Ind. Eng. Chem. Res. 1999, 38, 333-342
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APPLIED CHEMISTRY Application of Membranes and Compact, Porous Units for the Separation of Biopolymers Djuro Josic´ * Octapharma Produktionsges m.b.H., Oberlaaer Strasse 235, A-1100 Wien, Austria
Alesˇ S ˇ trancar BIA d.o.o., Teslova 30, SI-1111 Ljubljana, Slovenia
For fast separation of biopolymers, recently developed media have become increasingly widespread. They consist either of membranes or of compact, porous disks and tubes, both called Convective Interaction Media (CIM). Separation can be carried out in every mode, e.g., ionexchange, reversed-phase, hydrophobic-interaction, and affinity recognition. The units can be used for analytical as well as for preparative purposes. Such fast analytical units will allow separations within less than 10 s and can therefore be used for in-process analysis. The advantages and disadvantages of such analytical and preparative separations are discussed along with technical problems which have been solved. Introduction New developments in molecular and cell biology in the last quarter of this century have resulted in new technologies for the production of complex biomolecules which have the potential to increase the level of human life quality in the areas of diagnostics, prevention, and treatment of diseases. One of the most important and most cost-intensive steps in this production is the isolation and purification (downstream processing) of the target biomolecule.1 Precipitation, ultrafiltration, and chromatographic techniques are most widely used for these purposes. However, only liquid chromatography leads to a level of purity that is acknowledged to be safe for therapeutic use.1,2 As a rule, more than one liquid chromatographic step is necessary to isolate the product to the desired level of purity. A reduction in the number of separation steps and/or the period of time required for each step would result in a more cost-effective process. Consequently, only the concomitant development of molecular biology and more effective chromatographic techniques will help the production of complex biomolecules.2,3 Liquid chromatography of biopolymers on so-called soft supports is typically slow, often causing significant product degradation. It also requires expensive separation media and large volumes of solvents. Diffusional constraints, in particular, set an upper limit on separation speed, because they cause a rapid reduction in resolution with increasing elution velocity, when conventionally packed columns are used.1,4 Evolution of both column and packing design has to put strong emphasis on speeding up separations with the following objectives in mind: (1) on-line analysis of production and separation processes necessary for regulation, optimization, and regulatory purposes; (2) reduction of * To whom correspondence should be addressed. Fax: +43-1/610 32/330. E-mail:
[email protected].
manufacturing costs; (3) reduction of losses from degradation of the biopolymers; and (4) increase of the speed of the scaling-up processes. Apart from progress made in column chromatography with porous supports, new ways have been investigated in recent years for analytical and preparative separations of biopolymers. The new chromatographic media should allow fast separation, combined with high capacity and low nonspecific interaction. A uniform chromatographic behavior of the support is also important, allowing easy scalability. The first difficulty to be overcome is the rather slow diffusion which will impair the mass transfer of big and frequently irregularly shaped molecules such as biopolymers. The problem was first solved by using columns with nonporous supports. With such supports the ligands are located on the particle surfaces, not inside the pores. This allows a fast mass transfer, and the diffusion of macromolecules into the pores is no longer a limiting factor. By using nonporous supports, the chromatographic separation of biopolymers is cut from over 15 min to a few minutes.5-8 However, columns with nonporous supports have a rather low capacity. This is caused by the small surface of such media and consequently its low ligand quantity. Besides, when supports with particle diameters of between 1 and 2 µm are used, the backpressure in the column is very high, up to 25 MPa at higher flow rates. For these reasons, columns with nonporous supports are suitable only for fast, analytical separations. Scalingup and preparative purposes are rare exceptions to the rule. The introduction of supports for perfusion chromatography was another step taken toward the development of media for biopolymer chromatography.9,10 As the particles for perfusion chromatography have large, continuous canals, the eluent flows past all of the ligands of the support, those located on its surface as well as those found in its interior. Diffusion, which
10.1021/ie970600f CCC: $18.00 © 1999 American Chemical Society Published on Web 12/19/1998
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might otherwise impair fast mass transfer, is therefore reduced in perfusion chromatography. Because of the structure of the particles, backpressure is low and separations can be carried out at high flow rates. Moreover, media for perfusion chromatography have a much higher capacity than nonporous supports. Depending on the hydrophobicity of the polymer used, nonspecific bindings can also be reduced. All of these combined characteristics allow fast separation of biopolymers by perfusion chromatography. With neither capacity nor flow rate being a limiting factor, the scaling up of an analytical column to a preparative column is here no longer a problem. The separation behavior of a preparative column differs little from that of an analytical column. This, in turn, allows prompt monitoring of a preparative separation, using the same support. Small analytical columns containing immobilized antibodies, combined with reversed-phase microcolumns, can confirm within minutes the results obtained in a biopolymer purification, avoiding more complicated analyses such as SDS-PAGE.11 The same degree of performance has recently been observed with other, so-called fast separation media. These media, similar to perfusion media, allow fast mass transfer and separations, as fast as those carried out on micropellicular stationary phases.12,13 Here again, neither capacity nor flow rate constitutes a limiting factor, allowing simple upscaling. Results similar to those in perfusion chromatography are obtained with high-performance membrane chromatography (HPMC). The basis for the development of HPMC was a fast development of membrane technology in recent years. The production of membranes with defined, uniform pores has allowed a simple separation of components according to their sizes, such as fast desalination and concentration of protein solutions. These operations can be carried out in a short time.14 If the membranes also contain immobilized ligands, the process taking place on such a unit is not only filtration but also a specific interaction between the membranes and components of the sample (the ligates). As in membrane chromatography, the components of the sample are first retarded and subsequently eluted by a gradient, depending on the strength of the interaction.15,16 The supports for HPMC have similar surface chemistry and therefore can be used like bulk supports in all existing separation modes such as ion-exchange (IE), reversed-phase (RP), hydrophobic-interaction (HI), and affinity chromatography (AC).15-18 There are several techniques of membrane construction. One strategy is to bundle several thin membranes which are made of synthetic hollow fibers or cellulose. Another strategy involves compact, porous, disk- or tube-shaped units made of silica gel or polymer supports.16-18,21 Membranes and disks have several advantages over high-performance liquid chromatography (HPLC) columns. The chromatographic separation is carried out on a wide, thin disk, resulting in only a low-pressure drop even at high flow rates. The fact that separation on membranes can be carried out very quickly is due to the fast reaction kinetics in such systems.4,17,21,23-26 Chromatography on membranes, compact porous disks, tubes, and rods can be distinguished from particlebased chromatography by the fact that the interaction between ligate (biopolymer) and ligand, immobilized to the matrix, takes place in the through-pores of the separation unit, not in the dead-end pores of the
Figure 1. Comparison of particle-based chromatography and chromatography on compact, porous units. (Reprinted from ref 26 with permission.)
Figure 2. Comparison of a conventional porous particle medium (Mono Q) with a CIM disk with regard to their separation performance under fast gradient elution conditions: (A) a Mono Q anion exchange column 50 × 5 mm i.d.; (B) a QA anion exchange CIM disk 3 × 10 mm i.d. Conditions: buffer A, 10 mM Tris, pH 7.4; buffer B, buffer A + 0.5 M NaCl. Gradient time: (A) 0-100% buffer B in 4 min; (B) 0-100% buffer B in 6 s. Flow rate: (A) 1 mL/min; (B) 10 mL/min. Backpressure: (A) 0.9 MPa; (B) 0.8 MPa. Calibration solution: myoglobin (peak 1), conalbumin (peak 2), and soybean trypsin inhibitor (peak 3) in buffer A. (Reprinted from ref 38 with permission.)
particle, as shown in Figure 1. The main difference between the two chromatographic systems is of a hydrodynamic nature: the mass transport resistance in compact chromatographic units is reduced because pore diffusion is practically nonexistent. This means that film diffusion from the core of the mobile phase to the surface of the matrix in the interior of a through-pore is the only transport resistance.26,27 In Figure 2 it is shown that the period of time required for separation with such units is shorter by at least 1 order of magnitude because
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of faster mass transport, based on convective interaction. A comparison is made with porous bulk supports, where mass transfer is determined by diffusion. Such “fast” compact, porous separation media are therefore also called “Convective Interaction Media” (CIM). Technical problems have at first prevented further progress in the use of HPMC. The bundling of thin membranes often leads to leakages in the system, which in turn causes the mobile phase (and the sample) to overflow the rim and to miss the membranes. Another equally important problem is the distribution of the sample and the mobile phase, which are released out of a narrow capillary onto the wide disk surface. If the void volume is large enough, distribution is ensured before and after the disk. However, separation performance is impaired because of peak broadening.17,22 In recent years, technical problems have been solved satisfactorily, not only by designing piled-up membranes but also by developing other solutions for membrane systems and the appropriate hardware for compact disks and tubes.17,22,23,25-28 In the case of hollow-fiber membranes, there is the further option to fold the separation unit in such a way that it can be fitted into a filtration cartridge. Because of the rather large void volume, such cartidges are used preferably for preparative separations or for affinity chromatography.29-31 If the separation layer is higher than 15 mm, the tools are compact, porous rods with a continuous bed, also called monoliths.25,32-34 These units can be used analytically as well as preparatively. The preparative units allow both the axial flow31-34 and the radial flow mode.35 This review deals with the use of supports that have recently been developed as an alternative to traditional column chromatography with bulk supports. Among these are membranes, compact, porous disks, and tubes, and different continuous rods. They can all be used for fast analytical separations, but also on a preparative scale offering at least the same separation power. On both the preparative and analytical scales, the separation times are considerably reduced particularly through the use of compact, porous disks and tubes. Technical Problems: Construction of a Separation Unit If the membranes are used for affinity-based separations, e.g., for selective isolation or removal of a specific component following the all-or-nothing principle, the questions of sample distribution of peak broadening are not as critical as in the case of fast analytical separations. Sample distribution is guaranteed by a sufficiently large dead volume before the separation unit. When the radius of the capillary, through which the sample is released, is enlarged, the pressure of the incoming liquid is lowered, thus contributing to a better distribution of the sample. In this way the problem of sample distribution is solved by impairing the performance of the separation unit and by broadening the peaks. These separation units have been used primarily for solving special problems such as sample preparation for semipreparative and preparative chromatography. Figure 3 shows the limitations of separation units containing membranes or compact disks, if a rather large dead volume exists. Josic´ et al.17 and Reif and Freitag22 have demonstrated by simple experiments how the performance of an HPMC unit can be improved manifold by simply reducing the void volume.
A
B
Figure 3. Anion-exchange chromatography of standard proteins using a compact, porous disk: (A) with optimized distribution; (B) with a distribution plate, which does not provide the best distribution and subsequently causes a large void volume. Chromatographic conditions: unit, a DEAE poly(glycidyl methacrylate) CIM disk; d ) 25 mm; h ) 2 mm; flow rate, 3 mL/min, pressure 0.10.4 mPa. Standard proteins: (1) myoglobin, (2) conalbumin, (3) ovalbumin, (4) soybean trypsin inhibitor, (5) ferritin. (Reprinted from ref 17 with permission.)
Sˇ vec and Tennikova36 have overcome the difficulties concerning sample distribution by installing the chromatographic unit, that is, the disk made of poly(glycidyl methacrylate), at the bottom of a filtration device. The sample or, during rinsing and elution, the mobile phase is added and pumped through the porous disk. Once again a high dead volume accumulating in the separation unit proves to be the chief disadvantage of this method. If the sample is applied by an HPLC or peristaltic pump, it will inevitably be diluted by the ensuing mobile phase. However, this can be avoided by using an Amicon filtration device for the concentration of protein solutions.17,37 The chromatographic disk is installed at the bottom of the filtration device instead of an inert, porous membrane. The sample is transported through the porous disk by means of compressed air. The container is subsequently connected with the HPLC pump and filled with a sample buffer. In the next step the elution buffer is pumped into the container and stirred. This results in a gradient with an exponential slope. The disadvantage of this method lies in the difficulties arising with the complicated sample application. Reproducibility of the gradient is also rather poor.17 An advantage of the method lies in the fact that it allows fairly large amounts of diluted sample to be applied, together with a good sample distribution, thereby making use of the entire disk surface. This kind of sample application is recommended in preparative chromatog-
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raphy with its large sample volumes, e.g., for the separation of components from cell culture supernatants. It is also useful in affinity chromatography and generally in all kinds of chromatographic separations, where elution is carried out by a step gradient. However, this kind of gradient formation is not recommended for other chromatographic separations. The problem of sample distribution is even more critical when porous, compact disks are used, as opposed to the application of several thin membranes which are bundled. When single membranes are bundled, the ratio between the width of the separation unit and its thickness is much better. The capacity of a unit that consists of several bundled membranes equals the sum of the capacities found in the single subunits. The difficulties arising with the bundling of single thin membranes do not concern sample distribution but the frequent leakage of the system.17 With constructions such as that the seals have to be installed in such a way that the sample is pumped through the membranes and not through the empty space between the membranes and the cartridge. After initial difficulties, the problem was solved as far as MemSep cartridges (formerly made by Millipore) were concerned, e.g., by positioning appropriate seals between single membranes.20 Reif and Freitag22 have solved the problem of bundling equally well with membranes of other producers. When compact disks made of porous, synthetic materials, silica gel, or ceramics are used, bundling is less favorable. Polymerization allows the direct production of thicker layers.32-34 Leakage problems are easily avoided through the use of O-rings. As mentioned before, the problem of sample distribution is most acute when relatively thin but wide disks are used. The application of distribution plates, which in the case of HPLC columns are frequently used, is not possible. The jet of liquid coming out of a capillary with a maximum diameter of 1 mm cannot be evenly distributed on a surface which is 25 mm in diameter or more.17,27,35 Experiments showed that only a small portion of the disk, usually in its center, was available for binding or separating the sample, if distribution plates were used, which are common in HPLC columns. Apart from inadequate distribution, another problem arose. The extremely high pressure building up in the center of the disk shortened the life of the separation device considerably. Therefore, appropriate distribution plates had to be installed before and after the separation device.17,27 In chromatographic columns and even in those separation devices which contain bundled membranes, the ratio between the layer thickness and diameter is much better. Poor distribution on the surface is compensated through subsequent spreading in the first millimeters of the separation layer. Such compensation is impossible when the layer of a disk with a relatively large diameter is very thin, invariably resulting in lower capacity and poor separation. Figure 4 shows the chromatogram obtained by a separation unit containing a compact, porous disk, whose distributor was further optimized. As shown here, a separation of three standard proteins was achieved within less than 8 s. Apart from thin, compact disks, so-called continuous rods or monoliths have been developed from similar materials. Such media are produced by a simple molding process.25,33,34 The rods can be prepared directly within the housing for an HPLC column. Axial columns containing molded rods of porous polymer are usually used
Figure 4. Separation of standard proteins obtained by QA anionexchange compact, porous disk of 3 × 10 mm i.d. Chromatographic conditions: buffer A, 20 mM Tris‚HCl, pH 7.4; buffer B, 1 M NaCl in buffer A; detection, UV at 280 nm; gradient time, 0-50% buffer B in 6 s and then 3 s isocratic 50% buffer B; flow rate, 10 mL/min; backpressure, 0.8 MPa; room temperature. Calibration proteins: 39 µg of myoglobin (peak 1), 130 µg of ovalbumin (peak 2), and 260 µg of soybean trypsin inhibitor (peak 3) in 20 µL of buffer A. (Reprinted from ref 28 with permission.)
Figure 5. Separation of ribonuclease A (1), cytochrome c (2), myoglobin (3), and ovalbumin (4) by reversed-phase chromatography on the continuous poly(styrene-co-divinylbenzene) rod column. Conditions: column (rod), 150 × 4.6 mm i.d.; mobile phase, linear gradient from 20 to 60% acetonitrile in 0.1% aqueous trifluoroacetic acid within 2.4 min; flow rate, 5 mL/min. (Reprinted from ref 33 with permission.)
for analytical separations.25,33,34 However, semipreparative and preparative applications have also been reported.32,35,38 An analytical chromatogram made with such a rod is shown in Figure 5. To reduce the considerable loss of pressure that has to be expected in separations on axial columns with molded rods of porous polymers, so-called compact tubes were introduced. These separation units are constructed in a way similar to that of radial columns, as is shown in Figure 6. Because of the radial flow through the rather thin walls of the column, the backpressure is also much reduced. Such a separation unit was used successfully for preparative purification of plasma proteins, e.g., clotting factor VIII.35 Several technical problems have to be solved in connection with the use of monoliths and compact, porous tubes, especially on a preparative scale. This concerns the prevention of leakage between the body of the monolith and the column case and the above-
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Figure 6. Construction of the separation unit, which contains a compact, porous tube. The mobile phase or the sample is pumped from the inner side of the cylinder, flows through the wall, and is collected on the other side of the unit. The separation process takes place during flow through the porous wall of the tube, as in the case of a radial column. (Reprinted from ref 35 with permission.)
mentioned loss of pressure. In the case of larger monoliths, cracks may occur in the column during polymerization. In the case of compact, porous disks, tubes, and other thin separation units, such problems are less severe. In upscaling, however, an adequate sample and buffer distribution at the entrance to the separation unit must be provided.35,38 Applications Ion-Exchange Chromatography. Ion-exchange chromatography, above all anion-exchange (AE) chromatography on membranes, disks, and rods, is one of the most frequently used methods. Such units, either with quaternary amino groups or (diethylamino)ethyl (DEAE) groups as ligands, are used for analytical as well as preparative purposes.16,17,21,22,34-36,39-48 All of the aforementioned separation units can be used for fast analytical separations. Analysis can theoretically be carried out within a few seconds. However, the difficulties arising from the construction of the unit (cf. Figure 3A,B) can delay separation and impair its quality. If the unit is adequately designed, it will allow separations within seconds such as the DEAE disk shown in Figure 4. It can then be used for in-process control of biopolymer isolation. Since both micro- and macroseparation units are made from the same material, upscaling from an analytical to a preparative scale is easily achieved.17,35,38 Apart from the application of membranes and monoliths carrying AE ligands for the separation of serum proteins,17,22,40,42 the method is used for separating microbial proteins and enzymes,22,23 several proteins from the clotting cascade,28,35,42 membrane proteins,17,42,43 and cytokines.44 Moreover, its use is not limited to the separation of proteins but includes other complex biopolymers such as nucleic acids.45 The application of membranes, disks, tubes, and rods carrying cation-exchange (CE) ligands is not as widespread as that of AE ligands, although the method can be a very effective tool.20,22 Most reports deal with the isolation of monoclonal and polyclonal antibodies46,47 and recombinant proteins such as the 51-kDa-targeted immunodiffusion protein produced by Escherichia coli.48 Hydrophobic-Interaction and Reversed-Phase Chromatographies. HI and RP chromatographies
have rarely been carried out with membranes. Their use has so far been confined chiefly to disks28,36,44,49 and rods.33,51-53 An analytical separation on a HI disk can be achieved within seconds, comparable to that in ionexchange mode.28 Similar results were obtained with rods in RP mode.33,52 In HI mode, the hydrophobicity of the ligand can be adjusted through its density and the number of C atoms in its chain. Apart from propyl ligands, methyl, ethyl, and butyl ligands have been used as well.35,36,51 Disks and monoliths made of polystyrenedivinyl benzene are usually tools for RP separations of biopolymers, mainly proteins.25,33,50 The use of IE and affinity chromatography with membranes, disks, tubes, and monoliths has become quite widespread, and a growing number of applications are found in both the analytical and the preparative sphere. In contrast, the HI and RP modes of these special separation methods have not won general acceptance. In-process applications of RP and HI chromatographies have their problems, because the initial conditions are difficult to fulfill. In the case of HI chromatography, the sample has to be applied with a rather high salt concentration, whereas in the case of RP chromatography, the use of organic solvents presents a certain obstacle. However, as far as downstream processing is concerned, the use of disks, tubes, and monoliths in HI or RP mode will in some cases be the method of choice.25 In one such application, the chromatographic unit with a butyl ligand was used for purification of the recombinant tumor necrosis factor.44 Affinity Chromatography. Apart from IE chromatography, high-performance affinity chromatography on membranes, disks, tubes, and rods has been most widely used for separating biopolymers. Similar to porous or nonporous beads, all kinds of ligands can be immobilized to membranes, disks, tubes, or rods with active groups. A large number of substances has so far been successfully bound, including protein A and protein G,17,24,42,46 antibodies,4,17,42 lectins,55 receptors,4,29 heparin,56 collagen,17 gelatine,16 inhibitors,57,65 and a number of ligands with low molecular masses.37,58-65 In separation units such as membranes, disks, tubes, and rods, practically all of the ligands are bound on the surface and not inside the pores (cf. Figure 1 and ref 27). Therefore, interaction between the ligand and the components of the samples is very fast.4,24 The layers of both the membranes and the disks are comparatively thin, making elution from the unit easier.26,66 This is particularly helpful in cases where chromatographic separation is achieved by binding to the separation unit and subsequent elution with a step gradient. This separation schema applies to almost all cases of affinity chromatography. Because of the lack of smaller pores, the specific surface of the support is very small, reducing the possibility of nonspecific interaction. Nonspecific interaction with the support is a frequently occurring problem in affinity chromatography, especially if complex samples are separated. These very often contain hydrophobic and sticky components.17,56 Separation by affinity chromatography on membranes, disks, tubes, and rods is usually carried out after binding of the ligate(s) and washing out of any nonspecifically bound components by elution with a step gradient. Usually only one component is eluted, but it is possible to separate several components with different affinities from the ligand by choosing the appropriate step gradient.53
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Monoliths made of poly(glycidyl methacrylate) as well as hollow-fiber membranes, activated with epoxy or other groups, allow the immobilization of any kind of protein ligand.17,26,50 However, membranes made of cellulose fibers do not always provide a sufficient number of active groups for protein immobilization.38 In some cases adequate quantities of different proteins were immobilized to the separation unit, and the corresponding ligate was subsequently isolated on the basis of its affinity.26,42,54 The most frequent and so far most successful use of compact, porous units in affinity chromatography is the isolation of monoclonal and polyclonal antibodies with immobilized protein A or protein G.42,46 Isolated antibodies can subsequently be immobilized to the next epoxy-activated unit. In this way dipeptidyl peptidase IV (DPP IV) and the adhesion protein cellCAM were isolated, both glycoproteins from the plasma membranes of rat liver.55 The elution of antibodies and antigen from their respective separation units with immobilized ligands has to be carried out under rather sharp conditions. This almost always implies the use of buffers with high or low pH or of highly concentrated solutions of chaotropic substances. This will often cause the loss of biological activity of the isolated substances.55 In some cases, as in the isolation of antibodies, the rather short elution time has proved to be an advantage. Because of the fast elution, the isolated immunoglobulins have almost completely retained their bond to the corresponding antigen.42 Besides, the bound ligand is exposed to the denaturating buffer for only a very short time. Consequently, the life of such a unit is much longer than that of a column with the same ligand. A disk with immobilized monoclonal antibodies against the membrane protein DPP IV was used up to 20 times, a column with the same antibodies 4-6 times only.55 Compact, porous disks with immobilized ligands have low backpressure and allow high flow rates during separation. They can therefore be operated in tandems. For instance, several disks with immobilized single calcium-binding proteins from the group of the annexins were used for the isolation of monospecific, polyclonal antibodies against the annexin 65/67. The cross-reacting antibody from the antiserum was bound to the first disk containing immobilized annexins with low molecular masses and subsequently eluted.42 To retain the biological activity of the antigen after separation by immunoaffinity, the use of immobilized antibodies, which have a weaker affinity to the antigen, is recommended. Dissociation of the antigen from the antibody has to be carried out under much milder conditions, using chaotropic reagents (urea, KSCN) or simply choosing a higher pH between 4 and 6, at high salt concentrations. The idea of the mild conditions is to protect the antigen. Again the advantage of affinity chromatography on disks with immobilized, weakly binding antibodies lies in the fast elution provided by this method. Denaturing of the antigen usually requires a certain period of time. Therefore, fast elution increases the probability of the antigen retaining its natural configuration and to the same extent its biological activity. Affinity chromatography with immobilized lectins or with other immobilized ligands with high molecular masses is still rarely used. However, the method has at least the same potential as column affinity chromatography. Fast mass transfer and low nonspecific in-
teraction with the support have to be pointed out as important advantages of chromatography on such supports.55,75 Compact, porous disks and tubes with immobilized concanavalin A (ConA) and with wheat germ agglutinin (WGA) were used for purification of the plasma membrane enzymes 5′-nucleotidase and DPP IV as well as the cell-cell adhesion receptor cell-CAM from the Triton X-100 extract of plasma membranes from the liver. All three components are intrinsic plasma membrane glycoproteins and very sensitive to denaturing agents. Because of the mild isolation conditions, the activity of the two enzymes is retained. In a subsequent step, using a disk with immobilized collagen, the enzymatically active DPP IV can be bound. It is then separated from the other proteins and eluted with a salt gradient, retaining its enzymatic activity.55 Such a purification schema also allows multidimensional chromatography, that is, a sequential arrangement of separation units, similar to the isolation of monospecific, polyclonal antibodies against annexin 65/67.42,67 Practically all lectins can be bound to epoxy-activated disks. However, the capacity of some units containing activated groups, especially of some membranes, is rather low at present (Josic´ and Sˇ trancar, unpublished observation). Apart from disks and tubes, hollow-fiber membrane modules are used for the immobilization of large molecules such as receptors.4,29 Heparin chromatography on membranes and monoliths is another affinity technique suitable for the isolation of large molecules from complex mixtures. One of its applications is the separation of the enriched clotting factor IX from human plasma.56 However, heparin affinity chromatography has shown certain shortcomings, which were not expected after the initial successful experiments. The main reasons for these limitations are the sometimes poor quality of the heparin and considerable batch-to-batch differences. This may cause two separation units produced with heparin from other than the same batch to be substantially different in their respective separation behavior. The phenomenon is less important in the case of separations which follow the “all-or-nothing principle”, e.g., the isolation of antithrombin III from plasma. However, if several components have to be selectively eluted after their binding, e.g., if clotting factors are separated (cf. ref 56), the aforementioned variations in the quality of the heparin will cause considerable difficulties. Affinity Chromatography Using Ligands with Low Molecular Masses. It is much easier to bind ligands with low molecular masses to membranes, disks, tubes, and rods than ligands with high molecular masses. Therefore, this method is widely used. Almost all ligands with low molecular masses can be immobilized to activated materials. This includes dyes, inhibitors, co-enzymes, and other biologically active ligands, which can subsequently enter into specific interaction with components of the sample.37,58,59,62,65 The possibility of looking for adequate ligands in peptide libraries or producing them by combinatorial chemistry creates additional options for the use of this method. Affinity chromatography with ligands with low molecular masses follows rules which are slightly different from those applying to the use of ligands with high molecular masses. Therefore, the method is discussed under a separate heading.
Ind. Eng. Chem. Res., Vol. 38, No. 2, 1999 339 Table 1. Review of Some Commercially Available Fast Separation Materials
a
producer
commercial name for anion-exchange ligand
type of material
Pharmacia TosoHaas Perseptive Biosystems Merck Perseptive Biosystems Sartorius BioRad BIA
Resource Q DEAE NPR TSK Porous Q Fractosep TMA MemSep QMA Sartobind Q15 UNO Q-1 CIMa QA Disk
monodisperse porous particles nonporous particles perfusion particles hollow-fiber membranes staked membranes staked membranes continuous bed monolith
Convective Interaction Media.
An early application of affinity chromatography on small ligands was the isolation of the enzyme carboanhydrase from human erythrocytes.37 A compact, porous disk was used with immobilized p-(aminomethyl)benzolsulfonamide, an inhibitor to carboanhydrase. After binding at high pH and washing out of the nonspecifically bound components, the active enzyme was eluted with a buffer at pH 5.5. Another method with a considerable potential is immobilized metal affinity chromatography (IMAC). Metal-chelating ligands, in most cases iminodiacetic acid (IDA), can be bound to membranes or disks in the same way as to silicagel beads or beads made of natural or synthetic polymers. The groups can chelate metal ions. Usually Cu(II), Zn(II), Ni(II), and Co(II) are used. The chelated metal ions, in turn, are able to interact with histidine residues of proteins. The resulting molecules can be specifically recognized and bound by IMAC, allowing fast isolation from complex mixtures. Reif et al.59 have carried out extensive studies with IMAC. They found that membranes with immobilized metal ions are, in principle, similar to the other membranes and disks which have ligands with low molecular masses. They separated three standard proteins by IMAC, using immobilized Cu(II) ions. If the flow rate is increased from 1 to 15 or 35 mL/min, the separation time is reduced from 40 to 2 min or 60 s, respectively, without any deterioration in resolution. This experiment confirms earlier findings made with IE chromatography.17 There it was shown that separation time was considerably reduced and resolution improved at higher flow rates, not only in the case of membranes but also with compact, porous disks, tubes, and rods. Comparable to the functions of column chromatography, IMAC will be used in the future for the isolation of those recombinant proteins which carry a polyhistidine sequence at the end of a polypeptide chain. This was demonstrated by Reif et al.59 in the case of the recombinant fusion protein of E. coli. Immobilized dyes, above all Cibacron blue58,59,68 and other dyes,58 have been used for the purification of a number of proteins, among others proteins from yeast extract,58,63,68 microbial proteins,63,64,69 and plasma proteins.60 Freitag et al.60 have shown that a combination of AE and Cibacron blue units, applied as “controlled mixedmode interaction chromatography”, allows a better separation of antithrombin III from the contaminating plasma proteins transferrin and bovine serum albumin. Other ligands with low molecular masses such as amino acids can be used for “pseudo”affinity chromatography of diverse proteins. The compact, porous separation units with immobilized histidine are applied to the isolation of immunoglobulin G.62,70 Other immobilized amino acids have been used also, e.g., phen-
ylalanine65 and tryptophan.71 However, separation experiments with membranes such as this have chiefly been carried out with standard proteins. Other Applications. Compact, porous disks and rods have been used for the immobilization of enzymes and subsequent conversion of substrates with either low25,28,37,57 or high49 molecular masses. Such reactors allow a fast conversion of the substrate and can be used on small as well as on large scales. However, a broader practical use for this has so far not been found. Another important application is the removal of endotoxins from protein mixtures. Until now AE membranes72 with immobilized histidine70 have been used for this purpose. Commercially Available Fast Supports. Several fast separation media have recently appeared on the market, and here the development of media for perfusion chromatography can be regarded as a breakthrough in this field.9,10 Table 1 shows the fast media based on particles, membranes, and compact, porous supports. Table 2 contains data on capacity, backpressure, maximum flow rate, and pH stability of such media, as presented by Sˇ trancar.38 It follows from these data that the capacity of fast supports is on the average still lower than that of conventional porous particles (represented here by Mono Q and Mono P). Besides, the capacities of individual types of fast supports differ widely. However, lower capacity can be compensated by the up to 1 order of magnitude faster mass transport. Therefore, the use of these media promises a still more efficient separation process. In the case of CIM, especially compact, porous tubes, capacity has recently been improved by optimizing ligand density on the surface of the support, without any reduction in mass transport. Capacity is now comparable to that of optimized, porous bulk supports.35,75 Similar results have been obtained with the newly developed IE membranes of Sartorius (Barut et al., to be published). The newly developed porous, fast bulk materials (such as the “Resource” supports by Pharmacia and the “Porous” supports by Perseptive Biosystems) as well as membranes and CIM (disks and tubes) offer the possibility of making analytical as well as preparative separation units out of the same support materials. This means that in-process analyses can be performed under conditions similar to those used in preparative chromatography with the same support. This has been demonstrated for in-process analysis with CIM AE tubes, during preparative isolation of clotting factor VIII.28,35 Figure 7 shows some chromatograms carried out with separation units, which contain some fast chromatographic media. The nonporous media, in this case DEAE TSK NPR, are limited by high backpressure (20.5 MPa at a flow rate of 2 mL/min). Consequently, they do not
340 Ind. Eng. Chem. Res., Vol. 38, No. 2, 1999 Table 2. Comparison of Different Chromatographic Media
commercial name
type of material
Mono Q Mono P Resource Q DEAE NPR TSK Porous Q Porous PI Fractosep TMA MemSep QMA MemSep DEAE Sartobind Q15 Sartobind D15 UNO Q-I CIM QA Disk CIM DEAE Disk
porous particles porous particles monodisp. por. part. nonporous particles perfusion particles perfusion particles hollow-fiber membranes staked membranes staked membranes staked membranes staked membranes continuous bed monolith monolith
a
bed bed bed diameter height volumea (mm) (mm) (mL) 5 5 6.4 4.6 5 5 8 25 25 22 22 7 25 25
50 50 30 35 70 70 95 5 5 5 5 35 3 3
0.98 0.98 1.00 0.58 1.37 1.37 4.8 1.10 1.10 1.90 1.90 1.3 1.47 1.47
capacity backpressure (mg of SA/ at 1 mL/min max. flow max. allowed mL of bed of flow rate rate backpressure pH volume) (MPa) (mL/min) (MPa) stability 44 51 48 low 15 15 5 13 16 15 6 31 36 33
1.2 1.2 0.2 10.5 0.4 0.4
