immobilized metal affinity adsorption for antibody depletion from human serum with monosize beads

Immobilized Metal Affinity Adsorption for Antibody Depletion from HumanSerum with Monosize Beads

Evrim Banu Altıntas ,† Nalan Tu1zmen,‡ Lokman Uzun,† and Adil Denizli* ,†

Department of Chemistry, Biochemistry DiVision, Hacettepe UniVersity, Ankara, Turkey, and Department ofChemistry, Biochemistry DiVision, Dokuzeylu¨l UniVersity, Izmir, Turkey

Iminodiacetic acid (IDA)-functionalized adsorbents have attracted increasing interest in recent years forimmobilized metal-affinity chromatography (IMAC). In this study, IDA was covalently attached to nonporousmonosize poly(glycidyl methacrylate) [poly(GMA)] beads (1.6µm in diameter). Cu2+ ions were chelated viaIDA groups for affinity depletion of immunoglobulin G (IgG) from human serum. The monosize poly(GMA)beads were characterized by scanning electron microscopy. The Cu2+-chelated beads (628µmol/g) were usedin the IgG adsorption-elution studies. Studies to determine the effects of IgG concentration, pH, andtemperature on the adsorption efficiency of Cu2+-chelated beads were performed in a batch system. Nonspecificbinding of IgG to monosize beads in the absence of Cu2+ ions was very low (0.45 mg/g). The IgG adsorptionto chelated Cu2+ ions was 171.2 mg/g. The equilibrium IgG adsorption increased with increasing temperature.The negative change in Gibbs free energy (∆Go < 0) indicated that the adsorption of IgG on the Cu2+-chelated beads was a thermodynamically favorable process. The∆Sand∆H values were 172.1 J/mol‚K and-43.2 kJ/mol, respectively. A significant amount of the adsorbed IgG (up to 97.2%) was eluted in the elutionmedium containing 1.0 M NaCl in 1 h. The kinetics of the interactions suggest that the interactions could bebest represented by a mechanism based on second-order kinetics (k ) 9.8 × 10-5 to 118.9 × 10-5

g‚mg-1‚min-1). The adsorption followed the Langmuir isotherm model with monolayer adsorption capacityof 156.2-212.8 mg/g. Consecutive adsorption-elution experiments showed that the Cu2+-chelated beadscan be reused almost without any loss in the IgG adsorption capacity. To test the efficiency of IgG depletionfrom human serum, proteins in the serum and eluted portion were analyzed by two-dimensional gelelectrophoresis. The depletion efficiency for IgG was above 98.2%. Eluted proteins include mainly IgG anda negligible amount of non-albumin proteins such as apo-lipoprotein A1, sero-transferrin, haptoglobulin, andR1-antitrypsin. When anti-HSA-Sepharose adsorbent is used together with our metal-chelated monosize poly-(GMA) beads, IgG and HSA can be depleted in a single step.

1. Introduction

Serum plays a central role in clinical diagnosis. Serum isthought to contain tens of thousands of proteins along with theircleaved or modified forms. These proteins are a reflection ofongoing physiological or pathological events.1 Serum may oftenserve as an indicator of disease and is a rich source for biomarkerdiscovery. However, the large dynamic range of proteins inserum makes the analysis very challenging because highlyabundant proteins including albumin, immunoglobulins (IgG andIgA), antitrypsin, haptoglobin, and transferrin tend to mask thoseof lower abundance.2 Human serum albumin (HSA) and IgGrepresent over 80% of all proteins present in plasma, and theirhigh abundance masks the detection and determination of thelow-abundance proteins that are potential biomarkers for variousdiseases, e.g., cancer, and are, therefore, of great biologicalimportance in proteome studies.3-8

There are several removal strategies to deplete the higher-abundant proteins from serum, including ultracentrifugal filtra-tion, dye affinity, and immunoaffinity chromatography.9 Thedepletion of IgG is commonly achieved by Protein A/G affinityadsorbents, which bind to the Fc region of the IgG, but specificantibodies can also be used.10 The high specificity of thebioligands (i.e., Protein A/G) provides excellent selectivity.However, in spite of their high selectivities, Protein A/G or

antibody-carrying adsorbents also have some drawbacks thatare worth considering: (i) the cost of the ligands tend to bevery high; (ii) these bio-ligands are difficult to immobilize inthe proper orientation; and (iii) ligand may leak from thestationary phase and such contamination cannot, of course, betolerated in clinical applications. In addition, the depletion ofIgG in human serum is employed successfully for the treatmentof immune disorders including systemic lupus erythematosus,rheumatoid arthritis, myasthenia gravis, alloimmunization andcancer.11-14

Recently, immobilized metal affinity chromatography (IMAC)has shown great potential in the purification of proteins andpeptides15 and several types of IMAC columns have beenapplied to proteomics.16 One is for the enrichment of phospho-rylated peptides with Ga3+ or Fe3+ immobilized.17 The other isfor the selection of peptides with Cu(II) loaded on to thecolumns.18 The esterification-immobilized metal affinity selec-tion of peptides with Cu(II) loaded on to the columns.18 Theesterification-immobilized metal-affinity chromatography (IMAC)method has been successfully used to characterize>200phosphopeptides in the yeast proteome and human carcinomacell line.19,20 IMAC-based enrichment of phosphorylated pep-tides also has been demonstrated to work well on simplifiedmixtures. For instance, IMAC enrichment of phosphorylatedpeptides without prior chemical modification was used tocharacterize nearly 300 phosphopeptides after prefractionationwith anion-exchange chromatography21 in cultured plant cells.The first study using IMAC to analyze phosphorylation sitespresent in mammalian tissue has been published recently.22 In

* Corresponding author. E-mail: [email protected]. Tel.:+90312 297 7963. Fax:+90 312 297 6084.

† Hacettepe University.‡ Dokuzeylul University.

7802 Ind. Eng. Chem. Res.2007,46, 7802-7810

10.1021/ie061164c CCC: $37.00 © 2007 American Chemical SocietyPublished on Web 10/10/2007

this work, phosphorylated proteins and, subsequently, phos-phorylated peptides were enriched with IMAC. Recently, amethod has been reported for large-scale rapid analysis ofphosphoproteins in tissues or cells by combining IMAC withphage display cDNA library screening.23 The selection ofhistidine-containing peptides is an important aspect of proteom-ics in sample simplification and database search. ImmobilizedCu2+-affinity chromatography has been used in proteomics tosimplify sample mixtures by selecting histidine-containingpeptides from proteolytic digests.24 IMAC combined with massspectrometry has recently been employed for isolation ofnaturally occurring metal-binding proteins from total proteinsin human liver cells.25 Cu2+-charged alginate beads have beendirectly used as an IMAC medium for purification of IgG fromgoat serum.26 So far, to our knowledge, only one study has beenpublished using IMAC to deplete immunoglobulin G fromhuman serum for analyzing the proteome of human serum.27

We have used iminodiacetic acid (IDA) modified poly-(glycidyl methacrylate) [poly(GMA)] monosize beads withchelated Cu2+ ions as a model adsorbent capable of selectivebinding of IgG from human plasma. Poly(GMA) was used asthe basic matrix because of its known good mechanical strength,stability at neutral pH values even in wet conditions, and highreactivity of the epoxy groups for surface immobilization.28

Epoxy-derived adsorbents seem to be almost ideal systems todevelop very easy protocols for biomolecule immobilization.29

2. Experimental Section

2.1. Chemicals.Immunoglobulin G (IgG) (Sigma Cat. No.) 160101) and iminodiacetic acid disodium salt (IDA) werepurchased from Aldrich (Munich, Germany) and used withoutfurther purification. Glycidyl methacrylate (GMA, Fluka A.G.,Buchs, Switzerland) was purified by vacuum distillation andstored in a refrigerator until use. Azobisisobutyronitrile (AIBN)and poly(vinyl pyrrolidone) (MW) 30.000, BDH ChemicalsLtd., Poole, England) were selected as the initiator and the stericstabilizer, respectively. AIBN was recrystallized from methanol.Ethanol (Merck, Germany) was used as the diluent withoutfurther purification. All other chemicals were of reagent gradeand were purchased from Merck AG (Darmstadt, Germany).Laboratory glassware was kept overnight in a 5% nitric acidsolution. Before use, the glassware was rinsed with deionizedwater and dried in a dust-free environment. All water used inthe experiments was purified using a Barnstead (Dubuque, IA)ROpure LP reverse-osmosis unit with a high-flow celluloseacetate membrane (Barnstead D2731) followed by a BarnsteadD3804 NANOpure organic/colloid-removal and ion-exchangepacked-bed system.

2.2. Preparation of Poly(GMA) Beads.Poly(GMA) beadswere prepared as described elsewhere.30 Dispersion polymeri-zation was performed in a sealed polymerization reactor (volume) 500 mL) equipped with a temperature-control system. Atypical procedure applied for the dispersion polymerization ofGMA is given below. The monomer phase comprised 40 mLof GMA. AIBN (250 mg) was dissolved into the monomerphase. The resulting medium was sonicated for∼5 min at 200W within an ultrasonic water bath (Bransonic 2200, England)for the complete dissolution of AIBN in the polymerizationmedium. Poly(vinyl pyrrolidone) (4.0 g) was dissolved in ahomogeneous solution of ethanol (100 mL) and water (100 mL)placed in a polymerization reactor. The reactor content wasstirred at 500 rpm during the monomer addition, completedwithin ∼5 min, and the heating was started. Then, the mixturewas degassed by purging with nitrogen for∼20 min. Then, the

sealed reactor was placed in a shaking water bath at roomtemperature. The initial-polymerization time was defined whenthe reactor temperature was raised to 65°C. The polymerizationwas carried out at 65°C for 4 h with continuous strirring. Aftercompletion of the polymerization period, the reactor contentwas cooled down to room temperature and centrifuged at 5 000rpm for 10 min for the removal of dispersion medium. Thispolymerization reaction led to the formation of white beads.Poly(GMA) beads were redispersed within 10 mL of ethanoland centrifuged again under similar conditions. The ethanolwashing was repeated three times for complete removal ofunconverted monomers and other components. Finally, poly-(GMA) beads were redispersed within 10 mL of water (0.10%,by weight) and stored at room temperature.

2.3. IDA-Attached Beads.For coupling IDA, the reactionmixture (50 mL of 0.8 g IDA+ 2.0 M NaCO3, pH 11) andpoly(GMA) beads were incubated at 70°C in a heating mantleunder mild stirring for 12 h. After the coupling reaction, thebeads were washed with 5% acetic acid and deionized wateruntil the washing solutions were neutral. Afterward, the remain-ing epoxy groups were blocked with 2 M ethylene diamine atpH 10 for 16 h under gentle stirring. In order to remove thenonspecifically attached IDA molecules, an extensive cleaningprocedure was applied, which was as follows: The beads werefirst washed with deionized water. The monosize beads weredispersed in methanol, and the dispersion was sonicated for 2h in an ultrasonic bath. At the last stage, the beads were washedagain with deionized water. IDA-attached poly(GMA) beadswere stored at 4°C with 0.02% sodium azide to prohibitmicrobial contamination.

2.4. Chelation of Cu2+ Ions. Chelates of Cu2+ ions withIDA-modified poly(GMA) beads were prepared as follows: 1.0g of the IDA-modified beads were mixed with 50 mL of aqueoussolution containing 30 ppm Cu2+ ions, at constant pH of 4.1(adjusted with HCl and NaOH), which was the optimum pHfor Cu2+-chelate formation, and at room temperature. A 1000ppm atomic absorption standard solution (Cu(NO3)2 salt con-taining 10% HNO3) was used as the source of Cu2+ ions. Theflasks were stirred magnetically at 100 rpm for 1 h (sufficientto attain equilibrium). The concentration of the Cu2+ ions inthe resulting solutions was determined with a graphite furnaceatomic absorption spectrophotometer (AAS AA800, Perkin-Elmer, Bodenseewerk, Germany). The Cu2+-chelation step andother chemical modifications mentioned before (i.e., IDAattachment) are depicted in Figure 1. All instrumental conditionswere optimized for maximum sensitivity as described by themanufacturer. For each sample, the mean of 10 AAS measure-ments was recorded. The amount of adsorbed Cu2+ ions wascalculated using mass balance.

Cu2+ leakage from the IDA-modified beads was investigatedwith media pH (4.0-8.0) and also in a medium containing 1.0M NaCl. The bead suspensions were stirred for 24 h at roomtemperature. Cu2+-ion concentration was then determined in thesupernatants using an atomic absorption spectrophotometer. Itshould be also noted that immobilized metal-containing beadswere stored at 4°C in the 10 mM tris-HCl buffer (pH 7.4) with0.02% sodium azide to prevent microbial contamination.

2.5. Characterization of Monosize Beads.The amount ofattached IDA was determined using an elemental analysisinstrument (Leco, CHNS-932, U.S.A.). The amount of IDAattachment on the monosize beads was calculated by consideringthe nitrogen stoichiometry.

To confirm the effectiveness of IDA attachment on poly-(GMA) beads, the Fourier transform infrared (FTIR) spectra

Ind. Eng. Chem. Res., Vol. 46, No. 23, 20077803

was obtained through a Shimadzu FTIR 8000 series spectrom-eter in normal transmission mode using a KBr detector. Thesample beads were blended with KBr and pressed into discsfor FTIR scans. The spectra were derived from the average of64 scans with the 2 cm-1 resolution. All spectra were baselinecorrected and normalized to a thickness of 1µm. The polymerbeads were degassed overnight in a vacuum oven maintainedat 60°C before FTIR measurements.

Poly(GMA) beads were gold coated (∼100 Å thickness)under a high vacuum, 0.1 Torr, high voltage, 1.2 kV, and 50mA. Coated beads were examined using scanning electronmicroscopy of JEOL, JEM 1200 EX (Tokyo, Japan), tocharacterize the morphology and size of beads (see Figure 2).

The content of epoxy group in the poly(GMA) beads wasdetermined by the perchloric acid titration method. Poly(GMA)

beads were dispersed in 0.1 mol/L of tetraethylammoniumbromide in acetic acid solution and titrated with 0.1 mol/L ofperchloric acid solution until the crystal violet indicator changedto be blue-green.

2.6. IgG Adsorption from Aqueous Solutions.The effectsof IgG concentration, pH, and temperature on the adsorptioncapacity of Cu2+-chelated poly(GMA)/IDA beads were studied.The adsorption experiments were carried out batchwise in themedia at different pH values. The pH of the adsorption mediumwas varied between 4.0 and 8.0 using different buffer systems(0.1 M CH3COONa-CH3COOH for pH 4.0-6.0, 0.1 MK2HPO4-KH2PO4 for pH 7.0, and 0.1 M tris/HCl for pH 8.0).IgG concentration was varied between 0.5 and 3.0 mg/mL. Ina typical adsorption experiment, IgG was dissolved in 100 mLof buffer solution, and 250 mg of monosize beads were added.Then the adsorption experiments were performed for 2 h at 25°C at a stirring rate of 100 rpm. At the end of this equilibriumperiod, IgG adsorption was determined by measuring the initialand final concentrations of IgG within the adsorption mediumusing Coomassie Brilliant Blue as described by Bradford. Theprotein adsorption capacity was calculated by mass balance.

2.7. Elution and Repeated Use.Regeneration and reuse ofadsorbents are important aspects of adsorption studies. Theelution of IgG was carried out using 1 M NaCl at roomtemperature. IgG adsorbed beads (250 mg) were placed in theelution medium and stirred for 1 h, at 25°C, at a stirring rateof 100 rpm. The final IgG concentration within the elutionmedium was determined by using Coomassie Brilliant Blue asdescribed by Bradford. The elution ratio was calculated fromthe amount of IgG adsorbed on the monosize beads and theamount of IgG eluted into the medium.

In order to test the reusability of the metal-chelated beads,the IgG adsorption-elution procedure was repeated 10 times

Figure 1. Schematic diagram for the preparation of poly(GMA) metal-chelated beads.

Figure 2. SEM photograph of the monosize poly(GMA) beads.

7804 Ind. Eng. Chem. Res., Vol. 46, No. 23, 2007

by using the same affinity beads. In order to regenerate andsterilize, after elution, the beads were washed with 50 mMNaOH solution.

2.8. IgG Depletion from Human Serum.Affinity depletionwas carried out on a batch system. The blood is collected fromthoroughly controlled voluntary blood donors. Each unit wasseparately controlled and found to be negative for hepatit Bspecific antigen and HIV I, II, and hepatitis C antibodies. Nopreservatives are added to the samples. Blood samples werecentrifuged at 500 g for 3 min at room temperature to separatethe serum. The serum samples were filtered using 0.45µmcellulose acetate microspin filters (Alltech, Deerfield IL). Theoriginal serum of the healty donor contained 11.2 (mg of IgG)/mL as determined by nephelometric assay. Total protein contentsof crude and depleted serum samples were determined usingthe DC protein assay (Bio-Rad) according to the manufacturersinstructions with IgG as the standard (Pierce, Rockford, IL).The total protein concentration in the crude serum was 59.7mg/mL. In order to deplete human serum albumin (HSA), thefreshly separated human serum (100 mL) was loaded onto aanti-HSA antibody-sepharose column (10 cm× 1 cm insidediameter) equipped with a water jacket for temperature control.Equilibration of the anti-HSA antibody-sepharose column(Sigma) was performed by passing four column volumes ofsodium acetate buffer (pH) 5.2) before injection of the serum.When serum passed through the column, the HSA moleculesadsorbed on the anti-HSA antibody-sepharose adsorbent. Thealbumin-free serum that passed from the column consistedmainly of IgG and other serum proteins. After that, the serumwas ready for metal-chelate-affinity depletion of IgG. HSAconcentration was determined using Ciba Corning albuminreagent (catalog ref no.) 229241) based on bromocresol green(BCG) dye method. The concentration of HSA in crude serumwas determined to be 36.0 mg/mL. The concentration ofremaining HSA in the serum sample was very low. Thepercentage of albumin depletion was>99.6%. Then, 25 mL ofthe albumin-free serum was incubated with 250 mg of beadspre-equilibrated with acetate buffer (pH 5.0) for 2 h. Theseexperiments were conducted at 20°C. The amount of IgGadsorbed by metal-chelated beads was determined by measuringthe initial and final concentrations of IgG in the serum. Analysisof IgG was performed by a nephelometer assay (Beckman Array360, U.S.A.). Human serum was diluted with phosphate bufferedsaline (PBS, 1.9 mM NaH2PO4, 8.1 mM Na2HPO4, 154 mMNaCl, pH) 7.3). In order to test the binding performance, two-dimensional gel electrophoresis (2DE) was carried out asdescribed in detail previously.31

All sodium dodecylsulfate-polyacrylamide gel electrophore-sis (SDS/PAGE) analyses of the serum samples were performedon 10% separating minigels (9 cm× 7.5 cm) for 120 min at200 V. Stacking gels (6%) were stained with 0.25% (w/v)Coomassie Brillant R 250 in an acetic acid-methanol-watermixture (1:5:5, v/v/v) and destained in an ethanol-acetic acid-water mixture (1:4:6, v/v/v).

3. Results and Discussion

3.1. Characteristics of Monosize Poly(GMA) Beads.FTIRspectrums were undertaken to determine the structure of thepoly(GMA) and the IDA-attached poly(GMA) beads (Figure3). The FTIR spectrum of IDA-attached poly(GMA) beadsshowed characteristic peaks that appear at 2950 cm-1 (CH3

stretching vibration), 2132 cm-1 (C-N stretching vibration),and 1731 cm-1 (carbonyl stretching vibration) (Figure 3B). TheN-H peak that appears at 3626 cm-1 is associated with the

IDA. These data confirmed that the monosize beads weremodified with functional groups IDA.

Metal-chelating ligand IDA is covalently attached on poly-(GMA)beads, via the reaction between the epoxide groups ofthe GMA and the primer amine groups of the IDA. The highestIDA surface density obtained was 673 (µmol of IDA)/(g ofpolymer). The studies of IDA leakage from the poly(GMA)beads showed that there was no IDA leakage in any mediumused throughout this study, even for a long storage period oftime (>40 weeks).

Figure 3. FTIR spectrums: (A) poly(GMA) beads and (B) IDA-attachedpoly(GMA) beads.

Figure 4. Effect of IgG concentration on IgG adsorption: Cu2+ content) 628 µmol/g; pH ) 6.0.

Table 1. Some Properties of the Monosize Poly(GMA) Beads

particle diameter 1.6( 0.01µmpolydispersity index 1.006theoretical epoxy group content 3.8 mmol/gexperimental epoxy group content 3.0 mmol/gswelling ratio 45%wet density 1.09 g/mLIDA attachment 673µmol/gCu2+ content 628µmol/g

Table 2. Equilibrium Adsorption Constants and Free Energies

Langmuir model Freundlich Model

T (K) Qmax (mg/g) b (mL/g) KF 1/n ∆G (kJ/mol)

277 156.2 6.4 130.9 0.162 -90.8298 172.4 29.0 166.3 0.067 -94.5310 196.0 85.0 198.1 0.140 -96.5318 212.8 52.2 208.0 0.127 -97.9


The amount of Cu2+ present in the monosize beads was 628µmol/(g of poly(GMA) beads) (as determined by atomicabsorption spectroscopy). Note that the binding ratio of Cu2+

ions to conjugated IDA molecules was∼1 (see Table 1 for data).3.2. Depletion of IgG from Aqueous Solutions. 3.2.1.

Adsorption Isotherms. Figure 4 shows the effect of IgGconcentration on adsorption. As presented in this figure, theamount of IgG adsorption increased with increasing IgGconcentration up to 1.0 mg/mL. It reached almost saturationwhen the protein concentration was 1.5 mg/mL. The steep slopeof the initial part of the adsorption isotherm represents a highaffinity between IgG and chelated Cu2+ ions. A negligibleamount of IgG molecules adsorbed on the poly(GMA) beads,which was∼0.45 mg/g. The imidazole ring (side chains ofhistidine residue in protein structure) was known to have amixed-mode interaction mechanism by hydrophobic interaction,Van der Waals forces, electrostatic interactions, and hydrogenbonding. Cu2+ chelation significantly increased the IgG adsorp-tion capacity of the beads up to 171.2 mg/g (for 25°C).Transition metal ions have a high affinity for the peptide

sequences His-Gly, His, His-Tyr-NH2, and His-Trp.32 Thesignificant IgG adsorption onto metal-chelated beads could bedue to its greater number of histidine residues, which interactedwith the chelated Cu2+ ions. One surface histidine is reportedas sufficient for the adsorption on a Cu2+-IMAC adsorbent,and proteins varying by only one histidine can be separated.

Two important physicochemical aspects for evaluation of theadsorption process as a unit operation are the kinetics and theequilibria of adsorption. Modeling of the equilibrium data hasbeen done using the Langmuir and Freundlich isotherms.33 TheLangmuir and Freundlich isotherms are represented as followsby eq 1 and eq 2, respectively.

Here,b is the Langmuir isotherm constant,Ce is the equilibriumconcentration,qmax is the maximum adsorption capacity,KF isthe Freundlich constant, andn is the Freundlich exponent. 1/nis a measure of the surface heterogeneity ranging between 0and 1, becoming more heterogeneous as its value gets closer tozero. The ratio ofqe gives the theoretical monolayer saturationcapacity of monosize beads.

Some model parameters were determined by nonlinearregression with commercially available software and are shown

Figure 5. Pseudo-first-order kinetics of the experimental data for themonosize beads.

Figure 6. Pseudo-second-order kinetics of the experimental data for themonosize beads.

Table 3. First- and Second-Order Kinetic Constants for MonosizeBeads

first-order kinetics second-order kineticsIgGconc

(mg/mL)qexp

(mg/g)k1 × 10-3

(min-1)qe

(mg/g) R2k2 × 10-5

(g/mg‚min)qe

(mg/g) R2

0.5 50.0 58.4 77.9 0.951 118.9 57.1 0.9961.0 142.2 64.0 267.8 0.943 21.5 178.5 0.9721.5 160.6 44.9 231.6 0.871 11.0 222.2 0.9832.0 168.5 47.4 258.5 0.878 9.8 238.1 0.9793.0 171.2 56.4 238.5 0.946 18.2 217.3 0.977

Figure 7. SDS/PAGE of serum fractions. The serum fractions were assayedby SDS/PAGE using 10% separating gel (9× 7.5 cm), and 6% stackinggels were stained with 0.25% (w/v) Coomassie Brillant R 250 in aceticacid-methanol-water (1:5:5, v/v/v) and destained in ethanol-acetic acid-water (1:4:6, v/v/v). Lane 1, biomarker; Lane 2, 1:10 diluted serum; Lane3, 1:10 depleted serum. Equal amounts of samples were applied to eachline.

Table 4. IgG Depletion from Human Serum; IgG Concentrationbefore Dilution ) 11.2 mg/mL

dilution agent adsorption capacity (mg/g)

serum (undiluted) 97.5( 2.281/2 diluted seruma 69.5( 2.771/5 diluted seruma 55.8( 2.481/10 diluted seruma 38.3( 2.75

a Human serum was diluted with phosphate buffer (pH 6.5).

1/qe ) (1/qmax) + [1/(qmaxb)](1/Ce) (1)

ln qe ) 1/n(ln Ce) + ln KF (2)


in Table 2. Comparison of all theoretical approaches used inthis study shows that the Langmuir equation fits the experimentaldata best.

The thermodynamic parameters,∆G, ∆H, and ∆S, for theadsorption process are calculated and changes for the processcan be estimated using the following equation,

where Kd, the distribution coefficient of the adsorbate, isequal to (qe/Ce). The plot of ln Kd versus 1/T yields straightlines with the slope and the intercept giving values of∆Hand ∆S. These values could be used to compute∆G fromthe Gibbs relation,∆G ) ∆H - T∆S, at constant temperature.In deriving the values of the thermodynamic parameters,it is assumed that the enthalpy does not change with temp-erature.

The equilibrium adsorption of IgG onto the Cu2+-chelatedbeads significantly increased with increasing temperature. Apossible explanation for this behavior is as follows: chemicalinteraction between the chelated Cu2+ ions and the IgGmolecules increased with increasing temperature. The neg-ative change in free energy (∆Go < 0) indicated that theadsorption of IgG on the Cu2+-chelated beads was a thermo-dynamically favorable process. The∆S value for theadsorption of IgG to Cu2+-chelated monosize beads wascalculated as 172.1 J/mol‚K. The positive value for∆Sindicates an increase in the total disorder of the system duringadsorption. The calculated∆H value of the system for theinteraction of IgG with chelated Cu2+ ions was -43.2kJ/mol.

3.2.2. Adsorption Dynamics.In order to quantify the extentof uptake in adsorption kinetics, the kinetic models (pseudo-first- and second-order equations) can be used in this case,assuming that the measured concentrations are equal to adsor-bent surface concentrations.34 The first-order rate equation ofLagergren is one of the most widely used for the adsorption ofsolute from a liquid solution. It may be represented as follows,

where qe is the experimental amount of IgG adsorbed atequilibrium (mg/g);qt is the amount of IgG adsorbed at timet(mg/g); k1 is the equilibrium rate constant of first-order

adsorption (1/min); andqe is the adsorption capacity calculatedby the pseudo-first-order model (mg/g).

The rate constant for the second-order adsorption could beobtained from the following equation,

wherek2 is the equilibrium rate constant of pseudo-second-orderadsorption (g/mg‚min) and qe is the adsorption capacitycalculated by the pseudo-second-order kinetic model (mg/g).

Figure 5, Figure 6, and Table 3 show the results for boththe first-order and second-order kinetic models. On com-parison, it was found that the second-order kinetics based ont/qe versust (figure not shown here) yielded the best results.Therefore, chemisorption might be the rate-limiting stepthat controls the adsorption process. The rate-controlling mech-anism may vary during the course of the adsorption process;three possible mechanisms may be occurring.35 There is anexternal surface mass transfer or film diffusion process thatcontrols the early stages of the adsorption process. This maybe followed by a reaction or constant-rate stage and, finally,by a diffusion stage where the adsorption process slows downconsiderably.36

3.2.3. Regeneration of the Beads.In the last step of theaffinity separation, the main concern was to desorb the ad-sorbed protein in the shortest time and at the highest amountpossible. It was, thus, necessary to evaluate the regen-eration efficiency of the affinity adsorbents after each cycle.Elution of IgG from monosize beads was also carried outin a batch system, using 1 M NaCl. More than 95% of theadsorbed IgG molecules was eluted easily from the chelatingbeads in 30 min. With the elution data given, we concludedthat NaCl is a suitable elution agent for the Cu2+-chelatedbeads.

In order to show the reusability of the metal-chelated beads,the adsorption-elution cycle was repeated 20 times using thesame beads from aqueous IgG solution. Significant re-duction in adsorption capacity has not been observed with thereuse of the metal-chelated beads up to 20 times (decreasingratio ) 5%). It should also be noted that no obvious changesof the morphology of the beads were found in the recyclingprocess when the beads were examined visually. The resultfurther confirmed that the Cu2+-chelated beads have a goodstability.

Table 5. Comparison of the Adsorption Capacities for IgG of Various Adsorbents

adsorbent ligand qmax (mg/g) referencePHEMA L-histidine 44.8 37PHEMA methacryloylamidohistidine 73.8 38Eupergit, Affigel Protein A 20.1 39PHEMA Protein A 24.0 40polymethylmethacrylate Cu2+ 54.3 41poly(caprolactam) fibers Protein A 28.3 42poly(ethylene) membrane phenylalanine 50.0 43Sepharose 4B L-histidine 0.23 44poly(ethylene vinyl alcohol) L-histidine 77.7 45polysulfone Protein A 8.8 46Sartobind Protein A 0.51 47polymethylmethacrylate Protein A/G 6.6 48nylon membrane Protein A 13.2 49Sepharose CL 6B 3-aminophenol 52.0 50

4-amino-1-naphtholSepharose 6B biomimetic ligand 7.0 51Sepharose 4B biomimetic ligand 25.0 52PHEMA beads Cu2+, Ni2+, Zn2+, Co2+ 79.6 53PHEMA beads Concanavalin A 69.4 54PHEMA monolith histidine 96.5 55poly(hydroxypropyl methacrylate) Reactive Green HE 4BD 71.0 56poly(GMA) IDA/Cu2+ 171.2 in this study

ln Kd ) ∆S/R - ∆H/RT (3)

log(qe- qt) ) log(qe) - (k1t)/2.303 (4)

(t/qt) ) (1/k2qe2) + (1/qe)t (5)


The effects of metal-ion leakage and the resulting toxicityduring adsorption and elution are also significant issues to beevaluated. The reasons for metal-ion leakage at different stagesare not the same. At the adsorption stage, the unstablyimmobilized metal ions may be tightly captured by the proteinsand released to the solution. On the other hand, they are possiblydisplaced by salt ions in the elution buffer at the elution step.A higher salt concentration at the elution step is more effectivefor gaining high recoveries, but it may cause more severe metal-ion leakage. Reduction in the salt concentration could dim-inish the metal-ion leakage, but the adsorbed protein may notbe able to be completely eluted out of the adsorbent. Conse-quently, an appropriate salt concentration in the elution-buffer should be carefully selected. The use of NaCl for elutionoffers an economical manner for purification of proteinscomparing with imidazole. During the elution of IgG, no leakageof Cu2+ ions was observed from the Cu2+-chelated beads.

3.3. IgG Depletion from Human Serum. Depletionof additional abundant proteins can be beneficial in the an-alysis of serum proteins, and therefore, we attempted to -deplete the IgG class immunoglobulins from human serum. Inthe first step of this study, the depletion of HSA was achievedby using the anti-HSA antibody-sepharose 4B column. Thedepletion efficiency for HSA was 99.6% in the serum -sample. Then, in the second step, the depletion of IgG fromhuman serum was performed with metal-chelated beads -in the batch system. The depletion efficiencies for IgG were>90% for all studied concentrations. Results, shown in Table4, indicate that a large portion of the IgG was bound by themetal-chelated beads. To test the efficiency of IgG depletionfrom human serum, proteins in the serum and the eluted portionwere analyzed by two-dimensional gel electrophoresis. Proteinsthat were eluted from the metal-chelated beads include IgG anda small number of nonalbumin proteins. A negligible amountof relatively abundant proteins such as apo-lipoprotein A1, sero-transferrin, haptoglobulin, andR1-antitrypsin was bound by themetal-chelated beads. We reached up to 98.2% IgG depletionamount, and it may be concluded that metal-chelated beads aresufficient in terms of efficiency of IgG depletion.

In order to confirm that depletion occurred, the diluted serumand IgG-depleted serum were analyzed by SDS/PAGE, and theresults are shown in Figure 7. As expected, the depleted serumshowed no protein bands, confirming the depletion of high-abundance proteins, which were recovered in the depletedfraction; see Figure 7.

3.4. Comparison of Adsorption Capacity with OtherBioaffinity Adsorbents. A comparison of the maximumadsorption capacity,qmax, of the Cu2+-chelated monosize poly-(GMA) beads with those of some other bioaffinity adsorbentsreported in the literature is given in Table 5. The adsorptioncapacity of Cu2+-chelated monosize poly(GMA) beads wasrelatively high when compared with those of other adsorbents.Differences of IgG adsorption are due to the properties of eachadsorbent such as structure, functional groups, ligand loading,and surface area.

4. Conclusion

These results are consistent with published studies.9,57Bjorhallet al. used five different commercially available depletioncolumns including Aurum Serum Protein Minikit (Bio-Rad,Hercules, CA), ProteoExtract Albumin/IgG Removal kit(Merck, Darmstadt, Germany), Multiple Affinity RemovalColumn (Agilent Technologies, San Diego, CA), POROSAffinity Depletion Cartridges (Applied Biosystems, Framing-

ham, MA), and Albumin-IgG Removal Kit (AmershamBiosciences, Uppsala, Sweden).7 It should be noted that AurumSerum Protein Minikit (Bio-Rad, U.S.A.) and ProteoExtractAlbumin/IgG Removal kit contained Protein A as ligand,while Multiple Affinity Removal Column and Albumin-IgGRemoval Kit contained polyclonal antibodies as ligand. POROSAffinity Depletion Cartridges contained protein G. The de-pletion efficiencies were>90%, but because of the high dilutionfactor after the depletion procedure as compared with the crudeserum, the concentrations of remaining IgG in depleted serumsamples were below the detection limits for almost all samples.However, this was stated as a minimum depletion of 94% ofIgG in depleted serum samples by any affinity column. Jainand Gupta described a simple one-step method for IgG purifica-tion from goat serum using IMAC. They found a recovery ratioof 97.5% with an 8-fold purification.26 Sitnikov et al. appliedthe Multiple Affinity Removal Column (Agilent Technologies,San Diego, CA) for the depletion of blood plasma proteins undervolatile conditions. The percentage of IgG depletion was>99%.58 Babac et al. used concanavalin A attached poly-(AAM -AGE) monolithic cryogel for IgG depletion, and theyachieved∼85% of IgG depletion.59 Plavina et al. reported thedevelopment of a robust and relatively high-throughput methodconsisting of depletion of albumin and IgG with multi-lectinaffinity chromatography.60 The total protein recovery for deple-tion of abundant proteins was 96%. Karatas¸ et al. tested Cu2+-chelated magnetic beads for IgG depletion from human serum,and they achieved high depletion efficiency (99.4%).61 Theremoval of IgG was shown to be>99% using commerciallyavailable Applied Biosystems affinity depletion cartridgescarrying Protein G.62 We reached up to 98.2% IgG depletionamount. In the light of the above discussion, we believe thatthe metal-chelated monosize poly(GMA) beads offer a promis-ing strategy with good depletion specificity and efficiency ofIgG in combination with the anti-HSA antibody-Sepharosecolumn.

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ReceiVed for reView September 5, 2006ReVised manuscript receiVed July 26, 2007

AcceptedAugust 14, 2007

IE061164C


immobilized metal affinity adsorption for antibody depletion from human serum with monosize beads

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