Ind. Eng. Chem. Res. 2004, 43, 6507-6513
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Poly(ethylene dimethacrylate-glycidyl methacrylate) Monolith as a Stationary Phase in Dye-Affinity Chromatography Lokman Uzun,† Handan Yavuz,† Rıdvan Say,‡ Arzu Erso1 z,‡ and Adil Denizli*,†
Ind. Eng. Chem. Res. 2004.43:6507-6513. Downloaded from pubs.acs.org by UNIV OF WINNIPEG on 01/23/19. For personal use only.
Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara, Turkey, and Department of Chemistry, Anadolu University, Eskis¸ ehir, Turkey
Porous monolith was obtained by the bulk polymerization of ethylene dimethacrylate (EDMA) and glycidyl methacrylate (GMA) conducted in a glass tube. Poly(EDMA-GMA) monolith had a specific surface area of 98.7 m2/g. Poly(EDMA-GMA) monolith was characterized by swelling studies, FTIR, scanning electron microscopy, and elemental analysis. Poly(EDMA-GMA) monolith with a swelling ratio of 48%, and containing 45.7 µmol Cibacron Blue F3GA/g, were used in the adsorption/desorption of human serum albumin (HSA) from aqueous solutions and human plasma. The nonspecific adsorption of HSA was very low (0.8 mg/g). The maximum amount of HSA adsorption from aqueous solution in phosphate buffer was 22 mg/g at pH 5.0. Higher HSA adsorption value was obtained from human plasma (up to 53.2 mg/g) with a purity of 92%. Desorption of HSA from Cibacron Blue F3GA-attached poly(EDMA-GMA) monolith was obtained using 0.1 M Tris/HCl buffer containing 0.5 M NaCl. It was observed that HSA could be repeatedly adsorbed and desorbed with poly(EDMA-GMA) monolith without significant loss in the adsorption capacity. Introduction Human serum albumin (HSA) is the most abundant protein in the circulatory system.1 It consists of a single, nonglycosylated, polypeptide chain containing 585 amino acid residues and has many physiological functions which contribute significantly to colloid osmotic blood pressure and aid in the transport, distribution, and metabolism of many endogeneous and exogeneous substances including bile acids, bilirubin, long-chain fatty acids, amino acids (notably tryptophan, thyrosine, and cysteine), steroids (progesterone, testosterone, aldosterone, and cortisol), metal ions such as copper, zinc, calcium, and magnesium, and numerous pharmaceuticals.2 HSA commonly used for therapeutic purposes, such as for shock, heavy loss of blood, etc., requires relatively high purity for medical use. Research on HSA separation has attracted considerable attention for its great potential in blood protein manufacture. HSA is at present commonly isolated from human plasma by Cohn’s classical blood fractionation procedure.3 Cohn’s method involves precipitation of proteins using ethanol with varying pH, ionic strength, and temperature. But this technique, which is the oldest method of industrial fractionation of blood proteins, is not highly specific and can give partially denaturated proteins.4 Dye-ligand chromatography has been used extensively in laboratory- and large-scale protein purification.5-10 Dye-ligands are commercially available, inexpensive, and can be easily immobilized, especially on matrixes bearing hydroxyl groups. Although dyes are all synthetic in nature, they are still classified as affinity ligands because they interact with the active sites of many proteins mimicking the structure of the substrates, cofactors, or binding agents for those proteins. * To whom correspondence should be addressed. E-mail:
[email protected]. † Hacettepe University. ‡ Anadolu University.
A number of textile dyes, known as reactive dyes, have been used for protein purification. Most of these reactive dyes consist of a chromophore (either azo dyes, anthraquinone, or phathalocyanine) linked to a reactive group (often a mono- or dichloro-triazine ring). The interaction between the dye ligand and proteins can be by complex combination of electrostatic, hydrophobic, hydrogen bonding. Cibacron Blue F3GA is an anthraquinone textile dye that interacts specifically and reversibly with albumin.11 Conventional packed-bed columns possess some inherent limitations such as the slow diffusional mass transfer and the large void volume between the beads.12 Although some new stationary phases such as the nonporous polymeric beads13 and perfusion chromatography packings are designed to resolve these problems, these limitations cannot be overcome in essence.14 Recently, monolith materials have been considered as a novel generation of stationary phases in the separation science because of their easy preparations, excellent flow properties, and high performances compared to beads conventionally used fortheseparationof biomolecules.15-22 Porous monoliths are a very good alternative for protein separation, and they have many advantages. Several potential advantages of monoliths are large surface area, short diffusion path, low-pressure drop, and very short residence time for both adsorption and elution. This work reports on the purification of an HSA from human plasma by dye affinity chromatography with a novel monolith column. Poly(EDMA-GMA) monolith is a copolymer of ethylene dimethacrylate (EDMA) and glycidyl methacrylate (GMA) and was obtained by bulk polymerization. Poly(EDMA-GMA) monolith was characterized using FTIR, scanning electron microscopy (SEM), porosity measurements, elemental analysis, and swelling test. HSA adsorption on the poly(EDMA-GMA) monolith from aqueous solutions containing different amounts of HSA, at different pHs and ionic strengths, and also from human plasma was also performed. In
10.1021/ie040045z CCC: $27.50 © 2004 American Chemical Society Published on Web 08/27/2004
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the last part, desorption of HSA and stability of these materials was tested. Experimental Section Materials. Glycidyl methacrylate (GMA, Sigma Chemical Co., St. Louis, MO) and ethylene dimethacrylate (EDMA, Aldrich, Munich, Germany) were distilled under vacuum (100 mm Hg). Cibacron Blue F3GA was obtained from Polyscience (Warrington, PA) and used without further purification. Human serum albumin (HSA, 98% pure by gel electrophoresis, fatty acid free, 67 kDa) was purchased from Aldrich (Munich, Germany). All other chemicals were of the highest purity commercially available and were used without further purification. Coomassie Blue for the Bradford Protein assay was from BioRad (Richmond, CA). 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. Preparation of Poly(EDMA-GMA) Monoliths. The poly(ethylene dimethacrylate-glycidyl methacrylate) [poly(EDMA-GMA)] monolith was prepared by an in situ polymerization within a glass tube using benzoyl peroxide as the initiator. Toluene was included in the polymerization recipe as the diluent (as a pore former). Benzoyl peroxide (50 mg) was dissolved in the mixture of monomer (EDMA 2.0 mL and GMA 1.0 mL) and porogenic diluent (toluene 1.0 mL). The monomer mixture was sonicated to obtain a clear solution and was then purged with nitrogen for 15 min. The glass tube (20 × 10 mm i.d.) was filled with the above mixture and then sealed. The polymerization was allowed to proceed at 60 °C for 4 h, then at 80 °C for 2 h. The tube was then attached to a chromatographic system. Ethyl alcohol (50 mL) and water (50 mL) were pumped through the column at a flow rate of 1.0 mL/min to remove the unreacted monomers and porogenic diluents present in the monolith after the polymerization was completed. The monolith was stored in buffer containing 0.02% sodium azide at 4 °C until use. Cibacron Blue F3GA Immobilization. Cibacron Blue F3GA immobilization studies were carried out in a recirculating system equipped with a water jacket for temperature control. The monolith was washed with 30 mL of water. Then, 100 mL of Cibacron Blue F3GA solution (5 mg/mL) containing NaOH (5 g) was pumped through the column under recirculation at 80 °C for 2 h. Under these experimental conditions, a chemical reaction took place between the group of the Cibacron Blue F3GA containing chloride and the epoxide group of the poly(EDMA-GMA) monolith. The adsorption was followed by monitoring the decrease in UV absorbance at 280 nm. After incubation, the Cibacron Blue F3GAattached poly(EDMA-GMA) monolith was washed with distilled water and methanol until all the physically adsorbed Cibacron Blue F3GA was removed. The modified monolith was then stored at 4 °C with 0.02% sodium azide to prevent microbial contamination. Characterization of Monoliths. Water uptake ratio of the monolith was determined in distilled water. The experiment was conducted as follows: initially dry monolith was carefully weighed before being placed in a 50 mL vial containing distilled water. The vial was put into an isothermal water bath at 25 °C for 24 h. The monolith was taken out of the water, wiped using
a filter paper, and weighed. The mass ratio of dry and wet samples was recorded. The water content of the monolith was calculated using the following expression:
Water uptake ratio % )[(Ws - Wo)/Wo] × 100
(1)
where Wo and Ws are the mass of poly(EDMA-GMA) monoliths before and after uptake of water, respectively. The morphology of a cross section of the dried monolith was investigated by scanning electron microscope (Raster Electronen Microscopy, Leitz-AMR-1000, Germany). The monolith was dried in a vacuum oven at 50 °C for 24 h. Pore volumes and average pore diameter greater than 20 Å were determined by mercury porosimeter up to 2000 kg/cm2 using a Carlo Erba model 200 (Milano, Italy). The specific surface area of the monolith was determined in BET isoterm of nitrogen with an ASAP2000 instrument (USA, Micromeritics). FTIR spectra of the Cibacron Blue F3GA, the poly(EDMA-GMA) monolith, and Cibacron Blue F3GAattached poly(EDMA-GMA) monolith were obtained by using a FTIR spectrophotometer (FTIR 8000 Series, Shimadzu, Japan). The dry monolith (about 0.1 g) was thoroughly mixed with KBr (0.1 g, IR Grade, Merck, Germany), and pressed into a tablet, and the spectrum was then recorded. To evaluate Cibacron Blue F3GA immobilization amount, the poly(EDMA-GMA) monolith was subjected to elemental analysis using a Leco Elemental Analyzer (Model CHNS-932). Chromatographic Procedures. HSA-Adsorption from Aqueous Solutions. The HSA adsorption studies were carried out in a recirculating system equipped with a water jacket for temperature control. The monolith was washed with 30 mL of water and then equilibrated with 25 mM phosphate buffer containing 0.1 M NaCl (pH 7.4). Then, the prepared HSA solution (50 mL of the aqueous HSA solution) was pumped through the column under recirculation for 2 h. The adsorption was followed by monitoring the decrease in UV absorbance at 280 nm. Effects of HSA concentration, pH of the medium, and ionic strength on the adsorption capacity were studied. The flow rate of the solution was 1.0 mL/ min. To observe the effects of the initial concentration of HSA on adsorption, it was changed between 0.5 and 7.0 mg/mL. To determine the effect of pH on the adsorption, pH of the solution was changed between 4.0 and 8.0. To observe the effects of ionic strength, NaCl solution was used at ionic strength values of 0.01 and 0.1. Desorption and Repeated Use. In all cases adsorbed HSA molecules were desorbed using 0.1 M Tris/ HCl buffer containing 0.5 M NaCl. In a typical desorption experiment, 50 mL of the desorption agent was pumped through the monolithic column at a flow rate of 1.0 mL/min for 1 h. The final HSA concentration in the desorption medium was spectroscopically determined. When desorption was achieved, the monolith was cleaned with 1 M NaOH and then reequilibrated with 25 mM phosphate buffer containing 0.1 M NaCl (pH 7.4). The desorption ratio was calculated from the amount of HSA adsorbed on the monolith and the final HSA concentration in the desorption medium. To test the repeated use of poly(EDMA-GMA) monolith, the HSA adsorption-desorption cycle was repeated 10 times using the same monolithic column. To regenerate and sterilize the monolith after the desorption, it was washed with 1 M NaOH solution.
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Figure 1. Schematic diagram for the immobilization of dye molecules through the poly(EDMA-GMA) monolith.
Figure 2. SEM micrograph of poly(EDMA-GMA) monolith.
HSA Adsorption from Human Plasma. Human blood was collected into EDTA-containing vacutainers and red blood cells were separated from plasma by centrifugation at 4000g for 30 min at room temperature, then filtered (3-µm Sartorius filter) and frozen at -20 °C. Before use, the plasma was thawed for 1 h at 37 °C. Before application, the viscous sample was diluted with 25 mM phosphate buffer containing 0.1 M NaCl (pH 7.4). Dilution ratios were 1:2 and 1:10. A 50-mL portion of the human plasma with a HSA content of 37.7 mg/ mL was pumped through the monolith column at a flow rate of 1.0 mL/min for 1 h. Human serum albumin concentration was determined by using Ciba Corning albumin reagent (Ciba Corning Diagnostics Ltd., Halstead, Essex, England; Catalog Ref 229241) which is based on a bromocresol green (BCG) dye method.23 To show dye specificity, adsorption of other blood proteins (i.e., fibrinogen and γ-globulins) was also monitored. Total protein concentration was measured by using the total protein reagent (Ciba Corning Diagnostics; Catalog Ref 712076) at 540 nm which is based on a Biuret reaction.23 Chronometric determination of fibrinogen according to the Clauss method on plasma was performed by using a Fibrinogene Kit (Refs 68452 and 68582, bioMerieux Laboratory Reagents and Instruments, Marcy-l’Etoile, France).24 γ-Globulin concentration was determined from the difference. The purity of HSA was assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (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 acid-water (1:4:6, v/v/v). Electrophoresis was run for 2 h with a voltage of 110 V. Lysozyme and human serum albumin were used as standards.
contained mainly micropores. This pore diameter range is possibly available for diffusion of the HSA molecules. The general shape of HSA can be viewed as three tennis balls in a can or cylinder. The Stokes radius is 3.9 nm. On the basis of these data, it was concluded that the poly(EDMA-GMA) monolith had effective pore structures for liquid chromatographic separation of HSA. The water uptake ratio of the poly(EDMA-GMA) monolith was 48%. Specific surface area of the poly(EDMA-GMA) monolith is found to be almost same after Cibacron Blue F3GA attachment. Therefore, these pores were not blocked by the immobilized dye molecules. The cross-sectional structure of poly(EDMA-GMA) monolith is exemplified in Figure 2. It can be clearly seen that poly(EDMA-GMA) monolith is composed of much smaller particles. The particles are irregular. There are also many pores on the bulk structure of the particles. This also provides higher dye loading and HSA adsorption capacity. These pores reduce diffusional mass transfer resistance and facilitate convective transport because of high specific internal surface area. The back pressure drop was about 4.3 mPa at the flow-rate of 1.0 mL/min. So the poly(EDMA-GMA) monolith has good flow properties. Cibacron Blue F3GA was used as the biomimetic dyeligand for specific binding of HSA. Cibacron Blue F3GA has been found to interact with many proteins that possess a dinucleotide-binding domain.25 However, other proteins such as interferon26 and human serum albumin,27 which may not possess a dinucleotide-domain, have also been shown to bind to Cibacron Blue F3GA. The interaction between the dye and protein can be as follows. Dye molecules mimic natural ligands, and bind some proteins very specifically at their active points. However, under same conditions all proteins can be adsorbed onto dye-ligand affinity adsorbents, which means that these ligands provide numerous opportunities for other interactions with other parts of the proteins. Most proteins are bound nonspecifically by a complex combination of electrostatic, hydrophobic, hydrogen bonding, and charge-transfer interactions, all of which are possible considering the structural nature of the dyes. It is reported that Cibacron Blue F3GA has no side effect on biochemical systems.28 However, all commercial reactive dyes (including Cibacron Blue F3GA) contain various impurities which may affect their biochemical and related use. Reactive dyes have been
Results and Discussion The schematic diagram for the immobilization of Cibacron Blue F3GA molecules through the poly(EDMAGMA) monolith is given in Figure 1. According to mercury porosimetry data, the average pore size of the monolith was 800 nm. Specific surface area of the monolith was found to be 98.7 m2/g by BET method. The total pore volume was 3.4 mL/g and represented a porosity over 64%. This indicated that the monolith
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Figure 3. Effect of medium pH on the HSA adsorption: Cibacron Blue F3GA loading 45.7 µmol/g; HSA concentration 2.0 mg/mL; flow rate 1.0 mL/min; T 20 °C. Each data is an average of five parallel studies.
Figure 4. Effect of the concentration of HSA on the HSA adsorption: Cibacron Blue F3GA loading 45.7 µmol/g; pH 5.0; flow rate 1.0 mL/min; T 20 °C. Each data is an average of five parallel studies.
purified by a number of chromatographic procedures such as thin-layer chromatography, high-performance liquid chromatography, and column chromatography on Silica gel or sephadex. However, it is suggested that purification of reactive dyes is necessary only when free dyes are used.29 In cases where attached dyes are used, purification of the dye before immobilization is not likely to be necessary, because few of the contaminants will be attached on the support matrix, and proper washing of the matrix should remove adsorbed contaminants.30 Cibacron Blue F3GA is covalently attached on poly(EDMA-GMA) monolith, via the reaction between the chloride groups of the reactive dyes and the epoxide groups of the GMA. The FTIR bands observed at 1160 cm-1 were assigned to symmetric stretching of SdO, as also pointed out on the chemical structure of the Cibacron Blue F3GA (Figure 1). The band observed at 3500 cm-1 was assigned to the -OH functional group. After Cibacron Blue F3GA attachment, the intensity of the -OH band increases due to NH stretching. The split of the band at 3300-3500 cm-1 indicates also SO3H and NH2 groups. These bands show the attachment of Cibacron Blue F3GA within the poly(EDMA-GMA) monolith. The visual observations (the color of the monolith) ensured attachment of dye molecules. The dye loading was 45.7 µmol/g. Note that EDMA, GMA, and other chemicals in the polymerization formula do not contain sulfur. The sulfur amount determined by elemental analysis originated from only immobilized dye in the polymeric structure. The studies of Cibacron Blue F3GA leakage from the poly(EDMA-GMA) monolith showed that there was no dye leakage in any medium used throughout this study, even over a long period of time (more than 24 weeks). HSA Adsorption from Aqueous Solutions. Effects of pH. Figure 3 shows the effect of pH on the adsorption of HSA onto Cibacron Blue F3GA-attached poly(EDMA-GMA) monolith. The maximum adsorption of HSA was observed at pH 5.0, which is the isoelectric point of HSA. With the increase of pH above 5.0, and below pH 5.0, the HSA adsorption capacity decreased. The decrease in the HSA adsorption capacity can be attributed to electrostatic repulsion effects between the identically charged groups. At the isoelectric points, proteins have no net charge, and, therefore, the maximum protein adsorption from aqueous solution is usu-
ally observed at this point. In addition, these interactions between the dye and protein molecules may result both from the ionization states of several groups on both the ligands (i.e., Cibacron Blue F3GA) and amino acid side chains in human serum albumin structure, and from the conformational state of protein molecules at this pH. It should be noted also that nonspecific adsorption is independent of pH and it is observed at the same level at all the pH values studied. Adsorption Isotherm. Figure 4 shows the HSA adsorption isotherm of the plain and dye-affinity monoliths. Note that one of the main requirements in dyeaffinity chromatography is the specificity of the affinity adsorbent for the target molecule. The nonspecific interaction between the support, which is the poly(EDMA-GMA) monolith in the present case, and the molecules to be adsorbed, which are the HSA molecules here, should be minimum in order to consider the interaction as specific. As shown in this figure, a negligible amount of HSA was adsorbed nonspecifically on the poly(EDMA-GMA) monolith, which was 0.8 mg/ g; while dye-immobilization significantly increased the HSA coupling capacity of the monolith (up to 22 mg/g). The amount of HSA adsorbed per unit mass of the poly(EDMA-GMA) monolith increased first with the initial concentration of HSA then reached a plateau value which represents saturation of the active adsorption sites (which are available and accessible for HSA) on the monolith. This increase in the HSA coupling capacity may have resulted from a cooperative effect of different interaction mechanisms such as hydrophobic, electrostatic, and hydrogen bonding caused by the acidic groups and aromatic structures on the Cibacron Blue F3GA and by groups on the side chains of amino acids on the HSA molecules. It should be mentioned that Cibacron Blue F3GA is not very hydrophobic overall, but it has planar aromatic surfaces that prefer to interact with hydrophobic groups in HSA structure. Kinetic Analysis. To examine the controlling mechanism of adsorption process such as mass transfer and chemical reaction, kinetic models were used to test experimental data. The kinetic models (pseudo-first- and second-order equations) can be used in this case assuming that the measured concentrations are equal to adsorbent surface concentrations. The first-order rate equation of Lagergren is one of the most widely used
Ind. Eng. Chem. Res., Vol. 43, No. 20, 2004 6511 Table 1. First- and Second-Order Kinetic Constants for Poly(EDMA-GMA) Monolith first-order kinetic
second-order kinetic
initial exp. qeq k1 qeq R2 k2 qeq R2 concn (mg/mL) (mg/g) (1/min) (mg/g) (mg/g) (1/min) (mg/g) 0.5 1.0 2.0 3.0 4.0 5.0
13.75 16.18 17.03 20.07 21.17 21.90
0.050 0.044 0.045 0.041 0.052 0.050
15.26 17.14 17.65 19.93 19.76 18.56
0.978 0.979 0.989 0.997 0.975 0.986
0.0031 0.0024 0.0018 0.0017 0.0027 0.0040
19.34 19.34 21.51 24.63 24.39 24.01
0.997 0.997 0.963 0.992 0.994 0.998
for the adsorption of solute from a liquid solution. It may be represented as follows:
qt/dt ) k1(qeq - qt)
(2)
where k1 is the rate constant of pseudo-first-order adsorption (1/min) and qeq and qt denote the amounts of adsorbed protein at equilibrium and at time t (mg/g), respectively. After integration by applying boundary conditions, qt ) 0 at t ) 0 and qt ) qt at t ) t, gives
log[qeq/(qeq - qt)] ) (k1t)/2.303
(3)
Eq 3 can be rearranged to obtain a linear form
log(qeq - qt) ) log(qeq) - (k1t)/2.303
(4)
A plot of log(qeq) versus t should give a straight line to confirm the applicability of the kinetic model. In a true first-order process logqeq should be equal to the interception point of a plot of log(qeq - qt) via t. In addition, a pseudo-second-order equation based on equilibrium adsorption capacity may be expressed in the form
qt/dt ) k2 (qeq - qt)2
(5)
where k2 (g/mg‚min) is the rate constant of pseudo-firstorder adsorption process. Integrating eq 5 and applying the boundary conditions, qt ) 0 at t ) 0 and qt ) qt at t ) t, leads to
[1/(qeq - qt)] ) (1/qeq) + k2t
(6)
or equivalently for linear form
(t/qt) ) (1/k2qeq2) + (1/qeq)t
(7)
A plot of t/qt versus t should give a linear relationship for the applicability of the second-order kinetics. The rate constant (k2) and adsorption at equilibrium (qeq) can be obtained from the intercept and slope, respectively. The results of kinetic analysis are summarized in Table 1. A comparison of the experimental adsorption capacity and the theoretical values is presented in Table 1. The theoretical qe value estimated from the pseudo-firstorder and the pseudo-second-order kinetic model were very close to the experimental values and the correlation coefficients were very high. These results showed that this monolithic dye-adsorbent system was described both by the first-order and second-order kinetic models. Effect of NaCl Concentration. The effect of NaCl concentration on HSA adsorption is presented in Figure 5, which shows that the adsorption capacity decreases with increasing ionic strength of the binding acetate
Figure 5. Effect of the NaCl concentration on HSA adsorption: Cibacron Blue F3GA loading 45.7 µmol/g; HSA concentration 2.0 mg/mL; pH 5.0; Flow rate 1.0 mL/min; T 20 °C. Each data is an average of five parallel studies.
buffer. The adsorption amount of HSA decreased by about 75.7% as the NaCl concentration changed from 0.01 to 0.1 M. Increasing the NaCl concentration could promote the adsorption of the dye molecules to the polymer surface by hydrophobic interaction. Moreover, the hydrophobic interactions between the immobilized dye molecules themselves would also become strong, because it has been observed that the salt addition to a dye solution caused the stacking of the free dye molecules. Thus, the numbers of the immobilized dye molecules accessible to HSA would decrease as the ionic strength increased, and the adsorption of the HSA to immobilized dye became difficult. It is also suggested that an increase in NaCl concentration result in the reduction of electrostatic interactions.31 Desorption Studies. Desorption of HSA from the Cibacron Blue F3GA-attached poly(EDMA-GMA) monolith was also carried out in a continuous system. The desorptions of HSA are expressed in % of totally adsorbed HSA. Up to 97.2% of the adsorbed HSA was desorbed by using 0.1 M Tris/HCl buffer containing 0.5 M NaCl as elution agent. The addition of elution agent changed the charge of the peptide side groups due to their isoelectric points, resulting in the detachment of the HSA molecules from the immobilized dye molecules. Note that there was no Cibacron Blue F3GA release in this case which shows that dye molecules are bonded strongly to poly(EDMA-GMA) monolith. With the desorption data given above we concluded that 0.1 M Tris/ HCl buffer containing 0.5 M NaCl is a suitable desorption agent, and allows repeated use of the affinity monolith used in this study. To show the reusability of the Cibacron Blue F3GAattached poly(EDMA-GMA) monolith, the adsorptiondesorption cycle was repeated 10 times using the same dye-affinity poly(EDMA-GMA) monolith. There was no remarkable reduction in the adsorption capacity of the monolith (Figure 6). The HSA adsorption capacity decreased only 6.4% after 10 cycles. By taking into account the different experimental parameters studied above, it should be possible to scale-up the process of HSA separation by increasing the monolith size by dyeaffinity chromatography on Cibacron Blue F3GA-immobilized poly(EDMA-GMA) monolith. HSA Adsorption From Human Plasma. Table 2 shows the adsorption for human serum obtained from
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immunoglobulin G,20 polynucleotides,21 and lysozyme.34 The triazine dye Cibacron Blue F3GA immobilized to a poly(EDMA-GMA) monolith provided an efficient method to purify HSA, showing high binding capacity and high selectivity, ensuring a recovery of about 92%. It was possible to use the Cibacron Blue F3GA-attached poly(EDMA-GMA) monolith in a HSA adsorption-desorption cycle. Literature Cited
Figure 6. Repeated use of dye-attached poly(EDMA-GMA) monolith: Cibacron Blue F3GA loading 45.7 µmol/g; HSA concentration 2 mg/mL; pH 5.0; flow rate 1.0 mL/min; T 20 °C. Each data is an average of five parallel studies. Table 2. HSA Adsorption from the Plasma of a Healthy Donor (Cibacron Blue F3GA Loading 45.7 µmol/g; Flow Rate 1.0 mL/min; T 20 °C) dilution agent
adsorption capacity (mg/g)
plasma (undiluted) 1:2 diluted plasma 1:10 diluted plasma
53.2 ( 2.05 42.4 ( 1.76 35.9 ( 1.87
a healthy donor. There was a low adsorption of HSA (1.84 mg/g) on the poly(EDMA-GMA) monolith, while much higher adsorption values (53.2 mg/g) were obtained when the Cibacron Blue F3GA-attached poly(EDMA-GMA) monolith were used. The purity of HSA was assayed by SDS-PAGE. The purity of HSA obtained was found to be 92% after purification. It is worth noting that adsorption of HSA onto the Cibacron Blue F3GA-attached poly(EDMA-GMA) monolith was approximately 2.4-fold higher than those obtained in the studies in which aqueous solutions were used. This may be explained as follows: the conformational structure of HSA molecules within their native environment (i.e., human plasma) is much more suitable for specific interaction with the Cibacron Blue F3GA-attached poly(EDMA-GMA) monolith. Competitive protein adsorption was also carried out and interesting results were obtained in these studies. Adsorption capacities were achieved as 2.5 mg/g for fibrinogen and 6.9 mg/g for γ-globulin. The total protein adsorption was determined as 63.1 mg/g. It is worth noting that adsorption of other plasma proteins (i.e., fibrinogen and γ-globulin) on the Cibacron Blue F3GAattached poly(EDMA-GMA) monolith are negligible. It should be noted that HSA is the most abundant protein in plasma. It generally makes up more than half of the total plasma proteins. It may be resulted that this low adsorption of fibrinogen and γ-globulin is due to the high concentration of HSA. Conclusions Monoliths play an important role in advanced separation, e.g., high-performance affinity chromatography or capillary electrochromatography of proteins.32,33 Monolithic affinity adsorption is a developing bioseparation technique that has been studied for various proteins such as cytochrome C and bovine serum albumin,19
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Received for review February 9, 2004 Revised manuscript received July 8, 2004 Accepted July 10, 2004 IE040045Z
