Immobilized Metal Affinity Adsorption for Antibody Depletion from

Oct 10, 2007 - Immobilized Metal Affinity Adsorption for Antibody Depletion from Human. Serum with Monosize Beads. Evrim Banu Altıntas¸,† Nalan Tu...
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Immobilized Metal Affinity Adsorption for Antibody Depletion from Human Serum with Monosize Beads Evrim Banu Altıntas¸ ,† Nalan Tu1 zmen,‡ Lokman Uzun,† and Adil Denizli*,† Department of Chemistry, Biochemistry DiVision, Hacettepe UniVersity, Ankara, Turkey, and Department of Chemistry, Biochemistry DiVision, Dokuzeylu¨l UniVersity, I˙ zmir, Turkey

Iminodiacetic acid (IDA)-functionalized adsorbents have attracted increasing interest in recent years for immobilized metal-affinity chromatography (IMAC). In this study, IDA was covalently attached to nonporous monosize poly(glycidyl methacrylate) [poly(GMA)] beads (1.6 µm in diameter). Cu2+ ions were chelated via IDA 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 (628 µmol/g) were used in the IgG adsorption-elution studies. Studies to determine the effects of IgG concentration, pH, and temperature on the adsorption efficiency of Cu2+-chelated beads were performed in a batch system. Nonspecific binding of IgG to monosize beads in the absence of Cu2+ ions was very low (0.45 mg/g). The IgG adsorption to 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 ∆S and ∆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 elution medium containing 1.0 M NaCl in 1 h. The kinetics of the interactions suggest that the interactions could be best 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 capacity of 156.2-212.8 mg/g. Consecutive adsorption-elution experiments showed that the Cu2+-chelated beads can be reused almost without any loss in the IgG adsorption capacity. To test the efficiency of IgG depletion from human serum, proteins in the serum and eluted portion were analyzed by two-dimensional gel electrophoresis. The depletion efficiency for IgG was above 98.2%. Eluted proteins include mainly IgG and a negligible amount of non-albumin proteins such as apo-lipoprotein A1, sero-transferrin, haptoglobulin, and R1-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 is thought to contain tens of thousands of proteins along with their cleaved or modified forms. These proteins are a reflection of ongoing physiological or pathological events.1 Serum may often serve as an indicator of disease and is a rich source for biomarker discovery. However, the large dynamic range of proteins in serum makes the analysis very challenging because highly abundant proteins including albumin, immunoglobulins (IgG and IgA), antitrypsin, haptoglobin, and transferrin tend to mask those of lower abundance.2 Human serum albumin (HSA) and IgG represent over 80% of all proteins present in plasma, and their high abundance masks the detection and determination of the low-abundance proteins that are potential biomarkers for various diseases, e.g., cancer, and are, therefore, of great biological importance in proteome studies.3-8 There are several removal strategies to deplete the higherabundant proteins from serum, including ultracentrifugal filtration, dye affinity, and immunoaffinity chromatography.9 The depletion of IgG is commonly achieved by Protein A/G affinity adsorbents, which bind to the Fc region of the IgG, but specific antibodies can also be used.10 The high specificity of the bioligands (i.e., Protein A/G) provides excellent selectivity. However, in spite of their high selectivities, Protein A/G or * Corresponding author. E-mail: [email protected]. Tel.: +90 312 297 7963. Fax: +90 312 297 6084. † Hacettepe University. ‡ Dokuzeylu¨l University.

antibody-carrying adsorbents also have some drawbacks that are worth considering: (i) the cost of the ligands tend to be very high; (ii) these bio-ligands are difficult to immobilize in the proper orientation; and (iii) ligand may leak from the stationary phase and such contamination cannot, of course, be tolerated in clinical applications. In addition, the depletion of IgG in human serum is employed successfully for the treatment of immune disorders including systemic lupus erythematosus, rheumatoid arthritis, myasthenia gravis, alloimmunization and cancer.11-14 Recently, immobilized metal affinity chromatography (IMAC) has shown great potential in the purification of proteins and peptides15 and several types of IMAC columns have been applied to proteomics.16 One is for the enrichment of phosphorylated peptides with Ga3+ or Fe3+ immobilized.17 The other is for the selection of peptides with Cu(II) loaded on to the columns.18 The esterification-immobilized metal affinity selection of peptides with Cu(II) loaded on to the columns.18 The esterification-immobilized metal-affinity chromatography (IMAC) method has been successfully used to characterize >200 phosphopeptides in the yeast proteome and human carcinoma cell line.19,20 IMAC-based enrichment of phosphorylated peptides also has been demonstrated to work well on simplified mixtures. For instance, IMAC enrichment of phosphorylated peptides without prior chemical modification was used to characterize nearly 300 phosphopeptides after prefractionation with anion-exchange chromatography21 in cultured plant cells. The first study using IMAC to analyze phosphorylation sites present in mammalian tissue has been published recently.22 In

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this work, phosphorylated proteins and, subsequently, phosphorylated peptides were enriched with IMAC. Recently, a method has been reported for large-scale rapid analysis of phosphoproteins in tissues or cells by combining IMAC with phage display cDNA library screening.23 The selection of histidine-containing peptides is an important aspect of proteomics in sample simplification and database search. Immobilized Cu2+-affinity chromatography has been used in proteomics to simplify sample mixtures by selecting histidine-containing peptides from proteolytic digests.24 IMAC combined with mass spectrometry has recently been employed for isolation of naturally occurring metal-binding proteins from total proteins in human liver cells.25 Cu2+-charged alginate beads have been directly used as an IMAC medium for purification of IgG from goat serum.26 So far, to our knowledge, only one study has been published using IMAC to deplete immunoglobulin G from human serum for analyzing the proteome of human serum.27 We have used iminodiacetic acid (IDA) modified poly(glycidyl methacrylate) [poly(GMA)] monosize beads with chelated Cu2+ ions as a model adsorbent capable of selective binding of IgG from human plasma. Poly(GMA) was used as the basic matrix because of its known good mechanical strength, stability at neutral pH values even in wet conditions, and high reactivity of the epoxy groups for surface immobilization.28 Epoxy-derived adsorbents seem to be almost ideal systems to develop 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) were purchased from Aldrich (Munich, Germany) and used without further purification. Glycidyl methacrylate (GMA, Fluka A.G., Buchs, Switzerland) was purified by vacuum distillation and stored in a refrigerator until use. Azobisisobutyronitrile (AIBN) and poly(vinyl pyrrolidone) (MW ) 30.000, BDH Chemicals Ltd., Poole, England) were selected as the initiator and the steric stabilizer, respectively. AIBN was recrystallized from methanol. Ethanol (Merck, Germany) was used as the diluent without further purification. All other chemicals were of reagent grade and were purchased from Merck AG (Darmstadt, Germany). Laboratory glassware was kept overnight in a 5% nitric acid solution. Before use, the glassware was rinsed with deionized water and dried in a dust-free environment. All water used in the experiments was purified using a Barnstead (Dubuque, IA) ROpure LP reverse-osmosis unit with a high-flow cellulose acetate membrane (Barnstead D2731) followed by a Barnstead D3804 NANOpure organic/colloid-removal and ion-exchange packed-bed system. 2.2. Preparation of Poly(GMA) Beads. Poly(GMA) beads were prepared as described elsewhere.30 Dispersion polymerization was performed in a sealed polymerization reactor (volume ) 500 mL) equipped with a temperature-control system. A typical procedure applied for the dispersion polymerization of GMA is given below. The monomer phase comprised 40 mL of GMA. AIBN (250 mg) was dissolved into the monomer phase. The resulting medium was sonicated for ∼5 min at 200 W within an ultrasonic water bath (Bransonic 2200, England) for the complete dissolution of AIBN in the polymerization medium. Poly(vinyl pyrrolidone) (4.0 g) was dissolved in a homogeneous solution of ethanol (100 mL) and water (100 mL) placed in a polymerization reactor. The reactor content was stirred at 500 rpm during the monomer addition, completed within ∼5 min, and the heating was started. Then, the mixture was degassed by purging with nitrogen for ∼20 min. Then, the

sealed reactor was placed in a shaking water bath at room temperature. The initial-polymerization time was defined when the reactor temperature was raised to 65 °C. The polymerization was carried out at 65 °C for 4 h with continuous strirring. After completion of the polymerization period, the reactor content was cooled down to room temperature and centrifuged at 5 000 rpm for 10 min for the removal of dispersion medium. This polymerization reaction led to the formation of white beads. Poly(GMA) beads were redispersed within 10 mL of ethanol and centrifuged again under similar conditions. The ethanol washing was repeated three times for complete removal of unconverted 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 reaction mixture (50 mL of 0.8 g IDA + 2.0 M NaCO3, pH 11) and poly(GMA) beads were incubated at 70 °C in a heating mantle under mild stirring for 12 h. After the coupling reaction, the beads were washed with 5% acetic acid and deionized water until the washing solutions were neutral. Afterward, the remaining epoxy groups were blocked with 2 M ethylene diamine at pH 10 for 16 h under gentle stirring. In order to remove the nonspecifically attached IDA molecules, an extensive cleaning procedure was applied, which was as follows: The beads were first washed with deionized water. The monosize beads were dispersed in methanol, and the dispersion was sonicated for 2 h in an ultrasonic bath. At the last stage, the beads were washed again with deionized water. IDA-attached poly(GMA) beads were stored at 4 °C with 0.02% sodium azide to prohibit microbial contamination. 2.4. Chelation of Cu2+ Ions. Chelates of Cu2+ ions with IDA-modified poly(GMA) beads were prepared as follows: 1.0 g of the IDA-modified beads were mixed with 50 mL of aqueous solution containing 30 ppm Cu2+ ions, at constant pH of 4.1 (adjusted with HCl and NaOH), which was the optimum pH for Cu2+-chelate formation, and at room temperature. A 1000 ppm atomic absorption standard solution (Cu(NO3)2 salt containing 10% HNO3) was used as the source of Cu2+ ions. The flasks were stirred magnetically at 100 rpm for 1 h (sufficient to attain equilibrium). The concentration of the Cu2+ ions in the resulting solutions was determined with a graphite furnace atomic absorption spectrophotometer (AAS AA800, PerkinElmer, Bodenseewerk, Germany). The Cu2+-chelation step and other chemical modifications mentioned before (i.e., IDA attachment) are depicted in Figure 1. All instrumental conditions were optimized for maximum sensitivity as described by the manufacturer. For each sample, the mean of 10 AAS measurements was recorded. The amount of adsorbed Cu2+ ions was calculated using mass balance. Cu2+ leakage from the IDA-modified beads was investigated with media pH (4.0-8.0) and also in a medium containing 1.0 M NaCl. The bead suspensions were stirred for 24 h at room temperature. Cu2+-ion concentration was then determined in the supernatants using an atomic absorption spectrophotometer. It should be also noted that immobilized metal-containing beads were stored at 4 °C in the 10 mM tris-HCl buffer (pH 7.4) with 0.02% sodium azide to prevent microbial contamination. 2.5. Characterization of Monosize Beads. The amount of attached IDA was determined using an elemental analysis instrument (Leco, CHNS-932, U.S.A.). The amount of IDA attachment on the monosize beads was calculated by considering the nitrogen stoichiometry. To confirm the effectiveness of IDA attachment on poly(GMA) beads, the Fourier transform infrared (FTIR) spectra

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

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

was obtained through a Shimadzu FTIR 8000 series spectrometer in normal transmission mode using a KBr detector. The sample beads were blended with KBr and pressed into discs for FTIR scans. The spectra were derived from the average of 64 scans with the 2 cm-1 resolution. All spectra were baseline corrected and normalized to a thickness of 1 µm. The polymer beads were degassed overnight in a vacuum oven maintained at 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 50 mA. Coated beads were examined using scanning electron microscopy of JEOL, JEM 1200 EX (Tokyo, Japan), to characterize the morphology and size of beads (see Figure 2). The content of epoxy group in the poly(GMA) beads was determined by the perchloric acid titration method. Poly(GMA)

beads were dispersed in 0.1 mol/L of tetraethylammonium bromide in acetic acid solution and titrated with 0.1 mol/L of perchloric acid solution until the crystal violet indicator changed to be blue-green. 2.6. IgG Adsorption from Aqueous Solutions. The effects of IgG concentration, pH, and temperature on the adsorption capacity of Cu2+-chelated poly(GMA)/IDA beads were studied. The adsorption experiments were carried out batchwise in the media at different pH values. The pH of the adsorption medium was varied between 4.0 and 8.0 using different buffer systems (0.1 M CH3COONa-CH3COOH for pH 4.0-6.0, 0.1 M K2HPO4-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. In a typical adsorption experiment, IgG was dissolved in 100 mL of 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 equilibrium period, IgG adsorption was determined by measuring the initial and final concentrations of IgG within the adsorption medium using Coomassie Brilliant Blue as described by Bradford. The protein adsorption capacity was calculated by mass balance. 2.7. Elution and Repeated Use. Regeneration and reuse of adsorbents are important aspects of adsorption studies. The elution of IgG was carried out using 1 M NaCl at room temperature. IgG adsorbed beads (250 mg) were placed in the elution medium and stirred for 1 h, at 25 °C, at a stirring rate of 100 rpm. The final IgG concentration within the elution medium was determined by using Coomassie Brilliant Blue as described by Bradford. The elution ratio was calculated from the amount of IgG adsorbed on the monosize beads and the amount 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

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by using the same affinity beads. In order to regenerate and sterilize, after elution, the beads were washed with 50 mM NaOH solution. 2.8. IgG Depletion from Human Serum. Affinity depletion was carried out on a batch system. The blood is collected from thoroughly controlled voluntary blood donors. Each unit was separately controlled and found to be negative for hepatit B specific antigen and HIV I, II, and hepatitis C antibodies. No preservatives are added to the samples. Blood samples were centrifuged at 500 g for 3 min at room temperature to separate the serum. The serum samples were filtered using 0.45 µm cellulose acetate microspin filters (Alltech, Deerfield IL). The original serum of the healty donor contained 11.2 (mg of IgG)/ mL as determined by nephelometric assay. Total protein contents of crude and depleted serum samples were determined using the DC protein assay (Bio-Rad) according to the manufacturers instructions with IgG as the standard (Pierce, Rockford, IL). The total protein concentration in the crude serum was 59.7 mg/mL. In order to deplete human serum albumin (HSA), the freshly separated human serum (100 mL) was loaded onto a anti-HSA antibody-sepharose column (10 cm × 1 cm inside diameter) equipped with a water jacket for temperature control. Equilibration of the anti-HSA antibody-sepharose column (Sigma) was performed by passing four column volumes of sodium acetate buffer (pH ) 5.2) before injection of the serum. When serum passed through the column, the HSA molecules adsorbed on the anti-HSA antibody-sepharose adsorbent. The albumin-free serum that passed from the column consisted mainly of IgG and other serum proteins. After that, the serum was ready for metal-chelate-affinity depletion of IgG. HSA concentration was determined using Ciba Corning albumin reagent (catalog ref no. ) 229241) based on bromocresol green (BCG) dye method. The concentration of HSA in crude serum was determined to be 36.0 mg/mL. The concentration of remaining HSA in the serum sample was very low. The percentage of albumin depletion was >99.6%. Then, 25 mL of the albumin-free serum was incubated with 250 mg of beads pre-equilibrated with acetate buffer (pH 5.0) for 2 h. These experiments were conducted at 20 °C. The amount of IgG adsorbed by metal-chelated beads was determined by measuring the initial and final concentrations of IgG in the serum. Analysis of IgG was performed by a nephelometer assay (Beckman Array 360, U.S.A.). Human serum was diluted with phosphate buffered saline (PBS, 1.9 mM NaH2PO4, 8.1 mM Na2HPO4, 154 mM NaCl, pH ) 7.3). In order to test the binding performance, twodimensional gel electrophoresis (2DE) was carried out as described in detail previously.31 All sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS/PAGE) analyses of the serum samples were performed on 10% separating minigels (9 cm × 7.5 cm) for 120 min at 200 V. Stacking gels (6%) were stained with 0.25% (w/v) Coomassie Brillant R 250 in an acetic acid-methanol-water mixture (1:5:5, v/v/v) and destained in an ethanol-acetic acidwater mixture (1:4:6, v/v/v). 3. Results and Discussion 3.1. Characteristics of Monosize Poly(GMA) Beads. FTIR spectrums were undertaken to determine the structure of the poly(GMA) and the IDA-attached poly(GMA) beads (Figure 3). The FTIR spectrum of IDA-attached poly(GMA) beads showed 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). The N-H peak that appears at 3626 cm-1 is associated with the

Figure 3. FTIR spectrums: (A) poly(GMA) beads and (B) IDA-attached poly(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 1.6 ( 0.01 µm 1.006 3.8 mmol/g 3.0 mmol/g 45% 1.09 g/mL 673 µmol/g 628 µmol/g

particle diameter polydispersity index theoretical epoxy group content experimental epoxy group content swelling ratio wet density IDA attachment Cu2+ content

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)

277 298 310 318

156.2 172.4 196.0 212.8

6.4 29.0 85.0 52.2

130.9 166.3 198.1 208.0

0.162 0.067 0.140 0.127

-90.8 -94.5 -96.5 -97.9

IDA. These data confirmed that the monosize beads were modified with functional groups IDA. Metal-chelating ligand IDA is covalently attached on poly(GMA)beads, via the reaction between the epoxide groups of the GMA and the primer amine groups of the IDA. The highest IDA surface density obtained was 673 (µmol of IDA)/(g of polymer). The studies of IDA leakage from the poly(GMA) beads showed that there was no IDA leakage in any medium used throughout this study, even for a long storage period of time (>40 weeks).

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

Figure 7. SDS/PAGE of serum fractions. The serum fractions were assayed by SDS/PAGE using 10% separating gel (9 × 7.5 cm), and 6% stacking gels were stained with 0.25% (w/v) Coomassie Brillant R 250 in acetic acid-methanol-water (1:5:5, v/v/v) and destained in ethanol-acetic acidwater (1:4:6, v/v/v). Lane 1, biomarker; Lane 2, 1:10 diluted serum; Lane 3, 1:10 depleted serum. Equal amounts of samples were applied to each line. Figure 6. Pseudo-second-order kinetics of the experimental data for the monosize beads.

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

Table 3. First- and Second-Order Kinetic Constants for Monosize Beads first-order kinetics IgG conc qexp k1 × 10-3 qe (mg/mL) (mg/g) (min-1) (mg/g) R2 0.5 1.0 1.5 2.0 3.0

50.0 142.2 160.6 168.5 171.2

58.4 64.0 44.9 47.4 56.4

77.9 267.8 231.6 258.5 238.5

0.951 0.943 0.871 0.878 0.946

second-order kinetics k2 × 10-5 qe (g/mg‚min) (mg/g) 118.9 21.5 11.0 9.8 18.2

57.1 178.5 222.2 238.1 217.3

R2 0.996 0.972 0.983 0.979 0.977

The amount of Cu2+ present in the monosize beads was 628 µmol/(g of poly(GMA) beads) (as determined by atomic absorption 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 IgG concentration on adsorption. As presented in this figure, the amount of IgG adsorption increased with increasing IgG concentration up to 1.0 mg/mL. It reached almost saturation when the protein concentration was 1.5 mg/mL. The steep slope of the initial part of the adsorption isotherm represents a high affinity between IgG and chelated Cu2+ ions. A negligible amount of IgG molecules adsorbed on the poly(GMA) beads, which was ∼0.45 mg/g. The imidazole ring (side chains of histidine residue in protein structure) was known to have a mixed-mode interaction mechanism by hydrophobic interaction, Van der Waals forces, electrostatic interactions, and hydrogen bonding. Cu2+ chelation significantly increased the IgG adsorption capacity of the beads up to 171.2 mg/g (for 25 °C). Transition metal ions have a high affinity for the peptide

a

dilution agent

adsorption capacity (mg/g)

serum (undiluted) 1/2 diluted seruma 1/5 diluted seruma 1/10 diluted seruma

97.5 ( 2.28 69.5 ( 2.77 55.8 ( 2.48 38.3 ( 2.75

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

sequences His-Gly, His, His-Tyr-NH2, and His-Trp.32 The significant IgG adsorption onto metal-chelated beads could be due to its greater number of histidine residues, which interacted with the chelated Cu2+ ions. One surface histidine is reported as 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 the adsorption process as a unit operation are the kinetics and the equilibria of adsorption. Modeling of the equilibrium data has been done using the Langmuir and Freundlich isotherms.33 The Langmuir and Freundlich isotherms are represented as follows by eq 1 and eq 2, respectively.

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

(1)

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

(2)

Here, b is the Langmuir isotherm constant, Ce is the equilibrium concentration, qmax is the maximum adsorption capacity, KF is the Freundlich constant, and n is the Freundlich exponent. 1/n is a measure of the surface heterogeneity ranging between 0 and 1, becoming more heterogeneous as its value gets closer to zero. The ratio of qe gives the theoretical monolayer saturation capacity of monosize beads. Some model parameters were determined by nonlinear regression with commercially available software and are shown

Ind. Eng. Chem. Res., Vol. 46, No. 23, 2007 7807 Table 5. Comparison of the Adsorption Capacities for IgG of Various Adsorbents adsorbent PHEMA PHEMA Eupergit, Affigel PHEMA polymethylmethacrylate poly(caprolactam) fibers poly(ethylene) membrane Sepharose 4B poly(ethylene vinyl alcohol) polysulfone Sartobind polymethylmethacrylate nylon membrane Sepharose CL 6B Sepharose 6B Sepharose 4B PHEMA beads PHEMA beads PHEMA monolith poly(hydroxypropyl methacrylate) poly(GMA)

ligand L-histidine methacryloylamidohistidine Protein A Protein A Cu2+ Protein A phenylalanine L-histidine L-histidine Protein A Protein A Protein A/G Protein A 3-aminophenol 4-amino-1-naphthol biomimetic ligand biomimetic ligand Cu2+, Ni2+, Zn2+, Co2+ Concanavalin A histidine Reactive Green HE 4BD IDA/Cu2+

qmax (mg/g) 44.8 73.8 20.1 24.0 54.3 28.3 50.0 0.23 77.7 8.8 0.51 6.6 13.2 52.0

reference 37 38 39 40 41 42 43 44 45 46 47 48 49 50

7.0 25.0 79.6 69.4 96.5 71.0 171.2

51 52 53 54 55 56 in this study

in Table 2. Comparison of all theoretical approaches used in this study shows that the Langmuir equation fits the experimental data best. The thermodynamic parameters, ∆G, ∆H, and ∆S, for the adsorption process are calculated and changes for the process can be estimated using the following equation,

adsorption (1/min); and qe is the adsorption capacity calculated by the pseudo-first-order model (mg/g). The rate constant for the second-order adsorption could be obtained from the following equation,

ln Kd ) ∆S/R - ∆H/RT

where k2 is the equilibrium rate constant of pseudo-second-order adsorption (g/mg‚min) and qe is the adsorption capacity calculated by the pseudo-second-order kinetic model (mg/g). Figure 5, Figure 6, and Table 3 show the results for both the first-order and second-order kinetic models. On comparison, it was found that the second-order kinetics based on t/qe versus t (figure not shown here) yielded the best results. Therefore, chemisorption might be the rate-limiting step that controls the adsorption process. The rate-controlling mechanism may vary during the course of the adsorption process; three possible mechanisms may be occurring.35 There is an external surface mass transfer or film diffusion process that controls the early stages of the adsorption process. This may be followed by a reaction or constant-rate stage and, finally, by a diffusion stage where the adsorption process slows down considerably.36 3.2.3. Regeneration of the Beads. In the last step of the affinity separation, the main concern was to desorb the adsorbed protein in the shortest time and at the highest amount possible. It was, thus, necessary to evaluate the regeneration efficiency of the affinity adsorbents after each cycle. Elution of IgG from monosize beads was also carried out in a batch system, using 1 M NaCl. More than 95% of the adsorbed IgG molecules was eluted easily from the chelating beads in 30 min. With the elution data given, we concluded that NaCl is a suitable elution agent for the Cu2+-chelated beads. In order to show the reusability of the metal-chelated beads, the adsorption-elution cycle was repeated 20 times using the same beads from aqueous IgG solution. Significant reduction in adsorption capacity has not been observed with the reuse of the metal-chelated beads up to 20 times (decreasing ratio ) 5%). It should also be noted that no obvious changes of the morphology of the beads were found in the recycling process when the beads were examined visually. The result further confirmed that the Cu2+-chelated beads have a good stability.

(3)

where Kd, the distribution coefficient of the adsorbate, is equal to (qe/Ce). The plot of ln Kd versus 1/T yields straight lines with the slope and the intercept giving values of ∆H and ∆S. These values could be used to compute ∆G from the 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 temperature. The equilibrium adsorption of IgG onto the Cu2+-chelated beads significantly increased with increasing temperature. A possible explanation for this behavior is as follows: chemical interaction between the chelated Cu2+ ions and the IgG molecules increased with increasing temperature. The negative change in free energy (∆Go < 0) indicated that the adsorption of IgG on the Cu2+-chelated beads was a thermodynamically favorable process. The ∆S value for the adsorption of IgG to Cu2+-chelated monosize beads was calculated as 172.1 J/mol‚K. The positive value for ∆S indicates an increase in the total disorder of the system during adsorption. The calculated ∆H value of the system for the interaction of IgG with chelated Cu2+ ions was -43.2 kJ/mol. 3.2.2. Adsorption Dynamics. In order to quantify the extent of uptake in adsorption kinetics, the kinetic models (pseudofirst- and second-order equations) can be used in this case, assuming that the measured concentrations are equal to adsorbent surface concentrations.34 The first-order rate equation of Lagergren is one of the most widely used for the adsorption of solute from a liquid solution. It may be represented as follows,

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

(4)

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

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

(5)

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The effects of metal-ion leakage and the resulting toxicity during adsorption and elution are also significant issues to be evaluated. The reasons for metal-ion leakage at different stages are not the same. At the adsorption stage, the unstably immobilized metal ions may be tightly captured by the proteins and released to the solution. On the other hand, they are possibly displaced by salt ions in the elution buffer at the elution step. A higher salt concentration at the elution step is more effective for gaining high recoveries, but it may cause more severe metalion leakage. Reduction in the salt concentration could diminish the metal-ion leakage, but the adsorbed protein may not be able to be completely eluted out of the adsorbent. Consequently, an appropriate salt concentration in the elutionbuffer should be carefully selected. The use of NaCl for elution offers an economical manner for purification of proteins comparing with imidazole. During the elution of IgG, no leakage of Cu2+ ions was observed from the Cu2+-chelated beads. 3.3. IgG Depletion from Human Serum. Depletion of additional abundant proteins can be beneficial in the analysis of serum proteins, and therefore, we attempted to deplete the IgG class immunoglobulins from human serum. In the first step of this study, the depletion of HSA was achieved by using the anti-HSA antibody-sepharose 4B column. The depletion efficiency for HSA was 99.6% in the serum sample. Then, in the second step, the depletion of IgG from human 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 Table 4, indicate that a large portion of the IgG was bound by the metal-chelated beads. To test the efficiency of IgG depletion from human serum, proteins in the serum and the eluted portion were analyzed by two-dimensional gel electrophoresis. Proteins that were eluted from the metal-chelated beads include IgG and a small number of nonalbumin proteins. A negligible amount of relatively abundant proteins such as apo-lipoprotein A1, serotransferrin, haptoglobulin, and R1-antitrypsin was bound by the metal-chelated beads. We reached up to 98.2% IgG depletion amount, and it may be concluded that metal-chelated beads are sufficient in terms of efficiency of IgG depletion. In order to confirm that depletion occurred, the diluted serum and IgG-depleted serum were analyzed by SDS/PAGE, and the results are shown in Figure 7. As expected, the depleted serum showed no protein bands, confirming the depletion of highabundance proteins, which were recovered in the depleted fraction; see Figure 7. 3.4. Comparison of Adsorption Capacity with Other Bioaffinity Adsorbents. A comparison of the maximum adsorption capacity, qmax, of the Cu2+-chelated monosize poly(GMA) beads with those of some other bioaffinity adsorbents reported in the literature is given in Table 5. The adsorption capacity of Cu2+-chelated monosize poly(GMA) beads was relatively high when compared with those of other adsorbents. Differences of IgG adsorption are due to the properties of each adsorbent such as structure, functional groups, ligand loading, and surface area. 4. Conclusion These results are consistent with published studies.9,57 Bjo¨rhall et al. used five different commercially available depletion columns including Aurum Serum Protein Minikit (Bio-Rad, Hercules, CA), ProteoExtract Albumin/IgG Removal kit (Merck, Darmstadt, Germany), Multiple Affinity Removal Column (Agilent Technologies, San Diego, CA), POROS Affinity Depletion Cartridges (Applied Biosystems, Framing-

ham, MA), and Albumin-IgG Removal Kit (Amersham Biosciences, Uppsala, Sweden).7 It should be noted that Aurum Serum Protein Minikit (Bio-Rad, U.S.A.) and ProteoExtract Albumin/IgG Removal kit contained Protein A as ligand, while Multiple Affinity Removal Column and Albumin-IgG Removal Kit contained polyclonal antibodies as ligand. POROS Affinity Depletion Cartridges contained protein G. The depletion efficiencies were >90%, but because of the high dilution factor after the depletion procedure as compared with the crude serum, the concentrations of remaining IgG in depleted serum samples were below the detection limits for almost all samples. However, this was stated as a minimum depletion of 94% of IgG in depleted serum samples by any affinity column. Jain and Gupta described a simple one-step method for IgG purification from goat serum using IMAC. They found a recovery ratio of 97.5% with an 8-fold purification.26 Sitnikov et al. applied the Multiple Affinity Removal Column (Agilent Technologies, San Diego, CA) for the depletion of blood plasma proteins under volatile 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 they achieved ∼85% of IgG depletion.59 Plavina et al. reported the development of a robust and relatively high-throughput method consisting of depletion of albumin and IgG with multi-lectin affinity chromatography.60 The total protein recovery for depletion 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 The removal of IgG was shown to be >99% using commercially available Applied Biosystems affinity depletion cartridges carrying Protein G.62 We reached up to 98.2% IgG depletion amount. In the light of the above discussion, we believe that the metal-chelated monosize poly(GMA) beads offer a promising strategy with good depletion specificity and efficiency of IgG in combination with the anti-HSA antibody-Sepharose column. Literature Cited (1) Li, C.; Lee, K. H. Affinity Depletion of Albumin from Human Cerebrospinal Fluid Using Cibacron Blue 3G A Derivatized Photopatterned Copolymer in A Microfluidic Device. Anal. Biochem. 2004, 333, 381. (2) Kocourek, A.; Eyckerman, P.; Thome-Krome, B. The Combined Removal of Albumin and Immuno-Globulins from Human Serum. Bio. Tech. Int. 2005, 17, 24. (3) Steel, L. F.; Trotter, M. G.; Nakajima, P. B.; Mattu, T. S.; Gonye, G.; Block, T. Efficient and Specific Removal of Albumin from Human Serum Samples. Mol. Cell. Proteomics 2003, 2, 262. (4) Petric, T. C.; Brne, P.; Gabor, B.; Govednik, L.; Barut, M.; Strancar, A.; Kralj, L. Z. Anion-Exchange Chromatography Using Short Monolithic Columns as A Complementary Technique for Human Serum Albumin Depletion Prior to Human Plasma Proteome Analysis. J. Pharm. Biomed. Anal. 2007, 43, 243. (5) Altıntas¸ , E. B.; Denizli, A. Efficient Removal of Albumin from Human Serum by Monosize Dye-Affinity Beads. J. Chromatogr., B. 2006, 832, 216. (6) Zhou, M.; David, A.; Lucas, D. A.; Chan, K. C.; Issaq, H. J.; Petricoin, E. F., III; Liotta, A. A.; Veenstra, T. D.; Conrads, T. P. An Investigation into The Human Serum Interactome. Electrophoresis 2004, 25, 1289. (7) Bjo¨rhall, K.; Miliotis, T.; Davidsson, P. Comparison of Different Depletion Strategies for Improved Resolution in Proteomic Analysis of Human Serum Samples. Proteomics 2005, 5, 307. (8) Kocaurek, A.; Eyckerman, P.; Zeidler, R.; Taufmann, M.; Klatt, M.; Thome-Krome, B. An Albumin Removal Assay Improves The Proteomic Investigation of Human Serum. Bioforum Eur. 2004, 8, 49. (9) Ahmed, N.; Barker, G.; Oliva, K.; Garfin, D.; Talmadge, K.; Georgiou, H.; Quinn, M.; Rice, G. An Approach to Remove Albumin for The Proteomic Analysis of Low Abundance Biomarkers in Human Serum. Proteomics 2003, 3, 1980.

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ReceiVed for reView September 5, 2006 ReVised manuscript receiVed July 26, 2007 Accepted August 14, 2007 IE061164C