Biotechnol. frog. 199 I, 7 ' 4 12-4 1a
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Adsorption Characteristics of an Immobilized Metal Affinity Membrane Hideki Iwata, Kyoichi Saito,' and Shintaro Furusaki Department of Chemical Engineering, Faculty of Engineering, University of Tokyo, Hongo, Tokyo 113, Japan
Takanobu Sugo and Jiro Okamoto Japan Atomic Energy Research Institute, Takasaki Radiation Chemistry Research Establishment, Takasaki, Gunma 370-12, Japan
An immobilized metal affinity (IMA) hollow-fiber membrane was prepared by radiationinduced graft polymerization of glycidyl methacrylate (GMA) onto a porous polyethylene hollow fiber, followed by chemical conversion of the produced epoxide group into an iminodiacetate (IDA) group and its chelation with copper(I1) ion. The IDA hollow fiber, whose degree of GMA grafting was 12096, was found to retain 0.42 mol of Cu ion/kg of dry weight of the resulting IMA hollow fiber. The pure water flux of the affinity membrane was 0.90 m / h at a filtration pressure of 1 X lo5 Pa. The 0.1 g/L L-histidyl-L-leucine (His-Leu) solution permeated across the IMA hollow fiber, whose inner diameter and thickness were 0.78 and 0.365 mm, respectively, at a prescribed filtration pressure ranging from 0.2 X lo5 to 1.0 X lo5 Pa. The adsorption of His-Leu during permeation of the solution showed that the overall adsorption rate was independent of the filtration pressure, i.e., the residence time, because of the negligible diffusional resistance of His-Leu to the pseudobioaffinity ligand located on the pore surface of the membrane. No deterioration in the adsorption capacity was observed after five cycles of His-Leu adsorption, its elution, and reimmobilization of copper. The adsorption isotherm of bovine serum albumin (BSA) on the IMA hollow fiber was measured and compared with that for the conventional agarose-based bead containing the IDA-Cu ligand. The diol group formed by HzS04 treatment of the epoxide group remaining after conversion of epoxide into IDA functionality significantly reduced the nonselective adsorptivity of His-Leu and BSA on the membrane.
Introduction The technique of affinity chromatography has revolutionized the purification technology of biological macromolecules such as proteins, enzymes, and antibodies. Currently, columns charged with affinity beads have widespread use in affinity separations. The affinity chromatography column charged with the beads has the throughput/pressure trade-off. To overcome this disadvantage, Brandt et al. (1988) have proposed a membranebased affinity device and demonstrated its advantage whereby diffusional distances can be reduced by convecting protein mixtures through a microporous matrix. Private companies (Sepracor Inc., 1990; Millipore Co., 1990; Nygene Co., 1990) have commercialized ligandimmobilized porous membranes. Affinity ligands can be classified into biospecific and pseudobiospecific ligands (Vijayalakshmi, 1989). Antibodies are representative of biospecific affinity ligands to antigens, whereas dyes, immobilized metal, and amino acid belong to the pseudobiospecific affinity ligand group. Pseudobiospecific ligands are relatively effective in processing the biological fluids at a crude purification step because of their lower cost and higher chemical stability. The technique of immobilized metal affinity (IMA)chromatography was introduced by Porath et al. (1975) and has gained wide acceptance (Sulkowski, 1985; Porath, 1988). For example, IMA chromatography has proved to be an effective method for large-scale purification of recombinantproteins for pharmaceuticalapplications (Ho~
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* To whom correspondence should be addressed. 8758-7938/91/3007-0412$02.50/0
chuli, 1988). The immobilized metal ligand has a high affinity for a group of proteins whose exposed surfaces contain histidine, cysteine, or tryptophan amino acid residues. Several kinds of affinity beads containing the immobilized metals have been commercialized (Pharmacia LKB Biotech., 1990; Tosoh Co., 1990; Showa Denko Co., 1990). Metal ions such as Zn, Ni, Cu, and Fe were immobilized by chelation with an iminodiacetate group, which was covalently attached to agarose or a poly(viny1 alcohol) matrix (Sulkowski, 1985). Previous studies of IMA chromatography have focused on the interaction between immobilized metals and proteins and the performance of the affinity column charged with IMA beads (Porath and Olin, 1983; Porath, 1988). Recently, we developed a new type of hollow fiber that can collect metal ions during permeation of the solution (Saito et al., 1989; Sugo and Saito, 1989). The chelating hollow fiber was synthesized by radiation-induced graft polymerization of glycidyl methacrylate (GMA) onto a porous polyethylene hollow fiber, followed by chemical conversion of the epoxide group into a chelate-forming group. The hollow fibers have been mainly used as a form of filtration membrane because they provide a high ratio of surface area to volume. However, they will hereafter be used in novel applications as (1)bifunctional materials capable of filtration and metal collection, (2) functional materials with high mass transfer rates by transporting the ions or proteins in liquid to ion-exchange groups or affinity ligands by convection, and (3) packing materials having high surface areas and being operated with a lower
0 1991 American Chemical Society and American Instltute of Chemical Engineers
Bbtechnol. Rog., 1991, Vol. 7, No. 5
pressure drop than conventional ones of the bead form (Skiens, 1971; Saito et al., 1988; Uezu et al., 1988; Ding et al., 1989). In this study, we propose a novel affinity hollow-fiber membrane designed for IMA chromatography and discuss the adsorption characteristics, Le., adsorption rate, isotherm, and durability, of the biomolecules (peptide and protein) onto a pseudobiospecific affinity hollow-fiber membrane containing an iminodiacetate-copper ligand. The objectives of our study were 3-fold: (1)to evaluate the ligand density and flux of the IMA hollow-fiber membrane, (2) to examine the adsorption of a peptide during permeation of a solution across the affinity membrane, and (3) to examine both specific and nonselective binding of the biomolecules on the affinity membrane. We selected L-histidyl-L-leucineand bovine serum albumin as the model biomolecules having a high affinity for immobilized copper.
Experimental Procedures Preparation of Hollow Fiber Containing IDA Group. The hollow fiber for IMA chromatography can be prepared by the following four steps: (1)radiationinduced graft polymerization of glycidyl methacrylate (GMA)ontoa starting hollow fiber, (2) chemical conversion of the produced epoxide group into an iminodiacetate (IDA) group, (3) acid hydrolysis of the remaining epoxide group into a diol group, and (4) chelation of the IDAgroup with copper(I1) ion. A commercially available porous polyethylene hollow fiber (Asahi Chemical Industry Co., Japan) was used as the polymer matrix to immobilize the ligand. The inner and outer diameters of this hollow fiber were 0.62 and 1.24 mm. The hollow fiber has a nominally 0.18-pm-diameter pore size and 75% porosity. The GMA-grafted hollow fiber, with a 120-13095 degree of grafting, was prepared by radiation-induced graft polymerization as previously described (Saito et al., 1989). The degree of GMA grafting is defined to be degree of GMA grafting (76)= [ ( W ,- Wo)/Wo]lOO (1) where W Oand W Iare weights of the starting hollow fiber and the GMA-grafted hollow fiber, respectively. The epoxide group of the graft branches was changed into an IDA group by soaking the GMA-grafted hollow fiber in 0.425 M disodium iminodiacetate (IDA-2Na) solution at 353 K. The pH of the IDA-2Na solution ranged from 9.0 to 12.4 with 1M Na2C03/NaHC03 buffer (Hemdan and Porath, 1985). After the reaction, the hollow fiber was rinsed with deionized water, followed by sulfuric acid treatment. In this study, the H2S04 treatment meant that the fiber was immersed in 0.5 M sulfuric acid solution at 353 K for 2 h. By this treatment, the IDA group is converted from the Na to the H form and the remaining epoxide group is also hydrolyzed into a diol group (Hrudkova et al., 1977). After the treatment, the hollow fiber was rinsed repeatedly with deionized water and dried under reduced pressure. The weight of the hollow fiber, W2, was then measured. The conversion of the epoxide group into the iminodiacetic acid, X, can be related to the following weight change:
W o+ (Wl - W0)[x(142 + 133) + (1- X)(142 + l8)]/142 = W , (2) where the factors 142,133, and 18 correspond to the molecular weights of GMA, iminodiacetic acid, and water. Thus, the conversion and the density of the IDA group
413
can be calculated as conversion X = [142(W2- W,,)/(W,- Wo) - 160]/115
(3)
density of IDA group/kg of base polymer (mol/kg) = X [ ( W , - Wo)/142I/Wo (4) The resulting hollow fiber will be referred to as an IDA-C fiber. For comparison, a GMA-grafted hollow fiber was hydrolyzed with 0.5 M sulfuric acid. The resulting hollow fiber containing diol groups will be referred to as a GMAH-C fiber. Chelation of IDA Group with Copper. The IDA-C fiber was soaked in 0.01 M cupric sulfate solution with an excess molar ratio of copper ion in the solution to IDA group on the hollow fiber. After the prescribed period, the two hollow-fiber samples were taken out and washed with deionized water to remove any unbound copper ions. One sample was then immediately dried and the distribution of copper ion across the hollow fiber was determined by measuring the characteristic X-ray of copper with an electron probe X-ray microanalyzer (XMA). For the determination of the Cu immobilized on the hollow fiber, the other sample was eluted by agitating the hollow fiber in 1M hydrochloric acid at ambient temperature for 2 h. The eluate was collected and its copper content was measured with an atomic absorption spectrophotometer (Hitachi 170-10, Hitachi Co., Japan). The density of the metal chelate was calculated as the amount of metal ions eluted divided by the dry weight of the IMA hollow fiber. The resulting hollow fiber designed for IMA chromatography will be referred to here as IDA-Cu-BAM, where the term BAM stands for bioaffinity membrane. The preparation process and corresponding reaction conditions of the IDA-Cu-BAM are summarized in Figure 1and Table I. Properties of IDA-Cu-BAM. Pure water flux of the IDA-Cu-BAMwas determined by an established constantpressure method using the dead-end mode apparatus shownin Figure 2 (Yamagishi et al., 1988;Kim et al., 1991). Prior to the flux measurement, the dried hollow fiber was treated with methanol. Methanol treatment means that the hollow fiber was immersed for 10 min in methanol, which was then replaced with water. This treatment allows the pores to fill with water. The flux was calculated on the basis of the inner surface area of the hollow fiber. The dimensions of the IDA-Cu-BAM were measured with a microscope and a scale. After the hollow fiber was dried under reduced pressure, its specific surface area was measured by using Quantasorb (Yuasa Ionics Co., Japan) according to an established BET method. The cross section of the hollow fiber was also observed by using scanning electron micrography (SEM). Simultaneous Permeation and Adsorption. An apparatus similar to that used for flux measurement of the hollow fiber was used for simultaneous permeation and adsorption. An 11-cm-long IDA-Cu-BAM was set in a U-configuration. First, ultrapure water was forced to permeate across the IDA-Cu-BAM from the inside to the outside through its pores, and second, a buffer was applied for equilibration. Third, the His-Leu solution was made to permeate radially across the IDA-Cu-BAM under a constant filtration pressure by means of a flow controller (Chuo Rika Co., Japan). The effluent dropping from the outside of the hollow fiber was sampled continuously, and the content of His-Leu in each fraction was determined. The inlet concentration of His-Leu was 0.1 g/L. The
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E'
Grafting O f GMA
Coupling O f IDA
Hr.504
Treatment
Metal Chelation
Figure 1. Preparation process of IDA-Cu-BAM. (1) Grafting of glycidyl methacrylate onto a starting hollow fiber; (2) conversion of the epoxide group into an iminodiacetate group; (3) acid hydrolysis of the remaining epoxide group into a diol group; (4) chelation of the iminodiacetate group with the copper ion. Table 1. Preparation Conditions of IDA-Cu-BAM grafting dose reaction temp degree of GMA grafting introduction of IDA group concn of IDA-2Na PH reaction temp reaction time H2SOd treatment reaction temp reaction time metal chelation concn of CuSOl temp contact time
i
200 kGy 313 K 120-130% 0.425 M 11.9 353 K 24 h 353 K 2h 0.01 M
303 K 2h
I
1 Feed Tank 4 Hollow Fiber 5 Measuring Cylinder 2 Flow Controller 6 Drain Tank 3 Pressure Gauge Figure 2. Experimental apparatus for measuring flux. Pure water was forced to permeate from the inside to the outside of the hollow fiber. Also, adsorption of His-Leu during permeation across the hollow fiber was performed with this apparatus.
filtration pressure varied from 0.2 X 106 to 1X l o 6 Pa. All experiments were performed at 303 K. Adsorption Isotherm for Peptide. L-Histidyl-L-leucine (His-Leu, M, = 268) was purchased from Peptide Institute Inc. (grade AA). His-Leu was dissolved in 0.1 M sodium phosphate buffer (pH = 7.4) containing 0.1 M sodium chloride. The adsorption isotherm of His-Leu on the IDA-Cu-BAM was measured in a batchwise manner. The amount of His-Leu adsorbed nonselectively on the GMA-H-C fiber was also determined. The predetermined length of the hollow fiber previously rinsed with the buffer was immersed in the His-Leu solution and incubated in a thermobath maintained at 303 K. The amount of HisLeu adsorbed on the hollow fiber was calculated from the
decrease in the amount of His-Leu in the solution. The initial concentration of His-Leu ranged from 0.5 to 4 g/L. His-Leu was determined by a total organic carbon analyzer (TOC-5000,Shimadzu Co., Japan). The amount of copper migrating from the IDA-Cu-BAM into the solution was determined by atomic absorption spectroscopy. The chemical stability of the IDA-Cu-BAM for the repeated use of adsorption and elution was examined by the batchwise method. One cycle was defined as a series of procedures: adsorption, elution, and reimmobilization of copper. Repeated washing with buffer or deionized water was performed between procedures. Five cycleswere completed, and the amount of His-Leu adsorbed on the IDA-Cu-BAM was determined at each cycle. The initial concentration of His-Leu was about 2.5 g/L. The eluate was a mixture of 0.05 M disodium ethylenediaminetetraacetate and 0.5 M sodium chloride. Adsorption Isotherm for Protein. The adsorption isotherm was measured for bovine serum albumin (BSA) on the IDA-Cu-BAM by the batch method. BSA was purchased from the Sigma Chemical Co. (no. A-7511, essentially fatty acid free). BSA was dissolved in 0.05 M Tris-HC1 buffer (pH = 8) containing 0.15 M sodium chloride. Weighed quantities of the hollow fiber were placed in a test tube containing the BSA solution. The test tube was tightly sealed and incubated a t 303 K. The adsorption of BSA was also measured as a function of time under the same conditions as in the isotherm studies. One-day contact was sufficient to attain adsorption equilibrium. The BSA concentration was determined by the Biuret method. For comparison, agarose-based beads containing an IDA group (chelating Sepharose 6B) were purchased from Pharmacia LKB Biotech., Japan, and converted into the Cu form. The adsorption isotherm was measured under the same conditions as previously described.
Results and Discussion Preparation of IDA-Cu-BAM. An iminodiacetate (IDA) group-containing hollow fiber was prepared by coupling IDA-2Na with the GMA-grafted hollow fiber. The coupling efficiency, Le., the conversion of the epoxide group into an IDA group, was determined as a function of the solution pH. Between pH 11 and 12, the GMAgrafted hollow fiber exhibited maximum conversion. This tendency is almost consistent with the results obtained from the direct coupling of IDA-2Na to epichlorohydrinactivated agarose gel (Hemdan and Porath, 1985). The conversion at pH 11.9 leveled off after the coupling time of 20 h. Figure 3 (right) shows the distribution of copper across the hollow fiber as a function of contact time in the CuSOd solution as measured by XMA. At each contact time, the copper ion was found to be adsorbed symmetrically from both the outer and inner sides of the IDA-C fiber. After 2 h, a uniform distribution of copper was observed. This result indicates that the IDA-Cu ligand is located uniformly across the hollow fiber designed for the novel affinity adsorbent. The corresponding copper uptake curve is shown in Figure 3 (left). The hollow-fiber color changes from white to blue with an increasing amount of immobilized Cu. Properties of IDA-Cu-BAM. The properties of the IDA-Cu-BAM used for the measurement of flux and protein adsorption are summarized in Table 11. The degree of GMA grafting and the conversion calculated by eqs 1 and 3 were 120% and 0.2, respectively. The conversion of 0.2 corresponds to the density of the IDA-Cu ligand, 1.1
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120min
30min
.-6
c 0
L L
s d
1Omin
0 0
60
120
Time [minl
Figure 3. (Left) XMA profile of Cu across the IDA-Cu-BAM as a function of contact time. The IDA-Cu fiber was soaked in 0.01 M CuSOJ solution. (Right) Amount of Cu adsorbed as a function of contact time. The amount of copper eluted in 1 M HCI was determined. Table 11. Properties of IDA-Cu-BAM
degree of GMA grafting amount of GMA after grafting density of IDA group density of IDA-Cu ligand
120-130?5 8.459.15 mol/kg of B P 1.7-1.8 mol/kg of BP 1.1-1.2 mol/kg of BP 0.42-0.44 mol/kg of BAMb 0.18 mol/L of BAM 6.8-7.3 mol/kg of BP 0.42 kg (dry)/L (wet)
density of diol group apparent density of IDA-CU-BAM distribution of IDA-Cu ligand uniform dimensions inner diameter 0.78 mm 1.51 mm outer diameter pure water fluxC 0.90 m/h BP, base polymer. b BAM,bioaffhity membrane containing IDACu ligand. The weight of the base polymer ( WO)can be converted into that of the IDA-Cu-BAM (W3) as follows: W3/ WO= 1 + (dg) [61.5XY+ 115X + 160]/(142-100),wheredg,X,and Yarethedegree of GMA grafting, the conversion of the epoxide group into the IDA group, and the molar ratio of Cu immobilized to the IDA group. Filtration pressure = 1 X lo5 Pa.
H Figure 4. SEM picture of the IDA-Cu-BAM. The black part of the photograph indicates the pores (marker = 1 X lo4 m).
hollow fiber within feasible levels at the expense of the flux and specific surface area. Peptide Adsorption during Permeation. Figure 5 shows the breakthrough curves (BTCs)obtained by varying the filtration pressure at an inlet concentration of 0.1 g/L. The ordinate is the ratio in concentrration of the effluent to the inlet. The abscissa is the dimensionless time, T , defined as T
= (amount of His-Leu applied at Co)/(amount of His-Leu adsorbed in equilibrium with Co)
Q
mol/kg of dry weight of the starting hollow fiber. This ligand density is equivalent to 0.18 mol/L of wet volume of the IDA-Cu-BAM, which is about 5 times higher than that of a commercially available affinity bead (Belew et al., 1987a) containing the IDA-Cu ligand. The pure water flux of the affinity hollow fiber was about half that of the starting hollow fiber, 1.9 m/h, at a filtration pressure of 1X lo5Pa. Therefore, the IDA-Cu-BAM can still be used for an affinity membrane having micropores through which the solution is convected. After introduction of the IDA-Cu ligand into the hollow fiber, the inner and outer diameters of the fiber in the wet state were 0.78 and 1.51 mm. Thus, the IDA-Cu-BAM swelled in thickness by 3% when compared with the starting hollow fiber. The specific surface area of the IDACu-BAM, 11m2/g, had decreased when compared to that of the starting hollow fiber, 23 m2/g. Also, comparison of SEM pictures of the cross sections of both hollow fibers indicates that the graft branches formed on the pores and that the network structure of the resin phase swelled. Figure 4 is a SEM picture of the cross section of the IDACU-BAM. As a result, the pseudobiospecific affinity ligand could be incorporated into an existing porous polyethylene
where dj, do,and Pa are the inner and outer diameters and apparent density of the hollow fiber. COand 90 are the inlet concentration and the amount adsorbed in equilibrium with CO. uj and t are the flux, i.e., the superficial velocity based on the inner surface area of the hollow fiber, and the permeation time. The value of 90 can be calculated by
where V and W3 are the effluent volume and the weight of the IDA-Cu-BAM. V, is the effluent volume where the effluent concentration C reaches CO. The lack of dependence of the BTC shape on the filtration pressure demonstrates that the overall adsorption rate was not governed by the residence time of the solution across the hollow fiber through its pores. In other words, the convection of a peptide directly to the ligand reduced the mass transfer resistances. This is a significant advantage over conventional beads. The smooth BTC may be attributed to the distribution of the pore length, i.e., the tortuous structure of the IDA-Cu-BAM. Adsorption Isotherm for Peptide. Adsorption isotherms of His-Leu onto the IDA-Cu-BAM and GMA-H-C fibers are shown in Figure 6. The data fit the Langmuir equation well. The GMA-H-Cfiber containing exclusively the diol group yielded a negligible nonselective adsorption of His-Leu. The affinity of copper immobilized on the polymer matrix to His-Leu is attributed to the chelation of the nitrogen atom in the imidazole ring of His-Leu with immobilized Cu. The saturation level was determined as
Biorechnol. Prog., 1991, Vol. 7, NO. 5
416 'p
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Dimensionless time [-I Figure 5. Breakthrough curves of His-Leu as a function of the filtration pressure. The His-Leu solution permeated radially from the inside to the outside of the 11-cm-longIDA-Cu-BAM through ita pores. The filtration pressure ranged from 0.2 X 106 to 1.0 X 1V Pa. The inlet concentration of His-Leu was 0.1 g/L. The abscissa is dimensionless time, defined by eq 5.
f i 0.2
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