membrane chromatography: preparation and applications to protein...

REVIEW

Membrane Chromatography: Preparation and Applications toProtein Separation

Xianfang Zeng

Covance Biotechnology Services, Inc., 3000 Weston Parkway, Cary, North Carolina 27511

Eli Ruckenstein*

Department of Chemical Engineering, State University of New York at Buffalo, Amherst, New York 14260

As a result of the convective flow of solutes through porous membranes, membranechromatography has a higher capture efficiency and a higher productivity than columnchromatography and shows most promising industrial applications for the recovery,isolation, and purification of proteins and enzymes. This paper presents a compre-hensive review of the methods for preparation of adsorptive membranes (such assurface modification, in situ copolymerization, direct formation from hydrophilicmaterials, and functionalized particulate-entrapped membranes) and deals particularlywith novel macroporous chitin and chitosan membranes for protein separationsdeveloped by the authors.

Contents

1. Introduction 10032. Preparation Methods of Adsorptive

Membranes1004

2.1. Preparation of Basic Membranes 10042.2. Activation of the Membranes and

Coupling of Ligands1009

3. Membrane Chromatography forProtein Purification

1010

3.1. Applications of MacroporousChitin Membranes

1010

3.2. Applications of MacroporousChitosan Membranes

1013

1. Introduction

Membrane chromatography uses microporous or macro-porous membranes that contain functional ligands at-tached to their inner pore surface as adsorbents. As aresult of the convective flow of the solution through thepores, the mass transfer resistance is tremendouslyreduced, and the binding kinetics dominates the adsorp-tion process. This results in a rapid processing, whichgreatly improves the adsorption, washing, elution, andregeneration steps and decreases the probability ofinactivation of biomolecules. Compared to column chro-matography, which involves high pressure drops for smallbeads and compaction for soft gels at high flow rates,membrane chromatography has a lower pressure drop,

higher flow rate, and higher productivity as a result ofthe microporous/macroporous structure of the thin mem-brane. The easy packing and scale-up, as well as theunlikely fouling/clogging, provide additional advantages.Consequently, membrane chromatography is a promisinglarge-scale separation process for the isolation, purifica-tion, and recovery of proteins and enzymes (1-10).

Many publications have reported the performance ofadsorptive membranes (ion-exchange membranes, affin-ity membranes, reversed-phase, and hydrophobic inter-action membranes), as well as their theoretical descrip-tion and optimal design. Suen and Etzel (11) proposed atheoretical model that included convection, diffusion, andadsorption. The effects of axial diffusion, flow velocity,kinetics, and nonuniformity of the flat-sheet membraneswere analyzed. The porosity, thickness, ligand density,and pore size can dramatically affect the overall perfor-mance of the adsorptive membranes. Suen and Etzel (12)extended their model to multicomponent solutes. Liu andFried (13) reported a model for a hollow fiber that takesinto account the effects of axial dispersion and diffusionresistance. Shiosaki et al. (14), Goto and Hirose (15), andSridhar (16) investigated the breakthrough behavior byusing similar models. Assuming local equilibrium, Briefsand Kula (2) developed an analytical model for flatmembranes. Tejeda et al. (17, 18) used the Thomas modelto optimize the design of affinity membranes. A largenumber of experiments reported comparisons betweencolumn chromatography and membrane chromatographyand demonstrated that membrane chromatography hasa higher capture efficiency and a higher productivity (2,6-9).

Two reviews have described the fundamental mecha-nisms, operation, and applications of membrane chro-matography (3, 4). However, only a few publications

* Tel: (716) 645-2911, Ext. 2214. Fax: (716) 645-3822. Email:[email protected].

1003Biotechnol. Prog. 1999, 15, 1003−1019

10.1021/bp990120e CCC: $18.00 © 1999 American Chemical Society and American Institute of Chemical EngineersPublished on Web 10/29/1999

focused on the preparation of the adsorptive membranes.This review has the goal to present methods employedfor the preparation of adsorptive membranes and par-ticularly to report about new macroporous chitin andchitosan membranes for protein purification developedby the authors.

2. Preparation Methods of AdsorptiveMembranes

Three steps are usually involved in the preparation ofadsorptive membranes: (1) preparation of the basicmembrane, (2) activation of the basic membrane, and (3)coupling of ligands to the activated membrane. Amongthese, the preparation of the basic membrane is essentialfor the success of the separation process. Ideal basicmembranes should have (a) microporous or macroporousstructures for rapid flow, (b) available reactive groupsfor further coupling of functional ligands, (c) chemical andphysical stability under harsh conditions, and (d) ahydrophilic surface for preventing nonspecific binding.

2.1. Preparation of Basic Membrane. 2.1.1. Modi-fication of Commercial Microporous Membranes.Most commercial microporous membranes [such aspolysulfone, poly(ether sulfone), nylon, poly(vinylidenedifluoride) (PVDF), polyethylene, and polypropylene] areeither hydrophobic and/or relatively inert. They have tobe modified to acquire functional groups. So far two majormethods have been employed to achieve the above goal,namely, the coating method and grafting polymerization.

2.1.1.1. Coating Method. The coating method is thesimplest way to modify the membrane surface by intro-ducing hydrophilic and functional polymers. Microporouspolypropylene hollow fiber membranes were coated withpoly(vinyl alcohol) (PVA) aqueous solutions containing1 wt % divinyl sulfone (a cross-linking agent); subse-quently, a triazine dye Procion Blue MX-R was coupledto the PVA surface (19). The hydrophilic hydroxyethylcellulose was covalently bound to microporous polysulfonehollow fiber membranes (20), poly(ether sulfone)-poly-(ethylene oxide) blend membranes (21), or nylon mem-branes (22) via epoxy groups activated with ethyleneglycol diglycidyl ether. Titania microfiltration inorganicmembranes were coated with methanolic poly(ethylen-imine) solutions, then activated with 1,4-butanedioldiglycidyl ether, and finally coupled with Cibacron BlueF3GA (23). Sulfonated poly(ether sulfone) hollow fibermembranes (24) or microporous poly(ether sulfone) mem-branes (25) were coated with hydrophilic chitosan. Poly-aldehydes (such as polyacrolein or a copolymer of acroleinwith hydroxyethyl methacrylate) were coated on polysul-fone membranes (26). However, these methods could notprovide durable and stable coating layers, because thecoating could be subsequently leached off, thus adverselyaffecting the product quality. To overcome this drawback,an alternative procedure has been employed. This pro-cedure involved the coating of a membrane [poly(vi-nylidene fluoride), poly(tetrafluoroethylene), poly(ethersulfone), nylon, or polycarbonate membrane] with amixture containing a functional monomer (hydroxyalkylacrylate or methacrylate), a polymerization initiator(ammonium persulfate or potassium persulfate), and across-linking agent (difunctional acrylates, methacry-lates, or acrylamides, such as tetraethylene glycol dia-crylate, glycidyl acrylate, or methylene bisacrylamide).It was followed by radical polymerization under heatingand ultraviolet or γ radiation (27). For instance, a 0.2µm hydrophobic PVDF membrane was wet with metha-nol, rinsed with water, and then placed in a mixturecontaining 5.25 g of 2-hydroxyprop-1-yl acrylate, 1.75 gof 1-hydroxyprop-2-yl acrylate, 1 g of tetraethyleneglycoldiacrylate, 1 g of ammonium persulfate, and 91 g of waterfor 10 min. After the removal of the solution, themembrane was covered with two polyester sheets (oneon the top and the other on the bottom) and held againsta heated photodrier at 90 °C for 2 min. The membrane

Eli Ruckenstein, Distinguished Professor of ChemicalEngineering at the State University of New York at Buffalo,received all of his degrees from Polytechnic Institute ofBucharest, Romania, and was a professor there between1949 and 1969. In 1969 he was invited by the NationalScience Foundation as a senior scientist at Clarkson Uni-versity. He served as professor at the University of Delawarein the Department of Chemical Engineering from 1970 to1973 before joining the faculty of Buffalo. Professor Ruck-enstein’s research interests have covered most aspects ofchemical engineering, including transport phenomena, ca-talysis, surface phenomena, colloids, emulsions, and biocom-patible surfaces and materials. Professor Ruckenstein hasreceived numerous awards for his research, including boththe Alpha Chi Sigma (1977) and the William H. Walker(1988) awards from the American Institue of ChemicalEngineers; the Kendall Award (1986), the E. V. MurphreeAward in Industrial and Engineering Chemistry (1996), theSchoellkopf Medal (1991), and the Langmuir DistinguishedLecture Award (1994) from the American Chemical Society;and the Senior Humboldt Award of the German Governmentfor Research in Surfactants (1985). He is a member of theNational Academy of Engineering and has received theNational Medal of Science.

Xianfang Zeng received his Bachelor’s degree in 1987 andhis Master’s in 1990 from Nanjing University of ChemicalTechnology, Nanjing, China, and his Ph.D. in 1998 fromState University of New York at Buffalo, all in ChemicalEngineering. He conducted his doctorate thesis research inmembrane chromatography for protein purification underthe supervision of Professor Eli Ruckenstein. Currently heis working in the process development department at Co-vance Biotechnology Services, Inc. His main responsibilitiesinclude downstream process development for recombinantproteins from microbial fermentation or mammalian cellculture, using cell disruption, liquid/liquid extraction, processchromatography, microfiltration/ultrafiltration, precipita-tion, and unfolding and refolding techniques.

1004 Biotechnol. Prog., 1999, Vol. 15, No. 6

was rinsed with water and boiling methanol and dried.Thus, the modified PVDF membrane acquired hydro-philic and durable surfaces, as well as a low nonspecificprotein binding capacity; they have been widely used forsterile filtration in biological processes.

2.1.1.2. Graft Polymerization. Radiation-induced graftpolymerization is an effective procedure for the modifica-tion of the hydrophobic and inert polyethylene or polypro-pylene membranes. Microporous polyethylene and polypro-pylene membranes were grafted with various ion-exchange groups (-CH2OH, -NH2, -SO3H, -COOH)-containing monomers (such as sodium styrenesulfonates,vinyl acetate, or 2-hydroxyethyl methacrylate) to functionas ionic exchange membranes or with glycidyl methacry-late to acquire active epoxide groups, which can befurther modified with ionic exchange groups (diethyl-amino, hydroxyethylamino, or sulfonic acid groups),metal chelate groups (iminodiacetate), or hydrophobicgroups (phenyl, butyl, phenylalanine, or tryptophan) (28-42). These membranes have high water permeabilitiesand good mechanical strength. This method is, however,excessively energy-consuming.

Free radical grafting constitutes an alternative methodfor the modification of the hydrophobic membranes (suchas the nylon membranes). For instance, a 0.45 µm poresize nylon membrane was pretreated with a 6 N HClaqueous solution at room temperature for 5 min toacquire amino terminal groups. After it was rinsed withdeionized water, the treated nylon membrane was con-tacted with a 10% epoxy-containing polymer (such asglycidyl methacrylate) in dimethyl formamide, at roomtemperature, for 30 min. The epoxy groups could eitherbe oxidized to aldehyde groups, by treating the membranewith a 1.5 wt % sodium periodate solution for 1 h, or becoupled with ethylenediamine or hexane diamine togenerate a spacer with a terminal amino group. Afteractivation with glycidyl methacrylate and coupling withhexane diamine, the nylon membrane was treated for 6h at room temperature with a 0.25% glutaraldehyde in0.1 M sodium phosphate (pH 7.3) solution containing 1g/L sodium cyano borohydride to acquire aldehyde groups,which became available for further ligand coupling (withprotein G or amino acids) (43).

The hydrophobic PVDF membrane was transformedinto a hydrophilic one by its grafting with amino acids.When PVDF was treated with a strong basic solution,fluorine and hydrogen were eliminated from the chainand active sites were generated on the surface. Theseactive sites allowed the grafting of glycine molecules.Briefly, the PVDF membrane was wet with methanol andhot water for 10 min and then placed in a boiling glycinesolution (pH 13.5) containing 35 g of glycine and 50 mLof 6 N sodium hydroxide solution for 75 min. The glycine-grafted PVDF membranes possessed excellent hydro-philic surfaces and very low nonspecific protein bindingand maintained the strength and flexibility of the initialmembranes (44). They are excellent membranes fordownstream purification, blotting of proteins, immunoas-says, and solid-phase peptide and oligonucleotide se-quencing (45-48).

2.1.1.3. Other Approaches. Microporous glass hollowfiber membranes were activated with glycidoxypropyltrimethoxy silane to generate epoxide groups to which achelating agent, iminodiacetate, could be subsequentlycoupled (49). Rodemann and Staude (50) modified thepolysulfone membrane with metalation agents. Thepolysulfone was dissolved in tetrahydrofuran underargon, and an n-butyllithium solution in hexane wasadded dropwise with vigorous stirring. Twenty minutes

later, glycidyl-4-oxohexyl ether was introduced. Thesolution was then neutralized with 1 M HCl, and theactivated polysulfone was precipited in methanol anddried. The epoxidized polysulfone (15 wt %) was dissolvedin triethyl phosphate and cast on a glass plate. Activatedpolysulfone membranes were thus obtained via phaseinversion. Iminodiacetic acid (IDA) was then coupled tothe epoxidized polysulfone membranes. The IDA-polysul-fone membranes were used for separation of amino acidsor proteins (51).

The co-casting of a hydrophobic polymer and a hydro-philic polymer is another method to obtain hydrophilicsurfaces with high mechanical strength. Nylon was co-cast with a hydrophilic and functional copolymer thatcontained abundant pendant amine, imine, hydroxyl, orcarboxyl groups (52, 53). Hydrophilic and microporousnylon membranes with functional groups (possessingnegative or positive charges) were thus obtained, whichwere activated with various activating agents and coupledto biological ligands. These membranes have been usedas transfer membranes in immunodiagnostic applicationsor as adsorptive filters for endotoxin removal. Similarly,a composite membrane was obtained by co-casting of ablend of poly(ether sulfone) and poly(ethylene oxide)(PEO), the latter polymer providing the membrane witha low ability for nonprotein binding.

2.1.2. In Situ Copolymerization of Two FunctionalMonomers (54-61). The modification of commercialpolymer membranes usually involves multiple stepsunder harsh conditions. To overcome these shortcomings,an integrated procedure was developed, consisting in anin situ copolymerization of two functional monomers. Itled in a single step to porous membranes with mechanicalstrength and functional groups. One functional monomer(styrene, vinylpyridine, acrylamide, or glycidyl meth-acrylate) usually served as the core support material forfunctional groups; the other monomer (divinylbenzene,ethylene dimethacrylate, or methylenebisacrylamide)functioned as a cross-linking agent and ensured mechan-ical strength. A porogen, either an organic solvent or aninorganic salt, was also employed. This process is pre-sented in Figure 1. Two monomers, a porogen, and an

Figure 1. Formation of macroporous membrane via in situcopolymerization. Monomers, cross-linker, porogen, and initia-tors were dissolved in an appropriate solvent, and the solutionwas cast on a desired surface (in plate or rod shape) and allowedto completely polymerize. The porogen in the polymerizedmembranes was then extracted with water, a solvent, or anacidic/basic solution, and macroporous membranes or rods wereobtained.

Biotechnol. Prog., 1999, Vol. 15, No. 6 1005

initiator were dissolved in an appropriate solvent, andthe solution was cast on a desired surface (in plate orrod shape) and allowed to completely polymerize. Afterthe removal of the porogen from the polymerized mem-branes with water, a solvent, or an acidic/basic solution,macroporous membranes were obtained.

Macroporous poly(glycidyl methacrylate-co-ethene di-methacrylate) membranes or molded rods were preparedvia the in situ free radical copolymerization of a 24 vol% glycidyl methacrylate with 16 vol % ethylene di-methacrylate in the presence of 54 vol % cyclohexanoland 6 vol % dodecanol porogen. After 1 wt % (with respectto monomers) initiator (azobisisobutyronitrile, AIBN) wasadded, the system was heated under nitrogen at 55 °Cfor 12 h. After polymerization, the porogens (cyclohexanoland dodecanol) and other soluble compounds were ex-tracted by pumping tetrahydrofuran or methanol/waterthrough the membranes (54-58). These membranesacquired active epoxide groups, which could be easilyfurther modified using various ligands (such as diethyl-amine, ethylenediamine, propane sulfone, butyl, octyl, orphenyl groups) for ion-exchange separation, affinityseparation, or hydrophobic interaction separation. Asimilar method was also used to prepare macroporouspoly(styrene-co-divinylbenzene) rods (59, 60). A mixturecontaining 1 volume styrene, 1 volume divinylbenzene,8 volumes of a dodecyl alcohol-toluene (70: 30) mixture(porogen), and 1 wt % AIBN (with respect to themonomers) was purged with nitrogen for 15 min andintroduced into a stainless steel tube, which was thensealed with rubber nut plugs. After 24 h of polymerizationat 70 °C, the rubber plugs were replaced with columnend fittings, and tetrahydrofuran was pumped throughthe column to remove the porogen and solvent. This rodhas an average pore size of 10.5 µm and is useful forreversed-phase separation of peptides and amino acids.

Microporous poly(acrolein-co-acrylonitrile) membranescontaining active aldehyde groups were prepared via thesolution polymerization of acrolein and acrylonitrile (61).Poly(acrolein-co-acrylonitrile) was synthesized using 7 wt% acrolein, 92.6 wt % acrylonitrile, and 0.4 wt % AIBNin dimethyl sulfoxide (DMSO) by heating at 42-46 °Cfor 3 h. The poly(acrolein-co-acrylonitrile) solution wasvacuum evaporated, diluted with additional DMSO, andcast on a glass plate. Microporous poly(acrolein-co-acrylonitrile) membranes were obtained by the phaseinversion of the casting solution in 40-50 °C humid air.These membranes were, however, too hydrophilic andfragile and needed a support.

Macroporous poly(2-hydroxyethyl methacrylate) mem-branes were obtained via the free radical polymerizationof 2-hydroxyethyl methacrylate (HEMA) and ethylene-glycol dimethacrylate diester (EGDMA) (62-65). HEMA(24.88 wt %), 0.12 wt % EGDMA (cross-linking agent),and 75 wt % of water (porogen) were mixed and pouredinto a polymerization chamber of desired shape. Initiators(ammonium persulfate and sodium metabisulfide, 0.25wt % each with respect to the monomer) were added, andthe system was subjected to stirring. After polymerizationat 10 °C for 18 h, the formed membranes were soaked indistilled water for the removal of any unreacted mono-mer. The pore density and pore size distribution wasdependent on the water/HEMA weight ratio. Macroporousmembranes (5-20 µm pore size) were formed only whenthe ratio of water/HEMA was greater than 0.5. However,these membranes did not possess adequate mechanicalproperties.

Poly(ethylene-co-vinyl alcohol) hollow fiber membraneswere prepared via the copolymerization of ethylene and

vinyl alcohol (66-69). These membranes were activatedwith 1,4-butanedioldiglycidyl ether and coupled withhistidine for the IgG separation.

2.1.3. Other Methods. 2.1.3.1. Cellulose DerivativeMembranes. Regenerated cellulose and cellulose acetatemembranes can be obtained via phase inversion. Theyhave hydrophilic surfaces and low nonspecific proteinbinding, as well as reactive hydroxyl groups. Various ion-exchange groups (sulfonic acid, quaternary ammonium,carboxyl, or diethylamine groups) were coupled to cross-linked regenerated cellulose membranes (70, 71). How-ever, these membranes have small pores and a nonrigidstructure and need a support. Several methods weresuggested to overcome these drawbacks. Cross-linkedmacroporous cellulose membranes were prepared usingcoarse fibers as basic materials (72). Filter paper wasdispersed in a boiling sodium hydroxide solution contain-ing NaBH4 until a pulp was formed. The pulp was caston a glass plate, frozen at -30 °C for 45 min, andsubsequently thawed at room temperature. After immer-sion in a 10 wt % HCl aqueous solution for 1 h, thecellulose membrane was removed, rinsed with water, anddried. After cross-linking at 50 °C for 3 h with anepichlorohydrin solution containing 2.4 N NaOH, thecellulose membranes became stable chemically and me-chanically and acquired large pore sizes (1-2 µm).

Macroporous cellulose membranes were indirectlyobtained by deacetylating the cellulose acetate mem-branes with a methanolic KOH solution at room tem-perature for 6 h (73-75). These membranes are hydro-philic and pliable and exhibit little swelling at variouspHs. The hydroxyl groups on the cellulose surface wereoxidized to aldehyde groups, which provided sites forligand coupling. These membranes have pore sizesbetween 0.1 and 10 µm (mostly 3-5 µm). After proteinA or protein G was coupled to these membranes, theycould be used for the purification of monoclonal andpolyclonal IgG. Iminodiacetic acid (IDA), a metal-chelat-ing agent, could be also attached to the hydroxyl groupsof cellulose membranes (73). Cellulose membranes weretreated with a 6 N sodium hydroxide aqueous solutionat 2 °C for 30 min, with epichlorohydrin in 1 N NaOHsolution at 60 °C for 1 h, and finally with 0.2 N IDA insodium carbonate buffer at pH 11 and 80 °C overnight.The resulting IDA-cellulose membranes were treatedwith CuSO4, and then employed for serum proteinrecovery (such as bovine serum albumin and γ-globulin).

Composite macroporous cellulose membranes contain-ing acrylic sheaths as functional group carriers wereprepared via the grafting polymerization of glycidylmethacrylate to dispersed cellulosic fibers (76). Refinedcellulosic fiber pulps were well dispersed in water togenerate a slurry. Glycidyl methacrylate was poured, andammonium persulfate and sodium thiosulfate were addedto the slurry. After 1 h of reaction at 80-85 °C withstrong stirring, the slurry was cooled and cast onto aporous surface (such as a woven wire mesh), vacuumfiltered, and dried. These membranes possessed highporosity and good flow characteristics. Because of thepresence of active epoxides, these membranes could befurther modified with ion-exchange groups, such asdiethylaminoethyl (DEAE) or sulfopropyl (SP), and af-finity ligands (protein A or protein G) and used forpurification of various proteins (such as interleukin-1factor, recombinant protein, monoclonal antibodies, im-munoglobulins, tissue plasminogen, and clotting factors)(77-80).

2.1.3.2. Poly(ether-urethane-urea) Membranes (81). Po-rous poly(ether-urethane-urea) membranes were pre-


pared via an electrostatic spinning method. A solutionconsisting of 16 wt % poly(ether-urethane-urea) in amixture of N,N′-dimethylacetamide and methyl ethylketone (1.45:1, wt/wt) was introduced through an ap-propriate spinneret into an electric field to generatefilaments. The filaments were collected on a stainlesssteel rotating mandrel (diameter 5-10 cm, potential -10kV) in the form of membranes. These membranes con-sisted of fibrous networks with a pore size of 5-10 µmand large internal surface areas (4 m2/g). To acquire Cl,double bonds, or NdCdO groups, the membranes werefunctionalized using isocyanate agents (such as 2-chlo-roethyl isocyanate, 2-isocyanatoethyl methacrylate, hexa-methylene diisocyanate, or 2,4-toluene diisocyanate) inhexane at 25 °C for 1-5 days. The functionalizedmembranes were activated either via the free-radicalreaction with N-acryloxysuccinimide or N-succinimidemethylacrylamido-6-caproate in N,N′-dimethylacetamideor via the direct coupling with N-succinimidyl 3-(4-hydroxyphenyl)propionate, 1,1′-carbonyl diimidazole, or2-fluro-1-methylpyridinium toluene-4-sulfonate in aceto-nitrile. Affinity ligands (such as proteins A and G) werecoupled to the activated membranes.

2.1.3.3. Poly(vinyl chloride) and Poly(tetrafluoroethyl-ene) Microporous Composite Sheets. The FMC Corpora-tion developed a technology to prepare microporouspoly(vinyl chloride) (PVC) membranes in which particu-late or fibrous cellulose derivatives or functionalized silicabeads were embedded (82, 83). This technology consistedof the following steps: (1) blending PVC powders withparticulate cellulose derivatives (diethylaminoethyl, qua-ternary ammonium, carboxylmethyl, or sulfoxyethyl cel-lulose) or silica derivatives (carboxylmethyl or sulfopropyl-silica); (2) adding cyclohexanone (as organic solvent andporogen for the PVC) and water (as a swelling agent);(3) blending and extruding the mixture into a sheet form;and (4) extracting the organic solvent with hot water anddrying. These membranes possess a rigid structure, lownonspecific binding, and alkaline resistance, and theircellulose or silica derivatives are accessible to outsidesolutes. These membranes can be used for proteinpurification.

Poly(tetrafluoroethylene) (PTFE) microporous compos-ite sheets were prepared in a similar way, and styrene-divinylbenzene-based ion-exchange (quaternary NH4

+ orsulfonic acid) resins, iminodiacetate-cellulose resins, orsilica derivatives were enmeshed in the PTFE fibril

matrix (84, 85). The procedure consisted of (1) blendingone or more hydrophilic particulate materials and addingwater to form a damp mixture; (2) adding, under vigorousstirring, an aqueous emulsion of poly(tetrafluoroethylene)(which contained a nonionic surfactant, such as octylphe-nol polyoxyethylene or nonylphenol polyoxyethylene) andmixing vigorously at 50-100 °C to initiate the fibrillationof the PTFE particles; (3) biaxially calendering thePTFE-particulate mixtures to generate a self-supportingsheet; and (4) drying the resulting sheet. The uniformand porous composite sheets (0.45 µm pore size), consist-ing of 90 wt % uniformly distributed resins and 10%interentangled and fibrillated PTFE, could provide du-rable, tear-resistant, and water-swellable composite sheetfor protein separation or water purification.

Using a similar technique and molecular recognitiontechnology, IBC Advanced Technologies, Inc. commercial-ized the PTFE-based affinity membranes for metalremoval (86). Selective silica beads with macrocycleligands were enmeshed into PTFE membranes (Empore)without the use of binders or adhesives. These mem-branes are particularly useful for radioactive wastedisposal.

2.1.3.4. Macroporous Chitin and Chitosan Membranes(87-89). Macroporous chitin and chitosan membraneswith controlled pore sizes and good mechanical propertieswere prepared recently (87-89). Chitin, poly(2-amino-2-deoxy-D-glucose), which is present in the outershells oflobster, shrimp, and crab, is the second most abundantnatural biopolymer; chitosan is the deacetylated productof chitin (for their structures see Figure 2). Because oftheir availability, hydrophilicity, biocompatibility, non-toxicity, biodegradability, and chemical reactivity, chitinand chitosan could be excellent biomaterials (Table 1)(90-92). Chitosan is soluble in acidic solutions andinsoluble in basic solutions, whereas silica is insolublein acidic solutions and soluble in basic media. Theseopposite properties were employed to prepare macroporousmembranes (87). The macroporous chitosan membraneswere prepared as follows: (1) Chitosan was dissolved ina dilute aqueous acetic acid solution containing a certainamount of glycerol. (2) Silica particles of selected sizeswere added with vigorous stirring to the chitosan solu-tion. (3) The solution obtained was poured onto a rimmedglass plate, and the liquid (water) was allowed toevaporate. (4) The dried membrane was immersed in anaqueous NaOH solution in which the silica particles were

Figure 2. Structures of cellulose, chitin, and chitosan.


dissolved (chitosan is soluble in acidic solutions butinsoluble in alkaline ones), and macroporous structureswere formed. Heat treatment accelerated the dissolutionof silica, stabilized the pore structure of membranes, andeven improved the mechanical properties. (5) The porouschitosan membrane was rinsed with distilled water andthen either stored in ethanol/methanol or stored in drystate after an aqueous glycerol treatment (Figure 3). Thismethod provided a simple and effective method to obtainmacroporous chitosan membranes that possessed a strongmechanical strength and uniform three-dimensional porenetworks (Figure 4). One of the significant features wasthat by selecting a size and an amount of silica particlesone could easily control the pore size and the porosity ofthe membrane. Three kinds of silica particles (particleswith a broad size dispersion between 15 and 40 µm, andparticles with a narrow size and an average size of 10 or5 µm) were used for the preparation of the macroporouschitosan membranes. The larger silica particles providedthe larger pore sizes of the membranes. The average poresizes of the membranes (prepared with a weight ratio ofsilica to chitosan of 8:1) for the above three kinds of silicaparticles were 19.5 ( 13.0, 6.6 ( 3.7, and 2.5 ( 1.2 µm,respectively. The broad range of pore sizes is due to thedispersion of the silica particle sizes. The pore size of themembranes can be controlled by using a more narrowsize distribution of silica particles. By varying the weightratio of silica to chitosan one can easily control theporosity of the membrane. The higher the weight ratio,the higher was the porosity (Figure 4).

To prevent dissolution in acidic solutions, the chitosanmembranes were cross-linked with various agents. Forinstance, chitosan membranes were cross-linked throughtheir treatment with a 10 vol % ethylene glycol diglycidylether (EGDE) aqueous solution at 50 °C for 20 min.However, the cross-linking usually decreases the numberof functional groups (CH2OH and NH2) and hence the

possible ligand density. The amine groups of the chitosanmolecules are much more active than the CH2OH groupsand can be much more easily attacked by cross-linkersor by modifying agents (which provide functional groups)under mild conditions. To maintain the number of aminegroups, the chitosan membranes could be cross-linkedwith a 1 × 10-2 M epichlorohydrin aqueous solution atpH 10 and 50 °C for 2 h, which reacted mostly with theOH groups. The cross-linked membranes became in-

Table 1. Applications of Chitin and Chitosana

fields key properties applications

pharmaceutics biodegradable, easy film forming,cationic and linear polyelectrolytes,forming gels and capsules with anionic polymers

controlled drug delivery carriers,encapsules (microcapsules or

microspheres)biotechnology numerous reactive groups for activation and cross-linking,

entrapment and adsorption,porous beads/membranes for isolation and recovery

of biomolecules

enzymes and cells immobilization,matrix for affinity/ion-exchange

beads or membranes

biomedicalengineering

biological activities:antifungal/antimicrobial/antiinfectious,

immunoadjuvant activities:macrophage activation, cytokine production,suppression of Meth-A tumor growth,

adsorbable, flexible, adhesive membranes/fibers,cell binding, aggregation, and activating,biocompatible, exudate adsorbable,inhibit fibroplasia in wound healing and promote tissue growth,stimulate cell proliferation and reconstruction

wound dressings,bacteriostatic/hemostatic agents,antitumor/antivirus agents,blood anticoagulants,sutures/bandages/artifical skin,contact lenses, andartifical kidney

chemical industry metal chelation,flocculation with proteinaceous wastes, andseparation membranes

water purification, sludge treatment,gas separation, reverse osmosis,pervaporation, ultrafiltration, and

microfiltration membranesfood industry cationic polysaccharides,

hypocholesterolemic and hypolipidemic activity:chitosan binds cholesterol, fatty acids, andmonoglycerides, thus preventing their adsorption

anticholesterolemic agent, anddiet foods

cosmetics nontoxic cationic polymers, film forming,biodegradable and biocompatible,viscose and moisture-holding

hair spray, lotion, nail polish,shampoo, mousse, and moisturizer

agriculture protect plants,fungicides/herbicides/insecticides/pesticides,increase crop yields

seed coating

a See refs 90-92.

Figure 3. Preparation of macroporus chitosan membranes: (1)A 1 g portion of chitosan was dissolved in 100 mL of 1 vol %aqueous acetic acid solution, and silica particles of selected sizeswere added under vigorous stirring. (2) The silica suspensionin the chitosan solution was cast on a rimmed glass plate, andthe liquid (water) was allowed to evaporate. (3) The driedchitosan membrane was immersed in 5 wt % NaOH solution,the silica particles were dissolved (chitosan is soluble in acidicsolutions but insoluble in alkaline ones), and macroporouschitosan membranes with good mechanical properties were thusobtained. (4) The porous chitosan membranes was rinsed withdistilled water and then stored either in ethanol/methanol orin dry state after glycerol treatment. (b) silica particles; (O)pores.


soluble in 5 vol % aqueous acetic solution and alsoexhibited improved mechanical properties.

Because chitin is usually insoluble in acidic and basicsolutions, as well as in common organic solvents, it isdifficult to obtain macroporous chitin structures by theusual phase inversion method. However, macroporouschitin membranes could be easily obtained indirectly (87)via the acetylation of chitosan macroporous membranewith 5 vol % acetic anhydride in methanol at 50 °C for 1h (Figure 5). The acetylated chitosan membranes (chitinmembranes) have chemical stability and good mechanicalproperties. One significant feature of the chitin mem-branes is that they contain N-acetyl-D-glucosamine units,which are affinity ligands for lysozyme and wheat germagglutinin. Consequently, they could be directly utilizedfor the affinity purification of lysozyme or wheat germagglutinin without further chemical modifications.

2.2. Activation of the Membranes and Couplingof Ligands. Once the basic membranes are selected, theycan be activated (if the membranes have no activatedgroups) to acquire reactive groups for the coupling ofligands. The traditional activation methods used for

Figure 4. Morphologies of chitosan membranes prepared from 15-40 µm silica. Weight ratio of silica to chitosan: (a, top left) 20:1;(b, top right) 16:1; (c, bottom left) 8:1; (d, bottom right) 4:1.

Figure 5. Preparation of macroporous chitin membranes byacetylation of chitosan macroporous membranes with 5 vol %acetic anhydride in methanol at 50 °C for 1 h.


beads or gels in column chromatography (92-95) can beemployed for membrane activation. The most commonactivating reagents are carbonyl diimidazole (CDI), 2-flu-oro-3-methylpyridinium tosylate (FMP), cyanuric chloride(trichloro-s-triazine), N-hydroxy succinimide esters (NHS),1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC),and epoxy or bisoxirane.

CDI, bisoxiranes (such as 1,4-butanediol diglycidylether), cyanuric acid, NHS, and FMP are usually usedto activate the hydroxyl groups, whereas CDI and EDCare useful for the activation of carboxyl groups. Aminogroups can be activated with epichlorohydrine, glutaral-dehyde, epoxide, and bisoxiranes. Table 2 lists variousactivating and coupling methods.

After the membranes are activated, various ligands(metal chelates, dyes, ion-exchange ligands, hydrophobicinteraction ligands, or affinity ligands) (see Table 3) canbe coupled to them under mild conditions. Detailedcoupling conditions are provided in references (92-95).

3. Applications of Membrane Chromatographyfor Protein Purification

On the basis of different interactions between ligandsand ligates, various ion-exchange, affinity, hydrophobicinteraction, and reversed-phase membranes have beendeveloped for the purification of proteins, enzymes, andantibodies from various sources. Table 4 lists a numberof applications of membrane chromatography to proteinpurification. Detailed information is provided below onlyabout our recent research regarding the application ofchitin and chitosan membranes to protein purification.

3.1. Applications of Macroporous Chitin Mem-branes. The macroporous chitin membranes are verystable in both acidic and basic solutions and containnumerous N-acetyl-D-glucosamine moieties, which canselectively bind lysozyme and wheat germ agglutinin.Therefore, the chitin membranes can be excellent can-didates for the separation of lysozyme and wheat germagglutinin.

3.1.1. Lysozyme Separation from Egg White (96).

Table 2. Various Activation and Coupling Methodsa

functional groupson membranes activating agents activating conditions coupling conditions

reactivegroups

stabilityof bond

reagenttoxicity

OH CDI dioxane/acetonitrile15-30 min, rt

pH 4-10, 24 h, 4 °C NH2 good moderate

tresyl chloride acetone/pyridine10 min, rt

pH 7-10, 24 h, 4 °C NH2/SH excellent low

tosyl chloride acetone/pyridine10 min, rt

pH 9-11, 24 h, 4 °C NH2/SH excellent low

cyanuric chloride toluene, 2 h, 5 °C pH 8-10, 12-24 h NH2 good highcyanogen bromide 10-20 min NH2 poor highFMP acetronitrile/TEA

1 h, rtpH 7-9, rt NH2 excellent moderate

NH2 glutaraldehyde 1-3%, pH 7, 2 h, rt pH 5-9, 2 h, rt NH2/SH excellent moderatesuccinic anhydride 1%, pH 6 see carboxyl group activation and couplingdiglycolic anhydride dioxane/THF see carboxyl group activation and couplingthiophosgene 10% in CHCl3 pH 9-10, 24 h, rt NH2cyanuric chloride same as OH

COOH thionyl chloride 10% in CHCl3, 4 hr pH 8-9, 1 h, rt NH2CDI pH 2-5, 2-3 h, rt pH 9-10, 2-3 h, rt NH2/SH/OHhydroxysuccinimide dioxane/DCC, 4 h, rt pH 5-9, 2-24 h, rt NH2

a See ref 144. CDI, 1,1′-carbonyldiimidazole; FMP, 2-fluro-3-methylpyridinium tosylate; TEA, triethylamine; DCC, N,N′-dicyclohexyl-carboddiimide; rt, room temperature.

Table 3. List of Affinity Ligands and Corresponding Ligatesa

affinity ligands specificity ligates

concanavalin A R-D-glucosyl, R-D-mannosyl residues interferon, plasminogen activator, glycoproteins,polysaccharides, glycopeptides

lentil lectin similar to Con A glycoproteins, membrane glycoproteinswheat germ agglutinin N-acetyl-â-D-glucosaminyl residues glycoproteins, polysaccharidespeanut lectin N-acetyl-galactosamine/galactose residues glycoproteins, glycopeptidesprotein A Fc region of IgG IgA, IgG, IgM, antibody, insulin-like growth factorprotein G Fc region of IgG IgGheparin enzyme-activator interaction,

enzyme-inhibitor interactionhuman antithrombin polymerase, coagulation factor,

lipases and lipoproteinspolymyxin B endotoxinbenzamidine interaction with trypsin and

trypsin-like serine proteasesurokinase, trypsin, TPA, thrombin, plaminogen, kallikrein

Cibacron Blue F3GA/Procion Red HE-3B

NAD+ or NADP+ binding sites ofenzymes, or other specificities

interferon, kinases, dehydrogenases and albumins

metal chelates(iminodiacetic acid)

histidine-, typtophan-, and cysteine-containing proteins

calmodulin calcium-dependent proteins phosphdiesterases, ATPase, and calcinerinarginine and lysine biospecific or electrostatic interaction plasminogen, plasminogen activator, prothrombingelatin fibronectinantibody antigen-antibody interaction antigenantigen antigen-antibody interaction antibodyenzyme enzyme inhibitorenzyme inhibitor enzymeenzyme cofactor enzymehormone receptorDNA DNA/RNA polymerase

a NAD, nicotinamide adenine dinucleotide; NADP+, nicotinamide adenine dinucleotide phosphate.


Table 4. Applications of Membrane Chromatography for Protein Purificationa

basic membranes ligand ligate refs

modified cellulose protein G IgG 134STI trypsin 139PAB trypsin 137Cibacron Blue F3GA alkaline phosphatase 72histidine endotoxin 133

cross-linked regenerated cellulose Cibacron Blue F3GA lysozyme 13anti-BSA BSA 104S immunofusion protein 136S HSA 111S BSA 135anti-rat IgE IgE 106Q lysozyme/chymotrypsinogen/STI 111Q â-lactoglobulin/ovotransferrin 119S/Q lactoglobulin/lysozyme/conalbumin/

cytochrome c/chymotrypsinogen120

S/Q whey protein 125DEAE myoglobulin/ovalbumin 14DEAE lactoalbumin/transferrin/conalbumin/

ovalbumin/trypsin inhibitors103

CM chymotrypsinogen/cytochrome c/lysozyme

103

SP lactalbumin/BSA 122SP lysozyme/ovalbumin 119SP whey protein 124L-aspartate aspartase 130

cellulose acetate protein A/G IgG 75IDA BSA/bovine γ-globumin 73

cellulose/acrylic composite protein A/G IgG 77QAE kallikrein 118DEAE recombinant protein 80DEAE horse immunoglobumin 141DEAE albumins 143SP tPA 142SP human/bovine thrombin 79SP human interleukin 1â 140SP Mab 101IDA urokinase 138

chitin inherent GlcNac lysozyme 96Inherent GlcNac wheat germ agglutinin 97

chitosan inherent amino lysozyme/albumin 98inherent amino cytochrome c/STI 98inherent amino cytochrome c/HSA 98PAB trypsin 99Cibacron Blue F3GA HSA 100

nylon protein G IgG 43, 115nonproteinogenous ligand IgG 114Cibacron Blue F3GA FDHGCibacron Blue F3GA pyruvate decarboxylase/

adenylase kinase/MDHG6

Cibacron Blue F3GA L-alanine dehydrogenase 102IDA concanavalin A/ovalbumin/lysozyme 22protein G γ-globulin 43DEAE BSA/IgG 105

PVDF protein A IgG 48modified polysulfone protein A IgG 20

Trypsin STI 128DEAE â-galactosidase 117IDA histidine/tryptophan 51

modified poly(ether sulfone) (PES) Cibacron Blue F3GA HSA 25r-protein A IgG 24

Modified PES-poly(ethylene oxide) protein A IgG 21gelatin fibronectin 1

modified polyethylene L-phenylalanine bovine γ-globulin 28tryptophan bovine γ-globulin 31phenyl/butyl BSA 37diethylamino BSA 108ethanolamino BSA 8poly(L-lysine) heparin 131IDA BSA 33SP lysozyme 36STI trypsin 38

polypropylene Procion Blue MX-R creatine phosphokinase 19modified glass hollow fiber IDA cytochrome c/lysozyme/

ribonuclease A/chymotrypsinogen A

49


The maximum lysozyme adsorption capacity of macro-porous chitin membranes, from a 0.1 M phosphate buffer(pH 8) solution containing 1 M NaCl at 20 °C, is 40 mg/mL membrane, much larger than that of chitin-basedbeads [5 mg/mL for chitin-coated cellulose, and 2.5 mg/mL for magnetic chitin] and somewhat larger than thatof the commercial Fractogel TSK strong cation exchanger(30 mg/mL). This occurs because the chitin membraneshave a large surface area and provide a larger numberof accessible binding sites. Also, the macroporous chitinmembranes allow high flow rates. The dynamic capacityof chitin membranes was 19.1, 14.7, and 4.1 mg/mLmembrane at flow rates of 1, 5, and 15 mL/min, respec-tively.

A mixture containing 1 mg/mL lysozyme and 1 mg/mLovalbumin was used to investigate the selectivity of thechitin membranes for lysozyme or ovalbumin, and theresults are summarized in Table 5. A high puritylysozyme (>99%) with a high specific activity (∼48 000units/mg) was obtained. This high selectivity is due tothe hydrogen bonding as well as van der Waals interac-

tion between lysozyme and chitin oligosaccharide, (Glc-Nac)6. Ten cycles of adsorption/washing/desorption/regeneration were used, and the results indicated thatthe chitin membranes retained their adsorption capacity

Table 4 (Continued)

basic membrane ligand ligates refs

poly(GMA-co-EDMA) trypsin STI 58DEAE myoglobin/conalbumin/STI 116protein A IgG 109diethylamine chicken egg albumin 57diethylamine BSA/myoglobin/conalbumin/STI 56C4 ribonuclease/lysozyme/ovalbumin 54IgG protein G 121

poly(styrene-co-divinylbenzene) bradykinin/[D-Phe7]-bradykinin/cytochrome/myoglobin/ovalbumin

59

poly(acrolein-co-acrylonitrile) IgG 61poly(2-hydroxyethyl methacrylate) Cibacron Blue F3GA hydrogen peroxide oxidoreductase 64poly(ether-urethane-urea) protein A IgG 81poly(ethylene-co-vinyl alcohol) histidine IgG 66, 68, 107synthetic copolymer heparin antithrombin III 112

Cibacron Blue malate dehydrogenase/BSA 7S/Q Mab 129IDA HSA 113

PEI-coated titania Cibacron Blue F3GA HSA 23hydrazide hollow fiber LI-8/5B1 Mab interleukin-2 receptor/interleukin-2

human interferon-2-2 R110, 126,127

Immobilon AV membrane pepstatin A pepsin/chymosin 123Quick Disk Q tumor necrosis factor R 132PVC composite sheet functionalized cellulose BSA 82PTFE composite sheet functionalized

styrene-divinylbenzene145

a STI, soybean trypsin inhibitor; PAB, p-aminobenzamidine; PEVA, poly(ethylene vinyl alcohol); BSA, bovine serum albumin; HSA,human serum albumin; GPDHG, glucose-6-phosphate dehydrogenase; FDHG, formate dehydrogenase; MDHG, malate dehydrogenase;GMA, glycidyl methacrylate; EMDA, ethylene dimethacrylate; Mab, monoclonal antibody; IDA, iminodiacetic acid; S, sulfonic acid; Q,quaternary ammonium; SP, sulfopropyl; tPA, tissue plasminogen activator; DEAE, diethylamino ethyl.

Table 5. Separation of Lysozyme and Ovalbumin byChitin Membrane Cartridgea

no.flow rate(mL/min)

recirc.no.

loadingvol

(mL)

lysozymeeluted(mg)

purityb

(%)yieldc

(%)

specificactivityd

(units/mg)

1 1 1 100 39.7 >99 39.7 48 8662 1 1 40 17.8 >99 44.5 47 8653 5 2 40 18.4 >99 46 47 9344 15 3 40 24.6 >99 61.5 48 293

a Four membranes, 1 mm total thickness, and 1.4 mL totalvolume (96). Conditions: solution containing 1 mg/mL lysozymeand 1 mg/mL ovalbumin in buffer (0.1 M Na phosphate solutionplus 1 M NaCl, pH 8); washing with the buffer for 10 min at 15mL/min and elution with 0.1 M acetic acid aqueous solution for15 min at 5 mL/min. b Assayed on HPLC (weak cation-exchangecolumn, wide-pore CBx, 5 µm, 7.75 mm × 100 mm, J. T. Baker)c Yield of lysozyme is expressed as the ratio between the amountof lysozyme eluted and the amount of lysozyme loaded d Originalactivity of lysozyme was 50 000 units/mg

Figure 6. Isolation of lysozyme from egg white. A 6 mL portionof homogenized egg white was diluted with 60 mL of 0.1 M PBSbuffer, filtered, and centrifuged at 1600g for 20 min. Thesupernatant was loaded to a chitin membrane cartridge (fourstacked flat membranes, 1 mm total thickness and 1.4 mL totalmembrane volume) at 1 mL/min. It was followed by washingwith 0.1 M PBS buffer at 15 mL/min and elution with 0.1 Maqueous acetic acid solution at 5 mL/min.


at almost constant value. Macroporous chitin membraneswere also employed to separate lysozyme directly fromegg white. The operational conditions are presented inFigure 6. Eleven milligrams of lysozyme with a purity>98% and specific activity >54 000 units/mg was ob-tained from 6 mL of egg white. These results indicatethat macroporous chitin membranes are promising andeconomical matrixes for lysozyme separation.

3.1.2. Separation of Wheat Germ Agglutinin fromWheat Germ (97). Wheat germ agglutinin (WGA) is animportant and very expensive lectin useful in medicalstudies. Macroporous chitin membranes with large poresizes (average 18 µm) and high adsorption surface wereused recently to separate wheat germ agglutinin from awheat germ extract (97). Ten flat chitin membranes (6.9mL total membrane volume and 5 mm total thickness)were used. The maximum adsorption capacity of wheatgerm agglutinin on chitin membranes, from a 0.01 MTris-HCl, pH 8.5 solution, was 176.6 ( 8.5 mg WGA/gchitin membrane, almost 18 times greater than that onchitin beads (10 mg/g chitin).

The isolation of WGA from wheat germ and its puri-fication on chitin membranes are outlined in Figure 7,and the results are listed in Table 6. To obtain a highpurity wheat germ agglutinin, a two-step elution wasemployed. A 0.05 M HCl solution in the first elution step

could remove most contaminants (including the remain-ing lipids). [Although some wheat germ agglutinin wascoeluted in this step, the amount was relatively small(about 10% of the WGA eluted in the second step) andcould be recovered in a pure form after its dialysis against0.01 M Tris-HCl solution (pH 8.5).] The second elutionstep with 1 N CH3COOH could provide a high puritywheat germ agglutinin (Figure 8). A purification factorof 5.5 and an activity yield of 40% could be obtained.About 25 mg of pure wheat germ agglutinin could beobtained from 50 g of wheat germ.

3.2. Applications of Macroporous Chitosan Mem-branes. Because the chitosan membranes are hydro-philic, have mechanical strength, and contain numerousreactive amino groups, various affinity ligands (such asdyes and inhibitors) can react with the amino groups toprovide membranes with a high density of functionalgroups. Therefore, the chitosan membranes could serveas good ion-exchange membranes and affinity mem-branes for various protein purifications.

3.2.1. Cross-Linked Macroporous Chitosan Anion-Exchange Membranes for Protein Separation (98).One of the key properties of chitosan is the presence ofnumerous amine groups. At pH < 6.5, the chitosanmembranes are positively charged (pKb, where Kb)[NH3

+][OH-]/[NH2], is about 6.5-7) and can adsorb

Figure 7. Isolation and purification protocol for wheat germ agglutinin from wheat germ.

Table 6. Isolation and Purification of Wheat Germ Agglutinin from 50 g of Wheat Germ

steptotal protein

(mg)total activity

(units)specific activityb

(units/mg)activity recovery

(%)purification

factor

crude extract 296 ( 5.5 6623 ( 201 22.4 ( 1.1 100 1precipitation 193 ( 13 6273 ( 128 32.7 ( 1.6 94.9 ( 4.9 ∼1.5dialysis 51 ( 2 3228 ( 158 63.4 ( 5.6 48.9 ( 3.9 ∼3.2affinity stepc 24.7 ( 0.6d 2688 ( 26 109 ( 3.8 40.6 ( 0.8 ∼5.5

a See ref 97. b Specific activity of pure wheat germ agglutinin, 128 units/mg (assayed by hemagglutination method, using humanerythrocyte A+). c The crude wheat germ agglutinin solution after dialysis against buffer (0.01 M Tris-HCl, pH 8.5) was recirculatedthrough a chitin membrane cartridge (10 flat membranes, 5 mm total thickness, 6.9 total mL volume) for 1 h at a flow rate of 2 mL/min.d WGA eluted with 1 M CH3COOH plus WGA amount after dialysis of eluted WGA with 0.05 M HCl.


negatively charged proteins that have pI’s smaller thanabout 6. The chitosan membranes cross-linked withethylene glycol diglycidyl ether (EGDE) are very stable,maintain their strength even in acidic or basic solutions,and have a high anion-exchange capacity (0.83 mequiv/gdry membrane). Three proteins with low pI’s (ovalbumin,pI ) 4.6, human serum albumin, pI ) 4.8, soybeantrypsin inhibitor, pI ) 4.5) and two proteins with highpI’s (lysozyme, pI 11, and cytochrome c, pI 10.6) wereemployed as model proteins. The separation of proteinsby five flat chitosan membranes (3.45 mL total volume,5 mm total thickness) from various binary mixtures(ovalbumin-lysozyme, human serum albumin-cyto-chrome c, or soybean trypsin inhibitor-cytochrome c) wasinvestigated. A 20 mL sample containing 0.5 mg/mL ofeach of the two proteins was employed. Their effluentsfrom the membranes during adsorption, washing, andelution were collected, and their purity was assayed bythe SDS-PAGE method. All three mixtures were suc-cessfully separated, and highly pure products (>99%)with high yield were obtained (Table 7). These resultsindicate that the cross-linked chitosan membranes arestable, have high adsorption capacity, and can easilyseparate proteins in the anion-exchange mode. Thesemembranes could become excellent candidates for the

removal of endotoxins and nucleic acids, which havehighly negative charges, from recombinant proteins.

Figure 8. Purification of crude wheat germ agglutinin extract on a chitin membrane cartridge (10 stacked flat membranes, 5 mmtotal thickness and 6.9 mL total membrane volume). A 50 mL portion of crude wheat germ agglutinin extract solution (in 0.01 MTris-HCl buffer, pH 8.5) obtained from 50 g of wheat germ was loaded to the cartridge at a flow rate of 2 mL/min. This was followedby washing successively with 0.01 Tris buffer, 1 M NaCl in the buffer, and again with Tris buffer at 5 mL/min. The adsorbed proteinswere eluted at 5 mL/min stepwise with 0.05 M HCl to remove the impurities and with 1 M acetic acid aqueous solution to removethe strongly bound agglutinin.

Table 7. Protein Separation with a Cross-LinkedMacroporous Chitosan Membrane Cartridgea

protein mixture eluted protein (mg, purity)

lysozyme + ovalbumin ovalbumin (9.5, ∼99%)cytochrome c + HSA HSA (9.1, ∼99%)cytochrome c + STI STI (9.3, ∼99%)

aFive membranes, 5 mm total thickness, 3.45 mL total volume,ion-exchange capacity 0.83 mequiv/g dry membrane; from three20 mL samples of binary mixtures (10 mg for each protein) (98).

Figure 9. Scheme of the covalent coupling of p-aminobenza-midine (PAB) to macroporous chitosan membranes. Chitosanmembranes were cross-linked with 5 vol % ethylene glycoldiglycidyl ether aqueous solution at 50 °C and then coupled with1 wt % succinic anhydride in methanol at room temperaturefor 16 h. The unreacted amino groups of the chitosan mem-branes were blocked with 5 vol % acetic anhydride in methanolat 50 °C for 1 h. PAB was coupled to the succinylated chitosanmembranes with an excess amount of activating agent (1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, EDAC) in 0.1 M MESbuffer (pH 4.75) at room temperature for 16 h.


3.2.2. Trypsin Purification with p-Aminobenza-midine-Chitosan Affinity Membranes (99). p-Ami-nobenzamindine (PAB), a trypsin inhibitor, is muchcheaper than other inhibitors (such as soybean trypsininhibitor and ovomucoid) and widely used as ligand fortrypsin and trypsin-like enzymes (urokinases, plasmino-gen activators, and serine proteases) separation. PABwas coupled to chitosan membranes using succinic acidas spacer (Figure 9) (99). A high PAB density (2.7 × 10-3 mol PAB/g chitosan) was obtained. The maximumtrypsin adsorption capacity of PAB-chitosan membranewas as high as 78.4 ( 4.2 mg/g chitosan, hence muchgreater than that of PAB-Sartorius SM 16278 mem-brane (16 mg/g) (137).

Figure 10 presents the purification of trypsin by PAB-chitosan membranes (10 membranes, 3 mm total thick-ness, and 4.1 mL total volume) from a crude trypsinsolution (5 mL with 5 mg solid/mL, total protein 7.2 mg).The first 17 fractions (may contain digested trypsin,chymotrypsin, and other impurities) did not exhibittrypsin BAEE (NR-benzoyl-L-arginine ethyl ester) activity.Fractions 20-26 exhibited high trypsin activities. Bymixing fractions 20-43, the total product was about 2.1mg, with the high specific activity of 10 340 BAEE units/mg. Being stable, these membranes are suitable fortrypsin purification.

3.2.3. Dye-Coupled Chitosan Membranes for Hu-man Serum Albumin (HSA) Separation (100). Dye-ligand chromatography plays an important role in theseparation, purification, and recovery of proteins andenzymes because the dyes are inexpensive, stable, andgroup-specific. Cibacron Blue F3GA [which has a specificbinding for nicotinamide adenine dinucleotide (NAD+)-dependent enzymes] and Procion Red HE-3B [which hasa specific binding for nicotinamide adenine dinucleotidephosphate (NADP+)-dependent enzymes] were widelyused for the purification of various dehydrogenases,kinases, and albumins. Recently, monochloro triazinedyes (Cibacron Blue F3GA and Procion Red HE-3B) and

dichloro triazine dye (Procion Blue MX-R) were coupledto macroporous chitin and chitosan membranes, throughthe reaction between chlorine groups of the reactive dyesand CH2OH groups of chitin or CH2OH and NH2 groupsof chitosan under mild alkaline conditions (100). The dyecontent of the chitosan membranes (35.5 µmol/mL forCibacron Blue F3GA, 74.5 µmol/mL for Procion Red HE-3B, and 197 µmol/mL for Procion Blue MX-R) is muchhigher than the typical 1-10 µmol/mL bead for the cross-linked agarose beads, as result of the larger number ofreactive and accessible groups on chitosan and chitin. Thechitosan membranes have higher dye contents than thechitin membranes (7.3, 40, 14.5 µmol/mL for CibacronBlue F3GA, Procion Red HE-3B, and Procion Blue MX-R, respectively), because they possess both NH2 and CH2-OH groups, whereas the latter possess only CH2OHgroups. The adsorption capacities of these membranesfor HSA are Cibacron Blue F3GA > Procion Blue MX-R> Procion Red HE-3B, for both chitosan and chitinmembranes. Having a structure similar to that of biliru-bin, Cibacron Blue F3GA can bind more tightly to thebilirubin sites of the human serum albumin.

Cibacron Blue F3GA-chitosan membranes (10 mem-branes, 6.9 mL total volume, 35.5 µmol dye/mL mem-brane, maximum adsorption capacity 8.4 mg/mL mem-brane) were employed for the separation of human serumalbumin from human plasma. The separation protocol ispresented in Figure 11. From 1 mL of human plasma,12.5 mg of HSA with a very high purity (>96%) could beobtained. The Cibacron Blue F3GA-chitosan membranesexhibited reproducibility during four successive opera-tions on the same group of membranes. These resultsindicate that the Cibacron Blue F3GA-chitosan mem-brane has a high selectivity toward HSA and that a stableand efficient separation of HSA from human plasma canbe obtained.

Figure 10. Purification of trypsin from a crude trypsin solution by a p-aminobenzamidine (PAB)-chitosan membrane cartridge (10stacked flat membranes, 3 mm total thickness, 4.1 mL total volume, and PAB density of 2.7 mmol/g chitosan). Five milliliters of 5mg/mL crude trypsin solution (6500 BAEE units/mL) in 0.05 M Tris buffer (pH 8, containing 0.02 M CaCl2 and 0.1 M NaCl) wasloaded to the cartridge at a flow rate of 0.5 mL/min. This was followed by washing with 0.05 M Tris buffer at 2 mL/min and elutionwith 0.1 M acetic acid at 5 mL/min. Fractions were 2 mL each.


AcknowledgmentThis work was supported by the National Science

Foundation.

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Accepted September 24, 1999.

BP990120E


membrane chromatography: preparation and applications to protein...

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