Supported chitosan-dye affinity membranes and their protein adsorption

j o u r n a l of MEMBRANE SCIENCE ELSEVIER

Journal of Membrane Science 117 (1996) 271-278

Supported chitosan-dye affinity membranes and their protein adsorption Xianfang Zeng, Eli Ruckenstein * Department of Chemical Engineering, State University of New York at Buffalo, Amherst, NY 14260, USA Received 13 December 1995; revised 6 March 1996; accepted 14 March 1996

Abstract Chitosan-dye affinity microporous membranes supported on polyethersulfone are suggested to be used for protein separation. They were prepared via the phase inversion method, followed by the coupling of the Cibacron Blue F3GA to the membranes. The effect of temperature on the coupling, the adsorption of human serum albumin on the coupled membrane and the effect of the flow rate through the membrane on the amount adsorbed were investigated. Keywords: Affinity membrane; Chitosan microporous membrane; Dye membrane; Protein separation

1. Introduction The rapid development of biotechnology requires more reliable and efficient methods to isolate and purify the bioproducts (proteins, enzymes, peptides or nucleic acids). The affinity column chromatography is an effective and widely used method for protein/enzyme separation. However, it has a number of drawbacks, such as the compressibility of the beads, the plugging/fouling, and particularly the slow flow rate through the column. In recent years, the adsorptive membrane has emerged as an alternative to the traditional column chromatography, since it provides higher flow rates, much lower pressure drops, and it can provide higher productivities per unit time. While they have lower adsorption capacities, they can be cycled much more frequently, and, for this reason, can lead to higher * Corresponding author.

productivies per unit time. Some of the methods employed to prepare adsorptive membranes are: (1) the coating technique, which deposits hydrophilic layers [such as hydroxyethyl cellulose (HEC) [1], and polyethyleneimine (PEI) [2]] on hydrophobic ones; (2) copolymerization of two different monomers which contain reactive groups [such as poly(acrylonitrile-co-hydroxyalkyl methacrylate) [3] and p o l y ( g l y c i d y l rnethacrylate-co-ethylene dimethacrylate) [4]]; (3) the surface modification of the commercial microfiltration membranes (polyethylene [5], cellulose [6], nylon [7], polyvinylidene difluoride [8], polysulfone [9] and glass membrane [10]) via the attachment of functional groups, such as - O H , - N H 2, - S O 3 H , - C O O H , -CONH 2 or epoxy, by chemical modification or graft polymerization. Using the above membranes, affinity membranes were prepared, by introducing group-specific ligands, such as metal chelates, which bind strongly the proteins that contain histidine, cysteine or trypto-

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phan, or reactive dyes, which mimic the conformation of nicotinamide adenine dinucleotide (NAD) and bind various kinases and dehydrogenases. The immobilized metal affinity membranes were prepared by first introducing epoxy groups, either via the radiation-induced graft polymerization of glycidyl methacrylate to polyethylene membranes [5], or the silanization of glycidoxypropyl trimethoxy silane to glass membranes [10], or the metallization reaction between n-butyllithium and polysulfone membranes [9]. This was followed by the chemical reaction between the epoxy group and iminodiacetic acid (IDA) and the binding of metal ions (copper, nickel or zinc) to the acidic groups. Triazine dyes (Cibacron Blue F3GA or one of the Procions) were coupled, using polyethyleneimine (PEI) as a spacer, to nylon membranes by Kula et al. [11-13], and their adsorption capacities for lysozyme, bovine serum albumin (BSA), malate dehydrogenase (MDH) and glucose6-phosphate dehydrogenase (G6PDH) investigated. Liu and Fried [14] reported the breakthrough behavior of lysozyme through cellulose-Cibacron Blue 3GA membranes. Cibacron Blue F3GA and Active red K2BP were immobilized by Guo et al. [15] on microporous cellulose membranes and used to recover alkaline phosphatase. Spencer et al. [2] studied the affinity separation of human serum albumin (HSA) using Cibacron Blue F3GA grafted to a polyethyleneimine-coated titania microporous membrane. In the present study we report the use of dyemembranes based on chitosan [poly(2-amino-2-deoxy-D-glucose)], the deacetylated derivative of chitin. The latter compound is the most abundant natural biopolymer after cellulose. The chitosan molecules are hydrophilic because they contain a large number of reactive groups ( - O H and NH2), which can easily react with the chloride of the dyes to generate chitosan-dye membranes with high dye contents. Consequently, it is no longer necessary to increase the number of - N H 2 or - O H groups of the chitosan membrane. In contrast, the nylon membranes [1113], which are relatively hydrophobic, need additional reactive groups. In addition to its chemical reactivity, chitosan has an excellent film-forming ability. Chitosan microporous membranes were prepared through the phase inversion method and subsequently coupled with Cibacron Blue F3GA to gener-

ate chitosan-dye affinity membranes. Human serum albumin (HSA) was selected as a model protein and its adsorption on the chitosan based membrane investigated.

2. Experimental 2.1. Materials Polyethylene glycol (PEG, M w 35 000), chitosan (medium molecular weight, M w 750000), and Cibacron Blue F3GA were purchased from Fluka. Human serum albumin (HSA, fraction V), sodium chloride, sodium thiocyanate, sodium carbonate, sodium azide, sodium hydroxide and Tris-HC1 were obtained from Sigma, microporous polyethersulfone membranes (Supor-800, 47 mm) from Gelman Sciences Company and HC1 aqueous solution (36.5-38 wt%) from Fisher Scientific Company. The buffer for HSA contained 50 mM Tris-HC1 and 50 mM NaC1 at pH 8. The eluant was 0.5 N NaSCN in the buffer for HSA.

2.2. Apparatus An ultrafiltration cell ($2403, effective membrane area 11.3 cm 2, filter diameter 43 mm, Nuclepore, Costar Incorp.) was employed for the affinity separation. A peristaltic pump (ISCO) was used for the pumping of the protein solution, the buffer and the eluant through the membrane.

2.3. Assays The concentration of proteins was determined with an U V / V i s Spectrophotometer (DU-70, Beckman Instruments, Inc.) at 280 nm. The amount of Cibacron Blue F3GA coupled to the chitosan membrane was determined spectrophotometrically at the wavelength of maximum absorption (515 nm).

2.4. Preparation of chitosan microporous membranes The chitosan microporous membranes were prepared by the phase inversion method. A solution of chitosan was first obtained by dissolving 1 g chi-

X. Zeng, E. Ruckenstein / Journal of Membrane Science 117 (1996) 271-278

tosan in 100 ml of 1 vol% aqueous acetic acid solution containing 5 g polyethylene glycol as porogen. 2 ml of this solution was poured over a support [microporous polyether sulfone membrane (47 mm diameter, 150 /xm thickness and 0.8 /zm pore size)] placed in a plastic Petri dish (50 mm diameter), and allowed to evaporate for 1.5 h at room temperature. The wet chitosan layer supported on the polyether sulfone was immersed for 12 h into a NaOH solution containing 3 g NaOH in 100 ml distilled water, in order to extract the porogen and to generate a microporous membrane. This was followed by washing several times with distilled water and by wet storage for later use. The support provides better mechanical properties to the membrane. 2.5. The coupling of the Cibacron Blue F3GA to the chitosan membrane Cibacron Blue F3GA was covalently coupled to the chitosan membrane via the nucleophilic reaction between the chloride of its tfiazine ring and the hydroxyl or amino group of the chitosan molecule, under mild alkaline conditions. The coupling procedure [16] previously used for agarose gels was employed. First, the chitosan membrane was immersed, at 60°C, for 1 h, in 20 ml of an aqueous solution containing 1 g Cibacron Blue F3GA in 100 ml distilled water. Second, 1.2 g sodium chloride was added to the solution at 60°C in order to stimulate the adsorption of the dye. Since the NaC1 concentration is large (1.0 M), the electrostatic interactions between the dye and chitosan are negligible; the salting out of the dye is probably responsible for the stimulation of adsorption. Indeed, at large ionic strength, the water molecules are so well organized among themselves by the large number of ions, that the dye molecules become less compatible with water. After 2 h, the temperature of the solution was increased to 80°C and the pH increased from 8 to 10.5 by the addition of 0.4 g sodium carbonate. Under these conditions, a chemical reaction takes place between the group of the dye containing CI and one OH or NH 2 group of the chitosan, with the elimination of HC1, resulting in the coupling of Cibacron Blue F3GA to the chitosan membrane. 1 h later, the solution was cooled down to room temperature, and rinsed with warm distilled water to remove

273

Table 1 The characteristics of Cibacron Blue F3GA-chitosan membrane Thickness of the supported chitosandye layer in the wet state Thickness of supporting membrane (polyether sulfone) Effective membrane area Membrane volume in wet state of the chitosan supported layer Cibacron Blue F3GA coupling capacity Hydraulic permeability of pure water at 25°C

260/zm 150/zm l 1.3 cm 2 0.294 cm 3 0.248 g / g chitosan 4.94× 10 -2 m l / (s cm 2 atm)

the unbound dye, until the solution became colorless. In order to avoid the future leakage of the dye during protein adsorption, the membrane was washed with a large excess of buffer and eluant. Finally, the affinity membrane was stored in 0.4 wt% sodium azide aqueous solution to prevent biodegradation. The characteristics of the chitosan-dye membrane are presented in Table 1. The rejection of HSA by the chitosan-dye membrane in the presence of NaSCN was determined by passing 50 ml of 1 m g / m l HSA solution containing 50 mM NaC1 and 0.5 N NaSCN at a rate of 5 m l / m i n through the membrane. The presence of the eluant NaSCN in the aqueous solution prevented adsorption. More than 98% HSA was not adsorbed. This indicates that the pore sizes of the chitosan-dye membrane were large enough to allow HSA (molecular weight 68 500 Dalton) to permeate through the membrane. The permeability of the membrane for water at 20°C and 10 psi pressure drop was 4.94 × 10 -2 m l / ( s atm cm2). 2.6. The determination of the amount of Cibacron Blue F3GA bound to the chitosan membrane The amount of dye coupled to the membrane constitutes an important parameter in protein adsorption. The dye content of the membrane can be determined by immersing the chitosan-Cibacron Blue F3GA membrane in an appropriate medium and measuring the absorbance of the solution at the }~max (515 nm) of the dye. The hydrolysis in a 6 N HC1 aqueous solution at 40°C for 1 h [17], or the hydrolysis in 50 vol% acetic acid or formic acid in water at 100°C for 10 min [18] was used for agarose beads,

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and the dissolution in a phenol-methanol mixture for nylon membranes [11]. However, these methods were not suitable for the chitosan-Cibacron Blue F3GA membranes, because they could not release the dye molecules from the membrane. In the present study, a modified version of Chamber's method [17] was employed. The dye containing sample was introduced into 5 ml concentrated hydrochloric acid solution (12 N) at 80°C for 15 min, then the test tube was cooled down to 25°C, followed by dilution with 5 ml distilled water. The concentration of the dye was determined by measuring the absorbance at 515 nm, using a Beckman U V / V i s spectrophotometer and a calibration curve.

~

7

outlet Fig. 1. Apparatus for HSA adsorption and desorption: 1, protein solution; 2, washing buffer (50 mM Tris-HC1/50 mM NaCl, pH 8); 3, eluent (0.5 M NaSCN in buffer); 4, peristaltic pump; 5, ultrafiltration system; 6, UV/Vis detector; 7, profile recorder; 8, printer.

2.7. Equilibrium adsorption of protein The chitosan-Cibacron Blue F3GA membranes were immersed in 5 ml buffer for 30 min, then introduced in protein solutions of different concentrations, and shaken at 200 rpm for 12 h at 25°C to achieve adsorption equilibrium. Then, the membranes were removed, washed with buffer solution five times (4 ml each) to remove the weakly adherent protein molecules, and finally eluted five times successively with a solution 0.5 N NaSCN in buffer (4 ml each). SCN-, being a chaotropic ion, disorganizes the structure of water, thus stimulating the desorption of the protein. The eluates were collected and the concentration determined spectrophotometrically at 280 nm. The amount of protein adsorbed on the chitosan-dye membrane was obtained using a calibration curve of absorbance versus concentration.

2.8. Adsorption and desorption of HSA in the dynamic mode The dynamic adsorption experiment was carried out at 25°C in an ultrafiltration cell (Fig. 1) attached to an on-line U V / V i s detector (DU-70, Beckman) which measured continuously the absorbance of the eluates at 280 rim. The membrane was placed in the cell and 10 ml buffer (50 mM Tris-HC1/50 mM NaC1, pH 8) were pumped through the membrane. Then the buffer solution was replaced with a protein solution in buffer (0.5 m g / m l , 15 ml at various flow rates between 0.6 and 4.5 ml/min). After adsorption, a washing buffer (20 ml) at 1.3 m l / m i n was

used to remove the unbound protein until the absorbance of the eluate decayed to zero. Finally, 20 ml eluant (0.5 N NaSCN in buffer) at 1.3 m l / m i n was employed to elute the protein adsorbed by the membrane. The absorbance of the eluate was determined continuously at 280 nm, and the whole adsorption/washing/desorption profile obtained. The adsorption capacities at different flow rates were thus determined.

3. Results and discussion

3.1. Dependence of the flow rate through the chitosan-dye membrane on pressure drop The most significant feature of the affinity membranes is the fast flow rate and low pressure drop, in comparison with the slow flow rate and high pressure drop of the affinity column chromatography. The relationship between the pressure drop and the flow rate of water through the chitosan-dye membranes is given in Fig. 2. With the increase of pressure drop, the flow rate increases almost linearly at low pressure drops (less than 10 psi), but more slowly at high ones. This happens because the membrane is compressed at high pressure drops. One can see from Fig. 2 that relatively high flow rates are achieved at very low pressure drops. For instance, at a pressure drop of 10 psi, the flux is 2.12 m l / m i n / c m 2.

X. Zeng, E. Ruckenstein/ Journal of Membrane Science 117 (1996) 271-278

275

0.25

50

45 40

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0.15

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i

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15

20

25

Pressure drop(psi) Fig. 2. The relationship between flow rate and pressure drop of pure water through the chitosan-Cibacron Blue F3GA membrane.

Effect of temperature on the coupling Cibacron Blue F3GA to the chitosan membrane

3.2.

60

of

Cibacron Blue F3GA is a reactive dye, since its chloride can react with the -OH or - N H 2 groups of chitosan to generate a covalent bond (Fig. 3). One can see from Fig. 4 that with an increase in temperature from 50 to 80°C, the amount of dye immobilized on the membrane increases. This happens because the reaction rate at the surface increases as the temperature increases. For this reason all chitosan-

70

80

Temperature (*C)

30

Fig. 4. The relationship between the coupling temperature and the amount of Cibacron Blue F3GA coupled to the chitosan membrane.

dye membranes used for the latter experiments were prepared by coupling Cibacron Blue F3GA to the chitosan membranes at 80°C. 3.3. Protein adsorption equilibrium The adsorption isotherm of HSA at 25°C and pH 8 in a solution containing 50 mM Tris-HC1 and 50 mM NaC1 is presented in Fig. 5. The adsorption isotherm has a Langmuirian shape. With increasing 12

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C, HSA concentration in solution(mg/ml (el Fig. 3. Reaction scheme of the coupling of Cibacron Blue F3GA (a) to the chitosan membrane (b).

Fig. 5. Adsorption isotherm of the binding of HSA to the chitosan-Cibacron Blue F3GA membrane from HSA solutions containing 0.05 M Tris-HC1 and 0.05 M NaC1 at pH 8 and 25°C after 12h.

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Table 2 Equilibrium adsorption capacity of chitosan-Cibacron Blue F3GA membranes and undyed chitosan membrane in 10 ml 0.5 mg/ml HSA solution at 25°C Membrane a

HSA q (mg/cm 3)

I II III IV

8.88 9.23 8.58 2.62

(2)

C / q = C / q m -b K d / q m

The values of K d and qm can be determined from the straight-line plot of C / q against C by linear regression. One obtains K a = 0 . 1 1 5 m g / c m 3 and qm 10.04 m g / c m 3. Table 2 presents the values of q for three dyed and one undyed chitosan membranes, prepared under identical conditions (10 ml of 0.5 m g / m l H S A solution at 25°C and pH 8 for 12 h) and demonstrates that the membranes behave reproducibly. The small adsorption capacity of the undyed chitosan membrane is probably due to the low electrostatic attraction between the weakly cationic chitosan molecules and the anionic H S A molecules ( p I = 4.9). In order to investigate the influence of the support (polyethersulfone membrane) on the protein adsorption, Cibacron Blue F 3 G A was bound to a polyethersulfone membrane under the same conditions as the supported chitosan membrane and kept in 10 ml 0.5 m g / m l H S A solution at pH 8 for 12 h. The amount adsorbed by the membrane was 1 3 . 9 / z g / c m 2, which represents about 6% of the adsorption of the chit o s a n - d y e membrane supported on polyether sulfone (about 231 / z g / c m 2 ) . =

I, II, III: chitosan-Cibacron Blue F3GA membrane supported on polyether sulfone; IV: chitosan (undyed) membrane supported on polyether sulfone. a

protein concentration in solution, the amount per unit volume of H S A adsorbed by the membrane increases almost linearly at low concentrations, below about 0.1 m g / m l , then increases less rapidly and approaches saturation. It becomes constant when the protein concentration is greater than 1.0 m g / c m 3. The adsorption equilibrium can be described by the equation q = q m C / ( K d "q- C)

Eq. (1) can be rewritten in the form

(1)

where C ( m g / c m 3) is the concentration of H S A in solution, q is its concentration in the membrane ( r a g / c m 3 ) , qm is the maximum adsorption capacity ( m g / c m 3) and K d is a constant.

Table 3 Saturation protein/enzyme adsorption capacities on different affinity membranes Membrane Ligand, coupling capacity Proteins/ enzymes a HEC-coated PS PEI-coated titania Modified nylon-6 Modified nylon Sartobind Blue 2 Modified nylon Regenerated cellulose

Protein A, 2.4-3.1 mg/ml Cibacron Blue F3GA, 0.84 mg/ml Anti-BSA IgG, 0.04 mg/mg Cibacron Blue F3GA, 0.021 mg/mg Cibacron Blue F3GA Cibacron Blue F3GA, 0.42-5.88 mg/ml Cibacron Blue F3GA, 0.10 mg/mg

Poly(ether-urethane-urea) Sartobind-epoxy Cellulose acetate GMA-graftedPE Chitosan-coatedSPES Chitosan/PES

ProteinA Protein A, 5.9/zg/mg Protein A / G Phenylalanine, 0.025 -0.1 mg/mg Protein A, 6.43 mg/ml Cibacron Blue F3GA, 0.248 mg/mg

IgG HSA BSA Lysozyme G6PDH MDH Alkaline Phosphatase IgG IgG IgG BGG lgG HSA

Saturationadsorption capacity

Ref.

16 mg/cm 3 4 mg/cm 3, 40/zg/cm e

[ 1] [2] [7] [ 11] [ 12] [13] [ 15]

4.4-6.1 mg/cm 3, 87-122 /zg/cm 2 0.83 mg/cm 3, 17.5/zg/cm 2 10-20 mg/cm 3, 200-400/zg/cm 2 5.0-10 mg/cm 3, 150-300/zg/cm 2 4.8 mg/cm 3, 105/zg/cm 2 41 mg/cm 3 10.2 mg/cm 3, 265/zg/cm 2

[ 19] [20] [21] [22] [23] this study

IgG, immunoglobulinG; HSA, human serum albumin; BSA, bovine serum albumin; G6PDH, glucose-6-phosphatedehydrogenase; MDH, malate dehydrogenase; BGG, bovine gamma globulin; SPES, sulfonated polyethersulfone; HEC, hydroxyethyl cellulose; PS, polysulfone; PES, polyethersulfone; PE, polyethylene; GMA, glycidyl methacrylate; PEI, polyethyleneimine. a

X. Zeng, E. Ruckenstein / Journal of Membrane Science 117 (1996) 271-278

277

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1.5

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2.5

3

3.5

4

4.5

Flow rate(ml/min) 10

20

30

40

50

Tim~minutes) Fig. 6. The adsorption and desorption profile of HSA on Chitosan-Cibacron Blue F3GA membrane: (a) adsorption from a total flow of 15 ml of 0.5 m g / m l HSA solution at a flow rate of 0.6 m l / m i n ; (b) washing with 20 ml buffer (50 mM Tris-HC1/50 mM NaCI, pH 8) at a flow rate of 1.3 m l / m i n ; (c) elution with a total flow of 20 ml 0.5 M NaSCN in buffer at a flow rate of 1.3 ml/min.

Comparing with other results from literature (Table 3), one can see that chitosan has a high coupling capacity for Cibacron Blue F3GA, and that the chitosan-Cibacron Blue F3GA membrane exhibits a high affinity adsorption toward HSA. Table 4 provides a comparison between the total amounts of HSA adsorbed from HSA solutions and desorbed from a Cibacron Blue F3GA-chitosan membrane, and shows that they are near to one another.

Fig. 7. The relationship between the flow rate of the feed solution and the adsorption capacity of the chitosan-dye membrane for a total flow of 15 ml 0.5 m g / m l HSA solution.

buffer at pH 8. The adsorption/desorption of the protein was achieved in less than 1 h. From Fig. 6 one can calculate that the amount adsorbed is 2.34 mg, and the amount desorbed is 2.07 mg. The two are almost equal. The effect of the flow rate on the adsorption of HSA on chitosan-dye membrane was also investigated and the results are presented in Fig. 7. The adsorption capacity of the membrane decreases from 8.85 to 2.12 m g / c m 3 with the raise of the flow rate from 0 to 4.5 ml/min. The adsorption capacity decreases rapidly with increasing flow rate but is near to the equilibrium value for flow rates below 0.1 ml/min.

3.4. Effect of flow rate on the protein adsorption

4. Conclusions

The adsorption/desorption profile of HSA solution on the chitosan-Cibacron Blue F3GA membrane is presented in Fig. 6, which shows that the protein can be easily eluted with 0.5 N NaSCN in

Chitosan-Cibacron Blue F3GA affinity membranes were prepared by (1) dissolving the chitosan in a dilute acetic acid solution, (2) pouring the solution containing porogen (PEG) over a microp-

Table 4 Comparison between the total amounts of HSA adsorbed by and desorbed from a Cibacron Blue F3GA-chitosan membrane Amount of HSA introduced ( ~ g )

Amount of HSA remaining in solution after 12 h ( ~ g )

Membrane uptake ( ~ g )

Total amountof HSA desorbed ( ~ g )

5000 2500

2210 780

2790 1720

2430 1480

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X. Zeng, E. Ruckenstein / Journal of Membrane Science 117 (1996) 271-278

orous polyethersulfone membrane, and (3) coupling the membrane with the dye under mild alkaline condition. This dye-coupled membrane provides relatively large flow rates at low pressure drops. The adsorption of human serum albumin (HSA) in the chitosan-Cibacron Blue F3GA membrane was investigated under equilibrium and dynamic conditions. The dynamic adsorption to and desorption of HSA from this membrane were achieved in less than 1 h. Increasing the flow rate of the feed solution decreases the amount adsorbed, because of shorter residence times. The results show that the chitosanCibacron Blue F3GA affinity membranes have high adsorption capacities for human serum albumin.

[10]

[11]

[12]

[13]

[14]

[15]

References [1] E. Klein, E. Eichholz and D.H. Yeager, Affinity membranes prepared from hydrophilic coatings on microporous polysulfone hollow fibers, J. Membrane Sci., 90 (1994) 69-80. [2] Y. Li and H.G. Spencer, Dye-grafted, poly(ethylene imine)coated, formed-in-place class affinity membranes for selective separation of proteins, in W. Shalaby et al. (Eds.), Polymers of Biological and Biomedical Significance, American Chemistry Society, Washington, DC, 1994, pp. 297-305. [3] E. Klein and L. Silva, Hydrophilic semipermeable membranes based on copolymers of acrylonitrile and hydroxyalkyl esters of methacrylic acid, US Pat., 5,039,420 (1992). [4] T.B. Tennikova, M. Bleha, F. Svec, T.V. Almazova and B.G. Belenkii, High-performance membrane chromatography of proteins, a novel method of protein separation, J. Chromatogr., 555 (1991) 97-107. [5] H. lwata, K. Saito and S. Furusaki, Adsorption characteristics of an immobilized metal affinity membrane, Biotechnol. Progr., 7 (1991) 412-418. [6] J.L. Manganaro and B.S. Goldberg, Protein purification with novel porous sheets containing derivatized cellulose, Biotechnol. Progr., 9 (1993) 285-290. [7] K. Kugel, A. Moseley, G,B. Harding and E. Klein, Microporous poly(caprolactam) hollow fibers for therapeutic affinity adsorption, J. Membrane Sci., 74 (1992) 115-129. [8] A. Fabre, Hydrophilic PVDF semipermeable membrane, US Pat., 4,810,384. [9] K. Rodemann and E. Staude, Synthesis and characterization

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