Affinity membrane chromatography for the analysis and purification of proteins

J. Biochem. Biophys. Methods 49 Ž2001. 199–240 www.elsevier.comrlocaterjbbm

Review

Affinity membrane chromatography for the analysis and purification of proteins Hanfa Zou ) , Quanzhou Luo, Dongmei Zhou 1 National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116011, China

Abstract Affinity chromatography is unique among separation methods as it is the only technique that permits the purification of proteins based on biological functions rather than individual physical or chemical properties. The high specificity of affinity chromatography is due to the strong interaction between the ligand and the proteins of interest. Membrane separation allows the processing of a large amount of sample in a relatively short time owing to its structure, which provides a system with rapid reaction kinetics. The integration of membrane and affinity chromatography provides a number of advantages over traditional affinity chromatography with porous-bead packed columns, especially with regard to time and recovery of activity. This review gives detailed descriptions of materials used as membrane substrates, preparation of basic membranes, coupling of affinity ligands to membrane supports, and categories of affinity membrane cartridges. It also summarizes the applications of celluloserglycidyl methacrylate composite membranes for proteins separation developed in our laboratory. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Affinity chromatography; Membrane stationary phases; Monolithic columns; Proteins

1. Introduction Current limitations in bead-packed column-liquid chromatography include a relatively time-consuming and high-pressure packing process, a high-pressure drop in the columns and the slow diffusion of solutes within the pores of the bead matrix. Membrane chromatography is one of the significant chromatographic inventions during the past )

Corresponding author. Tel.: q86-411-3693409; fax: q86-411-3693407. E-mail address: [email protected] ŽH. Zou.. 1 Present address: College of Chemical Engineering, Dalian University of Science and Technology, Dalian 116011, China. 0165-022Xr01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 0 2 2 X Ž 0 1 . 0 0 2 0 0 - 7

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decade. The highly efficient process of membrane-chromatographic separation is based on the use of thin layers of finely organized and well-controlled macroporous polymeric stationary phases in the form of rigid disks of 2–3 mm thickness w1,2x. As a result of the convective flow of the solution through the pores, the mass transfer resistance is tremendously reduced. This results in rapid processing, which greatly improves the adsorption, washing, elution, and regeneration steps. Due to the macroporous structure of the membrane support, membrane chromatography has a lower pressure drop, higher flow-rate, and higher productivity than column chromatography. The additional advantages of easy packing and scale-up, as well as the unlikely foulingrclogging, make membrane chromatography an ideal large-scale separation process for the purification and recovery of proteins and enzymes w3–36x. A number of commercially available membrane devices with their respective manufactures and categorized by type of interactive chemistry or activity have been described w32x. Many publications have reported the performance of adsorptive membranes Žion-exchange membranes, affinity membranes, reversed-phase membranes, and hydrophobicinteraction membranes., as well as their theoretical description and optimal design w22,37–43x. Several reviews have described the fundamental mechanisms, operations, and applications of adsorptive membrane chromatography w14,31,32,44,45x. Roper and Lightfoot w32x reviewed the membrane substrates, coupling procedures, separation configuration or geometry, performance, operation, and application of adsorptive-membrane chromatography. Zeng and Ruckenstein w44x have presented methods for the preparation of adsorptive membranes and have particularly introduced the novel macroporous chitin and chitosan membranes for protein separations. Tennikova and Freitag w45x have summarized the state of the art in high-performance monolithic and especially high-performance monolithic disk chromatography ŽHPMDC., as well as the current understanding of the theory of protein separation by HPMDC. The basic differences between the monolithic disks, columns packed with conventional stationary phases Žincluding perfusion and micropellicular particles., and monolithic columns was outlined and the applications of HPDMC to protein isolation and analysis have also been presented. Recently, the gradient and isocratic high-performance membrane-chromatographic separation of small molecules with Convective Interaction Media ŽCIM. disks of different composition was reported and the mechanism was explained w43x. Rapid developments in biotechnology and the pharmaceutical potential of polypeptides, proteins and polynucleotides demand reliable, efficient methods for the purification of large amounts w46x. Affinity chromatography is unique among separation methods as it is the only technique that permits the purification of proteins based on biological functions rather than individual physical or chemical properties w47x. The high specificity of affinity chromatography is due to the strong interaction between the ligand and proteins of interest. Membrane separation allows the processing of a large amounts of sample in a relatively short time owing to its structure, which provides a system with rapid reaction kinetics w48x. The integration of membrane and affinity chromatography provides a number of advantages over traditional affinity chromatography with porousbead-packed columns, especially with regard to time and activity recovery w49,50x. Tejeda et al. w51x have developed a method for optimal affinity column design, based on the solution of the Thomas kinetic model for frontal analysis in membrane column

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adsorption. The method has allowed choosing suitable membrane operating conditions, column dimensions, and processing time to maximize the throughput when an operating capacity restriction in the range of 80–95% of the column capacity is used. In addition, the method for preparation of molecularly imprinted polymer ŽMIP. membranes, as well as their separation mechanisms and transport properties have been reviewed by Piletsky et al. w52x. The nature of the selectivity of microporous MIP membranes was discussed and a descriptive model for transport selectivity via specific AgatesB was developed. The application potential of MIP membranes, especially for affinity chromatography and membrane sensors, was also outlined. This review focuses on methods for the preparation of affinity membranes and, in particular, introduces the glycidyl methacrylate ŽGMA.rcellulose composite affinity membranes and their applications in the separation of proteins.

2. Preparation of affinity membranes Like in the preparation of affinity stationary phases for packed columns, three steps are usually involved in the preparation of affinity membranes: Ž1. preparation of the basic membranes, Ž2. activation of the basic membranes, and Ž3. coupling of affinity ligands to the activated membranes. Ideal membrane substrates used for affinity membrane chromatography should fulfil the following conditions: Ž1. proper pore structures and mechanical strength for use at high-flow rates and low back pressure, in rapid processing, Ž2. availability of reactive groups Žsuch as –OH, –NH 2 , –SH, –COOH. for the further coupling of spacer arms or ligands, Ž3. chemical and physical stability under harsh conditions of high temperature or chemical sterilization, Ž4. a hydrophilic surface for higher recovery of protein activity. There are many commercially available materials to choose from, and they include organic, polymeric, inorganic, and composite materials, as listed in Table 1. 2.1. Preparation of basic membranes 2.1.1. Cellulose and its deriÕatiÕe Cellulose and its derivatives are popular substrates used for the preparation of membranes. Cellulose is composed of linear units of b-1,4-linked D-glucose with occasional 1,6-bonds, as shown in Fig. 1. This makes the materials relatively strong, fibrous, and uniform. Native and derivatized cellulose membranes are soluble only in some strong acids w53,91x. The ion-exchange activity of residual carboxylic and aldehyde side groups in cellulose can be neutralized by borohydride reduction, and secondary hydrophobic interactions are reduced by the high level of hydration in these membranes. However, the application of native cellulose membranes to the purification of proteins is limited because their structure would be destroyed under the alkaline condition. In addition, the number and ratio of the reactive groups Ž –CH 2 OH. in cellulose molecules are much lower than in agarose, and this results in a rather low ligand density of cellulose membrane Žonly 1% of the ligand density of agarose..

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Table 1 Common geometries and materials of chromatographic membranes Geometry

Material

References

Thin sheet

Cellulose Regenerated cellulose CelluloserGMA composite PolyŽglycidyl methacrylate-co-ethylene dimethacrylate. PolyŽglycidy methacrylate. PolyŽmethyl methacrylate, acrylonitrile. Chitin and chitosan Nylon PolyŽHEMA. Polyamide Polyethylene PolyŽethylene vinyl alcohol. PolyŽpropylene. Silicon dioxide glass CelluloserGMA-DEAEMA copolymer PolyŽstyrene-co-divinylbenzene. PolyŽchloromethyl styrene-co-divinylbenzene. PolyŽglycidyl methacrylate-co-ethylene dimethacrylate. X PolyŽ N, N -methylenebisacrylamideco-acrylamide-co-butyl methacrylate

w11,25,28,53–55x w56x w57–60x w1,8,9,61–63x w64x w5x w65–69x w12,13,48,70,71x w72–77x w78–83x w7,84x w24x w6x w85x w86x w38,87,88x w39x w22,89,90x w40x

Hollow fiber

Spiral-wound Cross-linked rod

polyŽHEMA.: polyŽ2-hydroxylethyl methacrylate..

Regenerated cellulose and cellulose acetate membranes can be obtained through phase inversion. They present a hydrophilic surface and abundant reactive hydroxyl groups, as well as a low, non-specific adsorption. However, the mechanical strength of these membranes is poor. To overcome these shortcomings, several methods have recently been proposed w54,92,93x. Guo et al. w54x have described a method for the preparation of macroporous cellulose membranes. Filter paper was dispersed in a basic aqueous solution containing a certain amount of NaBH 4 . Then, the mixture was heated and kept boiling until a uniform pulp was obtained. The pulp was cast on a glass plate and frozen at y30 8C in a freezer for 45 min. After it had thawed at room temperature, the plate was immersed in 10% HCl for 1 h. The cellulose film was then rinsed with water until neutral, and was dried in air after being dipped in acetone for 2.5 h. Cross-linked cellulose membranes were prepared after the film had reacted with epoxy propane chloride at 50 8C for 3 h. Macroporous cellulose membranes were indirectly obtained by deacetylating the cellulose acetate membranes with a methanolic KOH solution at room temperature for 6 h w92,93x. These membranes were hydrophilic and pliable and hardly swelled in various solvents. The pore sizes of these membranes ranged from 0.1 to 10 mm Žmainly 3–5 mm.. Cellulose derivatives of chitin and chitosan have been used by Zeng et al. w66,68,69,94x to prepare macroporous membranes. It was observed that they have a finely controlled pore structure and good mechanical properties. Chitin and chitosan are good biological materials due to their easy availability, hydrophilicity, biocompatibility, and chemical reactivity. Chitin, a poly Ž2-amino-2-deoxy-D-glucose., which is presented in the outer

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Fig. 1. Structures of cellulose, chitin, and chitosan.

shells of lobsters, shrimps, and crabs, is the second most abundant natural polymer. Chitosan is the deacetylated product of chitin Žsee Fig. 1.. The preparation of a macroporous chitosan membrane involves five steps: Ž1. Chitosan is dissolved in a diluted acetic acid solution containing a certain amount of glycerol. Ž2. Silica particles of a selected size are added in the solution. Ž3. The solution is poured onto a glass plate, and the solvent evaporated. Ž4. The silica is removed by immersing the dried membrane in a NaOH solution Žchitosan is soluble in acidic solutions, but insoluble in alkali., and the macroporous structure is formed. Heat treatment stabilizes the pore structure and improves the mechanical properties. Ž5. The porous chitosan membrane is rinsed with distilled water and stored in either ethanolrmethanol or in the dry state after an aqueous glycerol treatment. This is a simple and effective method for obtaining macroporous chitosan membrane with good mechanical strength and a uniform three-dimensional pore network. The pore size and porosity of the chitosan membranes can easily be controlled by changing the amount and size of silica particles added. The silica particles used for the preparation of macroporous chitosan membranes have a broad size dispersion between 15 and 40 mm. Three kinds of membranes were obtained with the average pore sizes of 19.5 " 13.0, 6.6 " 3.7, and 2.5 " 1.2 mm, respectively. The broad dispersion of silica particle sizes leads to a broad range of pore sizes on membranes. By

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varying the weight ratio of silica to chitosan, the porosity of the membrane can be easily controlled. The higher the weight ratio of silica, the higher is the porosity obtained. Cross-linked chitosan is prepared to keep chitosan from dissolving in acidic solutions. However, the functional groups ŽCH 2 OH and NH 2 . are destroyed and, hence, the possible ligand density is decreased w67x. The amine groups on membrane react more easily with the cross-linking and modifying agents. Therefore, the reaction conditions should be chosen carefully to maintain the reactive amine groups. Macroporous chitin membranes can easily be obtained indirectly via the acetylation of chitosan membranes with acetic anhydride in methanol solution at 50 8C for 1 h. The acetylated chitosan membranes have good chemical stability and mechanical properties. Chitin membranes are stable in both acidic and basic solutions, as well as in common organic solvents. Another significant features of the chitin membranes is that they contain N-acetyl-D-glucosamine units, which are affinity ligands for lysozyme and wheat germ agglutinin. Thus they can be utilized directly for the affinity separation of these proteins without further chemical modification. 2.1.2. Composite cellulose Cellulose exhibits rather good mechanical strength, owing to its semicrystalline structure. This structure, however, impedes the introduction of enough functional groups into cellulose, and this results in cellulose membranes having a lower adsorption capacity than agarose beads. Although some modifications of cellulose, for example, Divicell, a commercially available bead-type cellulose, have been reported recently, the ligand density available on the surface of the modified cellulose is only 40–50 mmrml, and it displays a low adsorption capacity for proteins w95x. To overcome this shortcoming, each monomer containing an active group in a polymer was covalently coupled to a few activated hydroxyl groups in cellulose. As a result, not only the ligand surface density on cellulose could be increased greatly, but also the composite matrix based on the half-rigid cellulose materials showed much greater strength. This permits fast flow of liquids through the composite membrane for the purpose of fast purifications. The composite cellulose membranes were prepared by grafting cellulose with acrylic polymers formed by polymerizing glycidyl methacrylate w96–98x. This method can greatly improve the density of functional groups by the introduction of epoxide groups and increase the mechanical strength of membrane. Yang et al. w96x have reported procedures for the preparation of composite cellulose membranes as follows Žsee Fig. 2.: Ž1. Cellulose pulp of short cotton fiber was dispersed in water. Then glycidyl methacrylate monomer was added to a reactor at 70 8C, followed by ammonium persulfate and sodium thiosulfate as a redox catalyst. The polymerization reaction proceeded for 1–2 h. Ž2. The grafting reaction was performed for 1–2 h by raising the reaction temperature to 80 8C. Ž3. The resulting product was filtered and washed with a large volume of water. A composite cellulose matrix with epoxide groups as the reactive group for coupling ligands was thus prepared. The effects of the polymerization and grafting reaction temperatures and times on the properties of composite membranes were investigated. These reaction conditions notably affect the epoxide group density and the strength of the composite membrane. In polymerization at low temperatures, short chains are formed, resulting in lower epoxide group density on the membrane. The cellulose

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Fig. 2. Scheme for the preparation of composite cellulose membranes.

bearing epoxide groups, was subject to cross-linking by a ring-opening reaction at high grafting temperature. On the other hand, the strength of cellulose membrane, is also subject to the pulping degree of cellulose. The higher the pulping degree, the higher the strength of the membrane, but the lower the mass flux of the membrane. Therefore, it is essential to control the pulping degree of cellulose fiber to form membrane with higher mass flux. Furthermore, the character of the function groups derivatized on the surface of the cellulose also affects the strength of membrane. The strength of the membrane increases when it is coupled with hydrophilic groups, and decreases with hydrophobic groups. Zhou et al. w97x have prepared composite cellulose membranes with original cellulose fibers and fibers treated with alkaline solution. It was observed that alkali treatment of the cellulose fiber decreases the pressure resistance of the membrane to the mobile phases and greatly increases the volume accessible to the proteins, but it does not affect the immuno-adsorption capacity of human IgG on Protein A-immobilized membrane columns as much. This means that the alkali treatment of cellulose fibers only changes the void volume of the membrane greatly without apparent affect on the porosity and surface area of membrane. Alkali treatment of the cellulose fiber reduces the membrane-column efficiency significantly. Composite cellulose membranes were applied to prepare affinity columns by immobilization of metal ions, antigens, and antibodies, as well as dyes. The pore size distribution of the composite membrane ranges

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from 10 to 50 mm, which fully meets the requirements for fast mass transfer in bioprocessing w96x. 2.1.3. Polyamide and its deriÕatiÕe Due to their good mechanical and chemical stability, polyamide and nylon have gained wide acceptance in both laboratory and industrial use. Hydrolysis of nylon membranes is necessary to increase number of the active groups Ž –NH 2 . and to avoid non-specific binding between proteins and membrane substrates during separation. The residual carboxyl groups engendered by hydrolysis should be eliminated to prevent electrostatic interaction between the proteins and membranes. Hydrolysis conditions should be strictly controlled to obtain the maximum amount of primary amino groups, which provide reactive sites for further coupling of spacer arms or ligands. In general, the content of amino groups should be at least 20 mmrg of membrane substrate. Breifs and Kula w89x prepared affinity membranes with nylon-based membranes that had been treated to yield a hydrophilic surface. The affinity ligands, Cibacron Blue F3-GA, were chemically coupled with a nylon membrane for the purification of bovine serum albumin ŽBSA, Fraction V.. Pure BSA served as a model protein, whereas the enzyme pyruvate decarboxylase and formate dehydrogenase were recovered from crude extracts of Zymononas mobilis and Candida bodiniii, respectively. A commercially available nylon membrane was used by Birkenmeier and Dietze w12x to separate paraproteins from the plasma of patients suffering from paraproteinemia. In their work, the microporous nylon membrane, Immunodyne ŽPall, Glen Cove, NY, USA., was treated with aqueous alkali solution for 12 h at ambient temperature. The resulting membrane ware performed in a dead-end filtration mode to remove human monoclonal immunoglobulins, derived from paraproteinemic plasma. Unarska et al. w48x prepared a Protein A affinity membrane for the separation of g-globulin. Covalent immobilization of Protein A within the porous matrix of a nylon Loprodyne membrane ŽPall Bio Support, Portsmouth, UK. was achieved with modification of the 2-floro-1-methylpyridinium tolunene-4-sulphonate method. A detailed comparison between the membrane and agarose-bead-based affinity systems for the separation of human g-globulin was presented. Improvement was observed only when the solution of g-globulin was forced through the membrane pores. Charcosset w13x presented fractal dimension values for the kinetics of the binding of g-globulin molecules to Protein A immobilized on microporous nylon membranes and Sepharose beads for chromatographic applications. A nylon 66 microfiltration membrane was hydrolyzed with hydrochloric acid by Shang et al. w70x to prepare affinity material for endotoxin removal. Different activation agents for the immobilization of histidine ligand on the membrane were compared and evaluated. Membranes activated with 1,1X-carbonyldiimidazole achieved the highest histidine binding capacity. Although the hydrophobic surface of nylon membranes is improved by hydrolysis and chemical modification, their applications, especially in the separation of some hydrophobic proteins, are still limited by their hydrophobic surface w38x. Nylon membranes have a low concentration of terminal amino groups, and the direct activation of the nylon matrix leads to low ligand densities. To overcome these shortcomings, Beeskow et al. w99x prepared a composite membrane by coating hydroxyethyl cellulose ŽHEC. on the microporous polyamide membranes. The properties of chemical activity

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and low protein adsorption of HEC and those of microporous polyamide membranes, such as high consistency of the pore size distribution and mechanical rigidity, were combined to develop a new type of hydrophilic membrane. Immobilization of HEC on nylon membranes was expected to yield low non-specific adsorption of proteins and proper amount of active groups for affinity ligands. The initial activation of the nylon matrix by bisoxirane or formaldehyde was directed at the covalent immobilization of HEC on the membrane surface via amino end-groups or amide groups in the nylon chain. After the subsequent activation of the HEC hydroxyl groups by epichlorohydrin or bioxirane, iminodiacetic acid, a metal chelator, was immobilized on the HEC-coated membrane surface. The hydroxyl groups of HEC were suitable for further modification. The structure of the HEC polymer coated on the membrane surface. These coils were responsible not only for the reduction of the hydraulic permeability but also for 2- to 5-fold higher protein capacities of the affinity membranes than monolayer adsorption would yield. Formaldehyde-activated membranes produced a more compact structure of the HEC coating at close proximity to the nylon matrix than did bisoxirane-activated membranes. This results in slightly lower protein adsorption capacity and reduced accessibility of the immobilized ligands. A dextran-coated nylon membrane was prepared by Breifs et al. w89x. The ligands were first coupled with dextran and then the ligand-coupled dextran was attached in a partially covalent manner and partially adsorbed manner at the inner porous surface of the membrane that had previously been activated with trichloro-s-triazine ŽImmunodyne.. 2.1.4. Polysulfone and its deriÕatiÕe Polysulfone ŽPS. is suitable for the preparation of strong, thermally stable membranes. However, its strong hydrophobic and non-wettable surface is usually undesirable. The possibility of chemically modifying these surface properties is often precluded because of the absence of any convenient functional groups for linking surface modifiers permanently. This limitation applies, whether one seeks to change only the interfacial tension between the surface and the working solutions, or to modify the membrane surface with specific ligands for separation or catalytic purposes. Five different methods used to overcome this problem are described as follows: Ž1. Preparation of copolymers for membrane formation that have the desired functionality to confer both hydrophobic surfaces and accessible groups for derivative formation w100,101x. Ž2. Hydrophilic alloying polymers, which have some miscibility with the PS materials in the solid state, used to modify surface wettability w102,103x. Ž3. Strong hydrophobic bonding, achievable between selected amphiphilic polymers and the plastic w104x. Ž4. Using low degrees of substitution to introduce hydrophilic groups into the polymer backbone w105–107x. Ž5. Covalent techniques for surface coating on hydrophobic membranes w108,109x. Klein et al. w110x prepared cellulose-coated PS hollow-fiber membranes. The hydrophilic, derivatized microporous fibers combine the strong mechanical properties of PS and the chemical reactivity of cellulose coating. The combination could provide adequate binding capacity for a ligand protein and exhibit minimal non-specific binding. A combination method of adsorption and covalent linkage of the hydrophilic coating to PS microporous membranes was proposed. The hydrophilizing polymer was linked to

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the PS surface through covalent reaction with an activated end group of the polymer. The unmodified PS chain terminal groups were initially made to react with a diepoxide to produce a new reactive site. A hydrophilic polymer reacted with the activated site and served as the locus for the eventual immobilization of the ligands. The ligands of recombinant Protein A and an IgG-specific biomolecule were covalently linked either through amine or hydrazide functional groups, generated on the hydrophilic polymer coating. Higuchi et al. w106,107x modified the surface of the PS fibers with propane sulfone and SnCl 4 catalyst. The surface-modified fibers were immersed in acid solution after the surface reaction was completed. The immersion of the fibers in HCl solution yielded a lower molecular-weight cutoff than the originally surface-modified fiber. The fibers modified with propane sulfone showed less non-specific adsorption of proteins than the original and conventionally sulfonated fibers. In addition, hydroxide-type PS hollow fibers were obtained via reaction with propylene oxide and Friedel–Crafts catalyst by a one-step reaction that gives a hydrophilic surface. 2.1.5. Polyethylene and polypropylene Hydrophilic membranes such as cellulose acetate, poly Žvinyl alcohol., and polyacrylonitrile membranes have the superior characteristic of less non-specific adsorption of proteins. However, they do not usually have good thermal stability and are susceptible to chemical and bacteriological agents, whereas the hydrophobic membranes, such as polyethylene and polypropylene, have thermal stability and some chemical resistance. Surface modification of hydrophobic membranes that introduce hydrophilic segments on the surface may be an ideal method for combining both advantages of hydrophilic and hydrophobic membranes. Thermal stability and mechanical strength are maintained in the modified membranes due to the hydrophobic nature of polymer backbones by introducing transport characteristics of hydrophilic membranes, such as less non-specific adsorption of proteins w6,7,27,111x. Kim et al. w84x prepared a porous hollow-fiber membrane, containing L-phenylalanine as a pseudo-biospecific ligand, by radiation-induced grafting of glycidyl methacrylate onto polyethylene microfiltration hollow fibers, followed by coupling of the epoxide group produced with L-phenylalanine. A scheme for the coupling of the ligands to a porous polyethylene membrane was developed by Kiyohara et al. w112x. Two methods were employed for the introduction of activated groups, i.e., succinimide and epoxy groups, into a porous polyethylene hollow fiber by applying radiation-induced graft polymerization of acrylic acid ŽAAc. and glycidyl methacrylate ŽGMA., respectively, and subsequent chemical modifications. The succinimide group was attached via a reaction of the carboxyl group of the AAc-grafted polyethylene membrane with N-hydroxysuccinimide. The resultant membrane exhibited lower liquid permeability after the introduction of soybean trypsin inhibitor because the residual carboxyl groups on the graft chains induced a higher degree of extension of the graft chains. The epoxy group was introduced by grafting polymerization of epoxygroup-containing vinyl monomer ŽGMA.. The liquid permeability of the resultant membrane was retained at the original level when phenyalanine was introduced at a sufficiently high ligand density. The poly-GMA was found to be suitable for the introduction of the affinity ligands along with a hydrophilic group into a porous

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membrane, because it showed higher permeability and lower non-specific adsorption of proteins. 2.1.6. Poly(GMA-co-EDMA) monoliths Monolithic stationary phases have revolutionized protein chromatography, because they combine speed, capacity, and resolution in a unique manner. Since this kind of stationary phase does not contain particles but only flow-through pores, the mass transfer restrictions of the particle-packed column chromatography of large molecules do not apply and extremely fast separations become possible w18,113x. Thin discs of macroporous poly Žglycidyl methacrylate-co-ethylene dimethycralate. have been synthesized by free-radical polymerization of a mixture of a monovinyl monomer and a divinyl monomer, with heating w39,61,63x. The preparation procedures leads to finely controlled porous membranes with good mechanical strength and rich density of functional groups. Synthesis is initiated by azobisisobutyronitrile in the presence of porogenic solvents between two heated plates. Epoxy groups provide reactive sites for the further coupling of ligands. Sulfuric acid hydrolysis destroys residual underivatized groups to prevent secondary associations between proteins and substrates. Platonova et al. w62x developed an affinity-chromatographic method for the direct quantitative analysis of monospecific anti-peptide immunoglobulins Žantibodies. and, simultaneously, their semi-preparative isolation from blood serum of immunized animals. In their work, specifically prepared synthetic peptides with biological activity imitating that of the immunoglobulin binding sites of various proteins, were used as the selective ligands instead of native proteins. These ligands were immobilized by a single-step reaction that involved epoxy groups located on the pore surface of the porous polymer disc with amine groups of the peptide molecules. These novel immunosorbents, characterized by a large binding capacity, were well suited for high-throughput screening. The discs were used in a single-step enrichment of antibodies from both precipitated blood fractions and crude blood serum of immunized animals. Kasper et al. w10x proposed an affinity-chromatographic method for the fast, semi-preparative isolation of recombinant Protein G from E. coli cell lysate. Rigid, macroporous affinity discs based on a GMA-co-EDMA polymer were used as chromatographic supports. Human immunoglobulin G was immobilized by a single-step reaction. The globular affinity ligands were located directly on the pore wall surface and were therefore freely accessible to target molecules ŽProtein G. passing with the mobile phase through the pores. Giovannini et al. w8x prepared poly ŽGMA-co-EDMA. membranes for the separation of supercoiled plasmid DNA. Gradient and isocratic elution was investigated and high-performance membrane chromatography experiments were compared with similar ones performed on a conventional packed-bed column. Amatschek et al. w19x developed an affinity-chromatographic method in which peptides, derived from a combinatorial library, were used as immobilized ligands for the purification of human blood coagulation factor VIII. Trypsin, immobilized on molded macroporous poly ŽGMA-co-EDMA. carrier, exhibited significantly better mass-transfer characteristics than a conventional bead-packed column w22x. Viklund et al. w114x developed a photo-initiated, in situ polymerized system for the preparation of monolithic sorbents, based on glycidyl methacrylate and trimethylolpropane trimethacrylate. The ease of the preparation, the short time of the reaction, and

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the possibility of running the reaction at low temperature were some of the main advantages of the photo-initiated in situ polymerization compared to a thermally initiated polymerization. The effects of the parameters that control the properties and the characteristics of these new materials, predominantly with respect to their porous structures and flow characteristis, were investigated. The porous properties of the monoliths were a direct consequence of the quality of the porogenic solvents, as well as the percentage of cross-linking monomer and the ratio between the monomer and porogen phases. A mixture of isooctane and toluene was used as porogenic solvent. The higher the toluenerisooctane ratio, the higher the surface area and the smaller the pore size. The pore size distribution, which determines the specific surface area, could be controlled within a broad range by altering the thermodynamic quality of the porogen mixture w115–117x. The total amount of inert porogenic solvents in the polymerization mixture should be controlled to ensure the formation of a porous structure having both a pore size and a pore volume sufficiently large to allow flow-through operations at a reasonably low pressure. A decrease in porogen percentage in the system resulted in a decrease in the total pore volume. The use of the trivinyl monomer for the preparation of a monolithic material should provide a higher degree of cross-linking in the final polymeric sorbent and ensure the high rigidity and mechanical stability necessary for application in flow-through systems. The higher the ratio of the cross-linking agent to monomer, the higher will be the rigidity and mechanical stability of the final polymeric adsorbent. 2.1.7. Inorganic materials In addition to polymers, inorganic materials have also been used as membrane substrates. Li and Spencer w118x prepared an ion-exchange membrane with titanium dioxide. Polyethyleneimine ŽPEI., ion-paired with the oxide support, was subsequently cross-linked with glutaraldehyde to form a reactive film to which anion-exchange ligands were coupled. Titania is more alkali-stable than silica, and the adsorption of PEI on titania is straightforward. Alkaline conditions will not erode the PEI coating. Serafica et al. w85x prepared immobilized-metal-chelate affinity membranes by using glass hollow-fiber microfiltration membranes as substrate. The activation and coupling procedure for attaching the chelating agent, iminodiacetic acid, to the surface of the microporous glass hollow fiber membrane was optimized. The results were compared with those obtained with a commercial metal chelate adsorbent. The protein adsorption behavior of the metal chelate affinity membrane was also examined and compared with the results from a mathematical mode. 2.2. ActiÕation of basic membranes Once the basic membranes are prepared, they must be activated to acquire reactive groups for the coupling of ligands. The methods used for various affinity columns can be directly applied to membrane activation directly. 2.2.1. Cyanogen bromide actiÕation Cyanogen bromide activation introduced by Axen et al. w119x has become one of the most popular method. Activation is reasonably simple and involves attachment of the ligand via primary aromatic or aliphatic amino groups. However, cyanogen bromide has

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been reported to be strongly carcinogenic. Thus, proper precautions should be taken while handling it. All procedures should be carried out in a fume hood to avoid inhaling the poisonous gas. Cyanogen bromide activation is achieved by the following technique: the substrate was placed in 0.1 mM phosphate buffer ŽpH 7.2. and cooled to 4 8C. The solution is placed in an ice bath and cold aqueous cyanogen bromide is added. After gently shaking the solution for 10 min, it is filtered and the substrate is washed with cold water. 2.2.2. Epoxide actiÕation Reaction of OH groups with bioxiranes Žsuch as 1,4-butanediol diglycidyl ether., epichlorohydrin, or epoxy bromopropane will produce activated supports with oxiran groups w120x. Epoxide reagent is less toxic than cyanogen bromide and easily obtained. Because activation is simple, it plays an increasingly important role in the activation of separation media. Epoxide groups can be attached to the substrate by the following technique: the substrate is placed in 1 mM sodium hydroxide, containing 2 mgrml sodium borohydride, and 1,4-butanediol diglycidyl ether is added. The solution is mixed overnight at room temperature and finally the substrate is copiously washed with distilled water to obtain activated substrate. 2.2.3. Periodate oxidation Oxidation of polysaccharide supports has become a popular activation technique for the immobilization of proteins w121x. Reactive vicinal cis-hydroxyl groups within the matrix can be modified by treatment with sodium periodate. The result of this oxidation yields aldehydes that are easily converted into secondary amines by reductive amination or to hydrazides by reaction with dihydrazides. The reducing agent usually is a freshly prepared solution of sodium borohydride. Attachment of spacer arms or ligands is via primary amine groups. A great disadvantage of this technique is that undissolved salts and other compounds produced during the oxidation and reduction steps may destroy the micropores. A technique for performing periodate activation of biopolymers is as follows: the substrate, swollen in distilled water, is placed in 0.05 mM aqueous sodium periodate, and the mixture is stirred for 45 min at room temperature. Then, 2 mM ethylene glycol is added in and the mixture is rotated for a further 30 min. Now the activated substrate is ready to be used for coupling protein ligands. 2.2.4. Triazine actiÕation The triazine side chain is extensively used to bind chemically such reactive dyes as Cibacron Blue F3G-A to polysaccharide surfaces w122x. The technique involves activation of the matrix with 2-amino-4,6-trichloro-s-triazine during which a chlorine is replaced by solubilization groups. The ligand is covalently bound via a primary amino group in a second step during which another chlorine undergoes nucleophilic substitution. The reaction is allowed to proceed at room temperature, but the temperature may be raised to accelerate the reaction. A simplified technique for performing this process is as follows: the substrate is first swollen in distilled water and 12% Žwrv. NaOH. Then it is placed in 12% NaOH and cooled to 4 8C. A 0.5-mM solution of 2-amino-4,6-trichloro-s-triazeine in acetone is carefully added by running the solution gently down the side of the container at 4 8C. This should form a bilayer. The mixture is slowly stirred

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for 120 min at 4 8C. The activated substrate is washed with acetone and water for the further coupling of ligands. 2.2.5. Carbodiimide coupling Carbodiimide condensation can be used to form a peptide bond between free amino groups on the support surface and free carboxy groups on the ligand. During the coupling stage, the carbodiimide reacts with carboxyl groups to form O-cylisourea at pH 4–5. The activated carboxyl group then condenses with an amino group on the membrane support to form a peptide bond plus urea, which needs to be removed from the reaction mixture. Frequently, either 1-ethyl-3-Ž3-ethylamino-propyl. carbodiimide or 1-cyclohexyl-3-Ž2-morpholinoethyl. carbodiimide metho-p-toluene sulfonate is used for this reaction. The technique given below can be used to couple either spacer arms or protein ligands to any substrate which has free amino groups. Ligands are dissolved at a concentration of 1 mgrml in 0.1 M phosphate ŽpH 4.5.. The carbodiimide was dissolved at a concentration of 10 mgrml and the pH was adjusted to 4.5. The substrate swollen in 0.5 M NaCl is placed in a capped tube, an equal volume of ligand solution and carbodiimide solution is added, and the tube is incubated overnight in a mixer at room temperature. Lastly, the substrate is washed with 0.1 M phosphate buffer ŽpH 7.2. and then stored at 4 8C for further use w123x. 2.2.6. Carbonylation Carbonylation with 1,1X-carbonyl diimidazole can be performed on both glass and silica substrates. This safe, simple, and effective method is becoming increasingly popular. Reactive carbonyl diimidazole groups can easily be attached to amino groups on the substrates by the following procedure: the membrane substrate is added to a solution of N, N X-carbonyldiimidazole in dioxane. The mixture is incubated for 6–8 h at room temperature with constant mixing. The substrate is finally washed with dioxane and then dried on the air for protein coupling w91x. 2.2.7. Succinimide esters The most popular affinity reaction group is succinimide ester, which allows rapid and reliable attachment of protein ligands to a support matrix via amino groups. A basic method is as follows: the derivatized substrate is dissolved in a 10% chloroform solution of succinyl chloride. After 10% triethylamine has been added, the solution is refluxed for 30 min. The substrate is resuspended in an anhydrous dioxane solution containing 0.1 M N-hydroxysuccinimide and 0.1 M N, N X-dicyclohexylcarbodimide and the mixture is stirred for 2 h at room temperature w124x. 2.2.8. Other actiÕation methods In addition to the activation methods given above, still other methods have been developed w125x, e.g., the 2,2,2-trifluoroethanesulfonyl chloride, diglycolic anhydride method, glutaric dialdehyde method, sulfonic acid chloride method, sulfinyl chloride, and Woodward’s K method. They can be used for the activation of substrates that contain reactive groups such as –OH, –NH 2 , and –SH. The commonly used activation agents and their structure are given in Table 2. Some commercially available activated substrates are shown in Table 3.

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Table 2 List of various activation methods

2.3. Coupling of affinity ligands to the actiÕated membrane 2.3.1. Spacer arm After the membranes are activated, various ligands can be coupled to them. Occasionally, when the immobilized ligand is a small molecule, such as a receptor substrate or a chemical antigen, steric hindrance will occur between the immobilization support and

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Table 3 Activation methods, names, and producers of some commercially available activated substrates Activation methods and the names (1) Epoxide actiÕation Regenerated crosslinked cellulose membrane with 6-atom spacer arm ŽIndion. Cellulose mebrane stack ŽMemSep. Hydrophilic microporous nylon membrane Žimmunodyne. Microporous cellulose membrane ŽKF-C50. Microporous nylon 66 membrane with 6 atoms spacer arm ŽKF-N66-EP. (2) Glutaric dialdehyde actiÕation Cellulose filter membrane ŽNalgene U12, U38. Celluloseracrylamide polymer ŽZetaffinity. Crosslink-microporous cellulose membrane ŽKF-AD-C-50. Cellulose polymeric membrane Hydrophilic microporous PVC filter membrane device PVC membrane stack treated with polyvinyl imine ŽActi-Disk. (3) Vinylsulfone actiÕation Cellulose membrane separation device ŽMemSep. Acrylate coating microporous polyŽvinylenebifluorate. membrane Žimmobilon.

Producer Phoenix Domonick Hunter, Millipore Pall Rainin Dalian Institute of Chemistry and Physics, Chinese Academy of Sciences, China

Nalge Cuno Dalian Institute of Chemisty and Physics, Chinese Academy of Sciences, China Memtek FMC FMC

Domnick Hunter Tessek Millopore

the substance to be isolated. This phenomenon will cause a reduction or complete lack of specific binding. To overcome this problem, it is often necessary either to select a different support Ži.e., one with a spacer arm already attached. or to bind a spacer molecule to the support prior to attaching the ligand. The use of a spacer arm will ensure that a ligand is placed at a suitable distance from the surface of the support. This will allow the material to be isolated, with ample space for complete attachment to the immobilized ligand. Spacer arms can be attached directly to the support surface with a suitable ligand attachment to the reactive side-chain of the support, and the ligand can be attached to the spacer via a secondary reaction. An ideal spacer arm should have: Ž1. proper length Žat least three atoms.. Ž2. No active center which could cause non-specific adsorption between membrane and sample. Ž3. Bifunctional groups to react with both substrates and ligands. The spacer arm will bend under the column pressures and fold into the support surface. Its effective length will be greatly reduced in salt buffer when the spacer arm is too long. On the other hand, the access of a target substance to a ligand will be difficult, if the spacer arm is too short. Table 4 summarized some useful

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Table 4 List of some useful spacer arms and their structures Name

Structure

Alkylamine

Diamine Polypeptides

Polyamine

Polyether Amino acid

–OŽCH 2 . 2 OŽCH 2 . 2 OŽCH 2 . 2 OH NH 2 RCOOH

chemicals that can be used as spacer arms. Among them, compounds with diamine groups, such as hexanediamine, propane diamine, and ethylenediamine, are the most frequently used spacer arms in affinity chromatography w125x. Minobe et al. w126x prepared affinity adsorbents for pyrogen adsorption by immobilizing histamine to aminohexyl-Sepharose CL-4B. The effect of the chain length of the spacer on the

Table 5 Effect of chain length of spacer in adsorbents on adsorption of pyrogen Matrix

Spacer

Chain length

Coupled histamine Žmmolrml of adsorbent.

Concentration of pyrogen in effluent Žngrml.

CNBractivated Sepharose 4B

no

0 1.36

23.1 3.0

46 91.2

1.67

2.0

99.4

Epoxideactivated Sepharoes CL-4B

–CH 2 –CHŽOH. –CH 2 – –CH 2 CHŽOH CH 2 NHŽCH 2 . 4 .NHŽCH 2 .5 –CH 2 CHŽOH.CH 2 NHŽCH 2 . 6 NHŽCH 2 .5 –CH 2 CHŽOH.CH 2 NHŽCH 2 . 8 NHŽCH 2 .5 –CH 2 CHŽOH.CH 2 NHŽCH 2 .10 NHŽCH 2 .5 –CH 2 CHŽOH.CH 2 NHŽCH 2 .12 NHŽCH 2 .5

0.46 1.97 2.28 2.59 2.90 3.20

12.2 3.0 4.9 1.8 3.6 4.6

68.0 99.9 99.9 99.7 99.8 99.8

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affinity adsorbents for pyrogens was investigated. When the chain length of the spacer was 1.97–2.9 nm, the adsorbents showed the highest affinity for pyrogens Žsee Table 5.. 2.3.2. Coupling of ligands The selection of any ligands should meet the following conditions: Ž1. The ligand must specifically and reversibly bind the substances to be isolated and must contain groups that can be chemically modified to allow attachment to the support. Ž2. The chemical modification must not impair or damage the specific binding activity of the ligands w47x. The materials used as affinity ligands can be divided into two categories: Ž1. General ligands, such as dyes, amino acid, Protein A and G, lectin, coenzyme, and metal chelates, etc. Ž2. Specific ligands, such as enzymes and substrates, antibodies and antigens. Various ligands and corresponding ligates are summarized in Table 6.

Table 6 List of ligands and corresponding ligates Ligands

Ligate Cibacron Blue F3GA

Dyes

Procion Red HE3B K2BP

Amino acid

Trp Arg Lys His Phe Concanavalin A Lentil lectin

Protein A and G

Lectin Heparin Polymyxin B Metal chelates Gelatin Calmodulin Benzamidine Hormone DNA, RNA, ribose Antibody Antigen Enzyme Enzyme inhibitor Enzyme cofactor

Enzyme, calmodulin, serum albumin, lipoprotein, interferon, thrombin, synthetase, transferase, myoglubin, growth factor Enzyme, lipoprotein, cytotoxicity, carboxypeptidase G, kinase, dehydrogenase, alkaline phosphatase, polypeptide hormone Carboxypeptidase A Serine proteinase DNA, RNA Pyrogen, endotoxin, yeast proteinase g-Glubulin IgA, IgG, IgM, antibody, insulin-like growth factor Polysaccharide, glycoproteins, membrane glycoproteins, glucolipid, enzyme and coenzyme with glycosyl

Wheat germ lectin Peanut lectin Human antithrombin polymerase, coagulation factor Endotoxin Histidine-, typtophan-, and cysteine-containing proteins Fibronectin Phosphdiesterases, ATPase, and calcinerin Urokinase, trypsin, thrombin, kallikrein Receptor Nuclease, polymerase, necleotide Antigen Antibody Enzyme inhibitor Enzyme Enzyme

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3. Categories and characteristics of affinity membrane cartridges The affinity membrane cartridge is the core of a separation process. In principle, all kinds of the membrane cartridges used in ultrafiltration and millipore filtration can be used as affinity-membrane cartridges. However, the requirements of affinity membrane cartridges are stricter, because the preparation procedure involves several reactions under harsh conditions. By now, the cartridges used in affinity separation include sheets, hollow fibers, spiral-wound and Chromarod membranes. Membranes in the form of thin sheet or disks are convenient, inexpensive, and versatile. Sheets or disks may be mounted in conventional ultra-filtration or specially prepared cartridges. This permits rapid, low-pressure performance. Two technical problems have hampered more extensive use of membrane chromatography: Ž1. the distribution of sample or mobile phase and Ž2. the sealing of the bundling system. To solve these problems, the main factor is the design of the shell of the membrane cartridge. Guo et al. proposed a new kind of cartridge shell, including a distribution plate w55,127,128x. The construction of this kind of membrane cartridge is shown schematically in Fig. 3. It contains an adjustable cover from which various membranes stacking with different thickness could be adopted in the same cartridge, respectively. The circumference with the width of 1 mm on the edge of each individual membrane is saturated with an elastic glue. Up to 40 sheets of membrane may be packed and adhere together under a slight pressure applied by screwing down the adjustable cover. A cartridge prepared by this method is much preferable with regard to both sealing and separation efficiency. Recently, a new kind of membrane column, which is compatible with HPLC instruments, has been developed in our laboratory w57–60x. Membranes with the diameter of the column inner diameter are cut and packed into the column. Compared with the conventional membrane cartridges, this new kind of column has small dead volume and high column efficiency. It also avoids the leakage and non-homogeneous distribution of sample. The relative high lengthrdiameter ratio make this new kind of membrane column overcome the problems of conventional columns, and this makes the analysis of proteins in HPLC instruments simple.

Fig. 3. Schematic diagram of a membrane cartridge w56x. Ž1. Holder, Ž2. cover, Ž3. gasket, Ž4. distribution plate, Ž5. membrane stack and Ž6. bonding part.

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Table 7 Applications of affinity membrane chromatography for protein purification Basic membranes

Ligand

Ligate

References

Modified cellulose

Protein G STI Cibacron Blue F3GA Histidine Anti-BSA Anti-rat IgE L-Aspartate Protein ArG IDA Protein ArG IgG IgM IgG, IgM, Protein A IDA

IgG Trypsin Alkaline phosphatase Endotoxin BSA IgE Aspartate IgG BSArbovine g-Globulin IgG Protein A Protein A Protein A, IgG Bovin liver catalase BSA, HSA, IgG Lysozyme Wheat germ agglutinin Lysozymeralbumin Cytochrome crSTI Cytochrome crHAS HSA IgG IgG FDHG Pyruvate decarboxylaser Adenylase kinaserMDHG Concanavalin Ar ovalbuminrlysozyme g-Globulin Paraprotein Endotoxin Endotoxin IgG IgG STI Histidinertryptophan HSA IgG IgG Fibronectin Bovine g-globulin Bovine g-globulin Heparin BSA Trypsin Creatine phosphokinase Cytochrome crlysozymer ribonuclease Ar chymotrypsinogen A

w139x w53x w54x w140x w141x w25x w142x w93x w92x w143x w60x w144x w59x w58x w96x w145x w68x w67x w67x w67x w69x w146x w147x

Cross-linked regenerated cellulose Cellulose acetate CelluloserGMA composite

Chitin Chitosan

Nylon

Inherent GlcNac Inherent GlcNac Inherent amino Inherent amino Inherent amino Cibacron Blue F3GA Protein G Nonproteinogenous ligand Cibacron Blue F3GA Cibacron Blue F3GA IDA

PVDF Modified polysufone

Modified polyŽether sulfone.ŽPES. Modified PES-Poly Žethylene oxide. Modified polyethylene

Polypropylene Modified glass hollow fiber

Protein G Alkyl treatment His His Protein A Protein A Trypsin IDA Cibacron Blue F3GA r-Protein A Protein A Gelatin L-phenylalanine Tryptophan PolyŽL-lysine. IDA STI Procion Blue MX-R IDA

w33x w99x w146x w12x w125x w70,71x w148x w110x w50x w149x w150x w151x w152x w29x w84x w84x w153x w92x w112x w154x w85x

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Table 7 Ž continued . Basic membranes

Ligate

References

Trypsin Protein A IgG Cibacron blue F3GA Glucose oxidase Cibacron Blue F3GA Cibacron Blue F3GA Protein A Cibacron Blue F3GA Protien A Histidine

STI IgG Protein G Catalase Glocose Heavy metal ions BSA HIgG Lysozyme IgG IgG

w22x w155x w10x w72x w73x w74x w76x w77x w156x w157x w24,158x

PEI-coated titania Hydrazide hollow fiber

Heparin Cibacron Blue IDA Cibacron Blue F3GA LI-8r5BI Mab

w26x w35x w159x w118x w160x

Immobilon AV membrane

Pepstatin A

Antithrombin III Malate DehydrogenaserBSA HSA HSA Interleukin-2 receptorr Interleukin-2 Human interferon-2-2a Pepsinrchymosin

PolyŽGMA-co-EDTA.

PolyŽHEMA.

PolyŽether–urethane–urea. PolyŽethylene–co-vinyl alcohol. Synthetic copolymer

Ligand

w161x w162x

STI, soybean trypsin inhibitor; BSA, bovine serum albumin; HSA, human serum albumin; GPDHG, glucose6-phosphate dehydrogenase; FDHG, formate dehydrogenase; MDHG, malate dehydrogenase; GMA, glycidyl methacrylate; EDMA, ethylene dimethacrylate; Mab, monoclonal antibody; IDA, iminodiacetic acid; polyŽHEMA., polyŽ2-hydroxylethyl methacrylate..

Hollow fibers are usually bundled within a shell using potting material. Due to their cross-flow characteristics, the separation operation of hollow-fiber cartridges is simple, rapid, and inexpensive w84,85x. Spiral-wound and stacked-membrane cartridges bring some variations in porosity and membrane thickness which may decrease the average separation efficiency of single sheets w49x. They have a number of advantages relative to packed-bed chromatography. The cross-sectional dimension of membrane columns perpendicular to the flow direction is considerably longer than the flow path. In contrast, particle beds are commonly packed with a length-to-diameter ratio of 2.5 or greater. Consequently, residence time in membrane columns is greatly reduced and this reduces protein degradation by proteolysis and denaturation w30,32,129x. To increase the amount of target proteins that is adsorbed, the surface area of the affinity membrane should be as large as possible. Degen et al. developed a unique device with flat membranes, folded in a cylinder w98x. The activated membrane is formed by the reaction of a hydrophilic, microporous and skinless polyamide membrane with an activating agent to give a biologically active membrane having a large surface area. Affinity ligands, such as Protein A, lectins, enzyme substrates, and hormones are immobilized on the membranes to purify the corresponding ligates, such as monoclonal antibodies, polyclonal antibodies, glycoproteins, IgG classes of immunoglobulins, carrier proteins, and coagulation factors. Rapid separation process can be obtained due to the very low backpressure Ž- 0.5 MPa..

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Membrane-rod chromatography involves a column with attributes of both membrane adsorbers and packed-resin beds. The polymeric matrix is a macroporous structure Ž0.1–10 mm. producing a low back pressure. The pore-size distribution and the porosity of the rod can be easily controlled by selecting the proper porogen and varying the volume ratio of porogen to monomer. Linear decreases in efficiency due to intraparticle diffusion at increasing flow-rates are not observed w22,43,130–138x. A number of affinity-membrane cartridges are commercially available.

4. Applications Membrane chromatography in different separation modes, such as ion exchange, affinity, hydrophobic interaction, and reversed phase, has been widely used in the separation of various proteins. Table 7 lists a number of applications of affinity-membrane chromatography to protein purification. In this section, we focus on the applications of affinity-membrane chromatography and especially on the applications of the celluloserGMA composite membrane to protein purification in our laboratory. 4.1. Purification of alkaline phosphatase [54,128] Guo et al. w54,128x prepared an affinity-membrane medium from a chemically cross-linked cellulose film. The alkali-treated cellulose membrane has a higher porosity and larger pore size Ž1–2 mm. than commercially available cellulose microfiltration membranes. An affinity membrane cartridge with 80 sheets of cellulose membrane Žinner diameter, 40 mm. was prepared. The flux of the membrane cartridge can approach 5.0 mlrmin with a pressure drop of 0.1 MPa. The triazine dyes Cibacron Blue F3GA and Active Red K2BP were immobilized as affinity ligands, and a commercial alkaline phosphatase from calf intestine was purified, with the result shown in Fig. 4. The recovery of alkaline phosphatase activity was 60%, and a 40-fold purification was achieved. 4.2. Endotoxin remoÕal by membrane chromatography [55,70,71,127,140,163] Bacterial endotoxins are lipopolysaccharides ŽLPS. derived from the outer membranes of gram-negative bacteria. The highly pyrogenic nature of bacterial endotoxins made the terms of pyrogen, endotoxin, and LPS synonymous. Endotoxin is known to cause reactions in animals with symptoms of high fever, vasodilation, diarrhea, and in extreme cases, fatal shock. Shang et al. w70,71x prepared an affinity-membrane medium for endotoxin removal by immobilizing histidine on a Nylon 66 membrane substrate . The scheme for the preparation of the affinity membrane is shown in Fig. 5. The effects of different activation methods on the content of ligands immobilized on the Nylon 66 membrane were investigated with the results shown in Table 8. In addition, the effect of the spacer arm on endotoxin removal was also investigated. The optimal adsorption

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Fig. 4. Affinity chromatography of alkaline phosphatase on an Active Red K2BP-immobilized cellulose membrane column. Experimental conditions: 10.3 mg amount of alkaline phosphatase was dissolved in 2.0 ml of 0.1 M NaCl–0.1 M NaAc–HCl buffer ŽpH 7.5. and was applied to a 18=40 mm I.D. cartridge, containing 80 sheets of membrane, at a flow-rate of 0.1 mlrmin at 4 8C. The cartridge, at room temperature, was first equilibrated with 1.0 ml of 0.1 M NaCl–0.1 M NaAc–HCl buffer ŽpH 7.2. ŽSolution A., then the alkaline phosphatase and other impurities were eluted with 1.0 M NaCl–0.1 M NaAc–HCl buffer ŽpH 7.0. ŽSolution B. and 1.0 M NaCl–0.1 M NaAc–HCl buffer ŽpH 7.0., containing 60% ethylene glycol ŽSolution C., respectively. The flow-rate of the mobile phase was 0.8 mlrmin. The asterisk indicates alkaline phosphatase.

capacity of this affinity membrane was 0.2 mgrg E. coli 055 B5 endotoxin. Endotoxin could be successfully removed from bovine serum albumin, lysozyme, amino acid injection solutions, and human blood under optimal conditions. 4.3. Protein A affinity membrane cartridge for extracorporeal immunoadsorption therapy [57] Affinity chromatography allows extraction of substances from complex mixtures. Immunoadsorption therapy, based on this principle, is thus an attractive method, chiefly used for cleaning biological fluids, such as blood and plasma. Protein A is a cell-wall protein that binds to the Fc region of many mammalian IgG subclasses and has a high affinity for immune complexes. Systematic lupus erythematosus, autoimmmune hemolytic anemia and immune thrombocytopenic purpura, etc. are diseases of the immune system, related to antibodies and circulating immune complexes. A Protein A tangential-flow affinity-membrane chromatographic cartridge ŽTFAMC. for therapeutic

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Fig. 5. Scheme for the preparation of Nylon 66 affinity membranes.

extracorporeal immunoadsorption was prepared recently via coupling of Protein A to a composite celluloserGMA membrane. The TFAMC design is shown in Fig. 6. The membrane was spiral-wound around a perforated plastic center pole to form a tangential Table 8 Content of ligand on nylon 66 affinity membrane prepared with different activators Name

EE01 EE02 EE03 EE04 EE05 EE06 EE07 EE08 a

Synthesis method a

b

ECH –HDA –ECH–His GAD c –HDA–ECH–His ECH–HDA–GAD–His GAD–EDAd –ECH–His DBP e –HDA–GAD–His CDI f –HDA–GAD–His CDI–HDA–CDI–His GAD–His

Epichlorohydrin ŽECH.. Hexanediamine ŽHDA.. c Glutaric dialdehyde ŽGAD.. d Ethylenediamine ŽEDA.. e Dibromopropane ŽDBP.. X f 1,1 -carbonyldiimidazole ŽCDI.. b

Ligand content w ligand Ž10y3 .

M Žmmolrg.

6.00 5.78 5.02 7.94 5.44 15.1 29.1 27.6

39.0 37.2 32.3 51.2 35.0 97.3 188 178

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223

Fig. 6. Schematic diagram of tangential-flow affinity-membrane chromatographic cartridge Ždiameter 70=50 mm..

flow device. The flow characteristics of the immunoadsorption column affected its adsorption capacity and life. When the flow-rate was 130 mlrmin, the pressure drop on the TFAMC was 0.028, 0.036, and 0.093 MPa for water, plasma, and blood, respectively. When fresh human plasma Ž150 ml. was circulated through the TFAMC for 20 min at flow-rate of 32, 67, 92 and 132 mlrmin, the Protein A TFAMC adsorbed IgG in amount of 413.1, 394.8, 416.7, and 400.15 mg, respectively. Increasing the flow-rate from 32 to 132 mlrmin did not distinctly decrease the adsorption capacity for IgG, while the adsorption capacity for IgG was notably affected by circulating time. When fresh human plasma Ž150 ml. was circulated through the TFAMC for 10, 20, 40 and 60 min at a flow-rate of 64 mlrmin, Protein A TFAMC adsorbed IgG in amounts of 290, 413.1, 504.8, and 526.7 mg, respectively. The adsorption capacities of Protein A TFAMC for IgG from human plasma and blood were also measured. The cartridge with 139 mg Protein A immobilized on the matrix Ž6 mg Protein Arg dry matrix. adsorbed 55.3 mg IgG Ž23.8 mg IgGrg dry matrix. from human plasma and 499.4 mg IgG Ž21.5 mg IgGrg dry matrix. from human blood, respectively. The purity of IgG, eluted from Protein A TFAMC was analyzed by MALDI TOF-MS. The results showed that Protein A TFAMC adsorbed mainly IgG and very

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little of other plasma proteins. An extracorporeal circulation experiment in vivo on a dog was performed ŽFig. 7. and the results confirmed that the Protein A TFAMC had good biocompatibility and that the extracorporeal circulation system is safe and reliable. 4.4. Immobilized-metal affinity composite-cellulose membrane for the separation of proteins [96] One of the key advantages of composite celluloserGMA membrane is the abundance of epoxide groups, which provide reactive sites for the further coupling of ligands. The active group density on the matrix and the strength of the composite membranes were notably affected by the reaction conditions, such as the reaction temperature and the ratio of materials. The conditions for reaction of chelating agent Žimminodiacetic acid, IDA. with the composite cellulose membrane were also optimized. A higher IDA density on the surface of the composite membrane was obtained for the reaction at 75 8C ŽpH 11. for 4 h in the presence of 2–3% NaCl as promoter. The relationship between flow-rate and back pressure was almost linear for a cartridge of 16 mm I.D., stacked with 22 pieces of membranes. The back-pressure was only 1.17 = 10 5 Pa when the flow rate was 14 mlrmin. Human serum albumin ŽHSA., commonly used for therapeutic purposes, must be of high purity. Yang et al. w96x purified commercially available HSA solution with a Ni 2q-IDA membrane cartridge, and obtained the chromatogram shown in Fig. 8. The commercially available HSA solution and that purified on Ni 2q-IDA membrane cartridge were assayed by capillary zone electrophoresis Želectropherograms shown in Fig. 9.. The results showed that the metal-chelating membrane provided an efficiency comparable with agarose bead based metal immobilization affinity chromatography for HSA purification, but the performance of the membrane cartridge was 3–5 times faster

Fig. 7. Schematic diagram of the in vivo extracorporeal circulation system.

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225

Fig. 8. Chromatogram for the purification of commercial human serum albumin ŽHSA. solution on a Ni 2q-IDA composite membrane cartridge. Experimental conditions: sample eluted with Ž1. 0.02 M phosphate buffer ŽpH 7.8., containing 1 M NaCl; Ž2. above buffer at pH 6.0; Ž3. 0.05 M acetate buffer, containing 1 M NaCl. Fraction II is HSA solution.

than agarose bead packed column. Due to its macroporous structure and the convective flow of solution through the pores, fast assay of proteins could be performed by high-performance membrane chromatography.

Fig. 9. Capillary zone electrophoresis of Ža. commercial HSA solution and Žb. the same, purified by Ni 2q-IDA affinity-membrane chromatography.

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Fig. 10. Chromatograms of bovine liver catalase on Cu2q-IDA affinity membrane cartridge. Experimental conditions: Ža. analytical run with BLC in Peak III; Žb. preparative run. Eluents used for chromatography: Ž1. 20 mM phosphate buffer–1 M NaCl ŽpH 7.0.; Ž2. 50 mM acetate buffer ŽpH 5.5.; Ž3. 0 mM phosphate buffer–1 M NaCl ŽpH 7.0., containing 0.2 M imidazole. Žv . UV absorption at 280 nm; ŽB. enzyme activity.

Fig. 11. Scheme of two approaches for the immobilization of protein ligands on composite cellulose membranes.

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4.5. Cu 2 q-IDA chelating affinity-membrane chromatography for the purification of boÕine liÕer catalase [58] Catalase is a key enzyme in all aerobic cells and the enzyme from bovine liver is widely used in biochemical research and industrial processes. Bovine liver catalase

Fig. 12. Chromatogram for the immunoaffinity analysis of goat anti-HIgG and goat HIgM. Experimental conditions: sample solution was injected with loading buffer of 10 mM phosphate buffer, containing 0.15 M NaCl ŽpH 7.2. as the eluent, and 2 min after injection of the sample solution, elution buffer of 0.2 M glycine buffer at pH 2.3 used as eluent. Flow-rate, 1.0 mlrmin; detection, 280 nm at 0.5 aufs. Samples: Ža. 20 ml of solution, containing 100 mg crude powder of goat anti-HigG and Žb. 10 ml of 5-fold diluted goat plasma on a HIgM column.

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Table 9 Amount of ligands immobilized , adsorption capacity and affinity efficiency of columns and activity recovery of IgGs Ligands

Amount of ligands immobilized Žmgrcolumn. Amount of ligands immobilized Žmgrg of membrane. Adsorption capacity Žmgrcolumn. Adsorption capacity Žmgrg of membrane. Theoretical number of active sites in a free ligand molecule Affinity efficiency Ž%. Activity recovery Ž%.

Protein A immobilized via method a Žfor HIgG. 0.5

3.3

Protein A immobilized via method b Žfor HIgG. 2.0

13

HIgG immobilized via method a Žfor goat antiHIgG. 3.2

21

HIgM immobilized via method a Žfor goat antiHIgM. 2.5

17

2.3

0.43

0.67

0.56

15.3

2.9

4.5

3.7

5

5

1

5

24.3 96.0

1.1 98.2

20.9 95.9

22.4 96.3

ŽBLC. was purified by Cu2q-IDA composite-cellulose-membrane chromatography by Yang et al. w58x. The effect of pH on BLC binding capacity was investigated. The binding capacity of BLC on Cu2q-IDA composite cellulose membrane attained a

Fig. 13. Process monitoring of IgG in dog plasma on a Protein A column during immuno-adsorption therapy. Experimental conditions: the sample solution was injected with a loading buffer of 10 mM phosphate buffer, containing 0.15 M NaCl ŽpH 7.2. as the eluent, and 2 min after injection of the sample solution, elution buffer of 0.2 M glycine buffer at pH 2.3 used as eluent. Flow-rate, 1.0 mlrmin; detection, 280 nm at 0.5 aufs. Sample: 10 ml of 10-fold diluted dog plasma Ž1. before and after, Ž2. 30 min, Ž3. 60 min and Ž4. 90 min of immuno-adsorption therapy.

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maximum at pH 6.5–7.0. The effects of initial BLC concentration, chelator surface density, and flow-rate on BLC binding capacity were investigated. The affinity purification procedure of BLC on a Cu2q-IDA chelating membrane was performed both on an analytical and preparative scale Žsee Fig. 10.. An average 4.2-fold Ž7-fold for 85% of the fractions. purification of BLC with an enzyme activity recovery of 67.7% was obtained

Fig. 14. High-speed immunoaffinity analysis of IgG in human serum on a Protein A-immobilized affinity column. Experimental conditions: sample solution was injected with loading buffer of 10 mM phosphate buffer, containing 0.15 M NaCl ŽpH 7.2. as the eluent, and 20 s after injection of the sample solution, elution buffer of 0.15 M NaCl solution at pH 2.6 used as eluent. Flow-rate, 3.0 mlrmin; detection, 280 nm. Sample: Ža. 20 ml of human IgG solution Ž2.5 mgrml. and Žb. 10-fold diluted solution of human serum.

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in a single step. Two regeneration methods were investigated, and the results showed that the metal-chelating membrane adsorbent could easily be regenerated with EDTA buffer. 4.6. Membrane immunoaffinity chromatography for the analysis and purification of proteins [59,60,143,144,164] With the development of biotechnology in recent years, fast analysis and purification of proteins is becoming more and more indispensable for the optimization and control of biological process. Affinity membrane columns compatible with HPLC instruments were prepared in our laboratory by immobilization of various ligands of Protein A, human IgG, human IgM, DEAE, IDA-Cu2q, and pectinase on celluloserGMA composite membrane. Two approaches, shown in Fig. 11, were used for the immobilization of protein ligands on the celluloserGMA composite membranes. The influence of flow-rate on adsorption capacity of human IgG on Protein A column Ž40 = 4.6 mm I.D.. was investigated. The results showed that the adsorption capacity decreased slightly when the flow-rate changed from 0.2 to 0.5 mlrmin, and kept almost constant with a further increase of flow-rate to 3.0 mlrmin. Therefore, fast analysis and purification of biopolymers was feasible. The analysis of human IgG on a membrane column with immobilized Protein A can be completed within 3 min at a flow-rate of 1.0 mlrmin. The peak width of the retained human IgG is only ca. 0.1 min. Typical chromatograms for the analysis of goat anti-HIgG in a solution of crude powder and goat anti-HIgM are shown in Fig. 12. The amount of Protein A, human IgG, and IgM immobilized, the activity of the purified IgG recovered, the adsorption capacity, and the affinity efficiency of these affinity columns are summarized in Table 9. The activity recoveries of IgG on those affinity columns were all over 95%, which is very high compared to the conventional purification methods. The key factor in high activity recovery is that the operation can be performed very quickly. The affinity membrane cartridge could also be used for the rapid analysis and monitoring of biological processes. The immunoaffinity analysis of dog IgG in plasma in a clinical experiment in immuno-adsorption therapy was performed on a Protein A column Žchromatograms shown in Fig. 13.. The concentration of dog IgG at different stages of immuno-adsorption therapy experiment was determined. High-speed immunoaffinity analysis could be achieved by increasing the flow-rate of the mobile phase and decreasing the time for switching the mobile phases. Both human IgG and dog IgG in serum were determined within 30 s on a Protein A column at a flow-rate above 3.0 mlrmin. Fig. 14 shows the chromatograms for the fast analysis of human IgG on the Protein A column.

Fig. 15. Chromatograms for detection of polygalacturonase inhibiting proteins in fractions purified on a hydroxyapatite column with different phosphate buffers. Experimental conditions are the same as in Fig. 12. Samples: fraction of polygalacturonase inhibiting proteins purified by Ža. 0.01, Žb. 0.05 and Žc. 0.2 M phosphate buffers, respectively.

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Polygalacturonase inhibiting proteins in the fractions purified on the hydroxyapatite column by using different phosphate buffers was determined by affinity-membrane chromatography. Twenty microliters of each, concentrated and dialyzed sample solution, was injected into the immobilized pectinase column Žchromatograms shown in Fig. 15.. 4.7. On-line characterization of the actiÕity and reaction kinetics of immobilized trypsin by high-performance frontal analysis [165] A microreactor for trypsin immobilized on an activated glycidyl methacrylate ŽGMA.-modified cellulose-membrane-packed column was constructed by Jiang et al. w165x. A novel method for characterizing the activity and reaction kinetics of the immobilized enzyme has been developed, based on the frontal analysis of enzymatic reaction products. This was performed by on-line monitoring of the absorption at 410 nm of p-nitroaniline from the hydrolysis of N-a-Benzoyl-DL-arginine-p-nitroanilide ŽBAPNA.. The hydrolytic activity of the immobilized enzyme was 55.6% of free trypsin. The apparent Michaelis–Menten kinetics constant Ž K m . and Vmax values measured by the frontal analysis method were 0.12 mM and 0.079 mM mgrmin. enzyme, respectively. The former value is very close to that observed by the static and off-line detection methods, but the latter is about 15% higher. Inhibition of the immobilized trypsin by addition of benzamidine to substrate solution has been studied by the frontal analysis method. The apparent Michaelis–Menten constant of BAPNA Ž K m ., the inhibition constant of benzamidine Ž K i . and Vmax were determined. It was found that the interaction of BAPNA and benzamidine with trypsin is competitive, the K m value is affected, but the Vmax is unaffected by the benzamidine concentration. 4.8. Purification of human urinary kallikrein [166] Tissue kallikrein is a very important enzyme that cleaves a kininogen substrate to release the potent vasoactive kinin peptide. It is also an important agent in cardiovascular medicine and therapeutics for its blood pressure-lowering effect and the potential to dissolve blood clots. The purification of kallikrein is very difficult because of its low concentration Žabout 100 ngrml. in human urine. Wang et al. w166x developed a method for the purification of human urinary kallikrein ŽHUK.. The procedure consisted of three steps: ultra-dialysis, diethyl-Ž2-hydroxypropyl. amino ethyl ŽQAE. ion-exchange radial-flow membrane chromatography and affinity chromatography Žas shown in Table 10.. It was simple and suitable for large-scale purification. The product Table 10 Summary of purification steps of human urinary kallikrein Steps

Volume Žml.

Total activity ŽAu.a

Specific activity ŽAurA280.

Recovery Ž%.

Purification factor

Urine Ultradialysis Ion exchange Affinity

1.5=10 4 375 150 4.6

15.47 7.14 6.13 2.46

3.55=10y5 1.64=10y3 4.62=10y2 9.11

100 46.1 39.6 16.6

1 46.2 1301.4 2.5=10 5

a

Au s1rmol AMCrmin.

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was checked by SDS-PAGE and matrix-assisted laser desorption ionization mass spectrometry ŽMALDI., showing that 2.5 = 10 5-fold purification of HUK was achieved. The biological activity tested by enzyme-linked immunosorbent assay showed positive identity of the product compared with HUK standard.

5. Conclusion Affinity-membrane chromatography uses microporous or macroporous membranes that contain biospecific ligands, attached to their inner pore surface, as adsorbents. As a result of convective flow of the solution through the pores, the mass transfer resistance is tremendously reduced, and binding kinetics dominate the adsorption process. This results in rapid processing and greatly improves the adsorption, washing, elution, and regeneration steps, decreasing the probability of inactivation of proteins. The hydrophobicity, mechanical strength and functional ligand density of the membrane materials can be improved by various kinds of chemical modifications, which allow those materials to meet the requirements of affinity chromatography. Consequently, the combination of membrane chromatography with affinity interaction provides high selectivity and fast processing for the purification and analysis of proteins. Monolithic materials have quickly become well-established stationary phases for biological separation and purification. The simplicity of their in situ preparation methods and the large variety of readily available reaction schemes make the monolithic separation media an attractive alternative to liquid chromatography columns, packed with particulate and membrane materials.

Acknowledgements The financial support from the Natural Science Foundation of China ŽNo. 29635010. to Dr. Hanfa Zou is gratefully thanked. Dr. Hanfa Zou is recipient of the excellent young scientist award from the National Natural Science Foundation of China ŽNo. 29725512..

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