsorbent. Colloids Surf A2004;240:93–9.

34] Akgöl S, Bereli N, Denizli A. Magnetic dye affinity beads for the adsorption ofbeta-casein. Macromol Biosci 2005;5:786–94.

35] Chromy BA, Gonzales AD, Perkins J, Choi MW, Corzett MH, Chang BC, etal. Proteomic analysis of human serum by two-dimensional differential gelelectrophoresis after depletion of high-abundant proteins. J Proteome Res2004;3:1120–7.

36] Ahmed N, Barker G, Oliva K, Garfin D, Talmadge K, Georgiou H,et al. An approach to remove albumin for the proteomic analysis

of low abundance biomarkers in human serum. Proteomic 2003;3:1980–7.

37] Bereli N, Sener G, Altıntas EB, Yavuz H, Denizli A. Poly(glycidylmethacrylate) beads embedded cryogels for pseudo-specific affinitydepletion of albumin and immunoglobulin G. Mater Sci Eng C 2010;30:323–9.

[

[

istry 45 (2010) 1713–1719 1719

38] Altıntas EB, Tüzmen N, Uzun L, Denizli A. Immobilized metal affinity adsorptionfor antibody depletion from human serum with monosize beads. Ind Eng ChemRes 2007;46:7802–10.

39] Karatas M, Akgöl S, Yavuz H, Say R, Denizli A. Immunoglobulin G depletionfrom human serum with metal-chelated beads under magnetic field. Int J BiolMacromol 2007;40:254–60.

40] Li C, Lee KH. Affinity depletion of albumin from human cerebrospinal fluid usingCibacron-blue-3G-A-derivatized photopatterned copolymer in a microfluidicdevice. Anal Biochem 2004;333:381–8.

41] Zhou M, Lucas DA, Chan KC, Issaq HJ, Petricoin EF, Liotta LA, et al. An investiga-tion into the human serum “interactome”. Electrophoresis 2004;25:1289–98.

42] Akgöl S, Özkara S, Uzun L, Yılmaz F, Denizli A. Pseudospecific magnetic affinitybeads for immunoglobulin-g depletion from human serum. J Appl Polym Sci2007;106:2405–12.

43] Özkara S, Akgöl S, Canak Y, Denizli A. A novel magnetic adsorbent forimmunoglobulin G purification in magnetically stabilized fluidized bed.Biotechnol Prog 2004;20:1169–75.

44] Derazshamshir A, Baydemir G, Andac M, Say R, Galaev IY, Denizli A. Molecu-larly imprinted PHEMA-based cryogel for depletion of hemoglobin from humanblood. Macromol Chem Phys 2010;211:657–68.